Vesicle Cargo Research Articles

Unveiling Cryptosporidium parvum sporozoite-derived extracellular vesicles: profiling, origin, and protein composition.

Cryptosporidium parvum is a common cause of a zoonotic disease and a main cause of diarrhea in newborns. Effective drugs or vaccines are still lacking. Oocyst is the infective form of the parasite; after its ingestion, the oocyst excysts and releases four sporozoites into the host intestine that rapidly attack the enterocytes. The membrane protein CpRom1 is a large rhomboid protease that is expressed by sporozoites and recognized as antigen by the host immune system. In this study, we observed the release of CpRom1 with extracellular vesicles (EVs) that was not previously described. To investigate this phenomenon, we isolated and resolved EVs from the excystation medium by differential ultracentrifugation. Fluorescence flow cytometry and transmission electron microscopy (TEM) experiments identified two types of sporozoite-derived vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs). Nanoparticle tracking analysis (NTA) revealed mode diameter of 181 nm for LEVs and 105 nm for SEVs, respectively. Immunodetection experiments proved the presence of CpRom1 and the Golgi protein CpGRASP in LEVs, while immune-electron microscopy trials demonstrated the localization of CpRom1 on the LEVs surface. TEM and scanning electron microscopy (SEM) showed that LEVs were generated by means of the budding of the outer membrane of sporozoites; conversely, the origin of SEVs remained uncertain. Distinct protein compositions were observed between LEVs and SEVs as evidenced by their corresponding electrophoretic profiles. Indeed, a dedicated proteomic analysis identified 5 and 16 proteins unique for LEVs and SEVs, respectively. Overall, 60 proteins were identified in the proteome of both types of vesicles and most of these proteins (48 in number) were already identified in the molecular cargo of extracellular vesicles from other organisms. Noteworthy, we identified 12 proteins unique to Cryptosporidium spp. and this last group included the immunodominant parasite antigen glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.

Breast fat grafting and cancer: a systematic review of the science behind enhancements and concerns.

Autologous fat transfer (AFT) is gaining popularity in breast surgery, offering a natural-looking and minimally invasive approach for augmentation, reconstruction, and contouring. However, concerns about its impact on breast cancer necessitate an understanding of the interplay between transplanted adipose-derived stem cells (ADSCs) and the breast tissue microenvironment. Renowned for regeneration, ADSCs raise questions about their role in cancer promotion. This systematic review delves into the complex relationship between AFT and breast cancer, exploring how ADSCs may influence development, growth, and metastasis. A systematic search of electronic databases, including PubMed, Embase, and BVS was conducted to identify relevant studies. The search strategy employed a combination of keywords, including "breast augmentation", "fat grafting", "breast enhancement", "mammoplasty", "cancer", "neoplasm" and related terms. Two reviewers independently screened titles and abstracts. Full-text articles were then retrieved for further evaluation based on their potential contribution to the review objectives. Two hundred and forty records were identified. Among these, 104 duplicates were removed, resulting in 136 reports available for title and abstract screening. Subsequently, 54 papers were deemed potentially eligible for inclusion, and all reports were retrieved. In vitro studies reveal ADSCs dual role in breast cancer, influencing proliferation, migration, and drug resistance through complex signaling pathways. Animal studies highlight distinct ADSC subpopulations impacting tumor growth via direct interactions and extracellular vesicle cargo. In vivo, ADSC-enriched fat grafting is generally safe, showing no increased cancer recurrence risk compared to other methods. Notably, cases of invasive breast carcinoma warrant special attention. ADSC-enriched fat grafts exhibit potential benefits in graft retention and survival rates. Despite promising evidence, further studies are needed to comprehensively understand the intricate relationship between ADSCs and breast cancer for optimized clinical applications and potential therapeutic innovations.

