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PhD MSc BSc (hons) AFHEA
Department of Biological and Medical Sciences
Faculty of Health and Life Sciences
The scientific and clinical interest in extracellular vesicles (EV) has grown exponentially during the past 15 years. As most research indicates that EVs can be utilised in diagnostics, prognostics and therapeutics, we may be on the brink of establishing the clinical utility of EV measurement, but how can we make this a reality? If we are to introduce EVs as biomarkers into clinical laboratories it will be necessary to offer fully validated, International Organization for Standardization (ISO) standard 15189 assays. ISO 15189 defines the quality management system requirements particular to medical laboratories and is used internationally to determine accreditation. In order for a clinical laboratory to offer an accredited test for EVs, this assay must have been subjected to a thorough assay validation process. This process requires the generation of data related to defined performance characteristics, to ensure that an assay is performing in accordance with the needs of its clinical users. Each of the defined performance characteristics will be discussed in this review, along with the issues that specifically affect EV analysis. Accreditation is increasingly important for all clinical laboratories and the standards required to achieve this are becoming more and more stringent. Therefore, as companies seek to develop the best assays to detect EVs and their molecular contents for clinical utility, and as we move rapidly towards our goal of offering EV analysis in the diagnosis and monitoring of disease, it is timely to highlight the requirements for the clinical accreditation of such assays. It is essential to consider these parameters to ensure that we develop the highest quality assays possible and ultimately the best outcomes for patients.
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.
Background. The incidence of human papilloma virus positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) has increased rapidly in recent decades. These tumours have a favourable outcome compared to HPV‐negative (HPV−) OPSCC. However, HPV+ tumours are more likely to metastasise to distant sites, suggesting a difference in how these tumour subtypes interact with the metastatic niche. Extracellular vesicles (EVs) have emerged as important players in cell‐to‐cell communication and are a potential source of biomarkers for cancer diagnosis. This study aims to characterise the microRNA cargo of small EVs released by HPV+ and HPV− OPSCC cell lines. Methods. Extracellular vesicles produced by HPV+ (SCC2 and SCC90) and HPV− (SCC72 an SCC89) OPSCC cells were characterised by tunable resistive pulse sensing (TRPS) and western blotting. RNA was extracted from EVs and analysed by small RNA sequencing. A bioinformatics approach was used to identify EV miRNA signatures associated with HPV status. Results. HPV− OPSCC cells produced more EVs than HPV+ OPSCC cells. EVs were positive for the common EV markers CD63, CD9 and TSG101. Unbiased hierarchical clustering analysis of EV miRNA cargo revealed that samples clustered based on HPV status. 14 miRNA were enriched in HPV+ cell‐derived EVs, whereas 19 miRNA were enriched in EVs derived from HPV− cell lines. Conclusions. Here, we identify EV miRNA signatures indicative of the HPV status of the parent cell. This may provide a platform from which to validate salivary or blood‐based biomarkers with utility for early detection and stratifying risk in OPSCC patients.
This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.
Ovarian cancer is the deadliest gynaecological cancer. A major contributor to the poor survival rate is the development of chemoresistance to platinum-based therapies such as cisplatin and carboplatin. Here we aimed to test the role of miRNAs in the acquisition of drug resistance in ovarian cancer.
We used microarrays to measure miRNA levels in the ovarian cancer cell line A2780 and its cisplatin-resistant derivative CP70. The role of miRNAs and the mRNA targets were tested using transfected miRNA mimics and siRNAs, respectively. Potential in vivo significance was investigated by analysing RNA levels in cohorts of ovarian cancer patients.
We identified several miRNAs that are increased in cisplatin-resistant cells. We show that most of these do not directly contribute to cisplatin resistance. Interestingly, miR-21-3p, the passenger strand of the known oncomiR, directed increased resistance to cisplatin in a range of ovarian cell lines. This effect was specific to the star strand, as miR-21-5p had the opposite effect and actually increased sensitivity of A2780 cells to cisplatin. We identify NAV3 as a potential target of miR-21-3p and show that knockdown of NAV3 increases resistance. Exosomes released by CP70 cells were also capable of increasing resistance in A2780 cells, although this was independent of miR-21-3p. Finally, we use publically available transcriptomic data to demonstrate that miR-21-3p is raised, while NAV3 is reduced, in ovarian tumours that are resistant to platinum treatment.
