Small Cargo Research Articles

Abstract P3140: Extracellular Vesicle MicroRNA Cargo Drives Ventricular Arrhythmia In Heart Failure Patients By Recapitulating Developmental Genes

Heart Failure (HF) is the leading cause of death worldwide, of which arrhythmias account for 50%. The relationship between HF and arrhythmias is closely intertwined. Since novel biomarkers associated with HF that lead to these arrhythmogenic events are an area of unmet need, we aimed to identify the specific plasma Extracellular vesicles (EV)-driven microRNA cargo that could predispose ventricular cardiomyocytes to arrhythmia during HF as well as delineate targetable pathways to rescue the arrhythmic phenotype. We treated human pluripotent stem cell-derived ventricular cardiomyocytes (hPSC-VCM) with plasma-derived EVs from HF and control samples and observed significantly prolonged action potential duration in HF-treated hPSC-VCM (94.41 ± 16.13 msec) using optical voltage mapping. Small RNA cargo of the plasma EVs were sequenced to reveal 26 downregulated and 70 upregulated differentially expressed miRNAs between HF and control. Upon deconvolution analysis, many of these plasma miRNA transcripts were found to be enriched in the brain, RBCs, monocytes and other tissues. We also sequenced the long-RNA transcriptome of the recipient cardiomyocytes. We prioritized 33 targets for qRT-PCR validation based on predicted protein-coding targets of miRNA, and 10 of these mRNAs were different between HF and control EV-treated hPSC-VCMs. Interestingly, some mRNAs (EIF4E, NEDD4, ID3, ID1, CDKN1A, HSPD1) are known to be expressed during development. Pathway enrichment analysis of the validated targets converged in TGF beta signaling and Hippo pathways, implicated in cardiac developmental arrhythmogenic abnormalities. In conclusion, circulating EV small RNA cargo of HF patients can alter the electrophysiological properties of cardiomyocytes, many of which are derived, predominantly from the brain. More importantly, the arrhythmic phenotype is triggered when there is a recapitulation of developmental genes induced by the EV-derived miRNAs. While these miRNAs could be identified as putative biomarkers for SCD, targeting these developmental genes may be a fruitful therapeutic approach to rescue electrical remodeling in HF patients.

Ageing-associated small RNA cargo of extracellular vesicles

ABSTRACT Previous work on murine models and humans demonstrated global as well as tissue-specific molecular ageing trajectories of RNAs. Extracellular vesicles (EVs) are membrane vesicles mediating the horizontal transfer of genetic information between different tissues. We sequenced small regulatory RNAs (sncRNAs) in two mouse plasma fractions at five time points across the lifespan from 2–18 months: (1) sncRNAs that are free-circulating (fc-RNA) and (2) sncRNAs bound outside or inside EVs (EV-RNA). Different sncRNA classes exhibit unique ageing patterns that vary between the fcRNA and EV-RNA fractions. While tRNAs showed the highest correlation with ageing in both fractions, rRNAs exhibited inverse correlation trajectories between the EV- and fc-fractions. For miRNAs, the EV-RNA fraction was exceptionally strongly associated with ageing, especially the miR-29 family in adipose tissues. Sequencing of sncRNAs and coding genes in fat tissue of an independent cohort of aged mice up to 27 months highlighted the pivotal role of miR-29a-3p and miR-29b-3p in ageing-related gene regulation that we validated in a third cohort by RT-qPCR.

#3633 DIRECT EXOSOME TRANSFECTION WITH FLUORESCENTLY LABELED SMALL RNAS IS A USEFUL TOOL FOR EXOSOMAL CARGO TRAFFICKING AND RNAI IN CULTURED PODOCYTES

