Analysis of Schistosoma mansoni Extracellular Vesicles Surface Glycans Reveals Potential Immune Evasion Mechanism and New Insights on Their Origins of Biogenesis

Parasitic helminths are master manipulators of host immunity. Their strategy is complex and involves the release of excreted/secreted products, including extracellular vesicles (EVs). The protein and miRNA contents of EVs have been characterised for many parasitic helminths but, despite reports suggesting the importance of EV surface carbohydrate structures (glycans) in the interactions with target cells and thus subsequent effector functions, little is known about parasite EV glycomics. Using lectin microarrays, we identified several lectins that exhibit strong adhesion to Schistosoma mansoni EVs, suggesting the presence of multiple glycan structures on these vesicles. Interestingly, SNA-I, a lectin that recognises structures with terminal sialic acid, displayed strong affinity for S. mansoni EVs, which was completely abolished by neuraminidase treatment, suggesting sialylation in the EV sample. This finding is of interest, as sialic acids play important roles in the context of infection by aiding immune evasion, affecting target recognition, cell entry, etc., but are not thought to be synthesised by helminths. These data were validated by quantitative analysis of free sialic acid released from EVs following treatment with neuraminidase. Lectin histochemistry and fluorescence in situ hybridisation analyses on whole adult worms suggest the involvement of sub-tegumental cell bodies, as well as the digestive and excretory systems, in the release of EVs. These results support previous reports of EV biogenesis diversity in trematodes and potentially highlight new means of immune modulation and evasion employed by schistosomes.

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Comprehensive Proteomic Analysis Revealed a Large Number of Newly Identified Proteins in the Small Extracellular Vesicles of Milk from Late-Stage Lactating Cows

Animals ◽

10.3390/ani11092506 ◽

2021 ◽

Vol 11 (9) ◽

pp. 2506

Author(s):

Md. Matiur Rahman ◽

Shigeo Takashima ◽

Yuji O. Kamatari ◽

Kaori Shimizu ◽

Ayaka Okada ◽

...

Keyword(s):

Extracellular Vesicles ◽

Late Stage ◽

Immune Modulation ◽

Bioinformatics Analysis ◽

Bovine Milk ◽

Bioinformatic Analysis ◽

Target Cells ◽

Diverse Range ◽

Lactating Cows ◽

Chromatography Tandem Mass Spectrometry

Bovine milk contains small extracellular vesicles (sEVs) that provide proteins, miRNAs, mRNAs, DNAs, and lipids to target cells and play a role in intracellular communications. Previous studies have characterized proteins in milk sEVs from early- and mid-stage lactation. However, the proteins in milk sEVs from late-stage lactation are mostly unexplored. The aim of this study was to determine the proteomic profile of milk sEVs from late-stage lactating cows. A comprehensive nanoliquid chromatography–tandem mass spectrometry (nanoLC-MS/MS) approach was carried out to reveal the proteins in milk sEVs. Additionally, bioinformatics analysis was carried out to interpret the molecular signatures of newly identified proteins in milk sEVs from three late-stage lactating cows. NanoLC-MS/MS analysis revealed a total of 2225 proteins in milk sEVs from cows. Notably, after comparing these identified proteins with previously deposited datasets of proteins in bovine milk sEVs, 429 proteins were detected as newly identified. Bioinformatic analysis indicated that these newly identified proteins in milk sEVs were engaged in a diverse range of molecular phenomena relevant to mammary gland physiology, milk production, immunity, and immune response. These findings suggest that the newly identified proteins could expand the inventory application of molecular cargos, nutritional status, and immune modulation of sEVs in milk during the late-stage lactation.

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Siglec-7 Tetramers Characterize B Cell Subpopulations and Identify Immunomodulatory Ligands on Malignant Cells

Blood ◽

10.1182/blood.v116.21.1728.1728 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1728-1728

Author(s):

Philippa Mang ◽

Friederike Gieseke ◽

Susanne Viebahn ◽

Inga Gondesen ◽

Anne Kruchen ◽

...

