Search

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its Minimal Information for Studies of Extracellular Vesicles, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

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.

Human pregnancy is a highly orchestrated process requiring extensive cross-talk between the mother and the fetus. Extracellular vesicles released by the fetal tissue, particularly the placenta, are recognized as important mediators of this process. More recently, the importance of placental extracellular vesicle biodistribution studies in animal models has received increasing attention as identifying the organs to which extracellular vesicles are targeted to helps us understand more about this communication system. Placental extracellular vesicles are categorized based on their size into macro-, large-, and small-extracellular vesicles, and their biodistribution is dependent on the extracellular vesicle's particle size, the direction of blood flow, the recirculation of blood, as well as the retention capacity in organs. Macro-extracellular vesicles are exclusively localized to the lungs, while large- and small-extracellular vesicles show high levels of distribution to the lungs and liver, while there is inconsistency in the reporting of distribution to the spleen and kidneys. This inconsistency may be due to the differences in the methodologies employed between studies and their limitations. Future studies should incorporate analysis of placental extracellular vesicle biodistribution at the macroscopic level on whole animals and organs/tissues, as well as the microscopic cellular level. [ABSTRACT FROM AUTHOR]

Materials platforms that enable controlled isolation and subsequent release of chemical/biological entities are in great demand for a diverse range of practical applications. Current technologies lack good control and efficiency of the release, which is needed to preserve the captured targets of interest. Here, this need is addressed by providing a versatile, controllable, electrochemical capture/release interface. The interface consists of a highly porous electrospun membrane, electrodeposited with a thiol-functionalized 3,4-ethyl-enedioxythiophene (EDOT) conductive terpolymer, in which the thiol moiety undergoes oxidation/reduction cycles at moderate potentials (+1.0 and -0.8 V, respectively) to enable capture/release. The fast oxidative capture (1 min) and reductive release (2 min) of a model thiol molecule in a highly controllable manner, followed by successful capture/release of an antibody, are demon- strated. Then, femtosecond laser-patterning is used to fabricate an array of ≈30 µm pores on the electrospun membrane, subsequently coated with the conducting terpolymer, enabling the highly efficient (>90%), fast (20 min) and selective capture of MCF7 cancer cells with 33% release efficiency when polarized at -0.8 V. The released cells show a high level of viability, indicating the capture and release process does not affect cell survival. [ABSTRACT FROM AUTHOR]

The functional genomic approaches of transcriptomics, proteomics and metabolomics aim to measure the mRNA, protein or metabolite complement of a cell, tissue or organism. In this study we have investigated the compatibility of transcriptional analysis, using Reverse Transcription (RT)-PCR, and metabolite analysis, by high-resolution magic angle spinning (HRMAS) [sup.1]H NMR spectroscopy, in BT4C rat glioma following the induction of programmed cell death. The metabolite and transcriptional changes that accompanied apoptosis were examined at 0, 4 and 8 days of ganciclovir/thymidine kinase gene therapy. Despite the high spinning speeds employed during HRMAS [sup.1]H NMR spectroscopy of one-half of the tumor samples, RT-PCR analysis of the pro-apoptotic transcripts Bcl-2, BAK-1, caspase-9 and FAS was possible, producing similar results to those detected in the unspun half of the tumors. Furthermore, the expression of FAS was inversely correlated with some of the key metabolic changes across the time period examined including the increases CH = CH and CH = CHC[H.sub.2] lipid resonances which accompany apoptosis. This study demonstrates how combined transcriptomic and metabolomic studies of tumors can be used to understand the molecular events that accompany well documented metabolic perturbations during cell death processes.

The minimal information for studies of extracellular vesicles (EVs, MISEV) is a field‐consensus rigour initiative of the International Society for Extracellular Vesicles (ISEV). The last update to MISEV, MISEV2018, was informed by input from more than 400 scientists and made recommendations in the six broad topics of EV nomenclature, sample collection and pre‐processing, EV separation and concentration, characterization, functional studies, and reporting requirements/exceptions. To gather opinions on MISEV and ideas for new updates, the ISEV Board of Directors canvassed previous MISEV authors and society members. Here, we share conclusions that are relevant to the ongoing evolution of the MISEV initiative and other ISEV rigour and standardization efforts. [ABSTRACT FROM AUTHOR]

