Artificial Cargo Research Articles

Improving the mechanism of port handling complexes

The issue of modernization of cargo flow mechanization schemes, while optimizing logistics costs with the aim of increasing port capacity, was considered. The process of cargo transshipment using various lifting and transport machines is studied, the schemes of complex mechanization of port terminals are presented. The main schemes of complex mechanization in the port are analyzed on the example of transportation of artificial cargo - slab. The leading link of these schemes is the gantry crane, for which a dynamic 3D model has been developed, which allows you to create a scenario of the transport operation using simulation systems. With the help of the simulator, it is possible to evaluate indicators of performance and reliability of the crane. The results of the calculations of the characteristics of the warehouse, its capacity and the layout of the slabs are given. To build a logistics algorithm for cargo transportation operations, input data (structure of freight traffic, energy costs, logistics costs), objects (lifting and transport equipment, ships, railway transport) and output information in the form of a report with simulation results are determined. The paper proposes an algorithm for the simulation model of the port transshipment complex, which allows you to visualize the technological process of transshipment operations and optimize the parameters according to the specified quality criteria. The materials of the article can be useful to specialists of the mechanization services of sea and river ports, as well as used in the educational process as tasks for laboratory, practical and course work

Biomimicking Extracellular Vesicles with Fully Artificial Ones: A Rational Design of EV-BIOMIMETICS toward Effective Theranostic Tools in Nanomedicine.

Extracellular Vesicles (EVs) are the protagonists in cell communication and membrane trafficking, being responsible for the delivery of innumerable biomolecules and signaling moieties. At the moment, they are of paramount interest to researchers, as they naturally show incredibly high efficiency and specificity in delivering their cargo. For these reasons, EVs are employed or inspire the development of nanosized therapeutic delivery systems. In this Perspective, we propose an innovative strategy for the rational design of EV-mimicking vesicles (EV-biomimetics) for theranostic scopes. We first report on the current state-of-the-art use of EVs and their byproducts, such as surface-engineered EVs and EV-hybrids, having an artificial cargo (drug molecule, genetic content, nanoparticles, or dye incorporated in their lumen). Thereafter, we report on the new emerging field of EV-mimicking vesicles for theranostic scopes. We introduce an approach to prepare new, fully artificial EV-biomimetics, with particular attention to maintaining the natural reference lipidic composition. We overview those studies investigating natural EV membranes and the possible strategies to identify key proteins involved in site-selective natural homing, typical of EVs, and their cargo transfer to recipient cells. We propose the use also of molecular simulations, in particular of machine learning models, to approach the problem of lipid organization and self-assembly in natural EVs. We also discuss the beneficial feedback that could emerge combining the experimental tests with atomistic and molecular simulations when designing an EV-biomimetics lipid bilayer. The expectations from both research and industrial fields on fully artificial EV-biomimetics, having the same key functions of natural ones plus new diagnostic or therapeutic functions, could be enormous, as they can greatly expand the nanomedicine applications and guarantee on-demand and scalable production, off-the-shelf storage, high reproducibility of morphological and functional properties, and compliance with regulatory standards.

Cargo surface fluidity can reduce inter-motor mechanical interference, promote load-sharing and enhance processivity in teams of molecular motors.

In cells, multiple molecular motors work together as teams to carry cargoes such as vesicles and organelles over long distances to their destinations by stepping along a network of cytoskeletal filaments. How motors that typically mechanically interfere with each other, work together as teams is unclear. Here we explored the possibility that purely physical mechanisms, such as cargo surface fluidity, may potentially enhance teamwork, both at the single motor and cargo level. To explore these mechanisms, we developed a three dimensional simulation of cargo transport along microtubules by teams of kinesin-1 motors. We accounted for cargo membrane fluidity by explicitly simulating the Brownian dynamics of motors on the cargo surface and considered both the load and ATP dependence of single motor functioning. Our simulations show that surface fluidity could lead to the reduction of negative mechanical interference between kinesins and enhanced load sharing thereby increasing the average duration of single motors on the filament. This, along with a cooperative increase in on-rates as more motors bind leads to enhanced collective processivity. At the cargo level, surface fluidity makes more motors available for binding, which can act synergistically with the above effects to further increase transport distances though this effect is significant only at low ATP or high motor density. Additionally, the fluid surface allows for the clustering of motors at a well defined location on the surface relative to the microtubule and the fluid-coupled motors can exert more collective force per motor against loads. Our work on understanding how teamwork arises in cargo-coupled motors allows us to connect single motor properties to overall transport, sheds new light on cellular processes, reconciles existing observations, encourages new experimental validation efforts and can also suggest new ways of improving the transport of artificial cargo powered by motor teams.

