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Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths. Though chemotherapy is the main treatment option for advanced CRC, patients invariably acquire resistance to chemotherapeutic drugs and fail to respond to the therapy. Although understanding the mechanisms regulating chemoresistance has been a focus of intense research to manage this challenge, the pathways governing resistance to drugs are poorly understood. In this study, we provide evidence for the role of ubiquitin ligase NEDD4 in resistance developed against the most commonly used CRC chemotherapeutic drug 5-fluorouracil (5-FU). A marked reduction in NEDD4 protein abundance was observed in a panel of CRC cell lines and patient-derived xenograft samples that were resistant to 5-FU. Knockout of NEDD4 in CRC cells protected them from 5-FU-mediated apoptosis but not oxaliplatin or irinotecan. Furthermore, NEDD4 depletion in CRC cells reduced proliferation, colony-forming abilities and tumour growth in mice. Follow-up biochemical analysis highlighted the inhibition of the JNK signalling pathway in NEDD4-deficient cells. Treatment with the JNK activator hesperidin in NEDD4 knockout cells sensitised the CRC cells against 5-FU. Overall, we show that NEDD4 regulates cell proliferation, colony formation, tumour growth and 5-FU chemoresistance in CRC cells.

Abstract Cell culture‐conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM‐derived EVs (CCM‐EV). The CCM‐EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell‐specific recommendations may need to be established for non‐mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM‐EV research.

Abstract MiR-21 was identified as a gene whose expression correlated with the extent of metastasis of murine mammary tumours. Since miR-21 is recognised as being associated with poor prognosis in cancer, we investigated its contribution to mammary tumour growth and metastasis in tumours with capacity for spontaneous metastasis. Unexpectedly, we found that suppression of miR-21 activity in highly metastatic tumours resulted in regression of primary tumour growth in immunocompetent mice but did not impede growth in immunocompromised mice. Analysis of the immune infiltrate of the primary tumours at the time when the tumours started to regress revealed an influx of both CD4+ and CD8+ activated T cells and a reduction in PD-L1+ infiltrating monocytes, providing an explanation for the observed tumour regression. Loss of anti-tumour immune suppression caused by decreased miR-21 activity was confirmed by transcriptomic analysis of primary tumours. This analysis also revealed reduced expression of genes associated with cell cycle progression upon loss of miR-21 activity. A second activity of miR-21 was the promotion of metastasis as shown by the loss of metastatic capacity of miR-21 knockdown tumours established in immunocompromised mice, despite no impact on primary tumour growth. A proteomic analysis of tumour cells with altered miR-21 activity revealed deregulation of proteins known to be associated with tumour progression. The development of therapies targeting miR-21, possibly via targeted delivery to tumour cells, could be an effective therapy to combat primary tumour growth and suppress the development of metastatic disease.

Milk is a complex biological fluid that has high-quality proteins including growth factors and also contains extracellular vesicles (EVs). EVs are a lipid bilayer containing vesicles that contain proteins, metabolites and nucleic acids. Several studies have proposed that EVs in cow milk can survive the gut and can illicit cross-species communication in the consuming host organism. In this study, we isolated and characterized extracellular vesicles from the raw milk of the four species of the Bovidae family, namely cow, sheep, goat and buffalo, that contribute 99% of the total milk consumed globally. A comparative proteomic analysis of these vesicles was performed to pinpoint their potential functional role in health and disease. Vesicles sourced from buffalo and cow milk were particularly enriched with proteins implicated in modulating the immune system. Furthermore, functional studies were performed to determine the anti-cancer effects of these vesicles. The data obtained revealed that buffalo-milk-derived EVs induced significantly higher cell death in colon cancer cells. Overall, the results from this study highlight the potent immunoregulatory and anti-cancer nature of EVs derived from the milk of Bovidae family members.

Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.

Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection. Molecular interactions between SARS-CoV-2 and its host dictate the course of COVID-19 disease progression; this study identifies LRRC15 as a new fibroblast SARS-CoV-2 spike receptor that can block infection, activate cellular antiviral programs, and suppress collagen production.

Dietary extracellular vesicles (EVs) could potentially be absorbed by the intestinal tract of the host and exert multiple phenotypic changes. Here, the authors isolate and characterize EVs from raw and commercial bovine milk and show orally administered EVs to have a context specific role in promoting or suppressing primary tumor growth and metastasis in multiple mouse tumor models.