Abstract Background and Aims Podocytes, highly-specialized renal epithelial cells, display the outer aspect of the glomerular filtration barrier. Changes of their complex 3D morphology with interdigitating foot processes are the leading cause for 80% of chronic kidney disease (CKD) cases. During injury development podocytes release increased amounts of extracellular vesicles like exosomes with disease-specific small RNA cargo composition. Exosomes are referred to as important means of cell-cell communication and display an interesting tool for understanding intercellular communication in CKD pathogenesis. It is not clear if exosomes could serve as delivery system for specific small RNAs to podocytes as potential therapeutic approach. To address this, we investigated if isolated exosomes, directly transfected with fluorescently labeled small RNAs, are suitable for exosomal cargo trafficking and for functional delivery of small RNAs in cultured podocytes. Method We isolated exosomes from cultured, murine podocytes and transfected them directly with Cy3-labeled siRNAs and miRNAs using ExoFect siRNA/miRNA transfection kit. The transfected exosomes were characterized by transmission electron microscopy and Western blot. The fluorescently labeled exosomes were incubated with cultured, murine podocytes and exosome cargo uptake was observed time- and localization dependently by confocal laser-scanning microscopy. Isolated exosomes were also transfected with filamin A (FlnA)-siRNAs and pre-miR-21 and were co-incubated with cultured, murine podocytes. Transfection- and knockdown efficiency were confirmed by RT-qPCR, Western blot and immunofluorescence microscopy, respectively. Results The isolated exosomes displayed a typical shape and size of 20 nm revealed by transmission electron microscopy. This was also consistent after transfection. They also exhibited exosomal marker proteins CD9 and TSG101 before and after transfection. We could show that cultured, murine podocytes take up fluorescently labeled exosomal RNAs. We could observe an increasing amount of fluorescently labeled RNA intracellularly compared to a decreasing amount of fluorescently labeled RNAs in the periphery over 1 week of treatment indicating a time-dependent exosome uptake by podocytes. Transfection of exosomes with FlnA-siRNAs led to a decrease of FlnA-expression in podocytes co-cultured with transfected exosomes as revealed by immunofluorescence microscopy and Western blot. Podocytes incubated with pre-miR-21-transfected exosomes exhibited a 338-fold upregulation of miR-21 in RT-qPCR. Conclusion Here we show that direct transfection of exosomes with fluorescently labeled small RNAs is a useful tool for exosome cargo trafficking in podocytes. Additionally, we proved that small RNAs transfected into exosomes can be functionally used for RNAi in cultured podocytes. This might be a novel strategy for regulating protein and miRNA expression in cultured podocytes.

Framework Nucleic Acids: A Promising Vehicle for Small Molecular Cargos.

Framework nucleic acids (FNAs), which are a series of self-assembled DNA nanostructures, are highly versatile tools for engineering intelligent molecular delivery vehicles. Owing to their precise and controllable design and construction, excellent programmability and functionality, as well as favorable intercalation between DNA and small molecules, FNAs provide a promising approach for small molecule delivery. This review discusses the advantages, applications, and current challenges of FNAs for the delivery of small molecular cargo. First, the physicochemical and biological properties that make FNAs favorable for the transport of small molecules are introduced. Thereafter, the classification of loaded cargos and the mechanism of combination between small molecules and FNAs are summarized in detail, and recent research on FNA-based delivery systems and their applications are highlighted. Finally, the challenges and prospects of FNA nanocarriers are discussed to advance their exploitation and clinical adoption.

Glass multi-gap RPCs for photon-transmission imaging

In the present R&D, multi-gap resistive plate chambers (MRPCs) are studied for photon non-destructive inspection of small airport baggages and cargos. Six-gap RPCs for line-scan operations are constructed with soda-lime glass with a thicknesses of 0.53 mm. The typical detector characteristics such as the operation high voltage (HV), the efficiency, and the strip multiplicity are measured by using 661.7 keV photons emitted from a 137Cs source with a current activity of 4.74 GBq. The efficiency for the 661.7 keV photons measured by the MRPC detector at the HV applied to obtain the photon-transmission images is 5.5%, which is about 84% of that predicted by a GEANT simulation. To demonstrate the quality operation of the MRPC detectors, we conduct line-scan operations for various objects and produce the photon-transmission images that barely identify 2 mm-thick stainless steel objects when measured with a color depth (contrast) of the image pixels of about 100.

Abstract 2370: The role of extracellular vesicles in the glioblastoma tumor microenvironment