Our data suggest that miR-21-3p can induce cisplatin resistance in ovarian tumours, potentially by targeting the NAV3 gene.
Ovarian cancers have a high mortality rate; this is in part due to resistance to the platinum-based compounds used in chemotherapy. In this paper, we assess the role of microRNA-31 in the development of chemoresistance to cisplatin. We used previous data from microarray experiments to identify potential microRNAs (miRNAs) involved in chemoresistance. The functional significance of these microRNAs was tested using miRNA mimics. We used RNA-seq to identify pathways and genes de-regulated in the resistant cell line and then determined their role using RNAi. Analysis of publically available datasets reveals the potential clinical significance. Our data show that miR-31 is increased, whilst potassium channel calcium activated large conductance subfamily M alpha, member 1 (KCNMA1), a subunit of calcium-regulated big potassium (BK) channels, is reduced in resistant ovarian cells. Over-expression of miR-31 increased resistance, as did knockdown of KCNMA1 or inhibition of BK channels. This suggests that these genes directly modulate cisplatin response. Our data also suggest that miR-31 represses KCNMA1 expression. Comparing the levels of miR-31 and KCNMA1 to cisplatin resistance in the NCI60 panel or chemoresistance in cohorts of ovarian cancer tumours reveals correlations that support a role for these genes in vitro and in vivo. Here we show that miR-31 and KCNMA1 are involved in mediating cisplatin resistance in ovarian cancer. Our data gives a new insight into the potential mechanisms to therapeutically target in cisplatin resistance common to ovarian cancer.
Orexins are neuropeptides that regulate the sleep-wake cycle and feeding behaviour. QRFP is a newly discovered neuropeptide which exerts similar orexigenic activity, thus playing an important role in energy homeostasis and regulation of appetite. The exact expression and signalling characteristics and physiological actions of QRFP and its receptor GPR103 are poorly understood. Alzheimer's disease (AD) patients experience increased nocturnal activity, excessive daytime sleepiness, and weight loss. We hypothesised therefore that orexins and QRFP might be implicated in the pathophysiology of AD. We report that the down-regulation of hippocampal orexin receptors (OXRs) and GPR103 particularly in the cornu ammonis (CA) subfield from AD patients suffering from early onset familial AD (EOFAD) and late onset familial AD (LOAD). Using an in vitro model we demonstrate that this downregulation is due to to Aβ-plaque formation and tau hyper-phosphorylation. Transcriptomics revealed a neuroprotective role for both orexins and QRFP. Finally we provide conclusive evidence using BRET and FRET that OXRs and GPR103 form functional hetero-dimers to exert their effects involving activation of ERK1/2. Pharmacological intervention directed at the orexigenic system may prove to be an attractive avenue towards the discovery of novel therapeutics for diseases such as AD and improving neuroprotective signalling pathways.
Extracellular vesicles (EVs) are small vesicles released by donor cells that can be taken up by recipient cells. Despite their discovery decades ago, it has only recently become apparent that EVs play an important role in cell-to-cell communication. EVs can carry a range of nucleic acids and proteins which can have a significant impact on the phenotype of the recipient. For this phenotypic effect to occur, EVs need to fuse with target cell membranes, either directly with the plasma membrane or with the endosomal membrane after endocytic uptake. EVs are of therapeutic interest because they are deregulated in diseases such as cancer and they could be harnessed to deliver drugs to target cells. It is therefore important to understand the molecular mechanisms by which EVs are taken up into cells. This comprehensive review summarizes current knowledge of EV uptake mechanisms. Cells appear to take up EVs by a variety of endocytic pathways, including clathrin-dependent endocytosis, and clathrin-independent pathways such as caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft-mediated internalization. Indeed, it seems likely that a heterogeneous population of EVs may gain entry into a cell via more than one route. The uptake mechanism used by a given EV may depend on proteins and glycoproteins found on the surface of both the vesicle and the target cell. Further research is needed to understand the precise rules that underpin EV entry into cells.