In recent years, attention has turned to examining the biodistribution of EVs in recipient animals to bridge between knowledge of EV function in vitro and in vivo. We undertook a systematic review of the literature to summarize the biodistribution of EVs following administration into animals. There were time‐dependent changes in the biodistribution of small‐EVs which were most abundant in the liver. Detection peaked in the liver and kidney in the first hour after administration, while distribution to the lungs and spleen peaked between 2–12 h. Large‐EVs were most abundant in the lungs with localization peaking in the first hour following administration and decreased between 2–12 h. In contrast, large‐EV localization to the liver increased as the levels in the lungs decreased. There was moderate to low localization of large‐EVs to the kidneys while localization to the spleen was typically low. Regardless of the origin or size of the EVs or the recipient species into which the EVs were administered, the biodistribution of the EVs was largely to the liver, lungs, kidneys, and spleen. There was extreme variability in the methodology between studies and we recommend that guidelines should be developed to promote standardization where possible of future EV biodistribution studies. [ABSTRACT FROM AUTHOR]

Abstract Background The differential expression pattern of microRNAs (miRNAs) during mammary gland development might provide insights into their role in regulating the homeostasis of the mammary epithelium. Our aim was to analyse these regulatory functions by deriving a comprehensive tissue-specific combined miRNA and mRNA expression profile of post-natal mouse mammary gland development. We measured the expression of 318 individual murine miRNAs by bead-based flow-cytometric profiling of whole mouse mammary glands throughout a 16-point developmental time course, including juvenile, puberty, mature virgin, gestation, lactation, and involution stages. In parallel whole-genome mRNA expression data were obtained. Results One third (n = 102) of all murine miRNAs analysed were detected during mammary gland development. MicroRNAs were represented in seven temporally co-expressed clusters, which were enriched for both miRNAs belonging to the same family and breast cancer-associated miRNAs. Global miRNA and mRNA expression was significantly reduced during lactation and the early stages of involution after weaning. For most detected miRNA families we did not observe systematic changes in the expression of predicted targets. For miRNA families whose targets did show changes, we observed inverse patterns of miRNA and target expression. The data sets are made publicly available and the combined expression profiles represent an important community resource for mammary gland biology research. Conclusion MicroRNAs were expressed in likely co-regulated clusters during mammary gland development. Breast cancer-associated miRNAs were significantly enriched in these clusters. The mechanism and functional consequences of this miRNA co-regulation provide new avenues for research into mammary gland biology and generate candidates for functional validation.

Abstract Background Post-translational modification of histones resulting in chromatin remodelling plays a key role in the regulation of gene expression. Here we report characteristic patterns of expression of 12 members of 3 classes of chromatin modifier genes in 6 different cancer types: histone acetyltransferases (HATs)- EP300, CREBBP, and PCAF; histone deacetylases (HDACs)- HDAC1, HDAC2, HDAC4, HDAC5, HDAC7A, and SIRT1; and histone methyltransferases (HMTs)- SUV39H1and SUV39H2. Expression of each gene in 225 samples (135 primary tumours, 47 cancer cell lines, and 43 normal tissues) was analysedby QRT-PCR, normalized with 8 housekeeping genes, and given as a ratio by comparison with a universal reference RNA. Results This involved a total of 13,000 PCR assays allowing for rigorous analysis by fitting a linear regression model to the data. Mutation analysis of HDAC1, HDAC2, SUV39H1, and SUV39H2 revealed only two out of 181 cancer samples (both cell lines) with significant coding-sequence alterations. Supervised analysis and Independent Component Analysis showed that expression of many of these genes was able to discriminate tumour samples from their normal counterparts. Clustering based on the normalized expression ratios of the 12 genes also showed that most samples were grouped according to tissue type. Using a linear discriminant classifier and internal cross-validation revealed that with as few as 5 of the 12 genes, SIRT1, CREBBP, HDAC7A, HDAC5 and PCAF, most samples were correctly assigned. Conclusion The expression patterns of HATs, HDACs, and HMTs suggest these genes are important in neoplastic transformation and have characteristic patterns of expression depending on tissue of origin, with implications for potential clinical application.