RNA interference-mediated silencing of coat protein II (COPII) genes affects the gut homeostasis and cuticle development in Locusta migratoria

The coat protein II (COPII) complex consists of five primary soluble proteins, namely the small GTP-binding protein Sar1, the inner coat Sec23/Sec24 heterodimers, and the outer coat Sec13/Sec31 heterotetramers. COPII is essential for cellular protein and lipid trafficking through cargo sorting and vesicle formation at the endoplasmic reticulum. However, the roles of COPII assembly genes remain unknown in insects. In present study, we identified five COPII assembly genes (LmSar1, LmSec23, LmSec24, LmSec13 and LmSec31) in Locusta migratoria. RT-qPCR results revealed that these genes showed different expression patterns in multiple tissues and developmental days of fifth-instar nymphs. Injection of double-stranded RNA against each LmCOPII gene induced a high RNAi efficiency, and considerably suppressed feeding, and increased mortality to 100 %. Results from the micro-sectioning and hematoxylin–eosin staining of midguts showed that the brush border was severely damaged and the number of columnar cells was significantly reduced in dsLmCOPII-injected nymphs, as compared with the control. The dilated endoplasmic reticulum phenotype of columnar cells was observed by transmission electron microscopy. RT-qPCR results further indicated that silencing any of the five genes responsible for COPII complex assembly repressed the expression of genes involved in insulin/mTOR-associated nutritional pathway. Therefore, COPII assembly genes could be promising RNAi targets for insect pest management by disrupting gut and cuticle development.

Abstract 273: Extrinsic photoaging vs intrinsic aging in dermal fibroblasts: Its impact on the microenvironment and melanoma progression

Abstract Melanoma is the deadliest form of skin cancer, with chronological aging as a major negative prognostic factor due to the influence of intrinsic aging on the tumor microenvironment (TME) to promote tumor initiation and progression. Ultraviolet radiation (UVR) exposure, which is a major risk factor for melanoma, also can cause a physical premature aging phenotype, known as extrinsic photoaging, from chronic UVA irradiation of the underlying dermis. Photoaging of the skin often occurs in tandem with chronological aging, and contributes to similar physical alterations in normal skin structure. However, the molecular mechanisms of how photoaging compares to chronological aging in changing the TME and on tumor progression is still lacking. Here we found that extrinsically photoaged fibroblasts have a higher level of secretion of lysosomal proteases, Cathepsins, which have been shown to be secreted via lysosomal exocytosis and play a functional role in extracellular matrix (ECM) remodeling at neutral PH levels. We found that Cathepsins B and D are secreted at higher levels in both extrinsically photoaged young fibroblasts and in intrinsically aged fibroblasts and upregulated at the endogenous protein level. Cathepsin B has been shown to be highly expressed in metastatic but not primary melanoma patient samples, and cleaves ECM proteins such as fibronectin and tenascin. Immunofluorescent staining of in-vitro fibroblast derived matrices (CDMs) confirmed that photoaging disrupts subsequent deposited extracellular matrix proteins similarly to that of intrinsically aged healthy fibroblasts and these matrix changes influences the growth pattern of melanoma lines plated on these CDMs. Mass spectrometry analysis of secreted proteomics confirmed that aged fibroblasts secrete more cathepsins than intrinsically young fibroblasts, and Cathepsins B and D were also detected in extracellular vesicle (EV) cargo proteomics. Single-Particle Interferometric Reflectance Imaging (SP-IRIS) of EVs showed that UVA irradiation induced a significant increase in the total number of secreted of exosomes and in expression of tetraspanin protein CD63, which plays a role in cargo sorting and has been detected in high levels in the plasma of melanoma patients. We hypothesize that extrinsic photoaging of fibroblasts induces the secretion of exosomes packaged with Cathepsins that play an integral role in ECM remodeling and matrix degradation in a beneficial manner for melanoma invasion. Further studies will assess the influence on secreted cathepsins on melanomagenesis and melanoma invasion through both mechanical and biochemical changes in the TME with in-vitro and in-vivo studies. Citation Format: Vania Wang, Yash Chhabra, Laura Hueser, Ashani Weeraratna. Extrinsic photoaging vs intrinsic aging in dermal fibroblasts: Its impact on the microenvironment and melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 273.