Keyword(s):

T Cells ◽

Cell Surface ◽

B Cell ◽

Nk Cells ◽

Immune Evasion ◽

Nk Cell ◽

Sialic Acids ◽

Target Cells ◽

Malignant Cells ◽

Neuraminidase Treatment

Abstract Abstract 1728 Cell surface glycoconjugates have important functions in the modulation of immune cell maturation, activation and homeostasis. Glycans on cell surfaces are generally sialylated. Sialic acids are predominantly found on the non-reducing end of oligosaccharide chains of glycoconjugates. They are recognized by sialic acid-binding immunoglobulin-like lectins (siglecs). CD33-related siglecs are receptors predominantly expressed on immune cells. Most of the CD33-related siglecs display a tyrosine-based inhibitory motif (ITIM) known to downregulate effector cell functions. So far, identification of ligands for siglecs was hampered by low affinity between proteins and glycans, but by glycan array analysis α2,8-, α2,3- as well as α2,6-linked sialic acids were identified as the carbohydrate part of the ligand. In order to analyse the expression of physiological siglec ligands on different lymphocyte subpopulations, the extracellular three domains of siglec-7 were cloned. To allow for mammalian glycosylation, the protein was expressed in 293T cells. To overcome the low affinity of lectins, siglec-7 was enzymatically biotinylated and oligomerized using streptavidin in analogy to HLA tetramers. Flow cytometric analysis of lymphocytes revealed reproducible binding patterns of siglec-7 tetramers. Specificity was ensured by treatment of PBMC with neuraminidase, specifically cleaving terminal sialic acids in α2,3, α2,6 and α2,8 linkages. On T cells, the density of siglec-7 ligands increased 3.5-fold during activation. This is contradictory to results using plant lectins, like Maackia amurensis lectin and Sambucus nigra lectin, which indicated an 1.8- and 1.5-fold decrease, respectively. Activation of NK cells did not affect the siglec-7 ligand expression. Interestingly, B cells could be divided in a siglec-7 ligand positive and a siglec-7 ligand negative population. This may reflect different differentiation or activation stages, which were not found with any antibody combination directed against typical protein B cell markers. Malignant transformation is often associated with aberrant cell surface glycosylation. In acute lymphoblastic leukemia (ALL), blasts of the more aggressive T-ALL express 17 fold higher amounts of siglec-7 ligand than blasts of the B cell lineage (cALL), where the siglec-7 ligand was nearly not detectable. These findings may contribute to the poor prognosis of T-ALL by a possible immune escape achieved by siglec-ligand binding induced inhibition. Since immune evasion mechanisms are also described for other malignancies, we additionally analysed rhabdomyosarcoma (RMS) cells. Siglec-7 ligand could be found on all tested RMS cell lines in different expression levels, independent of the classification. Siglec-7 is a known inhibitory receptor expressed by NK cells and T cells. The effect of siglec-7 ligand expressed by malignant cells on NK cell cytotoxicity was analysed. By the removal of all terminal sialic acids on the surface of RMS cells by neuraminidase treatment, the NK cell-induced cytotoxicity could be increased by 20%. Coating of siglec-7 ligands on target cells by preincubation with recombinant siglec-7 protein also resulted in an increased cytotoxicity. These results show that sialic acids play an important role in the immune system as physiologic ligands. However, the expression of siglec-7 ligands on malignant cells may contribute to immune evasion mechanisms. Disclosures: No relevant conflicts of interest to declare.

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Trastuzumab Modulates the Protein Cargo of Extracellular Vesicles Released by ERBB2+ Breast Cancer Cells

Membranes ◽

10.3390/membranes11030199 ◽

2021 ◽

Vol 11 (3) ◽

pp. 199

Author(s):

Silvia Marconi ◽

Sara Santamaria ◽

Martina Bartolucci ◽

Sara Stigliani ◽

Cinzia Aiello ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Extracellular Vesicles ◽

