Pericytes protect rats and mice from sepsis-induced injuries by maintaining vascular reactivity and barrier function:implication of miRNAs and microvesicles

Background Vascular hyporeactivity and leakage are key pathophysiologic features that produce multi-organ damage upon sepsis.We hypothesized that pericytes,a group of pluripotent cells that maintain vascular integrity and tension,are protective against sepsis via regulating vascular reactivity and permeability.Methods We conducted a series of in vivo experiments using wild-type(WT),platelet-derived growth factor receptor-β(PDGFR-β)-Cre+mT/mG transgenic mice and Tie2-Cre+Cx43^(flox/flox)mice to examine the relative contribution of pericytes in sepsis,either induced by cecal ligation and puncture(CLP)or lipopolysaccharide(LPS)challenge.In a separate set of experiments with Sprague-Dawley(SD)rats,pericytes were depleted using CP-673451,a selective PDGFR-βinhibitor,at a dosage of 40 mg/(kg·d)for 7 consecutive days.Cultured pericytes,vascular endothelial cells(VECs)and vascular smooth muscle cells(VSMCs)were used for mechanistic investigations.The effects of pericytes and pericyte-derived microvesicles(PCMVs)and candidate miRNAs on vascular reactivity and barrier function were also examined.Results CLP and LPS induced severe injury/loss of pericytes,vascular hyporeactivity and leakage(P<0.05).Transplantation with exogenous pericytes protected vascular reactivity and barrier function via microvessel colonization(P<0.05).Cx43 knockout in either pericytes or VECs reduced pericyte colonization in microvessels(P<0.05).Additionally,PCMVs transferred miR-145 and miR-132 to VSMCs and VECs,respectively,exerting a protective effect on vascular reactivity and barrier function after sepsis(P<0.05).miR-145 primarily improved the contractile response of VSMCs by activating the sphingosine kinase 2(Sphk2)/sphingosine-1-phosphate receptor(S1PR)1/phosphorylation of myosin light chain 20 pathway,whereas miR-132 effectively improved the barrier function of VECs by activating the Sphk2/S1PR2/zonula occludens-1 and vascular endothelial-cadherin pathways.Conclusions Pericytes are protective against sepsis through regulating vascular reactivity and barrier function.Possible mechanisms include both direct colonization of microvasculature and secretion of PCMVs.

Microvesicles with mitochondrial content are increased in patients with sepsis and associated with inflammatory responses

BACKGROUND Endothelial activation plays an important role in sepsis-mediated inflammation,but the triggering factors have not been fully elucidated.Microvesicles carrying mitochondrial content(mitoMVs)have been implicated in several diseases and shown to induce endothelial activation.AIM To explore whether mitoMVs constitute a subset of MVs isolated from plasma of patients with sepsis and contribute to endothelial activation.METHODS MVs were isolated from human plasma and characterized by confocal microscopy and flow cytometry.Proinflammatory cytokines,including interleukin(IL)-6,IL-8 and tumour necrosis factor(TNF)-α,and soluble vascular cell adhesion molecule(sVCAM)-1 were detected by ELISA.Human umbilical vein endothelial cells(HUVECs)were stimulated with the circulating MVs to evaluate their effect on endothelial activation.RESULTS MitoMVs were observed in plasma from patients with sepsis.Compared with those in healthy controls,expression of MVs,mitoMVs,proinflammatory cytokines and sVCAM-1 was increased.The number of mitoMVs was positively associated with TNF-αand sVCAM-1.In vitro,compared with MVs isolated from the plasma of healthy controls,MVs isolated from the plasma of patients with sepsis induced expression of OAS2,RSAD2,and CXCL10 in HUVECs.MitoMVs were taken up by HUVECs,and sonication of MVs significantly reduced the uptake of mitoMVs by HUVECs and expression of the above three type I IFNdependent genes.CONCLUSION MitoMVs are increased in the plasma of patients with sepsis,which induces elevated expression of type I IFN-dependent genes.This suggests that circulating mitoMVs activate the type I IFN signalling pathway in endothelial cells and lead to endothelial activation.