Abstract Glioblastoma (GBM) is the most malignant form of gliomas and is the most common and lethal primary brain tumor in adults. Within the tumor, there is a subtype of cells deemed brain tumor initiating cells (BTICs), which contribute to tumor progression, therapy resistance, and tumoral heterogeneity. The tumor microenvironment, surrounding tumors, contains an ecosystem of cells in which BTICs can interact with and further promote tumor progression. In the lateral wall of the lateral ventricles, the neurogenic niche of the subventricular zone (SVZ), contains neural stem cells (NSCs) that self-renew and generate highly migratory neural progenitor cells (NPCs). Interestingly, GBM tumors proximal to the SVZ are more proliferative, migratory, and negatively impact the survival of patients. We have previously observed that the presence of GBM induces reciprocal changes to the SVZ altering its neurogenic capacity and resulting in a more proliferative tumor. BTICs share the similar self-renewal capacity to NPCs and may exert intercellular communication resulting in increase malignancy features. However, the exact mechanisms of how GSCs communicate with NPCs is not well understood. Cancer cells release higher amounts of extracellular vesicles (EVs) than non-malignant cells. Cancer EVs help communicate with other nearby cells, leading to promotion of tumorigenesis. EVs are a heterogenous group of cell-derived membranous structures that serve as means of intercellular communication, allowing for cells to exchange proteins, lipids, RNA, and other genetic material. They can be further characterized into two main subtypes, small EVs, that range from 30-150nm, and large EVs, that range from 50-1000nm. EVs derived from gliomas or non-glioma cells in the tumor microenvironment are involved in tumor cell proliferation, invasion, malignancy, and drug resistance. The overarching goal of this project is to characterize BTIC-derived small EV cargo to identify proteins and RNAs that modify the GBM tumor microenvironment. We hypothesize that GBM-derived small extracellular vesicles alter the phenotype of non-cancer cells in the tumor microenvironment through distinct cargo contained in small extracellular vesicles. The results of these experiments will help us to elucidate what exact small EV cargo impacts the tumor microenvironment, with the potential of revealing possible therapeutic targets for GBM.The optimized protocol gives us the ability to enrich our desired EV population of small EVs from BTICs. BTIC-derived small EVs influence recipient NPCs by significantly upregulating markers for stemness, tumor aggressiveness, and invasion, as well as increasing NPC migration. Future experiments will include trying to establish a cell-specific labeling system in vitro to label EVs from BTICs, to allow for direct co-culture of BTICs with NPCs and other cells in the tumor microenvironment. Citation Format: Marissa Russo. The role of extracellular vesicles in the glioblastoma tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2370.

Abstract 3746: Dynamic characterization of small RNAs in non small cell lung cancer exosomes under immune-checkpoint inhibitor treatments

Abstract Immunotherapy based on Immune Checkpoint blockade (ICB) has become a significant therapeutic option for advanced Non Small Cell Lung Cancer (NSCLC) patients. However, there is an urgent need to find novel biomarkers that to reliably stratify good responders to immunotherapy. Currently, the available biomarkers are not specific enough. Exosomes are small membrane vesicles with sizes of 30-100 nm secreted by most cell types including cancer cells. Exosomes operate as an intercellular communication system by sending proteins, mRNA and miRNAs among other relevant RNA molecules. Exosomes enriched with miRNAS are involved in proliferation, differentiation, maduration and immune cell activation. Moreover, in cancer cells miRNA and other small RNA molecule expressions are dysregulated. Exosomes produced from cancer patient's plasma have been shown to be accurate diagnostic tools for the disease. In this study we profiled miRNAs and other small RNA cargo by exosome of plasma samples from 77 Non Small Cell Lung Cancer (NSCLC) metastatic patients before and after the first cycle of immunotherapy to evaluate the potentiality of predicting response to immunotherapy. We perform exosomes together with RNA isolation and small RNAseq sequencing from plasma samples before and after the first ICB cycle. Two independent softwares were used to identify small RNAs (RNAtoolbox and mirMaster). Prior ICB treatment we did not find differentially expressed miRNAs or other small RNA between good and bad responders. Interestingly we identified 12 exosomal miRNA differentially expressed between good and bad responders after the first cycle of ICB. Intriguingly, levels of miR-134-5p, miR-142-3p, miR-143-3p among others previously associated with NSCLC were found to be considerably higher in the good responder group than the bad responder group. Regarding other smallRNA molecules we observed a great variety and variability of piRNA, rRNA, scaRNA, lncRNA, snoRNA, snRNA, miscRNA and circRNA. To address the function of miRNA and other differentially expressed RNA molecules, we consulted KEGG, GO and Reactome for gene regulatory networks. Interestingly, KEGG results show pathways in cancer as top hit and Reactome highlight Immune System and cancer hits. In conclusion, we observed that patients that have favorable response to ICB have distinctive plasma exosomal miRNA patterns that could be used as possible biomarkers for predicting the effectiveness of immunotherapy in advanced NSCLC patients Citation Format: Maria Garrido-Barros, Javier Oliver, Juan Luis Onieva, Beatriz Martinez-Galvez, Jaime Duddelman, Antonio Rueda, Elisabeth Perez, Emilio Alba, Inmaculada Ramos, Juan Zafra, Manuel Cobo, Isabel Barragán. Dynamic characterization of small RNAs in non small cell lung cancer exosomes under immune-checkpoint inhibitor treatments. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3746.