Analysis of the current state and development trends of the market of containerized and artificial cargo transportation in Ukraine

У статті авторами проаналізовано сучасний стан вітчизняного ринку перевезень тарно-штучних вантажів в Україні, визначені основні проблеми та переваги перевезення тарно-штучних вантажів різними видами транспорту, досліджені попит та пропозиції на ринку перевезення тарно-штучних вантажів, виокремлено стратегічні напрямки розвитку перевезення тарно-штучних вантажів різними видами транспорту в умовах пандемії COVID-19. Перспективами подальших досліджень у даному напрямі є дослідження світового ринку перевезень тарно-штучних вантажів у вигляді збірних вантажів за допомогою контейнеризації. Ключові слова: тарно-штучні вантажі, морський транспорт, авіатранспорт, автотранспорт, залізничний транспорт, ринок перевезень.

Tuneable Time Delay in the Burst Release from Oxidation‐Sensitive Polymersomes Made by PISA

Reactive polymersomes represent a versatile artificial cargo carrier system that can facilitate an immediate release in response to a specific stimulus. The herein presented oxidation‐sensitive polymersomes feature a time‐delayed release mechanism in an oxidative environment, which can be precisely adjusted by either tuning the membrane thickness or partial pre‐oxidation. These polymeric vesicles are conveniently prepared by PISA allowing the straightforward and effective in situ encapsulation of cargo molecules, as shown for dyes and enzymes. Kinetic studies revealed a critical degree of oxidation causing the destabilization of the membrane, while no release of the cargo is observed beforehand. The encapsulation of glucose oxidase directly transforms these polymersomes into glucose‐sensitive vesicles, as small molecules including sugars can passively penetrate their membrane. Considering the ease of preparation, these polymersomes represent a versatile platform for the confinement and burst release of cargo molecules after a precisely adjustable time span in the presence of specific triggers, such as H2O2 or glucose.

Recent progress of 3D-printed microneedles for transdermal drug delivery.

Microneedles as novel transdermal drug delivery systems have lately attracted extensive attention due to their distinguished properties, including improved patient compliance and self-administration, compared to traditional parenteral administrations such as intravenous injection, intramuscular injection and subcutaneous injection. However, the great difficulties of precisely manufacturing those microneedles and patches within micro scale have strongly retarded their commercialization and clinical applications, particularly for the personalized medicine. Recently, numerous researches of utilizing 3D-priting process to fabricate transdermal drug delivery systems have been reported, not only adopting versatile printing methodologies, but also utilizing with different formulation strategies, to fabricate both artificial cargo delivery systems and sophisticated bio-inspired microneedles. This review aims to summarize those lately reported studies and to elaborate their advantages and limitations, discussing promising potential applications as novel drug delivery systems.

Influence of inertial and geometrical parameters of vacuum capturing devices on permisable efforts for maintenance of containerized artificial cargo

Influence of inertial and geometrical parameters of vacuum capturing devices on permisable efforts for maintenance of containerized artificial cargo

Nanoerythrosome-functionalized biohybrid microswimmers.