Trophoblasts are unique epithelial cells found only in the placenta. It has been possible to isolate and maintain human trophoblasts in in vitro culture for many decades. During this period there have been a vast array of media and supplements reported for trophoblast culture and often the reasons for using the media and specific supplements employed in any given laboratory have been lost in the 'mists of time'. After a gradual development over many years this field has recently changed, with the publication of several reports of the isolation, growth and differentiation of human trophoblast stem or stem-like cells. This advance was made largely because of a greater understanding of the molecular pathways that control human trophoblasts and availability of media supplements that can be used to manipulate those pathways. We have searched the literature and here summarise many of the different media and supplements and describe how and why they were developed and are used to culture human trophoblasts. [ABSTRACT FROM AUTHOR]

New Findings: What is the central question of this study?Does short‐term high‐intensity interval training alter the composition of the microbiome and is this associated with exercise‐induced improvements in cardiorespiratory fitness and insulin sensitivity?What is the main finding and its importance?Although high‐intensity interval training increased insulin sensitivity and cardiovascular fitness, it did not alter the composition of the microbiome. This suggests that changes in the composition of the microbiome that occur with prolonged exercise training might be in response to changes in metabolic health rather than driving exercise training‐induced adaptations. Regular exercise reduces the risk of metabolic diseases, and the composition of the gut microbiome has been associated with metabolic function. We investigated whether short‐term high‐intensity interval training (HIIT) altered the diversity and composition of the bacterial community and whether there were associations with markers of insulin sensitivity or aerobic fitness. Cardiorespiratory fitness (V̇O2peak) and body composition (dual energy X‐ray absorptiometry scan) were assessed and faecal and fasted blood samples collected from 14 lean (fat mass 21 ± 2%, aged 29 ± 2 years) and 15 overweight (fat mass 33 ± 2%, aged 31 ± 2 years) men before and after 3 weeks of HIIT training (8–12 × 60 s cycle ergometer bouts at V̇O2peak power output interspersed by 75 s rest, three times per week). Gut microbiome composition was analysed by 16S rRNA gene amplicon sequencing. The HIIT significantly increased the aerobic fitness of both groups (P < 0.001) and improved markers of insulin sensitivity (lowered fasted insulin and HOMA‐IR; P < 0.001) in the overweight group. Despite differences in the abundance of several bacterial taxa being evident between the lean and overweight group, HIIT did not affect the overall bacterial diversity or community structure (α‐diversity or β‐diversity). No associations were found between the top 50 most abundant bacterial genera and cardiorespiratory fitness markers; however, significant associations (P < 0.05) were observed between the abundance of the bacterial species Coprococcus_3, Blautia, Lachnospiraceae_ge and Dorea and insulin sensitivity markers in the overweight group. Our results suggest that short‐term HIIT does not greatly impact the overall composition of the gut microbiome, but that certain microbiome genera are associated with insulin sensitivity markers that were improved by HIIT in overweight participants. [ABSTRACT FROM AUTHOR]

Bacteria release nano-sized extracellular vesicles (EVs) into the extracellular milieu. Bacterial EVs contain molecular cargo originating from the parent bacterium and have important roles in bacterial survival and pathogenesis. Using 8-plex iTRAQ approaches, we profiled the EV proteome of two Escherichia coli strains, uropathogenic (UPEC) 536 and probiotic Nissle 1917. For these strains, we compared the proteome of crude input EVs prepared by ultracentrifugation alone with EVs purified by either density gradient centrifugation (DGC) or size exclusion chromatography (SEC). We further compared the proteome of EVs from bacterial cultures that were grown in iron-restricted (R) and iron-supplemented (RF) conditions. Overall, outer membrane components were highly enriched, and bacterial inner membrane components were significantly depleted in both UPEC and Nissle EVs, in keeping with an outer membrane origin. In addition, we found enrichment of ribosome-related Gene Ontology terms in UPEC EVs and proteins involved in glycolytic processes and ligase activity in Nissle EVs. We have identified that three proteins (RbsB of UPEC in R; YoeA of UPEC in RF; BamA of Nissle in R) were consistently enriched in the DGC- and SEC-purified EV samples in comparison to their crude input EV, whereas conversely the 60 kDa chaperonin GroEL was enriched in the crude input EVs for both UPEC and Nissle in R condition. Such proteins may have utility as technical markers for assessing the purity of E. coli EV preparations. Several proteins were changed in their abundance depending on the iron availability in the media. Data are available via ProteomeXchange with identifier PXD011345. In summary, we have undertaken a comprehensive characterization of the protein content of E. coli EVs and found evidence of specific EV cargos for physiological activity and conserved protein cargo that may find utility as markers in the future. Abbreviation: DGC: density gradient centrifugation; DTT: 1,4-dithiothreitol; EV: extracellular vesicles; FDR: false discovery rate; GO: Gene Ontology; R: iron-restricted; RF: iron-supplemented; iTRAQ: isobaric tags for relative and absolute quantitation; OMV: outer membrane vesicle; SWATH-MS: sequential window acquisition of all theoretical mass spectra; SEC: size exclusion chromatography. [ABSTRACT FROM AUTHOR]