Dynein Dysfunction Prevents Maintenance of High Concentrations of Slow Axonal Transport Cargos at the Axon Terminal: A Computational Study.

Here, we report computational studies of bidirectional transport in an axon, specifically focusing on predictions when the retrograde motor becomes dysfunctional. We are motivated by reports that mutations in dynein-encoding genes can cause diseases associated with peripheral motor and sensory neurons, such as type 2O Charcot-Marie-Tooth disease. We use two different models to simulate bidirectional transport in an axon: an anterograde-retrograde model, which neglects passive transport by diffusion in the cytosol, and a full slow transport model, which includes passive transport by diffusion in the cytosol. As dynein is a retrograde motor, its dysfunction should not directly influence anterograde transport. However, our modeling results unexpectedly predict that slow axonal transport fails to transport cargos against their concentration gradient without dynein. The reason is the lack of a physical mechanism for the reverse information flow from the axon terminal, which is required so that the cargo concentration at the terminal could influence the cargo concentration distribution in the axon. Mathematically speaking, to achieve a prescribed concentration at the terminal, equations governing cargo transport must allow for the imposition of a boundary condition postulating the cargo concentration at the terminal. Perturbation analysis for the case when the retrograde motor velocity becomes close to zero predicts uniform cargo distributions along the axon. The obtained results explain why slow axonal transport must be bidirectional to allow for the maintenance of concentration gradients along the axon length. Our result is limited to small cargo diffusivity, which is a reasonable assumption for many slow axonal transport cargos (such as cytosolic and cytoskeletal proteins, neurofilaments, actin, and microtubules) which are transported as large multiprotein complexes or polymers.

Solutions to the Problem of Freight Transport Flows in Urban Logistics

The operation of a city’s logistics system is associated with many problematic issues, for example, intense pollution and negative impacts on the environment, uneven intensity of traffic flows, and other problems. This article focuses on heavy transport because it causes disruptions in the supply system and affects timely customer service. Optimization processes are associated with route selection, deployment of logistics centers, and the need for cargo consolidation–de-consolidation, which leads to the possibility of using the PPP (public–private partnership) method in practice. A unique aspect of this research is related to the problems of the first and last mile and the use of elements of the “sharing economy”. Therefore, this article aims to analyze the problematic challenges of the first and last mile and the role and significance of heavy transport in urban logistics. For that purpose, in addition to an analysis of scientific sources, an expert survey was conducted and responses related to the questions on optimizing heavy traffic flows in city logistics were analyzed. The study data were processed using Kendall’s correlation coefficient, the concordance method, and the consistency coefficient. During analysis, using the average rank transformation to weights (ARTIW) method, the subjective normalized weights of the main factors that have the greatest influence on a city’s logistics system were determined. Groups of interested parties are also named separately, for whom the results of the study and the formulated decision may be relevant. Based on the results of the research, a recommendation was formulated for the development of small cargo collection and distribution services through self-service terminals located on the outskirts of cities. Proposals are presented to reorganize the system, provide details of new infrastructure elements and suburban terminals, and encourage for the use of environmentally friendly vehicles with a lower carrying capacity.

Combining stimuli-responsive colloids and focused ultrasound for drug delivery

Over the years my research group has engineered colloids that respond to changes in pressure or temperature locally induced by focused ultrasound. We designed thermosensitive liposomes that released small or large molecular cargo rapidly when heated above normal body temperature by only a few degrees. Potentially, these liposomes can be combined with MR-guided focused ultrasound for noninvasive and image-guided triggered drug delivery. Separately, we developed liquid perfluorocarbon nanoemulsions that can be vaporized with high amplitude acoustic pulses. The resultant bubbles can then be used to facilitate cavitation-mediated permeation of biological interfaces, including cell membranes and the blood-brain barrier. This presentation will review some of our key observations and will discuss the potential for translating these technologies.

Modular Chemical Construction of IgG-like Mono- and Bispecific Synthetic Antibodies (SynAbs).