Cancer derived exosomes are reported to promote metastatic dissemination. Here the authors show that cytokines in the tumor microenvironment bind to exosomes via glycosaminoglycan side chains of proteoglycans, and these exosomes are preferentially taken up by specific cell lineages and organs to promote metastasis.

Abstract Extracellular vesicles (EVs) are important mediators of intercellular communication. However, EV biogenesis remains poorly understood. We previously defined a role for Arrdc4 (Arrestin domain containing protein 4), an adaptor for Nedd4 family ubiquitin ligases, in the biogenesis of EVs. Here we report that ubiquitination of Arrdc4 is critical for its role in EV secretion. We identified five potential ubiquitinated lysine residues in Arrdc4 using mass spectrometry. By analysing Arrdc4 lysine mutants we discovered that lysine 270 (K270) is critical for Arrdc4 function in EV biogenesis. Arrdc4K270R mutation caused a decrease in the number of EVs released by cells compared to Arrdc4WT, and a reduction in trafficking of divalent metal transporter (DMT1) into EVs. Furthermore, we also observed a decrease in DMT1 activity and an increase in its intracellular degradation in the presence of Arrdc4K270R. K270 was found to be ubiquitinated with K‐29 polyubiquitin chains by the ubiquitin ligase Nedd4‐2. Thus, our results uncover a novel role of K‐29 polyubiquitin chains in Arrdc4‐mediated EV biogenesis and protein trafficking.

Extracellular vesicles (EVs) are small packages that contain proteins, lipids and nucleic acids and are released by various cell types [...]

Extracellular vesicles (EVs) are particles that are released from cells into the extracellular space both under pathological and normal conditions. It is now well established that cancer cells secrete more EVs compared to non-cancerous cells and that, captivatingly, several proteins that are involved in EV biogenesis and secretion are upregulated in various tumours. Recent studies have revealed that EVs facilitate the interaction between cancer cells and their microenvironment and play a substantial role in the growth of tumours. As EVs are involved in several aspects of cancer progression including angiogenesis, organotropism, pre-metastatic niche formation, fostering of metastasis, and chemoresistance, inhibiting the release of EVs from cancer and the surrounding tumour microenvironment cells has been proposed as an ideal strategy to treat cancer and associated paraneoplastic syndromes. Lately, EVs have shown immense benefits in preclinical settings as a novel drug delivery vehicle. This review provides a brief overview of the role of EVs in various hallmarks of cancer, focusing on (i) strategies to treat cancer by therapeutically targeting the release of tumour-derived EVs and (ii) EVs as valuable drug delivery vehicles. Furthermore, we also outline the drawbacks of the existing anti-cancer treatments and the future prospective of EV-based therapeutics.

Histone deacetylase 3 (HDAC3) is a regulator of lipid homeostasis in several tissues, however, its role in intestinal lipid metabolism was not yet known. Here the authors study intestine specific HDAC3 knock out mice and report that these animals have increased fatty acid oxidation and undergo remodeling of the intestinal epithelial cell lipidome.

Subtypes of cancer associated fibroblasts can both promote and suppress tumorigenesis. Here, the authors investigate how p53 status in pancreatic cancer cells affects their interaction with cancer associated fibroblasts, and report perlecan as a mediator of the pro-metastatic environment.

Abstract Extracellular vesicles (EVs) are important players in cell to cell communication in reproductive systems. Notably, EVs have been found and characterized in the male reproductive tract, however, direct functional evidence for their importance in mediating sperm function is lacking. We have previously demonstrated that Arrdc4, a member of the α‐arrestin protein family, is involved in extracellular vesicle biogenesis and release. Here we show that Arrdc4‐mediated extracellular vesicle biogenesis is required for proper sperm function. Sperm from Arrdc4–/– mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as observed by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and two‐cell embryo production. We found a significant reduction in extracellular vesicle production by Arrdc4–/‐ epididymal epithelial cells, and further, supplementation of Arrdc4–/– sperm with additional vesicles dampened the acrosome reaction defect and restored zona pellucida binding. These results indicate that Arrdc4 is important for proper sperm maturation through the control of extracellular vesicle biogenesis.