Emerging studies implicate the signalling of the mammalian target of rapamycin (mTOR) in a number of reproductive functions. To this date, there are no data regarding the expression of mTOR signalling components in the human myometrium during pregnancy. We hypothesized that mTOR-related genes might be differentially expressed in term or preterm labour as well as in labour or non-labour myometria during pregnancy. Using quantitative RT-PCR we demonstrate for first time that there is a significant downregulation of mTOR, DEPTOR, and Raptor in preterm labouring myometria when compared to non-pregnant tissues taken from the same area (lower segment). We used an immortalised myometrial cell line (ULTR) as an in vitro model to dissect further mTOR signalling. In ULTR cells DEPTOR and Rictor had a cytoplasmic distribution, whereas mTOR and Raptor were detected in the cytoplasm and the nucleus, indicative of mTORC1 shuttling. Treatment with inflammatory cytokines caused only minor changes in gene expression of these components, whereas progesterone caused significant down-regulation. We performed a non-biased gene expression analysis of ULTR cells using Nimblegen human gene expression microarray (n=3), and selected genes were validated by quantitative RT-PCR in progesterone treated myometrial cells. Progesterone significantly down-regulated key components of the mTOR pathway. We conclude that the human myometrium differentially expresses mTOR signalling components and they can be regulated by progesterone.
Butterflies are popular model organisms to study physiological mechanisms underlying variability in oogenesis and egg provisioning in response to environmental conditions. Nothing is known, however, about; the developmental mechanisms governing butterfly oogenesis, how polarity in the oocyte is established, or which particular maternal effect genes regulate early embryogenesis. To gain insights into these developmental mechanisms and to identify the conserved and divergent aspects of butterfly oogenesis, we analysed a de novo ovarian transcriptome of the Speckled Wood butterfly Pararge aegeria (L.), and compared the results with known model organisms such as Drosophila melanogaster and Bombyx mori.
Results A total of 17306 contigs were annotated, with 30% possibly novel or highly divergent sequences observed. Pararge aegeria females expressed 74.5% of the genes that are known to be essential for D. melanogaster oogenesis. We discuss the genes involved in all aspects of oogenesis, including vitellogenesis and choriogenesis, plus those implicated in hormonal control of oogenesis and transgenerational hormonal effects in great detail. Compared to other insects, a number of significant differences were observed in; the genes involved in stem cell maintenance and differentiation in the germarium, establishment of oocyte polarity, and in several aspects of maternal regulation of zygotic development.
Conclusions This study provides valuable resources to investigate a number of divergent aspects of butterfly oogenesis requiring further research. In order to fully unscramble butterfly oogenesis, we also now also have the resources to investigate expression patterns of oogenesis genes under a range of environmental conditions, and to establish their function
A pseudogene arises when a gene loses the ability to produce a protein, which can be due to mutation or inaccurate duplication. Previous dogma has dictated that because the pseudogene no longer produces a protein it becomes functionless and evolutionarily inert, being neither conserved nor removed. However, recent evidence has forced a re-evaluation of this view. Some pseudogenes, although not translated into protein, are at least transcribed into RNA. In some cases, these pseudogene transcripts are capable of influencing the activity of other genes that code for proteins, thereby altering expression and in turn affecting the phenotype of the organism. In the present chapter, we will define pseudogenes, describe the evidence that they are transcribed into non-coding RNAs and outline the mechanisms by which they are able to influence the machinery of the eukaryotic cell.
Organisms are often exposed to environmental pressures that affect homeostasis, so it is important to understand the biological basis of stress-response. Various biological mechanisms have evolved to help cells cope with potentially cytotoxic changes in their environment. miRNAs are small non-coding RNAs which are able to regulate mRNA stability. It has been suggested that miRNAs may tip the balance between continued cytorepair and induction of apoptosis in response to stress. There is a wealth of data in the literature showing the effect of environmental stress on miRNAs, but it is scattered in a large number of disparate publications. Meta-analyses of this data would produce added insight into the molecular mechanisms of stress-response. To facilitate this we created and manually curated the miRStress database, which describes the changes in miRNA levels following an array of stress types in eukaryotic cells. Here we describe this database and validate the miRStress tool for analysing miRNAs that are regulated by stress. To validate the database we performed a cross-species analysis to identify miRNAs that respond to radiation. The analysis tool confirms miR-21 and miR-34a as frequently deregulated in response to radiation, but also identifies novel candidates as potentially important players in this stress response, including miR-15b, miR-19b, and miR-106a. Similarly, we used the miRStress tool to analyse hypoxia-responsive miRNAs. The most frequently deregulated miRNAs were miR-210 and miR-21, as expected. Several other miRNAs were also found to be associated with hypoxia, including miR-181b, miR-26a/b, miR-106a, miR-213 and miR-192. Therefore the miRStress tool has identified miRNAs with hitherto unknown or under-appreciated roles in the response to specific stress types. The miRStress tool, which can be used to uncover new insight into the biological roles of miRNAs, and also has the potential to unearth potential biomarkers for therapeutic response, is freely available at http://mudshark.brookes.ac.uk/MirStress.