Background: Mesenteric lymph (ML) has been implicated in the development of multiple organ dysfunction syndrome in critical illness. Extracellular RNAs play a role in cell-to-cell communication during physiological and disease processes but they are rarely studied in ML. We aimed at examining the RNA profiles of peripheral plasma, ML, and ML's extracellular vesicle (ML-EV) and triglyceride-rich lipoprotein (ML-TRL) fractions, obtained from rodent models of critical illness. Methods and Results: We collected ML for 5 hours from rodent models of critical illness [Acute Pancreatitis, Cecal Ligation and Incision (CLI), Gut Ischemia-Reperfusion (IR)] and matching Sham control rats. ML-EV and ML-TRL fractions were also isolated. RNA sequencing was performed on the RNA extracted from ML, ML-EV, ML-TRL, and plasma by using the Ion Torrent Personal Genome Machine platform. RNA sequences were searched using the Basic Local Alignment Search Tool against rat genome and RefSeq, microRNA (miRNA), genomic tRNA, functional RNA, and Genbank nucleotide databases, and the read counts were analyzed. Each sample type had a distinct RNA profile. ML contained more RNA per volume and a larger proportion of tRNA fragments than plasma. ML-EVs were enriched with miRNA, whereas ML-TRLs contained low absolute amounts of RNA. The RNA size profiles for CLI and Gut IR were different from Sham. ML carried intestinal RNAs and in a CLI model it was significantly enriched with bacterial RNA sequences. Conclusions: We found the distinct but diverse RNA profiles of ML and its compartments, and their different profiles in critical illness. Intestinal-derived small RNAs in ML may have a direct role in critical illness and utility as potential biomarkers. [ABSTRACT FROM AUTHOR]

In human beings the immature brain is highly plastic and depending on the stage of gestation is particularly vulnerable to a range of insults that if sufficiently severe, can result in long-term motor, cognitive and behavioral impairment. With improved neonatal care, the incidence of major motor deficits such as cerebral palsy has declined with prematurity. Unfortunately, however, milder forms of injury characterized by diffuse non-cystic white matter lesions within the periventricular region and surrounding white matter, involving loss of oligodendrocyte progenitors and subsequent axonal hypomyelination as the brain matures have not. Existing therapeutic options for treatment of preterm infants have proved inadequate, partly owing to an incomplete understanding of underlying post-injury cellular and molecular changes that lead to poor neurodevelopmental outcomes. This has reinforced the need to improve our understanding of brain plasticity, explore novel solutions for the development of protective strategies, and identify biomarkers. Compelling evidence exists supporting the involvement of microRNAs (miRNAs), a class of small non-coding RNAs, as important post-transcriptional regulators of gene expression with functions including cell fate specification and plasticity of synaptic connections. Importantly, miRNAs are differentially expressed following brain injury, and can be packaged within exosomes/extracellular vesicles, which play a pivotal role in assuring their intercellular communication and passage across the blood–brain barrier. Indeed, an increasing number of investigations have examined the roles of specific miRNAs following injury and regeneration and it is apparent that this field of research could potentially identify protective therapeutic strategies to ameliorate perinatal brain injury. In this review, we discuss the most recent findings of some important miRNAs in relation to the development of the brain, their dysregulation, functions and regulatory roles following brain injury, and discuss how these can be targeted either as biomarkers of injury or neuroprotective agents. [ABSTRACT FROM AUTHOR]

Background: The human placenta extrudes a variety of extracellular vesicles (EVs) into the maternal blood daily. These vesicles may be crucial to the adaptation of the maternal cardiovascular and immune systems to pregnancy. Quantifying the EVs that are released in early gestation is important to our understanding of how placental EVs may contribute to the regulation of maternal physiology.Methods: EVs were isolated from first trimester placental explants and separated into micro- and nano-vesicles by differential centrifugation. The numbers of each type of EVs extruded from each milligram of placentae between gestational weeks 8 and 12 was determined by Nanoparticle Tracking Analysis. The total protein or DNA content of the vesicles was determined by BCA assay or Qubit® 2.0.Results: Neither the number of micro- nor nano-EVs/mg explant (n = 49), nor the total protein (n = 19) and DNA content (n = 29) of these EVs changed significantly between 8 and 12 weeks of gestation. When the increasing placental weight with gestation was accounted for, the daily number of placental EVs extruded into the maternal blood increased by more than 100 fold between 8 and 12 weeks (micro-EVs 6.23 X 1014 and nano-EVs 1.84 X 1014 at 12 weeks, p = 0.0003).Discussion: Constant production of micro- and nano-EVs per-milligram placenta, regardless of gestational age, and the increased daily burden of EVs across gestational age indicate these EVs have the potential to regulate maternal physiology from early pregnancy. Since total EV protein content, like EV numbers was, constant, this is a potentially reliable surrogate for quantifying EVs. [ABSTRACT FROM AUTHOR]