The innate immune system in mammals include pattern recognition receptors (PRRs), which initiate immune responses to microbial infection via several mechanisms. These PRRs include cell surface Toll-like receptors (TLRs) and cytosolic Nod-like receptors (NLRs) that recognizes extracellular and intracellular danger signals respectively. NLRs are poised to respond specifically to pathogens that access the host cell cytosol. The molecular mechanisms by which NLRs are activated to form inflammasomes and exert downstream inflammatory responses remain poorly understood. Additionally, very little is known about the regulation of cytosolic pathogen sensory NLR family members, except for NLRP3. Recently a deubiquitinase known as STAMBP has been implicated as a regulator of NLRP7 inflammasome assembly. We have investigated the role of STAMBP in regulation of other inflammasome components and its broader role in inflammation using genetic removal of STAMBP protein from cells using CRISPR/Cas9 gene editing and challenging these gene edited cells with an inflammatory stimuli. Our study demonstrated that STAMBP has a critical role in inflammation both in the context of NLR pathway, through NLRP stabilization and TLR pathway, through JNK signaling and downstream cytokine production. The findings indicate that STAMBP has a wider role in inflammation than previously thought to be the case.

Extracellular vesicles (EVs) represent a system for the coordinated secretion of a variety of molecular cargo including proteins, lipids, nucleic acids, and metabolites. They have an essential role in intercellular communication in multicellular organisms and have more recently been implicated in host-pathogen interactions. Study of the role for EVs in fungal biology has focused on pathogenic yeasts that are major pathogens in humans. In this study we have expanded the investigation of fungal EVs to plant pathogens, specifically the major cotton pathogen Fusarium oxysporum f. sp. vasinfectum. EVs isolated from F. oxysporum f. sp. vasinfectum culture medium have a morphology and size distribution similar to EVs from yeasts such as Candida albicans and Cryptococcus neoformans. A unique feature of the EVs from F. oxysporum f. sp. vasinfectum is their purple color, which is predicted to arise from a napthoquinone pigment being packaged into the EVs. Proteomic analysis of F. oxysporum f. sp. vasinfectum EVs revealed that they are enriched in proteins that function in synthesis of polyketides as well as proteases and proteins that function in basic cellular processes. Infiltration of F. oxysporum f. sp. vasinfectum EVs into the leaves of cotton or N. benthamiana plants led to a phytotoxic response. These observations lead to the hypothesis that F. oxysporum f. sp. vasinfectum EVs are likely to play a crucial role in the infection process.

Clinical management of cancer-associated cachexia, a multi-organ wasting syndrome, has been challenging without effective treatment strategies. An effective treatment that directly targets cancer-induced wasting is desperately needed to improve the quality of life and the survival of cancer patients. Recently, an antibiotic SFX was shown to have anti-tumour and anti-metastatic effects in mouse models of breast cancer. Hence, in this study, we examined the efficacy of SFX in the treatment of cancer-induced cachexia. C26 cachexic mice models were administered with SFX, and the tumour volume and body weight were regularly measured. Blood glucose, skeletal muscles, and adipose tissue were examined at the endpoint. Contrary to a previous study, SFX did not reduce the tumour volume in mice bearing C26 cells. Administration of SFX neither revealed any survival benefit nor rescued C26 cachectic mice from muscle wasting. Interestingly, SFX administration partially rescued (~10%) tumour-induced weight loss by preserving both the subcutaneous and intestinal fat mass. Together, these results suggest that the administration of SFX could partially rescue cancer-induced weight loss by inhibiting lipolysis. As anti-cachexia therapies are scarce, the results could facilitate the design of combinatorial therapies involving SFX, standard-of-care chemotherapeutics, and drugs that inhibit muscle atrophy for the treatment of cancer cachexia.

Abstract Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. They contain cell-state specific cargos which often reflects the (patho)physiological condition of the cells/organism. Milk contains high amounts of exosomes and it is unclear whether their cargo is altered based on the lactation stage of the organism. Here, we isolated exosomes from bovine milk that were obtained at various stages of lactation and examined the content by quantitative proteomics. Exosomes were isolated by OptiPrep density gradient centrifugation from milk obtained from cow after 24, 48 and 72 h post calving. As control, exosomes were also isolated from cows during mid-lactation period which has been referred to as mature milk (MM). Biochemical and biophysical characterization of exosomes revealed the high abundance of exosomes in colostrum and MM samples. Quantitative proteomics analysis highlighted the change in the proteomic cargo of exosomes based on the lactation state of the cow. Functional enrichment analysis revealed that exosomes from colostrum are significantly enriched with proteins that can potentially regulate the immune response and growth. This study highlights the importance of exosomes in colostrum and hence opens up new avenues to exploit these vesicles in the regulation of the immune response and growth.