Biohybrid microswimmers, which are realized through the integration of motile microscopic organisms with artificial cargo carriers, have a significant potential to revolutionize autonomous targeted cargo delivery applications in medicine. Nonetheless, there are many open challenges, such as motility performance and immunogenicity of the biological segment of the microswimmers, which should be overcome before their successful transition to the clinic. Here, we present the design and characterization of a biohybrid microswimmer, which is composed of a genetically engineered peritrichously flagellated Escherichia coli species integrated with red blood cell-derived nanoliposomes, also known as nanoerythrosomes. Initially, we demonstrated nanoerythrosome fabrication using the cell extrusion technique and characterization of their size and functional cell membrane proteins with dynamic light scattering and flow cytometry analyses, respectively. Then, we showed the construction of biohybrid microswimmers through the conjugation of streptavidin-modified bacteria with biotin-modified nanoerythrosomes by using non-covalent streptavidin interaction. Finally, we investigated the motility performance of the nanoerythrosome-functionalized biohybrid microswimmers and compared it with the free-swimming bacteria. The microswimmer design approach presented here could lead to the fabrication of personalized biohybrid microswimmers from patients' own cells with high fabrication efficiencies and motility performances.

GmGPA3 is involved in post-Golgi trafficking of storage proteins and cell growth in soybean cotyledons

As the major nutritional component in soybean seeds storage proteins are initially synthesized on the endoplasmic reticulum as precursors and subsequently delivered to protein storage vacuoles (PSVs) via the Golgi-mediated pathway where they are converted into mature subunits and accumulated. However, the molecular machinery required for storage protein trafficking in soybean remains largely unknown. In this study, we cloned the sole soybean homolog of OsGPA3 that encodes a plant-unique kelch-repeat regulator of post-Golgi vesicular traffic for rice storage protein sorting. A complementation test showed that GmGPA3 could rescue the rice gpa3 mutant. Biochemical assays verified that GmGPA3 physically interacts with GmRab5 and its guanine exchange factor (GEF) GmVPS9. Expression of GmGPA3 had no obvious effect on the GEF activity of GmVPS9 toward GmRab5a. Notably, knock-down of GmGPA3 disrupted the trafficking of mmRFP-CT10 (an artificial cargo destined for PSVs) in developing soybean cotyledons. We identified two putative GmGPA3 interacting partners (GmGMG3 and GmGMG11) by screening a yeast cDNA library. Overexpression of GmGPA3 or GmGMG3 caused shrunken cotyledon cells. Our overall results suggested that GmGPA3 plays an important role in cell growth and development, in addition to its conserved role in mediating storage protein trafficking in soybean cotyledons.

The Role of the Cover-Neck Bundle in Multi-Motor Transport against Load in Cells

Kinesin motor proteins share a highly-conserved catalytic motor domain responsible for coupling the chemical energy of ATP hydrolysis to force generation and microtubule-directed functions. Previous studies of kinesin-1 suggested that ATP binding to the motor domain induces the formation of the cover-neck bundle (CNB), a beta-sheet between β0 at the N-terminus (the coverstrand) and β9 in the first half of the necklinker. The CNB was shown to contribute to force generation by single kinesin-1 motors under purified conditions and has been predicted to function in force generation across the kinesin superfamily. However, differences in coverstrand and necklinker sequences make the role of the CNB across different kinesin families unclear. We adapted artificial cargo systems to delineate key features of the CNB during multi-motor transport against low and high loads in cells. Our results indicate that in teams, kinesin-1 motors can robustly transport cargo against high- and low-load in cells. Molecular modeling was used to predict mutations in the coverstrand and necklinker that disrupt and shorten beta-sheet formation. Experimental testing of these mutations shows that impaired beta-sheet formation compromised the capacity for teams of kinesin-1 motors to transport high-load cargo but not low-load cargo to the periphery of cells. Surprisingly, truncation of the region N-terminal to the coverstrand, predicted to have a minimal role in CNB formation, severely crippled the capacity for teams of kinesin-1 motors to transport both low- and high-load cargoes in cells. We plan to extend these studies to additional kinesin families to identify distinct motility dependencies on formation of the CNB.