Research progress of the relationship between microvesicles derived from stem cells and nervous system diseases

Microvesicles, also called microparticles, are membranous vesicles released from the cell membrane surface or by exocytose. Almost any type of cells can secrete vesicles, especially stem cells. Recent years, stem cells are becoming a research hotspot of cytotherapy for their capacity of self-renewing, expansion and proliferation in vitro and the microvesicles derived from the conditioned medium of stem cells have been widely used to regenerative medicine because they are safer, easily obtained, measurable and cause no obvious immune rejection. Stem cells derived microvesicles have been confirmed to be closely related to the progress and treatment of atherosclerosis, diabetes, inflammation and tumor. This review focuses on the new progress of stem cells derived microvesicles treating various nervous system diseases and its application in biological therapy and the behind molecule mechanisms.

Release of exosomes and microvesicles harbouring specific RNAs and glycosylphosphatidylinositol-anchored proteins from rat and human adipocytes is controlled by histone methylation

The transfer of proteins and nucleic acids from donor to acceptor cells via small membrane vesicles has been implicated with (patho)physiological consequences. Previously the upregulation of esterification and downregulation of lipolysis in small rat adipocytes upon incubation with exosomes and microvesicles (EMVs) released from large adipocytes and harbouring the glycosylphosphatidylinositol (GPI)-anchored proteins, Gce1 and CD73, transcripts specific for FSP27 and GPAT3, and microRNAs, miR-16 and miR-222 was demonstrated. Here the release of EMVs from large (but not small) primary and differentiated and human rat adipocytes in response to palmitate, H2O2 and the anti-diabetic sulfonylurea, glimepiride, is shown to be significantly reduced upon inhibition of histone H3 lysine9 methyltransferase G9a by trans-2-phenylcyclopropylamine (tPCPA) and histone H3 lysine4 demethylase LSD1 by BIX01294. Inhibition of EMV release by tPCPA and BIX01294 was not caused by apoptosis but accompanied by upregulation of the H2O2-induced stimulation of lipid synthesis and downregulation of lipolysis in large (but not small) primary and differentiated rat and human adipocytes. In contrast, the simultaneous presence of tPCPA and BIX-01294 had almost no effect on the induced release of EMVs and lipid metabolism. These findings argue for regulation of the release of EMVs harbouring specific GPI-anchored proteins, transcripts and microRNAs from rat and human adipocytes by histone H3 methylation at lysines 4 and 9 in interdependent fashion. Thus the EMV-mediated transfer of lipogenic and anti-lipolytic information between large and small adipocytes in response to certain physiological and pharmacological stimuli seems to be controlled by epigenetic mechanisms.

Microvesicles in Gliomas and Medulloblastomas: An Overview

Microvesicles (MVs) or shedding membrane vesicles have recently been described as a novel model of intercellular communication. Previously, MVs were considered as unnecessary or secreted cellular debris, but MVs have lately been described as having roles in a variety of biological functions, such as cell homeostasis and the cellular processes involved in the oncogenesis of many types of tumors. Carrying several key molecules that contribute to tumor development and progression, similar to mRNAs, microRNAs and other non-coding RNAs, DNA and even small proteins, MVs can be considered as a ubiquitous form of novel cell communication that is present in most somatic cells. Although tumor-derived MVs have been demonstrated in different types of cancers, the literature data on MVs in primary central nervous system (CNS) tumors are relatively scarce. In this review, we address the involvement of MVs in diffuse astrocytomas, particularly glioblastomas, as well as oligodendrogliomas and medulloblastomas. We placed particular focus on the cellular crosstalk between tumor and “normal” cells, the putative mechanisms how the tumor microenvironment is modulated and the spread of aggressive phenotypes. Additionally, a better understanding of the participation of tumor-derived MVs in the regulation of key cancer pathways will offer new insights into tumor pathogenesis and the mechanisms of multidrug resistance, and may help to develop new strategies for novel therapies against these infiltrative CNS tumors.

Interferon-γ Alters the Immune-related miRNA Expression of Microvesicles Derived from Mesenchymal Stem Cells

Increasing studies have demonstrated that interferon gamma（IFN-γ）,which serves as a critical inflammatory cytokine,is essential to induce the immunosuppressive effects of mesenchymal stem cells（MSCs）.However,the mechanisms underlying the enhanced immunosuppressive effects of IFN-γ-stimulated MSCs（γMSCs） are not fully understood.MSC-derived microvesicles（MSC-MVs） have been viewed as potential pivotal mediators of the immunosuppressive effects of MSCs.Moreover,micro RNAs（miR NAs） are important regulators of immunological processes and can be shuttled from cell to cell by MVs.The aim of our study was to analyze the the mi RNA expression signature of MVs derived from γMSCs（γMSC-MVs）,which may provide better understanding of the immunosuppressive property of their parent cells.Through mi RNA microarray and bioinformatics analysis,we found 62 significantly differentially expressed miR NAs（DEMs） in γMSC-MVs compared with MSC-MVs.And the potential target genes and signaling pathways regulated by DEMs were predicted and analyzed.Interestingly,many DEMs and predicted signaling pathways had been demonstrated to be involved in immunoregulation.Furthermore,the network between immunoregulation-related pathways and relevant DEMs was constructed.Collectively,our research on the mi RNA repertoires of γMSC-MVs not only provides new perspectives into the mechanisms underlying the enhanced immunosuppressive property of γMSCs,but also paves the way to clinical application of these potent organelles in the future.