In recent years there has been rising interest in the field of protein-protein conjugation, especially related to bispecific antibodies (bsAbs) and their therapeutic applications. These constructs contain two paratopes capable of binding two distinct epitopes on target molecules and are thus able to perform complex biological functions (mechanisms of action) not available to monospecific mAbs. Traditionally these bsAbs have been constructed through protein engineering, but recently chemical methods for their construction have started to (re)emerge. While these have been shown to offer increased modularity, speed, and for some methods even the inherent capacity for further functionalization (e.g., with small molecule cargo), most of these approaches lacked the ability to include a fragment crystallizable (Fc) modality. The Fc component of IgG antibodies offers effector function and increased half-life. Here we report a first-in-class disulfide rebridging and click-chemistry-based method for the generation of Fc-containing, IgG-like mono- and bispecific antibodies. These are in the FcZ-(FabX)-FabY format, i.e., two distinct Fabs and an Fc, potentially all from different antibodies, attached in a homogeneous and covalent manner. We have dubbed these molecules synthetic antibodies (SynAbs). We have constructed a T cell-engager (TCE) SynAb, FcCD20-(FabHER2)-FabCD3, and have confirmed that it exhibits the expected biological functions, including the ability to kill HER2+ target cells in a coculture assay with T cells.

Effects of motor mobility in cargo transport.

Long-range intracellular transport is mediated by motor proteins, kinesin and dynein, transporting cargos along a complex microtubule network. Here, we investigate the effects of network complexity on kinesin mediated cargo transport. We compare transport ability of single kinesin to small cargos (quantum dots) with rigid motor attachments. The network complexity comes from different densities of microtubule intersections and random networks of filaments. These studies will help us understand how motors can intrinsically navigate complex networks of filaments in live cells.

Small inland shipping in the Netherlands: Operating below the radar?

As the need for more sustainable and flexible transport continues to grow, inland shipping is emerging as a sector of interest. This paper is about small inland shipping in the Netherlands with a focus on dry cargo ships. There is currently only a limited number of studies specifically focussed on small dry cargo shipping. It has been at the centre of a societal and political debate in the Netherlands in which it has alternately attracted both positive and negative views. The recurring themes in this debate have been the shrinking of the fleet, the age of ships, the lack of entrepreneurial spirit among skippers and the sustainability of small inland shipping. Despite these discussions, there is currently no analysis of the dry cargo shipping market in the Netherlands.This paper presents an analysis of small dry cargo shipping in the Netherlands from three perspectives: fleet, cargo transport and inland ports. The analysis revealed that the size of the Dutch active small fleet is decreasing significantly. The transport performance also follows a negative development trend. However, there are also positive developments such as a slight increase in the number of trips, the average number of tonnes per loaded trip, the average number of kilometres per loaded trip and the average load factor. Another positive development is the increase in cargo throughput at inland ports situated along small waterways. Previous studies used different market definitions of small dry cargo shipping. This has an impact on the assessment of small dry cargo shipping’s development.

Distinct non-coding RNA cargo of extracellular vesicles from M1 and M2 human primary macrophages.

Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) carrying functional cargo including non-coding RNAs. Macrophages can be broadly classified into M1 'classical' and M2 'alternatively-activated' macrophages. M1 macrophages have been linked with inflammation-associated pathologies, whereas a switch towards an M2 phenotype indicates resolution of inflammation and tissue regeneration. Here, we provide the first comprehensive analysis of the small RNA cargo of EVs from human M1 and M2 primary macrophages. Using small RNA sequencing, we identified several types of small non-coding RNAs in M1 and M2 macrophage EVs including miRNAs, isomiRs, tRNA fragments, piRNA, snRNA, snoRNA and Y-RNA fragments. Distinct differences were observed between M1 and M2 EVs, with higher relative abundance of miRNAs, and lower abundance of tRNA fragments in M1 compared to M2 EVs. MicroRNA-target enrichment analysis identified several gene targets involved in gene expression and inflammatory signalling pathways. EVs were also enriched in tRNA fragments, primarily originating from the 5' end or the internal region of the full length tRNAs, many of which were differentially abundant in M1 and M2 EVs. Similarly, several other small non-coding RNAs, namely snRNAs, snoRNAs and Y-RNA fragments, were differentially enriched in M1 and M2 EVs; we discuss their putative roles in macrophage EVs. In conclusion, we show that M1 and M2 macrophages release EVs with distinct RNA cargo, which has the potential to contribute to the unique effect of these cell subsets on their microenvironment.