We report the results of a pilot project for clinical DNA sequencing in New Zealand. This project aimed to estimate the diagnostic yield of next generation sequencing in the New Zealand clinical environment. Trio whole exome sequencing (WES) was performed on germline DNA of 40 individuals from 12 families with presumptive Mendelian disorders. In addition, both WES and deep targeted sequencing (DTS) was performed on tumours, metastases and corresponding normal blood leukocytes from two cancer patients. For the rare Mendelian disorder cohort, the diagnostic yield was 6/12, including previously recognised pathogenic mutations and novel mutations. In tumour sequence analysis, WES identified somatic single nucleotide mutations and copy number aberrations in both cancer patients; however, DTS was required to obtain clinically informative information. This study showed that diagnostic germline and tumour WES and DTS could be easily undertaken in New Zealand, and identified specific infrastructural challenges that must be solved to facilitate its clinical use. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) regulate gene expression via transcript degradation and translational inhibition, and they may also function as long distance signaling molecules. Circulatory miRNAs are either protein-bound or packaged within vesicles (exosomes). Ten young men (24.6 ± 4.0 yr) underwent a single bout of high-intensity interval cycling exercise. Vastus lateralis biopsies and plasma were collected immediately before and after exercise, as well as 4 h following the exercise bout. Twenty-nine miRNAs previously reported to be regulated by acute exercise were assessed within muscle, venous plasma, and enriched circulatory exosomes via qRT-PCR. Of the 29 targeted miRNAs, 11 were altered in muscle, 8 in plasma, and 9 in the exosome fraction. Although changes in muscle and plasma expression were bidirectional, all regulated exosomal miRNAs increased following exercise. Three miRNAs were altered in all three sample pools (miR-1-3p, -16-5p, and -222-3p), three in both muscle and plasma (miR-21-5p, -134-3p, and -107), three in both muscle and exosomes (miR-23a-3p, -208a-3p, and -150-5p), and three in both plasma and exosomes (miR-486-5p, -126-3p, and -378a-5p). There was a marked discrepancy between the observed alterations between sample pools. A subset of exosomal miRNAs increased in abundance following exercise, suggesting an exercise-induced release of exosomes enriched in specific miRNAs. The uniqueness of the exosomal miRNA response suggests its relevance as a sample pool that needs to be further explored in better understanding biological functions. [ABSTRACT FROM AUTHOR]

Bacteria secrete RNAs, some of which have effects on other cells and on other species as signalling RNAs. Prokaryotic membrane vesicles (MVs) contain a range of RNA types. The MV structure offers protection from degradation as well as receptors to facilitate delivery to target cells. Microscopic imaging and molecular biology analyses have provided evidence to demonstrate that bacterial MVs deliver their RNA into eukaryotic cells. Moreover, in some cases the RNA cargo is demonstrably functional and phenotypic changes can be identified in MV-RNA treated target cells. The challenge now is to dissect the effect of MV-RNA on target cells away from the effects of non-RNA components of the MV such as lipopolysaccharide that can co-purify with RNA. [ABSTRACT FROM AUTHOR]

