Contribution of Extracellular Particles Isolated from Morus sp. (Mulberry) Fruit to Their Reported Protective Health Benefits: An In Vitro Study.

Morus sp. (mulberry) has a long tradition of use as a medicinal treatment, including for cardiovascular disease and type 2 diabetes, being shown to have antioxidant properties and to promote wound healing. Extracellular vesicles (EVs) are sub-micron, membrane-enclosed particles that were first identified in mammalian bodily fluids. EV-like particles have been described in plants (PDVs) and shown to have similar characteristics to mammalian EVs. We hypothesised that some of the health benefits previously attributed to the fruit of Morus sp. could be due to the release of PDVs. We isolated PDVs from Morus nigra and Morus alba via ultracentrifugation and incubated THP-1 monocytes, differentiated THP-1 macrophages, or HMEC-1 endothelial cells with pro-oxidant compounds DMNQ (THP-1) and glucose oxidase (HMEC-1) or lipopolysaccharide (LPS) in the presence of different fractions of mulberry EVs. Mulberry EVs augmented ROS production with DMNQ in THP-1 and caused the downregulation of ROS in HMEC-1. Mulberry EVs increased LPS-induced IL-1ß secretion but reduced CCL2 and TGF-ß secretion in THP-1 macrophages. In scratch wound assays, mulberry EVs inhibited HMEC-1 migration but increased proliferation in both low and high serum conditions, suggesting that they have opposing effects in these two important aspects of wound healing. One of the limitations of plant-derived therapeutics has been overcoming the low bioavailability of isolated compounds. We propose that PDVs could provide the link between physiological dose and therapeutic benefit by protecting plant active compounds in the GIT as well as potentially delivering genetic material or proteins that contribute to previously observed health benefits.

In sickness and in health: The functional role of extracellular vesicles in physiology and pathology in vivo: Part I: Health and Normal Physiology: Part I: Health and Normal Physiology.

Previously thought to be nothing more than cellular debris, extracellular vesicles (EVs) are now known to mediate physiological and pathological functions throughout the body. We now understand more about their capacity to transfer nucleic acids and proteins between distant organs, the interaction of their surface proteins with target cells, and the role of vesicle-bound lipids in health and disease. To date, most observations have been made in reductionist cell culture systems, or as snapshots from patient cohorts. The heterogenous population of vesicles produced in vivo likely act in concert to mediate both beneficial and detrimental effects. EVs play crucial roles in both the pathogenesis of diseases, from cancer to neurodegenerative disease, as well as in the maintenance of system and organ homeostasis. This two-part review draws on the expertise of researchers working in the field of EV biology and aims to cover the functional role of EVs in physiology and pathology. Part I will outline the role of EVs in normal physiology.

In sickness and in health: The functional role of extracellular vesicles in physiology and pathology in vivo: Part II: Pathology: Part II: Pathology.

It is clear from Part I of this series that extracellular vesicles (EVs) play a critical role in maintaining the homeostasis of most, if not all, normal physiological systems. However, the majority of our knowledge about EV signalling has come from studying them in disease. Indeed, EVs have consistently been associated with propagating disease pathophysiology. The analysis of EVs in biofluids, obtained in the clinic, has been an essential of the work to improve our understanding of their role in disease. However, to interfere with EV signalling for therapeutic gain, a more fundamental understanding of the mechanisms by which they contribute to pathogenic processes is required. Only by discovering how the EV populations in different biofluids change-size, number, and physicochemical composition-in clinical samples, may we then begin to unravel their functional roles in translational models in vitro and in vivo, which can then feedback to the clinic. In Part II of this review series, the functional role of EVs in pathology and disease will be discussed, with a focus on in vivo evidence and their potential to be used as both biomarkers and points of therapeutic intervention.

AKR1D1 knockout mice develop a sex-dependent metabolic phenotype.

Steroid 5ß-reductase (AKR1D1) plays important role in hepatic bile acid synthesis and glucocorticoid clearance. Bile acids and glucocorticoids are potent metabolic regulators, but whether AKR1D1 controls metabolic phenotype in vivo is unknown. Akr1d1-/- mice were generated on a C57BL/6 background. Liquid chromatography/mass spectrometry, metabolomic and transcriptomic approaches were used to determine effects on glucocorticoid and bile acid homeostasis. Metabolic phenotypes including body weight and composition, lipid homeostasis, glucose tolerance and insulin tolerance were evaluated. Molecular changes were assessed by RNA-Seq and Western blotting. Male Akr1d1-/- mice were challenged with a high fat diet (60% kcal from fat) for 20 weeks. Akr1d1-/- mice had a sex-specific metabolic phenotype. At 30 weeks of age, male, but not female, Akr1d1-/- mice were more insulin tolerant and had reduced lipid accumulation in the liver and adipose tissue yet had hypertriglyceridemia and increased intramuscular triacylglycerol. This phenotype was associated with sexually dimorphic changes in bile acid metabolism and composition but without overt effects on circulating glucocorticoid levels or glucocorticoid-regulated gene expression in the liver. Male Akr1d1-/- mice were not protected against diet-induced obesity and insulin resistance. In conclusion, this study shows that AKR1D1 controls bile acid homeostasis in vivo and that altering its activity can affect insulin tolerance and lipid homeostasis in a sex-dependent manner.