Abstract 2130: Glioblastoma-derived extracellular vesicle cargo as a reporter of drug delivery and effect

Abstract The selectivity of the blood-brain-barrier (BBB) can prevent therapeutics from reaching the brain, and this may contribute to the high failure rate of new drugs for tumors like glioblastoma (GBM) and brain metastasis. The capacity to detect BBB penetrance in real-time in a noninvasive manner would arguably aid the rate of therapeutic development. Extracellular vesicles, which are membrane bound particles secreted by all cell types in the body, have potential as a neuro pharmacodynamic reporter given that their protein and RNA cargo reflect the originating cell population. We hypothesized that the exposure of GBM cells to therapy results in altered EV cargo, and that the detection of this shift can be leveraged as a liquid biopsy from patients during early clinical trials to determine BBB penetrance and tumor engagement. To identify cargo shifts indicative of treatment, we exposed four short-term patient derived glioma cell lines to arsenic trioxide (an idiopathic toxin), MLN4924 (a neddylation inhibitor), and the alkylating agent temozolomide, then harvested EVs for unbiased mass spectrometry and RNA sequencing. Interestingly, we identified conserved upregulated and downregulated protein and RNA species across cell lines and across drug treatments, despite the diverse mechanisms of action. Candidate proteins were further validated by mesoscale immunoassays and exoview evaluation of tetraspanin-captured EVs. RNA species were validated by qRT-PCR. Overall, we demonstrate that GBM-derived EV cargo changes in response to therapy, supporting the development of EVs as a reporter of drug delivery in GBM. Ongoing and future work is focused on identifying enrichment handles in order to selectively evaluate GBM-derived EV cargo from patient biofluids. Thus far, B7H3, PTPRZ1, EGFRvII and IL13Ra2 have been screened as putative handles. Success in this pursuit could substantially enhance findings and accelerate drug testing in early-stage clinical trials in brain tumor patients by reporting drug accessibility to the tumor and engagement with drug targets. Citation Format: Valerie DeLuca, Nanyun Tang, Nathaniel Hansen, Anjali Raju, Charles Shaffer, Yue Hao, Terry C. Burns, Jann N. Sarkaria, Ian Parney, Patrick Pirrotte, Kendall Van Keuren-Jensen, Michael E. Berens. Glioblastoma-derived extracellular vesicle cargo as a reporter of drug delivery and effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2130.

Proteomics of mouse brain endothelium uncovers dysregulation of vesicular transport pathways during aging.

Age-related decline in brain endothelial cell (BEC) function contributes critically to neurological disease. Comprehensive atlases of the BEC transcriptome have become available, but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting-based mouse BEC enrichment protocol compatible with proteomics and resolved the profiles of protein abundance changes during aging. Unsupervised cluster analysis revealed a segregation of age-related protein dynamics with biological functions, including a downregulation of vesicle-mediated transport. We found a dysregulation of key regulators of endocytosis and receptor recycling (most prominently Arf6), macropinocytosis and lysosomal degradation. In gene deletion and overexpression experiments, Arf6 affected endocytosis pathways in endothelial cells. Our approach uncovered changes not picked up by transcriptomic studies, such as accumulation of vesicle cargo and receptor ligands, including Apoe. Proteomic analysis of BECs from Apoe-deficient mice revealed a signature of accelerated aging. Our findings provide a resource for analysing BEC function during aging.

Abstract 4270: Differential expression of the αVβ3 integrin affects small extracellular vesicle cargo and promotes small extracellular vesicle pro-tumorigenic activity in vivo via its downstream effector, NgR2