Maggots, through their excretions and secretions (ES), promote wound healing by removing necrotic tissue, counter bacterial infection, and activate wound associated cells. We investigated the effects of a physiological dose of maggot ES on four wound-associated cell types in vitro with Affymetrix gene expression arrays; keratinocytes, endothelial cells, fibroblasts, and monocytes. Keratinocytes showed the fewest ( n = 5; p < 0.05, fold-change ±2) and smallest fold-changes (up to 2.32×) in gene expression and conversely THP1 monocytes had the most ( n = 233) and greatest magnitude (up to 44.3×). There were no genes that were altered in all four cell-lines. Gene pathway analysis identified an enrichment of immune response pathways in three of the treated cell-lines. Analyses by quantitative RT-PCR found many genes dynamically expressed in ES dose dependent manner during the three day treatments. Phenotype analyses, however, found no effects of ES on cell viability, proliferation, migration and angiogenesis. ES was 100× less potent at triggering IL-8 secretion than fibroblasts treated with purified bacterial lipopolysaccharide (LPS; in equivalent amounts to that found in ES; ∼40 EU/ml). Furthermore, co-treatment with LPS and ES decreased the LPS-alone triggered IL-8 secretion by 13%. Although ES had no direct effect on wound cell phenotypes it did partially reduce the immune response to bacterial LPS exposure. These observations were consistent with the profile of transcriptional responses that were dominated by modulation of immune response genes. Maggot therapy may therefore improve wound healing through the secondary effects of these gene changes in the wound cells. [ABSTRACT FROM AUTHOR]

Background: Bacterium-to-host signalling during infection is a complex process involving proteins, lipids and other diffusible signals that manipulate host cell biology for pathogen survival. Bacteria also release membrane vesicles (MV) that can carry a cargo of effector molecules directly into host cells. Supported by recent publications, we hypothesised that these MVs also associate with RNA, which may be directly involved in the modulation of the host response to infection. Methods and Results: Using the uropathogenic Escherichia coli (UPEC) strain 536, we have isolated MVs and found they carry a range of RNA species. Density gradient centrifugation further fractionated and characterised the MV preparation and confirmed that the isolated RNA was associated with the highest particle and protein containing fractions. Using a new approach, RNA-sequencing of libraries derived from three different ‘size’ RNA populations (<50nt, 50-200nt and 200nt+) isolated from MVs has enabled us to now report the first example of a complete bacterial MV-RNA profile. These data show that MVs carry rRNA, tRNAs, other small RNAs as well as full-length protein coding mRNAs. Confocal microscopy visualised the delivery of lipid labelled MVs into cultured bladder epithelial cells and showed their RNA cargo labelled with 5-EU (5-ethynyl uridine), was transported into the host cell cytoplasm and nucleus. MV RNA uptake by the cells was confirmed by droplet digital RT-PCR of csrC. It was estimated that 1% of MV RNA cargo is delivered into cultured cells. Conclusions: These data add to the growing evidence of pathogenic bacterial MV being associated a wide range of RNAs. It further raises the plausibility for MV-RNA-mediated cross-kingdom communication whereby they influence host cell function during the infection process. [ABSTRACT FROM AUTHOR]

Background: The green bottle fly maggot, Lucilia sericata, is a species with importance in medicine, agriculture and forensics. Improved understanding of this species’ biology is of great potential benefit to many research communities. MicroRNAs (miRNA) are a short non-protein coding regulatory RNA, which directly regulate a host of protein coding genes at the translational level. They have been shown to have developmental and tissue specific distributions where they impact directly on gene regulation. In order to improve understanding of the biology of L. sericata maggots we have performed small RNA-sequencing of their secretions and tissue at different developmental stages. Results: We have successfully isolated RNA from the secretions of L. sericata maggots. Illumina small RNA-sequencing of these secretions and the three tissues (crop, salivary gland, gut) revealed that the most common small RNA fragments were derived from ribosomal RNA and transfer RNAs of both insect and bacterial origins. These RNA fragments were highly specific, with the most common tRNAs, such as GlyGCC, predominantly represented by reads derived from the 5’ end of the mature maggot tRNA. Each library also had a unique profile of miRNAs with a high abundance of miR-10-5p in the maggot secretions and gut and miR-8 in the food storage organ the crop and salivary glands. The pattern of small RNAs in the bioactive maggot secretions suggests they originate from a combination of saliva, foregut and hindgut tissues. Droplet digital RT-PCR validation of the RNA-sequencing data shows that not only are there differences in the tissue profiles for miRNAs and small RNA fragments but that these are also modulated through developmental stages of the insect. Conclusions: We have identified the small-RNAome of the medicinal maggots L. sericata and shown that there are distinct subsets of miRNAs expressed in specific tissues that also alter during the development of the insect. Furthermore there are very specific RNA fragments derived from other non-coding RNAs present in tissues and in the secretions. This new knowledge has applicability in diverse research fields including wound healing, agriculture and forensics. [ABSTRACT FROM AUTHOR]