Relationships of APOE Genotypes With Small RNA and Protein Cargo of Brain Tissue Extracellular Vesicles From Patients With Late-Stage AD.

Variants of the apolipoprotein E (APOE) gene are the greatest known risk factors for sporadic Alzheimer disease (AD). Three major APOE isoform alleles, ε2, ε3, and ε4, encode and produce proteins that differ by only 1-2 amino acids but have different binding partner interactions. Whereas APOE ε2 is protective against AD relative to ε3, ε4 is associated with an increased risk for AD development. However, the role of APOE in gene regulation in AD pathogenesis has remained largely undetermined. Extracellular vesicles (EVs) are lipid bilayer-delimited particles released by cells to dispose of unwanted materials and mediate intercellular communication, and they are implicated in AD pathophysiology. Brain-derived EVs (bdEVs) could act locally in the tissue and reflect cellular changes. To reveal whether APOE genotype affects EV components in AD brains, bdEVs were separated from patients with AD with different APOE genotypes for parallel small RNA and protein profile. bdEVs from late-stage AD brains (BRAAK stages 5-6) from patients with APOE genotypes ε2/3 (n = 5), ε3/3 (n = 5), ε3/4 (n = 6), and ε4/4 (n = 6) were separated using our published protocol into a 10,000g pelleted extracellular fraction (10K) and a further purified EV fraction. Counting, sizing, and multiomic characterization by small RNA sequencing and proteomic analysis were performed for 10K, EVs, and source tissue. Comparing APOE genotypes, no significant differences in bdEV total particle concentration or morphology were observed. Overall small RNA and protein profiles of 10K, EVs, and source tissue also did not differ substantially between different APOE genotypes. However, several differences in individual RNAs (including miRNAs and tRNAs) and proteins in 10K and EVs were observed when comparing the highest and lowest risk groups (ε4/4 and ε2/3). Bioinformatic analysis and previous publications indicate a potential regulatory role of these molecules in AD. For patients with late-stage AD in this study, only a few moderate differences were observed for small RNA and protein profiles between APOE genotypes. Among these, several newly identified 10K and EV-associated molecules may play roles in AD progression. Possibly, larger genotype-related differences exist and are more apparent in or before earlier disease stages.

Retro-2 alters Golgi structure

Retro-2 directly interacts with an ER exit site protein, Sec16A, inhibiting ER exit of a Golgi tSNARE, Syntaxin5, which results in rapid re-distribution of Syntaxin5 to the ER. Recently, it was shown that SARS-CoV-2 infection disrupts the Golgi apparatus within 6–12 h, while its replication was effectively inhibited by Retro-2 in cultured human lung cells. Yet, exactly how Retro-2 may influence ultrastructure of the Golgi apparatus have not been thoroughly investigated. In this study, we characterized the effect of Retro-2 treatment on ultrastructure of the Golgi apparatus using electron microscopy and EM tomography. Our initial results on protein secretion showed that Retro-2 treatment does not significantly influence secretion of either small or large cargos. Ultra-structural study of the Golgi, however, revealed rapid accumulation of COPI-like vesicular profiles in the perinuclear area and a partial disassembly of the Golgi stack under electron microscope within 3–5 h, suggesting altered Golgi organization in these cells. Retro-2 treatment in cells depleted of GRASP65/55, the two well-known Golgi structural proteins, induced complete and rapid disassembly of the Golgi into individual cisterna. Taken together, these results suggest that Retro-2 profoundly alters Golgi structure to a much greater extent than previously anticipated.

The sorting of cargo proteins in the plant trans-Golgi network.

Membrane trafficking contributes to distinct protein compositions of organelles and is essential for proper organellar maintenance and functions. The trans-Golgi network (TGN) acts as a sorting station where various cargo proteins are sorted and directed to post-Golgi compartments, such as the multivesicular body or pre-vacuolar compartment, vacuoles, and plasma membrane. The spatial and temporal segregation of cargo proteins within the TGN, which is mediated with different sets of regulators including small GTPases and cargo adaptors, is a fundamental process in the sorting machinery. Recent studies with powerful imaging technologies have suggested that the TGN possesses spatially distinct subdomains or zones for different trafficking pathways. In this review, we will summarize the spatially and dynamically characteristic features of the plant TGN and their relation to cargo protein trafficking.