Long‐Range Transport of Giant Vesicles along Microtubule Networks

We report on a minimal system to mimic intracellular transport of membrane-bounded, vesicular cargo. In a cell-free assay, purified kinesin-1 motor proteins were directly anchored to the membrane of giant unilamellar vesicles, and their movement studied along two-dimensional microtubule networks. Motion-tracking of vesicles with diameters of 1-3 μm revealed traveling distances up to the millimeter range. The transport velocities were identical to velocities of cargo-free motors. Using total internal reflection fluorescence (TIRF) microscopy, we were able to estimate the number of GFP-labeled motors involved in the transport of a single vesicle. We found that the vesicles were transported by the cooperative activity of typically 5-10 motor molecules. The presented assay is expected to open up further applications in the field of synthetic biology, aiming at the in vitro reconstitution of sub-cellular multi-motor transport systems. It may also find applications in bionanotechnology, where the controlled long-range transport of artificial cargo is a promising means to advance current lab-on-a-chip systems.

Effect of spastic paraplegia mutations in KIF5A kinesin on transport activity

Hereditary spastic paraplegia (HSP) is a neurodegenerative disease caused by motoneuron degeneration. It is linked to at least 30 loci, among them SPG10, which causes dominant forms and originates in point mutations in the neuronal Kinesin-1 gene (KIF5A). Here, we investigate the motility of KIF5A and four HSP mutants. All mutations are single amino-acid exchanges and located in kinesin's motor or neck domain. The mutation in the neck (A361V) did not change the gliding properties in vitro, the others either reduced microtubule affinity or gliding velocity or both. In laser-trapping assays, none of the mutants moved more than a few steps along microtubules. Motility assays with mixtures of homodimeric wild-type, homodimeric mutant and heterodimeric wild-type/mutant motors revealed that only one mutant (N256S) reduces the gliding velocity at ratios present in heterozygous patients, whereas the others (K253N, R280C) do not. Attached to quantum dots as artificial cargo, mixtures involving N256S mutants produced slower cargo populations lagging behind in transport, whereas mixtures with the other mutants led to populations of quantum dots that rarely bound to microtubules. These differences indicate that the dominant inheritance of SPG10 is caused by two different mechanisms that both reduce the gross cargo flux, leading to deficient supply of the synapse.

Quantum dots provide an optical signal specific to full collapse fusion of synaptic vesicles

Synaptic vesicles are responsible for releasing neurotransmitters and are thus essential to brain function. The classical mode of vesicle recycling includes full collapse of the vesicle into the plasma membrane and clathrin-mediated regeneration of a new vesicle. In contrast, a nonclassical mode known as "kiss-and-run" features fusion by a transient fusion pore without complete loss of vesicle identity and offers possible advantages for increasing the throughput of neurotransmission. Studies of vesicular traffic have benefited greatly from fluorescent probes like FM dyes and synaptopHluorin. However, intrinsic properties of these probes limit their ability to provide a simple and precise distinction between classical and nonclassical modes. Here we report a novel optical probe specific to full collapse fusion, capitalizing on the size and superior photo-properties of photoluminescent quantum dots (Qdots). Qdots with exposed carboxyl groups were readily taken up by synaptic vesicles in an activity-, Ca(2+)-, and clathrin-dependent manner. Electron microscopy showed that Qdots were harbored within individual vesicles in a 1:1 ratio. The release of Qdots was activity- and Ca(2+)-dependent, similar to FM dyes. As artificial cargo, approximately 15 nm in diameter, Qdots will not escape vesicles during kiss-and-run but only with full collapse fusion. Strikingly, Qdots unloaded with kinetics substantially slower than destaining of FM dye, indicating that full-collapse fusion contributed only a fraction of all fusion events. As a full-collapse-fusion-responsive reporter, Qdots will likely promote better understanding of vesicle recycling at small CNS nerve terminals.

Core-like Particles of an Enveloped Animal Virus Can Self-Assemble Efficiently on Artificial Templates

Alphaviruses are animal viruses holding great promise for biomedical applications as drug delivery vectors, functional imaging probes, and nanoparticle delivery vesicles because of their efficient in vitro self-assembly properties. However, due to their complex structure, with a protein capsid encapsulating the genome and an outer membrane composed of lipids and glycoproteins, the in-vitro self-assembly of virus-like particles, which have the functional virus coat but carry an artificial cargo, can be challenging. Fabrication of such alphavirus-like particles is likely to require a two-step process: first, the assembly of a capsid structure around an artificial core, second the addition of the membrane layer. Here we report progress made on the first step: the efficient self-assembly of the alphavirus capsid around a functionalized nanoparticle core.