Communication between irradiated and un-irradiated (bystander) cells can cause damage in cells that are not directly targeted by ionizing radiation, a process known as the bystander effect. Bystander effects can also lead to chromosomal/genomic instability within the progeny of bystander cells, similar to the progeny of directly irradiated cells. The factors that mediate this cellular communication can be transferred between cells via gap junctions or released into the extracellular media following irradiation, but their nature has not been fully characterized. In this study we tested the hypothesis that the bystander effect mediator contains an RNA molecule that may be carried by exosomes. MCF7 cells were irradiated with 2 Gy of X rays and the extracellular media was harvested. RNase treatment abrogated the ability of the media to induce early and late chromosomal damage in bystander cells. Furthermore, treatment of bystander cells with exosomes isolated from this media increased the levels of genomic damage. These results suggest that the bystander effect, and genomic instability, are at least in part mediated by exosomes and implicate a role for RNA.
Pseudogenes have long been labeled as "junk'' DNA, failed copies of genes that arise during the evolution of genomes. However, recent results are challenging this moniker; indeed, some pseudogenes appear to harbor the potential to regulate their protein-coding cousins. Far from being silent relics, many pseudogenes are transcribed into RNA, some exhibiting a tissue-specific pattern of activation. Pseudogene transcripts can be processed into short interfering RNAs that regulate coding genes through the RNAi pathway. In another remarkable discovery, it has been shown that pseudogenes are capable of regulating tumor suppressors and oncogenes by acting as microRNA decoys. The finding that pseudogenes are often deregulated during cancer progression warrants further investigation into the true extent of pseudogene function. In this review, we describe the ways in which pseudogenes exert their effect on coding genes and explore the role of pseudogenes in the increasingly complex web of noncoding RNA that contributes to normal cellular regulation.
The incidence of squamous cancer of the esophagus varies up to a hundredfold in different regions of the world. In Transkei, South Africa, a particularly high incidence of the disease is observed. We have previously proposed an association between a maize-rich diet and elevated levels of intragastric prostaglandin E2 production (PGE2). Here we investigate the molecular mechanisms by which a high-maize diet could lead to increased incidence of squamous cancer of the esophagus.We confirm that levels of PGE2 are high (606.8 pg/ml) in the gastric fluid of individuals from Transkei. We also show that treatment of esophageal cells with linoleic acid, which is found at high levels in maize and is a precursor to PGE2, leads to increased cell proliferation. Similarly, treatment of cells with PGE2 or with gastric fluid from Transkeians also leads to increased proliferation.Our data suggest that the high levels of PGE2 associated with a maize-rich diet stimulate cell division and induce the enzyme COX 2, resulting in a positive feedback mechanism that predisposes the esophagus to carcinoma.
The way in which the genome of a multicellular organism can orchestrate the differentiation of trillions of cells and many organs, all from a single fertilized egg, is the subject of intense study. Different cell types can be defined by the networks of genes they express. This differential expression is regulated at the epigenetic level by chromatin modifications, such as DNA and histone methylation, which interact with structural and enzymatic proteins, resulting in the activation or silencing of any given gene. While detailed mechanisms are emerging on the role of different chromatin modifications and how these functions are effected at the molecular level, it is still unclear how their deposition across the epigenomic landscape is regulated in different cells. A raft of recent evidence is accumulating that implicates long noncoding RNAs (lncRNAs) in these processes. Most genomes studied to date undergo widespread transcription, the majority of which is not translated into proteins. In this review, we will describe recent work suggesting that lncRNAs are more than transcriptional "noise", but instead play a functional role by acting as tethers and guides to bind proteins responsible for modifying chromatin and mediating their deposition at specific genomic locations. We suggest that lncRNAs are at the heart of developmental regulation, determining the epigenetic status and transcriptional network in any given cell type, and that they provide a means to integrate external differentiation cues with dynamic nuclear responses through the regulation of a metastable epigenome. Better characterization of the lncRNA-protein "interactome" may eventually lead to a new molecular toolkit, allowing researchers and clinicians to modulate the genome at the epigenetic level to treat conditions such as cancer.