Mutations in β-catenin, especially at the residues critical for its degradation, render it constitutively active. Here, we show that mutant β-catenin can be transported via extracellular vesicles (EVs) and activate Wnt signalling pathway in the recipient cells. An integrative proteogenomic analysis identified the presence of mutated β-catenin in EVs secreted by colorectal cancer (CRC) cells. Follow-up experiments established that EVs released from LIM1215 CRC cells stimulated Wnt signalling pathway in the recipient cells with wild-type β-catenin. SILAC-based quantitative proteomics analysis confirmed the transfer of mutant β-catenin to the nucleus of the recipient cells. In vivo tracking of DiR-labelled EVs in mouse implanted with RKO CRC cells revealed its bio-distribution, confirmed the activation of Wnt signalling pathway in tumour cells and increased the tumour burden. Overall, for the first time, this study reveals that EVs can transfer mutant β-catenin to the recipient cells and promote cancer progression.

Neuroblastoma accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is a well-established poor prognostic marker for neuroblastoma. Whilst N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressiveness of the disease is poorly understood. Exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. Hence, characterising the exosomal protein components from N-Myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. In this study, a comparative proteomic analysis of exosomes isolated from cells with varying N-Myc amplification status was performed. Label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the neuroblastoma cells. Gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in N-Myc amplified exosomes. Treatment of SH-SY5Y cells with N-Myc amplified SK-N-BE2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. Incubation of exosomes from N-Myc knocked down SK-N-BE2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. These findings suggest that exosomes could play a pivotal role in N-Myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. Abbreviations: RNA = ribonucleic acid; DNA = deoxyribonucleic acid; FCS = foetal calf serum; NTA = nanoparticle tracking analysis; LC-MS = liquid chromatography–mass spectrometry; KD = knockdown; LTQ = linear trap quadropole; TEM = transmission electron microscopy

Summary: Billions of cells undergo apoptosis daily and often fragment into small, membrane-bound extracellular vesicles termed apoptotic bodies (ApoBDs). We demonstrate that apoptotic monocytes undergo a highly coordinated disassembly process and form long, beaded protrusions (coined as beaded apoptopodia), which fragment to release ApoBDs. Here, we find that the protein plexin B2 (PlexB2), a transmembrane receptor that regulates axonal guidance in neurons, is enriched in the ApoBDs of THP1 monocytes and is a caspase 3/7 substrate. To determine whether PlexB2 is involved in the disassembly of apoptotic monocytes, we generate PlexB2-deficient THP1 monocytes and demonstrate that lack of PlexB2 impairs the formation of beaded apoptopodia and ApoBDs. Consequently, the loss of PlexB2 in apoptotic THP1 monocytes impairs their uptake by both professional and non-professional phagocytes. Altogether, these data identify PlexB2 as a positive regulator of apoptotic monocyte disassembly and demonstrate the importance of this process in apoptotic cell clearance. : Atkin-Smith et al. examine the role of Plexin B2, a membrane receptor, in the disassembly of apoptotic monocytes. Apoptosis induces caspase cleavage of Plexin B2 and cleaved Plexin B2 is enriched in apoptotic fragments. Genetic deletion of Plexin B2 impairs apoptotic monocyte disassembly and compromises apoptotic debris uptake by phagocytes. Keywords: apoptotic cell disassembly, plexin B2, apoptotic bodies, apoptotic cell clearance, apoptosis, apoptopodia

Neuroblastoma (NBL) is a pediatric cancer that accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc occurs in 20% of NBL patients and is considered high risk as it correlates with aggressiveness, treatment resistance and poor prognosis. Even though the treatment strategies have improved in the recent years, the survival rate of high-risk NBL patients remain poor. Hence, it is crucial to explore new therapeutic avenues to sensitise NBL. Recently, bovine milk-derived extracellular vesicles (MEVs) have been proposed to contain anti-cancer properties. However, the impact of MEVs on NBL cells is not understood. In this study, we characterised MEVs using Western blotting, NTA and TEM. Importantly, treatment of NBL cells with MEVs decreased the proliferation and increased the sensitivity of NBL cells to doxorubicin. Temporal label-free quantitative proteomics of NBL cells highlighted the depletion of proteins involved in cell metabolism, cell growth and Wnt signalling upon treatment with MEVs. Furthermore, proteins implicated in cellular senescence and apoptosis were enriched in NBL cells treated with MEVs. For the first time, this study highlights the temporal proteomic profile that occurs in cancer cells upon MEVs treatment.