Dynactin drives dynein without microtubule binding

Dynactin might bind to and organize microtubules. But it doesn't need to bind microtubules to jump-start dynein motoring, Kim et al. report on page 641. Figure 1 Organelle cargo distribution is similar in wild-type cells (left) and cells expressing nonmicrotubule-binding dynactin (right), indicating that transport remains intact. Dynactin retrieves microtubule motors such as dynein from the cytoplasm and docks them onto their cargo. Dynactin also anchors the minus ends of microtubules on the centrosome. One dynactin isoform that is found in human neurons lacks its microtubule-binding domain (MBD). Kim and colleagues thus supposed that dynactin might be functional even without locking onto microtubules. They found that indeed cargo transport does not rely on the MBD. With or without a functional MBD, dynactin helped move four different physiological cargos, including vesicles and proteins, just as far along microtubules and at the same velocity and frequency. Dynactin's MBD was previously thought to increase dynein's processivity by giving it a second hand to hang onto microtubules. But this idea was based largely on studies of the transport of dynein-coated beads. This artificial cargo, or the way dynein and dynactin were attached to it, might have altered the normal relations between dynein and dynactin observed in a cell. Not all dynactin activities were unaffected by the loss of the MBD, however. Cells expressing the MBD-deleted form commonly arrested in prometaphase with multipolar spindles, suggesting a failure of microtubules to correctly conjoin in centrosomes and thus a role of dynactin in latching together microtubules.

Evaluation of an LC8-Binding Peptide for the Attachment of Artificial Cargo to Dynein

The limited cytoplasmic mobility of nonviral gene carriers is likely to contribute to their low transfection efficiency. This limitation could be overcome by mimicking the viral strategy of recruiting the dynein motor complex for efficient transport toward the host cell nucleus. A promising approach for attaching artificial cargo to dynein is through an adaptor peptide that binds the 8 kDa light chain (LC8) found in the cargo-binding region of the dynein complex. Several viral proteins that bind LC8 have in common an LC8-binding motif defined by (K/R)XTQT. Short peptides containing this motif have also been shown to bind recombinant LC8 in vitro. However, since the majority of intracellular LC8 exists outside of the dynein complex, it remains unclear whether peptides displaying this LC8-binding motif can access and bind to dynein-associated LC8. In this study, we employed biochemical analysis to investigate the feasibility of attaching artificial cargo to the dynein motor complex using a peptide displaying the well-characterized LC8-binding motif. We report that free intracellular LC8 bound specifically to an LC8-binding (TQT) peptide and not to a control peptide with a mutated LC8-binding motif. However, a similar binding interaction between the TQT peptide and intracellular dynein was not detected. To determine whether dynein binding of the TQT peptide was prevented by competition with free intracellular LC8 or due to the inability of the peptide to access its LC8 binding site in the dynein complex, the TQT peptide was evaluated for its ability to bind either purified LC8 or purified dynein. Our results demonstrate that, while the TQT peptide readily binds free LC8, it cannot bind to dynein-associated LC8. The results emphasize the need to identify functional dynein-binding peptides and highlight the importance of designing peptides that bind to the intact dynein motor complex.

Kinesin's tail domain is an inhibitory regulator of the motor domain.

When not bound to cargo, the motor protein kinesin is in an inhibited state that has low microtubule-stimulated ATPase activity. Inhibition serves to minimize the dissipation of ATP and to prevent mislocalization of kinesin in the cell. Here we show that this inhibition is relieved when kinesin binds to an artificial cargo. Inhibition is mediated by kinesin's tail domain: deletion of the tail activates the ATPase without need of cargo binding, and inhibition is re-established by addition of exogenous tall peptide. Both ATPase and motility assays indicate that the tail does not prevent kinesin from binding to microtubules, but rather reduces the motor's stepping rate.