Molecular basis for maize as a risk factor for esophageal cancer in a South African population via a prostaglandin E2 positive feedback mechanism.

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 (PGE(2)). 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 PGE(2) 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 PGE(2), leads to increased cell proliferation. Similarly, treatment of cells with PGE(2) or with gastric fluid from Transkeians also leads to increased proliferation. Our data suggest that the high levels of PGE(2) 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.

Long noncoding RNAs, chromatin, and development.

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.

Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia.

ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia. This study investigated the effects of these ANKH mutants on cellular localisation and associated biochemistry. We generated four ANKH overexpression-plasmids containing either calcium pyrophosphate deposition disease or craniometaphyseal dysplasia linked mutations: P5L, E490del and S375del, G389R. They were transfected into CH-8 articular chondrocytes and HEK293 cells. The ANKH mutants dynamic differential localisations were imaged and we investigated the interactions with the autophagy marker LC3. Extracellular inorganic pyrophosphate, mineralization, ENPP1 activity expression of ENPP1, TNAP and PIT-1 were measured. P5L delayed cell membrane localisation but once recruited into the membrane it increased extracellular inorganic pyrophosphate, mineralization, and ENPP1 activity. E490del remained mostly cytoplasmic, forming punctate co-localisations with LC3, increased mineralization, ENPP1 and ENPP1 activity with an initial but unsustained increase in TNAP and PIT-1. S375del trended to decrease extracellular inorganic pyrophosphate, increase mineralization. G389R delayed cell membrane localisation, trended to decrease extracellular inorganic pyrophosphate, increased mineralization and co-localised with LC3. Our results demonstrate a link between pathological localisation of ANKH mutants with different degrees in mineralization. Furthermore, mutant ANKH functions are related to synthesis of defective proteins, inorganic pyrophosphate transport, ENPP1 activity and expression of ENPP1, TNAP and PIT-1.

An intergenic non-coding RNA promoter required for histone modifications in the human ß-globin chromatin domain.

Transcriptome analyses show a surprisingly large proportion of the mammalian genome is transcribed; much more than can be accounted for by genes and introns alone. Most of this transcription is non-coding in nature and arises from intergenic regions, often overlapping known protein-coding genes in sense or antisense orientation. The functional relevance of this widespread transcription is unknown. Here we characterize a promoter responsible for initiation of an intergenic transcript located approximately 3.3 kb and 10.7 kb upstream of the adult-specific human ß-globin genes. Mutational analyses in ß-YAC transgenic mice show that alteration of intergenic promoter activity results in ablation of H3K4 di- and tri-methylation and H3 hyperacetylation extending over a 30 kb region immediately downstream of the initiation site, containing the adult δ- and ß-globin genes. This results in dramatically decreased expression of the adult genes through position effect variegation in which the vast majority of definitive erythroid cells harbor inactive adult globin genes. In contrast, expression of the neighboring ε- and γ-globin genes is completely normal in embryonic erythroid cells, indicating a developmentally specific variegation of the adult domain. Our results demonstrate a role for intergenic non-coding RNA transcription in the propagation of histone modifications over chromatin domains and epigenetic control of ß-like globin gene transcription during development.

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017.

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.

The human myometrium differentially expresses mTOR signalling components before and during pregnancy: evidence for regulation by progesterone.

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 immortalized 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. This article is part of a Special Issue entitled 'Pregnancy and Steroids'.

Pseudogenes as regulators of biological 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.

Possible role of exosomes containing RNA in mediating nontargeted effect of ionizing radiation.

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.

Analysis of ß-globin chromatin micro-environment using a novel 3C variant, 4Cv.

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 ß-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 ß-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 ß-globin locus in mice and show that a significant proportion of the interactions of ß-globin are inter-chromosomal. Furthermore, our data show that in the liver, where the gene is active, ß-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 ß-globin locus leads to a change in nuclear position relative to the chromosome territory.