Abstract It is known that small extracellular vesicles (sEVs) are released from cancer cells and contribute to cancer progression via crosstalk with recipient cells. These sEVs are being investigated as powerful biomarkers and possible therapeutic agents for prostate cancer (PrCa), which remains the leading cause of cancer death among men in the United States. We have previously reported that sEVs expressing the αVβ3 integrin, a protein upregulated in aggressive neuroendocrine prostate cancer (NEPrCa), contribute to neuroendocrine differentiation (NED) in recipient cells. sEVs used in this study were isolated by iodixanol density gradient and characterized by nanoparticle tracking analysis, immunoblotting, and single vesicle analysis. Our proteomic profile of sEVs containing αVβ3, when compared to control sEVs, shows a downregulation of typical effectors involved in apoptosis and necrosis, and an upregulation of tumor cell survival factors. This subset also contains an αVβ3 downstream effector, NgR2, a member of the Nogo receptor family and a novel marker for NEPrCa. We have previously shown that NgR2 promotes NED and cell motility, and upregulates RhoA, a protein associated with aggressive phenotypes of PrCa. In this study, we show that the expression of NgR2 increases tumor growth in vivo. We also show that the downregulation of NgR2 inhibits tumor growth and NED. Furthermore, we report that sEVs containing αVβ3 are loaded with higher amounts of NgR2 as compared to sEVs that do not express αVβ3. Mechanistically, we demonstrate that sEVs containing NgR2 do not affect the sEV marker profile, but, when injected in vivo intratumorally, they promote tumor growth and induce NED. We confirm that the transfer of NgR2 into recipient cells is sufficient to promote tumor growth and show that NgR2 mimics the effect of sEVs containing αVβ3 since it displays increased growth of NgR2 transfectants in vivo, as compared to control cells. Overall, our results describe a new mechanism mediated by sEVs containing αVβ3 and NgR2 that promotes cancer progression. Acknowledgements: NIH R01-CA224769; DoD W81XWH2210826 Citation Format: Cecilia E. Verrillo, Fabio Quaglia, Nicole Naranjo, Anna Testa, Lucia R. Languino. Differential expression of the αVβ3 integrin affects small extracellular vesicle cargo and promotes small extracellular vesicle pro-tumorigenic activity in vivo via its downstream effector, NgR2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4270.

Monitoring the electroactive cargo of extracellular vesicles can differentiate various cancer cell lines

Extracellular vesicles (EVs) are pivotal in cell-to-cell communication due to the array of cargo contained within these vesicles. EVs are considered important biomarkers for identification of disease, however most measurement approaches have focused on monitoring specific surface macromolecular targets. Our study focuses on exploring the electroactive component present within cargo from EVs obtained from various cancer and non-cancer cell lines using a disk carbon fiber microelectrode. Variations in the presence of oxidizable components were observed when the total cargo from EVs were measured, with the highest current detected in EVs from MCF7 cells. There were differences observed in the types of oxidizable species present within EVs from MCF7 and A549 cells. Single entity measurements showed clear spikes due to the detection of oxidizable cargo within EVs from MCF7 and A549 cells. These studies highlight the promise of monitoring EVs through the presence of varying electroactive components within the cargo and can drive a wave of new strategies towards specific detection of EVs for diagnosis and prognosis of various diseases.

Next-generation sequencing reveals that miR-16-5p, miR-19a-3p, miR-451a, and miR-25-3p cargo in plasma extracellular vesicles differentiates sedentary young males from athletes.

A sedentary lifestyle and Olympic participation are contrary risk factors for global mortality and incidence of cancer and cardiovascular disease. Extracellular vesicle miRNAs have been described to respond to exercise. No molecular characterization of young male sedentary people versus athletes is available; so, our aim was to identify the extracellular vesicle miRNA profile of chronically trained young endurance and resistance male athletes compared to their sedentary counterparts. A descriptive case-control design was used with 16 sedentary young men, 16 Olympic male endurance athletes, and 16 Olympic male resistance athletes. Next-generation sequencing and RT-qPCR and external and internal validation were performed in order to analyze extracellular vesicle miRNA profiles. Endurance and resistance athletes had significant lower levels of miR-16-5p, miR-19a-3p, and miR-451a compared to sedentary people. Taking all together, exercise-trained miRNA profile in extracellular vesicles provides a differential signature of athletes irrespective of the type of exercise compared to sedentary people. Besides, miR-25-3p levels were specifically lower in endurance athletes which defines its role as a specific responder in this type of athletes. In silico analysis of this profile suggests a role in adaptive energy metabolism in this context that needs to be experimentally validated. Therefore, this study provides for the first time basal levels of circulating miRNA in extracellular vesicles emerge as relevant players in intertissue communication in response to chronic exercise exposure in young elite male athletes.

Membrane trafficking alterations in breast cancer progression.