Breast Cancer Cells ◽

Culture Supernatant ◽

High Resolution Mass Spectrometry ◽

Recombinant Antibody ◽

Target Cells ◽

Cellular Processes ◽

Erbb2 Signaling

Cancers overexpressing the ERBB2 oncogene are aggressive and associated with a poor prognosis. Trastuzumab is an ERBB2 specific recombinant antibody employed for the treatment of these diseases since it blocks ERBB2 signaling causing growth arrest and survival inhibition. While the effects of Trastuzumab on ERBB2 cancer cells are well known, those on the extracellular vesicles (EVs) released from these cells are scarce. This study focused on ERBB2+ breast cancer cells and aimed to establish what type of EVs they release and whether Trastuzumab affects their morphology and molecular composition. To these aims, we performed immunoelectron microscopy, immunoblot, and high-resolution mass spectrometry analyses on EVs purified by differential centrifugation of culture supernatant. Here, we show that EVs released from ERBB2+ breast cancer cells are polymorphic in size and appearance and that ERBB2 is preferentially associated with large (120 nm) EVs. Moreover, we report that Trastuzumab (Tz) induces the expression of a specific glycosylated 50 kDa isoform of the CD63 tetraspanin and modulates the expression of 51 EVs proteins, including TOP1. Because these proteins are functionally associated with organelle organization, cytokinesis, and response to lipids, we suggest that Tz may influence these cellular processes in target cells at distant sites via modified EVs.

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Rapid Multi-Hybridisation FISH Screening for Balanced Porcine Reciprocal Translocations Suggests a Much Higher Abnormality Rate Than Previously Appreciated

Cells ◽

10.3390/cells10020250 ◽

2021 ◽

Vol 10 (2) ◽

pp. 250

Author(s):

Rebecca E O’Connor ◽

Lucas G Kiazim ◽

Claudia C Rathje ◽

Rebecca L Jennings ◽

Darren K Griffin

Keyword(s):

Semen Analysis ◽

In Situ Hybridisation ◽

Economic Loss ◽

Environmental Damage ◽

Fluorescence In Situ Hybridisation ◽

Reciprocal Translocations ◽

Chromosome Translocations ◽

Farrowing Rate ◽

Significant Economic Loss

With demand rising, pigs are the world’s leading source of meat protein; however significant economic loss and environmental damage can be incurred if boars used for artificial insemination (AI) are hypoprolific (sub-fertile). Growing evidence suggests that semen analysis is an unreliable tool for diagnosing hypoprolificacy, with litter size and farrowing rate being more applicable. Once such data are available, however, any affected boar will have been in service for some time, with significant financial and environmental losses incurred. Reciprocal translocations (RTs) are the leading cause of porcine hypoprolificacy, reportedly present in 0.47% of AI boars. Traditional standard karyotyping, however, relies on animal specific expertise and does not detect more subtle (cryptic) translocations. Previously, we reported development of a multiple hybridisation fluorescence in situ hybridisation (FISH) strategy; here, we report on its use in 1641 AI boars. A total of 15 different RTs were identified in 69 boars, with four further animals XX/XY chimeric. Therefore, 4.5% had a chromosome abnormality (4.2% with an RT), a 0.88% incidence. Revisiting cases with both karyotype and FISH information, we reanalysed captured images, asking whether the translocation was detectable by karyotyping alone. The results suggest that chromosome translocations in boars may be significantly under-reported, thereby highlighting the need for pre-emptive screening by this method before a boar enters a breeding programme.

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Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing

Nature Communications ◽

10.1038/s41467-021-22203-2 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 2

Author(s):

Caitlin M. Singleton ◽

Francesca Petriglieri ◽

Jannie M. Kristensen ◽

Rasmus H. Kirkegaard ◽

Thomas Y. Michaelsen ◽

...

Keyword(s):

16S Rrna ◽

Wastewater Treatment Plants ◽

In Situ Hybridisation ◽

Amplicon Sequencing ◽

Rrna Genes ◽

Fluorescence In Situ Hybridisation ◽

Sequencing Data ◽

High Quality ◽

16S Rrna Amplicon Sequencing ◽

Long Read

AbstractMicroorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups.

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Delivery of Therapeutic Agents to the Central Nervous System and the Promise of Extracellular Vesicles

Pharmaceutics ◽

10.3390/pharmaceutics13040492 ◽

2021 ◽

Vol 13 (4) ◽

pp. 492

Author(s):

Charlotte A. René ◽

Robin J. Parks

Keyword(s):

Central Nervous System ◽

Endothelial Cells ◽

Nervous System ◽

Blood Brain Barrier ◽

Extracellular Vesicles ◽

Therapeutic Agents ◽

Target Cells ◽

Significant Barrier ◽

The Central Nervous System ◽

Delivery Vehicles

The central nervous system (CNS) is surrounded by the blood–brain barrier (BBB), a semipermeable border of endothelial cells that prevents pathogens, solutes and most molecules from non-selectively crossing into the CNS. Thus, the BBB acts to protect the CNS from potentially deleterious insults. Unfortunately, the BBB also frequently presents a significant barrier to therapies, impeding passage of drugs and biologicals to target cells within the CNS. This review provides an overview of different approaches to deliver therapeutics across the BBB, with an emphasis in extracellular vesicles as delivery vehicles to the CNS.