Higher order chromatin folding is critical to a number of developmental processes, including the regulation of gene expression. Recently developed biochemical techniques such as RNA TRAP and chromosome conformation capture (3C) have provided us with the tools to probe chromosomal structures. These techniques have been applied to the β-globin locus, revealing a complex pattern of interactions with regions along the chromosome that the gene resides on. However, biochemical and microscopy data on the nature of β-globin interactions with other chromosomes is contradictory. Therefore we developed a novel 4C variant, Complete-genome 3C by vectorette amplification (4Cv), which allows an unbiased and quantitative method to examine chromosomal structure. We have used 4Cv to study the microenvironment of the β-globin locus in mice and show that a significant proportion of the interactions of β-globin are inter-chromosomal. Furthermore, our data show that in the liver, where the gene is active, β-globin is more likely to interact with other chromosomes, compared to the brain where the gene is silent and is more likely to interact with other regions along the same chromosome. Our data suggest that transcriptional activation of the β-globin locus leads to a change in nuclear position relative to the chromosome territory.
Higher order chromatin folding is critical to a number of developmental processes, including the regulation of gene expression. Recently developed biochemical techniques such as RNA TRAP and chromosome conformation capture (3C) have provided us with the tools to probe chromosomal structures. These techniques have been applied to the Î²-globin locus, revealing a complex pattern of interactions with regions along the chromosome that the gene resides on. However, biochemical and microscopy data on the nature of Î²-globin interactions with other chromosomes is contradictory. Therefore we developed a novel 4C variant, Complete-genome 3C by vectorette amplification (4Cv), which allows an unbiased and quantitative method to examine chromosomal structure. We have used 4Cv to study the microenvironment of the Î²-globin locus in mice and show that a significant proportion of the interactions of Î²-globin are inter-chromosomal. Furthermore, our data show that in the liver, where the gene is active, Î²-globin is more likely to interact with other chromosomes, compared to the brain where the gene is silent and is more likely to interact with other regions along the same chromosome. Our data suggest that transcriptional activation of the Î²-globin locus leads to a change in nuclear position relative to the chromosome territory.
One of the hallmarks of ageing is the increased susceptibility to develop age-related diseases such as infections and cancers. This is believed to be favoured by a decline in the function of the innate and adaptive immune systems, a process known as immunosenescence. The current greying of the world population leads to the potential dilemma of increasing life expectancy without the concomitant increase in the quality of life with more individuals acquiring age-related disorders. Ultimately this may have profound socio-economic implications for those in positions of government and strategic policy makers. With this in mind, this chapter reviews the current understanding of some of the causes of immunosenescence and looks to address the question, “Can immunosenescence be corrected or reversed?”
Ovarian cancer (OC) is the deadliest gynecological malignancy. Most patients are diagnosed when they are already in the later stages of the disease. Earlier detection of OC dramatically improves the overall survival, but this is rarely achieved as there is a lack of clinically implemented biomarkers of early disease. Extracellular vesicles (EVs) are small cell-derived vesicles that have been extensively studied in recent years. They contribute to various aspects of cancer pathology, including tumour growth, angiogenesis and metastasis. EVs are released from all cell types and the macromolecular cargo they carry reflects the content of the cells from which they were derived. Cancer cells release EVs with altered cargo into biofluids, and so they represent an excellent potential source of novel biomarkers for the disease. In this review we describe the latest developments in EVs as potential biomarkers for earlier detection of OC. The field is still relatively young, but a number of studies have shown that EVs and the cargo they carry, including miRNAs and proteins, can be used to detect OC. They could also give insight into the stage of the disease and predict the likely therapeutic outcome. There remain a number of challenges to the use of EVs as biomarkers, but through ongoing research and innovation in this exciting field there is great potential for the development of diagnostic assays in the clinic that could improve patient outcome.