Breast cancer (BC) is the most common type of cancer in women, and remains one of the major causes of death in women worldwide. It is now well established that alterations in membrane trafficking are implicated in BC progression. Indeed, membrane trafficking pathways regulate BC cell proliferation, migration, invasion, and metastasis. The 22 members of the ADP-ribosylation factor (ARF) and the >60 members of the rat sarcoma (RAS)-related in brain (RAB) families of small GTP-binding proteins (GTPases), which belong to the RAS superfamily, are master regulators of membrane trafficking pathways. ARF-like (ARL) subfamily members are involved in various processes, including vesicle budding and cargo selection. Moreover, ARFs regulate cytoskeleton organization and signal transduction. RABs are key regulators of all steps of membrane trafficking. Interestingly, the activity and/or expression of some of these proteins is found dysregulated in BC. Here, we review how the processes regulated by ARFs and RABs are subverted in BC, including secretion/exocytosis, endocytosis/recycling, autophagy/lysosome trafficking, cytoskeleton dynamics, integrin-mediated signaling, among others. Thus, we provide a comprehensive overview of the roles played by ARF and RAB family members, as well as their regulators in BC progression, aiming to lay the foundation for future research in this field. This research should focus on further dissecting the molecular mechanisms regulated by ARFs and RABs that are subverted in BC, and exploring their use as therapeutic targets or prognostic markers.

TRNAGlu-derived fragments from embryonic extracellular vesicles modulate bovine embryo hatching

Transfer RNA-derived small RNAs (tsRNAs) have been shown to be involved in early embryo development and repression of endogenous retroelements in embryos and stem cells. However, it is unknown whether tsRNAs also regulate embryo hatching. In this study, we mined the sequencing data of a previous experiment in which we demonstrated that the microRNA (miRNA) cargo of preimplantation embryonic extracellular vesicles (EVs) influences embryo development. We thus profiled the tsRNA cargo of EVs secreted by blastocysts and non-blastocysts. The majority of tsRNAs was identified as tRNA halves originating from the 5´ ends of tRNAs. Among the 148 differentially expressed tsRNAs, the 19 nt tRNA fragment (tRF) tDR-14:32-Glu-CTC-1 was found to be significantly up-regulated in EVs derived from non-blastocysts. RT-qPCR assays confirmed its significant up-regulation in non-blastocyst embryos and their conditioned medium compared to the blastocyst group (P < 0.05). Inhibition of tDR-14:32-Glu-CTC-1 by supplementing antagomirs to the conditioned medium improved embryo hatching (P < 0.05). Transcriptomic analysis of embryos treated with tDR-14:32-Glu-CTC-1 antagomirs further showed differential expression of genes that are associated with embryo hatching and implantation. In summary, tDR-14:32-Glu-CTC-1 is up-regulated in non-blastocyst embryos and their secretions, and inhibition of tDR-14:32-Glu-CTC-1 promotes embryo hatching, while influencing embryo implantation-related genes and pathways. These results indicate that embryonic EVs containing specific tRFs may regulate preimplantation embryo development.

Clinical Insights into MicroRNAs in Depression: Bridging Molecular Discoveries and Therapeutic Potential.

Depression is a major contributor to the overall global burden of disease. The discovery of biomarkers for diagnosis or prediction of treatment responses and as therapeutic agents is a current priority. Previous studies have demonstrated the importance of short RNA molecules in the etiology of depression. The most extensively researched of these are microRNAs, a major component of cellular gene regulation and function. MicroRNAs function in a temporal and tissue-specific manner to regulate and modify the post-transcriptional expression of target mRNAs. They can also be shuttled as cargo of extracellular vesicles between the brain and the blood, thus informing about relevant mechanisms in the CNS through the periphery. In fact, studies have already shown that microRNAs identified peripherally are dysregulated in the pathological phenotypes seen in depression. Our article aims to review the existing evidence on microRNA dysregulation in depression and to summarize and evaluate the growing body of evidence for the use of microRNAs as a target for diagnostics and RNA-based therapies.