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Cell-to-Cell Communication by Host-Released Extracellular Vesicles in the Gut: Implications in Health and Disease

International Journal of Molecular Sciences ◽

10.3390/ijms22042213 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2213

Author(s):

Natalia Diaz-Garrido ◽

Cecilia Cordero ◽

Yenifer Olivo-Martinez ◽

Josefa Badia ◽

Laura Baldomà

Keyword(s):

Extracellular Vesicles ◽

Intestinal Mucosa ◽

Current Knowledge ◽

Cell Communication ◽

Bioactive Molecules ◽

Target Cells ◽

Immune Functions ◽

Intestinal Homeostasis ◽

General Terms ◽

Health And Disease

Communication between cells is crucial to preserve body homeostasis and health. Tightly controlled intercellular dialog is particularly relevant in the gut, where cells of the intestinal mucosa are constantly exposed to millions of microbes that have great impact on intestinal homeostasis by controlling barrier and immune functions. Recent knowledge involves extracellular vesicles (EVs) as mediators of such communication by transferring messenger bioactive molecules including proteins, lipids, and miRNAs between cells and tissues. The specific functions of EVs principally depend on the internal cargo, which upon delivery to target cells trigger signal events that modulate cellular functions. The vesicular cargo is greatly influenced by genetic, pathological, and environmental factors. This finding provides the basis for investigating potential clinical applications of EVs as therapeutic targets or diagnostic biomarkers. Here, we review current knowledge on the biogenesis and cargo composition of EVs in general terms. We then focus the attention to EVs released by cells of the intestinal mucosa and their impact on intestinal homeostasis in health and disease. We specifically highlight their role on epithelial barrier integrity, wound healing of epithelial cells, immunity, and microbiota shaping. Microbiota-derived EVs are not reviewed here.

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Effects of the diploidisation process upon the 5S and 35S rDNA sequences in the allopolyploid species of the Dilatata group of Paspalum (Poaceae, Paniceae)

Australian Journal of Botany ◽

10.1071/bt18236 ◽

2019 ◽

Vol 67 (7) ◽

pp. 521

Author(s):

Magdalena Vaio ◽

Cristina Mazzella ◽

Marcelo Guerra ◽

Pablo Speranza

Keyword(s):

Evolutionary Dynamics ◽

In Situ Hybridisation ◽

5S Rdna ◽

Its Region ◽

Variable Number ◽

Rrna Genes ◽

Fluorescence In Situ Hybridisation ◽

Rdna Sites ◽

35S Rdna ◽

Genome Restructuring

The Dilatata group of Paspalum includes species and biotypes native to temperate South America. Among them, five sexual allotetraploids (x = 10) share the same IIJJ genome formula: P. urvillei Steud, P. dasypleurum Kunze ex Desv., P. dilatatum subsp. flavescens Roseng., B.R. Arrill. & Izag., and two biotypes P. dilatatum Vacaria and P. dilatatum Virasoro. Previous studies suggested P. intermedium Munro ex Morong & Britton and P. juergensii Hack. or related species as their putative progenitors and donors of the I and J genome, respectively, and pointed to a narrow genetic base for their maternal origin. It has not yet been established whether the various members of the Dilatata group are the result of a single or of multiple allopolyploid formations. Here, we aimed to study the evolutionary dynamics of rRNA genes after allopolyploidisation in the Dilatata group of Paspalum and shed some light into the genome restructuring of the tetraploid taxa with the same genome formula. We used double target fluorescence in situ hybridisation of 35S and 5S rDNA probes and sequenced the nrDNA internal transcribed spacer (ITS) region. A variable number of loci at the chromosome ends were observed for the 35S rDNA, from 2 to 6, suggesting gain and loss of sites. For the 5S rDNA, only one centromeric pair of signals was observed, indicating a remarkable loss after polyploidisation. All ITS sequences generated were near identical to the one found for P. intermedium. Although sequences showed a directional homogeneisation towards the putative paternal progenitor in all tetraploid species, the observed differences in the number and loss of rDNA sites suggest independent ongoing diploidisation processes in all taxa and genome restructuring following polyploidy.

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