Background: The nucleic acid-binding protein YB-1, a member of the cold-shock domain protein family, has been implicated in the progression of breast cancer and is associated with poor patient survival. YB-1 has sequence similarity to LIN28, another cold-shock protein family member, which has a role in the regulation of small noncoding RNAs (sncRNAs) including microRNAs (miRNAs). Therefore, to investigate whether there is an association between YB-1 and sncRNAs in breast cancer, we investigated whether sncRNAs were bound by YB-1 in two breast cancer cell lines (luminal A-like and basal cell-like), and whether the abundance of sncRNAs and mRNAs changed in response to experimental reduction of YB-1 expression. Results: RNA-immunoprecipitation with an anti-YB-1 antibody showed that several sncRNAs are bound by YB-1. Some of these were bound by YB-1 in both breast cancer cell lines; others were cell-line specific. The small RNAs bound by YB-1 were derived from various sncRNA families including miRNAs such as let-7 and miR-320, transfer RNAs, ribosomal RNAs and small nucleolar RNAs (snoRNA). Reducing YB-1 expression altered the abundance of a number of transcripts encoding miRNA biogenesis and processing proteins but did not alter the abundance of mature or precursor miRNAs. Conclusions: YB-1 binds to specific miRNAs, snoRNAs and tRNA-derived fragments and appears to regulate the expression of miRNA biogenesis and processing machinery. We propose that some of the oncogenic effects of YB-1 in breast cancer may be mediated through its interactions with sncRNAs. [ABSTRACT FROM AUTHOR]

During axon pathfinding, growth cones commonly show changes in sensitivity to guidance cues that follow a cell-intrinsic timetable. The cellular timer mechanisms that regulate such changes are, however, poorly understood. Here we have investigated microRNAs (miRNAs) in the timing control of sensitivity to the semaphorin Sema3A in Xenopus laevis retinal ganglion cell (RGC) growth cones. A developmental profiling screen identified miR-124 as a candidate timer. Loss of miR-124 delayed the onset of Sema3A sensitivity and concomitant neuropilin-1 (NRP1) receptor expression and caused cell-autonomous pathfinding errors. CoREST, a cofactor of a NRP1 repressor, was newly identified as a target and mediator of miR-124 for this highly specific temporal aspect of RGC growth cone responsiveness. Our findings indicate that miR-124 is important in regulating the intrinsic temporal changes in RGC growth cone sensitivity and suggest that miRNAs may act broadly as linear timers in vertebrate neuronal development. [ABSTRACT FROM AUTHOR]

Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and luciferase assays, that validation by targeted proteomics substantially enriched for biologically relevant let-7 interactors. For example, we found that the zinc finger protein ZTF-7 was a bona fide let-7 miRNA target. We also validated predicted miR-58 targets, demonstrating that this approach is adaptable to other miRNAs. We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational methods or in large-scale experiments, and that the described strategy should be readily adaptable to other organisms. [ABSTRACT FROM AUTHOR]

Over the last 10 years there has been an explosion of information about the molecular biology of cancer. A challenge in oncology is to translate this information into advances in patient care. While there are well-formed routes for translating new molecular information into drug therapy, the routes for translating new information into sensitive and specific diagnostic, prognostic and predictive tests are still being developed. Similarly, the science of using tumor molecular profiles to select clinical trial participants or to optimize therapy for individual patients is still in its infancy. This review will summarize the current technologies for predicting treatment response and prognosis in cancer medicine, and outline what the future may hold. It will also highlight the potential importance of methods that can integrate molecular, histopathological and clinical information into a synergistic understanding of tumor progression. While these possibilities are without doubt exciting, significant challenges remain if we are to implement them with a strong evidence base in a widely available and cost-effective manner. [ABSTRACT FROM PUBLISHER]

Background: The differential expression pattern of microRNAs (miRNAs) during mammary gland development might provide insights into their role in regulating the homeostasis of the mammary epithelium. Our aim was to analyse these regulatory functions by deriving a comprehensive tissue-specific combined miRNA and mRNA expression profile of post-natal mouse mammary gland development. We measured the expression of 318 individual murine miRNAs by bead-based flow-cytometric profiling of whole mouse mammary glands throughout a 16-point developmental time course, including juvenile, puberty, mature virgin, gestation, lactation, and involution stages. In parallel whole-genome mRNA expression data were obtained. Results: One third (n = 102) of all murine miRNAs analysed were detected during mammary gland development. MicroRNAs were represented in seven temporally co-expressed clusters, which were enriched for both miRNAs belonging to the same family and breast cancer-associated miRNAs. Global miRNA and mRNA expression was significantly reduced during lactation and the early stages of involution after weaning. For most detected miRNA families we did not observe systematic changes in the expression of predicted targets. For miRNA families whose targets did show changes, we observed inverse patterns of miRNA and target expression. The data sets are made publicly available and the combined expression profiles represent an important community resource for mammary gland biology research. Conclusion: MicroRNAs were expressed in likely co-regulated clusters during mammary gland development. Breast cancer-associated miRNAs were significantly enriched in these clusters. The mechanism and functional consequences of this miRNA co-regulation provide new avenues for research into mammary gland biology and generate candidates for functional validation. [ABSTRACT FROM AUTHOR]