The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform “Coursera” and is free of charge.

Physical activity has systemic effects on the body, affecting almost every organ. It is important not only for general health and wellbeing, but also in the prevention of diseases. The mechanisms behind the therapeutic effects of physical activity are not completely understood; however, studies indicate these benefits are not confined to simply managing energy balance and body weight. They also include systemic factors which are released into the circulation during exercise and which appear to underlie the myriad of benefits exercise can elicit. It was shown that along with a number of classical cytokines, active tissues also engage in inter-tissue communication via extracellular vesicles (EVs), specifically exosomes and other small EVs, which are able to deliver biomolecules to cells and alter their metabolism. Thus, EVs may play a role in the acute and systemic adaptations that take place during and after physical activity, and may be therapeutically useful in the treatment of a range of diseases, including metabolic disorders such as type 2 diabetes and obesity; and the focus of this review, neurological disorders such as Alzheimer’s disease.

Currently, 5-fluorouracil (5-FU)-based combination chemotherapy is the mainstay in the treatment of metastatic colorectal cancer (CRC), which benefits approximately 50% of the patients. However, these tumors inevitably acquire chemoresistance resulting in treatment failure. The molecular mechanisms driving acquired chemotherapeutic drug resistance in CRC is fundamental for the development of novel strategies for circumventing resistance. However, the specific phenomenon that drives the cancer cells to acquire resistance is poorly understood. Understanding the molecular mechanisms that regulate chemoresistance will uncover new avenues for the treatment of CRC. Among the various mechanisms of acquired chemoresistance, defects in the drug metabolism pathways could play a major role. In the case of 5-FU, it gets converted into various active metabolites, which, directly or indirectly, interferes with the replication and transcription of dividing cells causing DNA and RNA damage. In this project, we developed a high-resolution mass spectrometry-based method to effectively extract and quantify levels of the 5-FU metabolites in cell lysates and media of parental and 5-FU resistant LIM1215 CRC cells. The analysis highlighted that the levels of 5-FU metabolites are significantly reduced in 5-FU resistant cells. Specifically, the level of the nucleotide fluorodeoxyuridine monophosphate (FdUMP) is reduced with treatment of 5-FU clarifying the compromised 5-FU metabolism in resistant cells. Corroborating the metabolomic analysis, treatment of the resistant cells with FdUMP, an active metabolite of 5-FU, resulted in effective killing of the resistant cells. Overall, in this study, an effective protocol was developed for comparative quantitation of polar metabolites and nucleotide analogues from the adherent cells efficiently. Furthermore, the utility of FdUMP as an alternative for CRC therapy is highlighted.

Milk is considered as more than a source of nutrition for infants and is a vector involved in the transfer of bioactive compounds and cells. Milk contains abundant quantities of extracellular vesicles (EVs) that may originate from multiple cellular sources. These nanosized vesicles have been well characterized and are known to carry a diverse cargo of proteins, nucleic acids, lipids and other biomolecules. Milk-derived EVs have been demonstrated to survive harsh and degrading conditions in gut, taken up by various cell types, cross biological barriers and reach peripheral tissues. The cargo carried by these dietary EVs has been suggested to have a role in cell growth, development, immune modulation and regulation. Hence, there is considerable interest in understanding the role of milk-derived EVs in mediating inter-organismal and cross-species communication. Furthermore, various attributes such as it being a natural source, as well as its abundance, scalability, economic viability and lack of unwarranted immunologic reactions, has generated significant interest in deploying milk-derived EVs for clinical applications such as drug delivery and disease therapy. In this review, the role of milk-derived EVs in inter-organismal, cross-species communication and in drug delivery is discussed.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture “what the community needs in a tool”. Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs.

The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane. While the TIM22 machinery has been well characterised in yeast, the human complex remains poorly characterised. Here, we identify Tim29 (C19orf52) as a novel, metazoan-specific subunit of the human TIM22 complex. The protein is integrated into the mitochondrial inner membrane with it’s C-terminus exposed to the intermembrane space. Tim29 is required for the stability of the TIM22 complex and functions in the assembly of hTim22. Furthermore, Tim29 contacts the Translocase of the Outer Mitochondrial Membrane, TOM complex, enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space. Identification of Tim29 highlights the significance of analysing mitochondrial import systems across phylogenetic boundaries, which can reveal novel components and mechanisms in higher organisms.

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.