Changes in proteomic cargo of circulating extracellular vesicles in response to lifestyle intervention in adolescents with hepatic steatosis

BackgroundRecent studies suggest that proteomic cargo of extracellular vesicles (EVs) may play a role in metabolic improvements following lifestyle interventions. However, the relationship between changes in liver fat and circulating EV-derived protein cargo following intervention remains unexplored. MethodsThe study cohort comprised 18 Latino adolescents with obesity and hepatic steatosis (12 males/6 females; average age 13.3 ±1.2 y) who underwent a six-month lifestyle intervention. EV size distribution and concentration were determined by light scattering intensity; EV protein composition was characterized by liquid chromatography tandem-mass spectrometry. ResultsAverage hepatic fat fraction (HFF) decreased 23% by the end of the intervention (12.5% [5.5] to 9.6% [4.9]; P = 0.0077). Mean EV size was smaller post-intervention compared to baseline (120.2 ± 16.4 nm to 128.4 ± 16.5 nm; P = 0.031), although the difference in mean EV concentration (1.1E+09 ± 4.1E+08 particles/mL to 1.1E+09 ± 1.8E+08 particles/mL; P = 0.656)) remained unchanged. A total of 462 proteins were identified by proteomic analysis of plasma-derived EVs from participants pre- and post- intervention, with 113 proteins showing differential abundance (56 higher and 57 lower) between the two timepoints (adj-p <0.05). Pathway analysis revealed enrichment in complement cascade, initial triggering of complement, creation of C4 and C2 activators, and regulation of complement cascade. Hepatocyte-specific EV affinity purification identified 40 proteins with suggestive (p < 0.05) differential abundance between pre- and post-intervention samples. ConclusionsCirculating EV-derived proteins, particularly those associated with the complement cascade, may contribute to improvements in liver fat in response to lifestyle intervention.

Protein Profiling of Placental Extracellular Vesicles in Gestational Diabetes Mellitus.

Throughout pregnancy, some degree of insulin resistance is necessary to divert glucose towards the developing foetus. In gestational diabetes mellitus (GDM), insulin resistance is exacerbated in combination with insulin deficiency, causing new-onset maternal hyperglycaemia. The rapid reversal of insulin resistance following delivery strongly implicates the placenta in GDM pathogenesis. In this case-control study, we investigated the proteomic cargo of human syncytiotrophoblast-derived extracellular vesicles (STBEVs), which facilitate maternal-fetal signalling during pregnancy, in a UK-based cohort comprising patients with a gestational age of 38-40 weeks. Medium/large (m/l) and small (s) STBEVs were isolated from GDM (n = 4) and normal (n = 5) placentae using ex vivo dual-lobe perfusion and subjected to mass spectrometry. Bioinformatics were used to identify differentially carried proteins and mechanistic pathways. In m/lSTBEVs, 56 proteins were differently expressed while in sSTBEVs, no proteins reached statistical difference. Differences were also observed in the proteomic cargo between m/lSTBEVs and sSTBEVs, indicating that the two subtypes of STBEVs may have divergent modes of action and downstream effects. In silico functional enrichment analysis of differentially expressed proteins in m/lSTBEVs from GDM and normal pregnancy found positive regulation of cytoskeleton organisation as the most significantly enriched biological process. This work presents the first comparison of two populations of STBEVs' protein cargos (m/l and sSTBEVs) from GDM and normal pregnancy isolated using placenta perfusion. Further investigation of differentially expressed proteins may contribute to an understanding of GDM pathogenesis and the development of novel diagnostic and therapeutic tools.

Extracellular vesicles in parasitic diseases – from pathogenesis to future diagnostic tools

Parasitic diseases are still a major public health problem especially among individuals of low socioeconomic status in underdeveloped countries. In recent years it has been demonstrated that parasites can release extracellular vesicles that participate in the host-parasite communication, immune evasion, and in governing processes associated with host infection. Extracellular vesicles are membrane-bound structures released into the extracellular space that can carry several types of biomolecules, including proteins, lipids, nucleic acids, and metabolites, which directly impact the target cells. Extracellular vesicles have attracted wide attention due to their relevance in host-parasite communication and for their potential value in applications such as in the diagnostic biomarker discovery. This review of the literature aimed to join the current knowledge on the role of extracellular vesicles in host-parasite interaction and summarize its molecular content, providing information for the acquisition of new tools that can be used in the diagnosis of parasitic diseases. These findings shed light to the potential of extracellular vesicle cargo derived from protozoan parasites as novel diagnostic tools.