The functional genomic approaches of transcriptomics, proteomics and metabolomics aim to measure the mRNA, protein or metabolite complement of a cell, tissue or organism. In this study we have investigated the compatibility of transcriptional analysis, using Reverse Transcription (RT)-PCR, and metabolite analysis, by high-resolution magic angle spinning (HRMAS) ¹H NMR spectroscopy, in BT4C rat glioma following the induction of programmed cell death. The metabolite and transcriptional changes that accompanied apoptosis were examined at 0, 4 and 8 days of ganciclovir/thymidine kinase gene therapy. Despite the high spinning speeds employed during HRMAS ¹H NMR spectroscopy of one-half of the tumor samples, RT-PCR analysis of the pro-apoptotic transcripts Bcl-2, BAK-1, caspase-9 and FAS was possible, producing similar results to those detected in the unspun half of the tumors. Furthermore, the expression of FAS was inversely correlated with some of the key metabolic changes across the time period examined including the increases CH=CH and CH=CHCH2 lipid resonances which accompany apoptosis. This study demonstrates how combined transcriptomic and metabolomic studies of tumors can be used to understand the molecular events that accompany well documented metabolic perturbations during cell death processes. [ABSTRACT FROM AUTHOR]

Milk has been shown to contain a specific fraction of extracellular particles that are reported to resist digestion and are purposefully packaged with lipids, proteins, and nucleic acids to exert specific biological effects. These findings suggest that these particles may have a role in the quality of infant nutrition, particularly in the early phase of life when many of the foundations of an infant's potential for health and overall wellness are established. However, much of the current research focuses on human or cow milk only, and there is a knowledge gap in how milk from other species, which may be more commonly consumed in different regions, could also have these reported biological effects. Our review provides a summary of the studies into the extracellular particle fraction of milk from a wider range of ruminants and pseudo-ruminants, focusing on how this fraction is isolated and characterised, the stability and uptake of the fraction, and the reported biological effects of these fractions in a range of model systems. As the individual composition of milk from different species is known to differ, we propose that the extracellular particle fraction of milk from non-traditional and minority species may also have important and distinct biological properties that warrant further study. [ABSTRACT FROM AUTHOR]

Simple Summary: Medical autopsies have a long history, but are currently experiencing a revival in order to progress modern cancer evolution research. Research autopsies represent an unparalleled opportunity to collect large volumes of cancer tissue accessible from multiple sites in a metastatic cancer patient's body—not usually feasible during the standard clinical course. These collections enable researchers to unravel tumour evolution and heterogeneity questions. Many institutions around the world have recognised the value of these tissues, and have established rapid cancer autopsy programmes. Our article discusses a comprehensive collection of 24 rapid cancer autopsy programmes from across the globe. Outstanding questions plaguing oncologists, centred around tumour evolution and heterogeneity, include the development of treatment resistance, immune evasion, and optimal drug targeting strategies. Such questions are difficult to study in limited cancer tissues collected during a patient's routine clinical care, and may be better investigated in the breadth of cancer tissues that may be permissible to collect during autopsies. We are starting to better understand key tumour evolution challenges based on advances facilitated by autopsy studies completed to date. This review article explores the great progress in understanding that cancer tissues collected at autopsy have already enabled, including the shared origin of metastatic cells, the importance of early whole-genome doubling events for amplifying genes needed for tumour survival, and the creation of a wealth of tissue resources powered to answer future questions, including patient-derived xenografts, cell lines, and a wide range of banked tissues. We also highlight the future role of these programmes in advancing our understanding of cancer evolution. The research autopsy provides a special opportunity for cancer patients to give the ultimate gift—to selflessly donate their tissues towards better cancer care. [ABSTRACT FROM AUTHOR]