Life‐cycle energy and environmental emissions of cargo ships

AbstractMaritime shipping is under increasing pressure to alleviate its environmental impact. To this end, life‐cycle footprint accounting provides a foundation for taking targeted measures in the green transition of shipping. This study used the process‐based hybrid life‐cycle inventory (LCI) modeling approach to estimate the “cradle‐to‐propeller” footprint of ships, including primary energy consumption, carbon dioxide emissions, and sulfur dioxide emissions. We used the input–output LCI model to calculate the embodied energy and emissions associated with the material and fuel use of ship manufacturing. We used a bottom‐up emission model and global marine traffic data to estimate the operational footprint of different types of ships. Based on 382 cargo ships (including bulk carriers, container ships, and general cargo ships) constructed in mainland China between 2011 and 2015, we estimated that the embodied footprint accounted for <10% of the cradle‐to‐propeller footprint under the pre‐2019 policy scenario. In terms of life‐cycle energy intensity (MJ/nm/1000 deadweight tonnage [DWT]), the large bulk carrier (>100,000 DWT) establishes the lowest value (46), followed by the small (0–100,000 DWT) bulk carrier (96), the large container ship (133), the small container ship (196), and the small general cargo (238). The bulk carrier was identified as the most energy efficient among the three ship types, and ships with larger capacities (i.e., DWT) had higher energy efficiencies than ships with lower capacities. Our study provides a comprehensive understanding of the life‐cycle footprints of cargo ships, thus enabling better evidence‐based policymaking to transition the global marine‐shipping industry to a future of greener energy.

Base editing in human cells with monomeric DddA-TALE fusion deaminases

Inter-bacterial toxin DddA-derived cytosine base editors (DdCBEs) enable targeted C-to-T conversions in nuclear and organellar DNA. DddAtox, the deaminase catalytic domain derived from Burkholderia cenocepacia, is split into two inactive halves to avoid its cytotoxicity in eukaryotic cells, when fused to transcription activator-like effector (TALE) DNA-binding proteins to make DdCBEs. As a result, DdCBEs function as pairs, which hampers gene delivery via viral vectors with a small cargo size. Here, we present non-toxic, full-length DddAtox variants to make monomeric DdCBEs (mDdCBEs), enabling mitochondrial DNA editing with high efficiencies of up to 50%, when transiently expressed in human cells. We demonstrate that mDdCBEs expressed via AAV in cultured human cells can achieve nearly homoplasmic C-to-T editing in mitochondrial DNA. Interestingly, mDdCBEs often produce mutation patterns different from those obtained with conventional dimeric DdCBEs. Furthermore, mDdCBEs allow base editing at sites for which only one TALE protein can be designed. We also show that transfection of mDdCBE-encoding mRNA, rather than plasmid, can reduce off-target editing in human mitochondrial DNA.

Cell-derived nanovesicles prepared by membrane extrusion are good substitutes for natural extracellular vesicles

Extracellular vesicles (EV) as drug delivery nanocarriers are under intense investigation. Although clinical-grade EVs have been produced on a large-scale, low yield and high production costs of natural EVs (nEV) limit the relevant industrial translation. Recent studies show that mechanical extrusion of cells can generate nEV-like cell-derived nanovesicles (CNV) which can also be used as drug nanocarriers. Moreover, in comparison with nEVs, CNVs have similar physicochemical properties. Nevertheless, a comprehensive comparison of cargo between nEVs and CNVs has not been investigated yet. Therefore, the aim of this study is to profile and compare CNVs to nEVs. Our results show that no significant difference was found in size, morphology, and classical markers between nEVs and CNVs derived from MDA-MB-231 cells. Protein sequencing data reveals the similarity of membrane proteins between the two groups was ∼71%, while it was ∼21% when pertaining to total protein cargo. Notably, a high similarity of membrane proteins was also found between nEVs and CNVs derived from eight additional cancer cell lines. Moreover, analysis of the top 1000 small RNAs with RNA sequencing showed a ∼65% similarity between the two groups. Altogether, we infer from the high similarity of membrane proteins and small RNA cargo that CNVs can be a good substitute for nEVs. In brief, our findings support previous studies with a notion that CNVs yield comparable performance with nEVs and could pave the way for clinical implementation of CNV-based therapeutics in the future.