Involvement of Extracellular Vesicles in the Proinflammatory Response to Clozapine: Implications for Clozapine-Induced Agranulocytosis.

Most idiosyncratic drug reactions (IDRs) appear to be immune-mediated, but mechanistic events preceding severe reaction onset remain poorly defined. Damage-associated molecular patterns (DAMPs) may contribute to both innate and adaptive immune phases of IDRs, and changes in extracellular vesicle (EV) cargo have been detected post-exposure to several IDR-associated drugs. To explore the hypothesis that EVs are also a source of DAMPs in the induction of the immune response preceding drug-induced agranulocytosis, the proteome and immunogenicity of clozapine- (agranulocytosis-associated drug) and olanzapine- (non-agranulocytosis-associated drug) exposed EVs were compared in two preclinical models: THP-1 macrophages and Sprague-Dawley rats. Compared with olanzapine, clozapine induced a greater increase in the concentration of EVs enriched from both cell culture media and rat serum. Moreover, treatment of drug-naïve THP-1 cells with clozapine-exposed EVs induced an inflammasome-dependent response, supporting a potential role for EVs in immune activation. Proteomic and bioinformatic analyses demonstrated an increased number of differentially expressed proteins with clozapine that were enriched in pathways related to inflammation, myeloid cell chemotaxis, wounding, transforming growth factor-β signaling, and negative regulation of stimuli response. These data indicate that, although clozapine and olanzapine exposure both alter the protein cargo of EVs, clozapine-exposed EVs carry mediators that exhibit significantly greater immunogenicity. Ultimately, this supports the working hypothesis that drugs associated with a risk of IDRs induce cell stress, release of proinflammatory mediators, and early immune activation that precedes severe reaction onset. Further studies characterizing EVs may elucidate biomarkers that predict IDR risk during development of drug candidates. SIGNIFICANCE STATEMENT: This work demonstrates that clozapine, an idiosyncratic drug-induced agranulocytosis (IDIAG)-associated drug, but not olanzapine, a safer structural analogue, induces an acute proinflammatory response and increases extracellular vesicle (EV) release in two preclinical models. Moreover, clozapine-exposed EVs are more immunogenic, as measured by their ability to activate inflammasomes, and contain more differentially expressed proteins, highlighting a novel role for EVs during the early immune response to clozapine and enhancing our mechanistic understanding of IDIAG and other idiosyncratic reactions.

Comparative analysis of two Caenorhabditis elegans kinesins KLP-6 and UNC-104 reveals a common and distinct activation mechanism in kinesin-3

Kinesin-3 is a family of microtubule-dependent motor proteins that transport various cargos within the cell. However, the mechanism underlying kinesin-3 activations remains largely elusive. In this study, we compared the biochemical properties of two Caenorhabditis elegans kinesin-3 family proteins, KLP-6 and UNC-104. Both KLP-6 and UNC-104 are predominantly monomeric in solution. As previously shown for UNC-104, non-processive KLP-6 monomer is converted to a processive motor when artificially dimerized. We present evidence that releasing the autoinhibition is sufficient to trigger dimerization of monomeric UNC-104 at nanomolar concentrations, which results in processive movement of UNC-104 on microtubules, although it has long been thought that enrichment in the phospholipid microdomain on cargo vesicles is required for the dimerization and processive movement of UNC-104. In contrast, KLP-6 remains to be a non-processive monomer even when its autoinhibition is unlocked, suggesting a requirement of other factors for full activation. By examining the differences between KLP-6 and UNC-104, we identified a coiled-coil domain called coiled-coil 2 (CC2) that is required for the efficient dimerization and processive movement of UNC-104. Our results suggest a common activation mechanism for kinesin-3 family members, while also highlighting their diversification.

Comparative analysis of two Caenorhabditis elegans kinesins KLP-6 and UNC-104 reveals a common and distinct activation mechanism in kinesin-3.

Kinesin-3 is a family of microtubule-dependent motor proteins that transport various cargos within the cell. However, the mechanism underlying kinesin-3 activations remains largely elusive. In this study, we compared the biochemical properties of two Caenorhabditis elegans kinesin-3 family proteins, KLP-6 and UNC-104. Both KLP-6 and UNC-104 are predominantly monomeric in solution. As previously shown for UNC-104, non-processive KLP-6 monomer is converted to a processive motor when artificially dimerized. We present evidence that releasing the autoinhibition is sufficient to trigger dimerization of monomeric UNC-104 at nanomolar concentrations, which results in processive movement of UNC-104 on microtubules, although it has long been thought that enrichment in the phospholipid microdomain on cargo vesicles is required for the dimerization and processive movement of UNC-104. In contrast, KLP-6 remains to be a non-processive monomer even when its autoinhibition is unlocked, suggesting a requirement of other factors for full activation. By examining the differences between KLP-6 and UNC-104, we identified a coiled-coil domain called coiled-coil 2 (CC2) that is required for the efficient dimerization and processive movement of UNC-104. Our results suggest a common activation mechanism for kinesin-3 family members, while also highlighting their diversification.

Proteomic analysis of extracellular vesicle cargoes mirror the cardioprotective effects of rivaroxaban in patients with venous thromboembolism.

Venous thromboembolism (VTE) remains a significant cause of morbidity and mortality worldwide. Rivaroxaban, a direct oral factor Xa inhibitor, mediates anti-inflammatory and cardiovascular-protective effects besides its well-established anticoagulant properties; yet, these remain poorly characterized. Extracellular vesicles (EVs) are considered proinflammatory messengers regulating a myriad of (patho)physiological processes and may be highly relevant to the pathophysiology of VTE. The effects of Rivaroxaban on circulating EVs in VTE patients remain unknown. We have established that differential EV biosignatures are found in patients with non-valvular atrial fibrillation anticoagulated with Rivaroxaban versus warfarin. Here, we investigated whether differential proteomic profiles of circulating EVs could also be found in patients with VTE. We performed comparative label-free quantitative proteomic profiling of enriched plasma EVs from VTE patients anticoagulated with either Rivaroxaban or warfarin using a tandem mass spectrometry approach. Of the 182 quantified proteins, six were found to be either exclusive to, or enriched in, Rivaroxaban-treated patients. Intriguingly, these proteins are involved in negative feedback regulation of inflammatory and coagulation pathways, suggesting that EV proteomic signatures may reflect both Rivaroxaban's anti-coagulatory and anti-inflammatory potential. These differences suggest Rivaroxaban may have pleiotropic effects, supporting the reports of its emerging anti-inflammatory and cardiovascular-protective characteristics relative to warfarin.

Myosin Va: Capturing cAMP for synaptic plasticity.

The plus-end directed actin-dependent motor protein, myosin Va, is of particular relevance for outward vesicular protein trafficking and for restraining specific cargo vesicles within the actin cortex. The latter is a preferred site of cAMP production, and the specificity of cAMP signaling is largely mediated through the formation of microdomains that spatially couple localized metabotropic receptor activity and cAMP production to selected effectors and downstream targets. This review summarizes the core literature on the role of myosin Va for the creation of such a cAMP microdomain at the mammalian nerve-muscle synapse that serves the activity-dependent recycling of nicotinic acetylcholine receptors (nAChRs)-a principal ligand-gated ion channel which is imperative for voluntary muscle contraction. It is discussed that i) the nerve-muscle synapse is a site with a unique actin-dependent microstructure, ii) myosin Va and protein kinase A regulatory subunit Iα as well as nAChR and its constitutive binding partner, rapsyn, colocalize in endocytic/recycling vesicles near the postsynaptic membrane, and iii) impairment of myosin Va or displacement of protein kinase A regulatory subunit Iα leads to the loss of nAChR stability. Regulation of this signaling process and underlying basic pieces of machinery were covered in previous articles, to which the present review refers.