Comparison of microRNA Expression Profiles in HCC-derived Microvesicles and the Parental Cells and Evaluation of Their Roles in HCC

Summary： To determine whether the microRNAs （miRNAs） contained in cancer-derived microvesi- cles （MVs） mirror those of the parental tumor cells, we compared the miRNA expression profiles of MVs derived from their parental hepatocellular carcinoma ,（HCC） cells. The presence and levels of 888 miRNAs from SMMC-7721 cells and MVs were detected by Agilent miRNA microarray analy- sis. Four selected miRNAs were verified by real time qRT-PCR. Furthermore, the genes of the miRNAs were bioinformatically identified to explore potential roles of the miRNAs in HCC micro- environment. Our results showed that miRNAs expression profiles of MVs derived from HCC were significantly changed. Of all the miRNAs tested, 148 miRNAs were co-expressed in MVs and SMMC-7721 cells, only 121 and 15 miRNAs were detected in MVs and SMMC-7721 cells, respec- tively. Among the 148 co-expressing miRNAs, 48 miRNAs had the similar expression level and 6 of them were supposed to be oncogenic or suppressive miRNAs. According to the target prediction by Quantile Algorithm method, these miRNAs may regulate 3831 genes which were closely related to cell cycle, apoptosis and oncogenesis, and 78 were known tumor suppressors or oncogenes. Gene ontology （GO） analysis indicated that 3831 genes were mainly associated with nucleic acid binding, cell death, cell adhesion. MVs containing miRNAs, released into the HCC microenvironment, bear the characteristic miRNAs of the original cells and might participate in cancer progression.

Immune modulatory function of abundant immune-related microRNAs in microvesicles from bovine colostrum

Colostrum provides essential nutrients and immunologically active factors that are beneficial to newborns.Our previous work demonstrated that milk contains large amounts of miRNA that is largely stored in milk-derived microvesicles(MVs).In the present study,we found that the MVs from colostrum contain signifi cantly higher levels of several immune-related miRNAs.We hypothesized that the colostrum MVs may transfer the immune-related miR-NAs into cells,which contribute to its immune modulatory feature.We isolated colostrum MVs by ultracentrifugation and demonstrated several immune modulation features associated with miRNAs.We also provide evidence that the physical structure of milk-derived MVs is essential for transfer miRNAs and following immune modulation effect.Moreover,we found that colostrum powder-derived MVs also contains higher levels of immune-related miRNAs that display similar immune modulation effects.Taken together,these results show that MV-containing immunerelated miRNAs may be a novel mechanism by which co-lostrum modulates body immune response.

Designer cell-self-implemented labeling of microvesicles in situ with the intracellular-synthesized quantum dots

Cell-derived microvesicles(MVs) are secreted from almost all kinds of mammalian cells into the extracellular space, and play crucial roles in intercellular communication and transporting biomolecules between cells. However, there is a great challenge for visualizing and monitoring of MVs’ bio-behaviors due to the limitations of existing labeling methods. Herein, we report the first paradigm of designer cell-self-implemented labeling of MVs secreted from living mammalian MCF-7 cells in situ using the intracellular-synthesized fluorescent quantum dots(QDs) during the formation of MVs. By elaborately coupling intracellular biochemical reactions and metabolism pathways, the MCF-7 cells can be illuminated brightly by intracellular-biosynthesized fluorescent CdSe QDs. Simultaneously, intracellular-synthesized QDs can be in situ encapsulated by the secreted MVs budding from the plasma membrane of the fluorescing cells to label the MVs with an efficiency of up to 89.9%. The whole labeling process skillfully combines designer precise cell-tuned intricate synthesis of CdSe QDs with mild in-situ labeling via cell-selfimplementation just after feeding the cell with suitable chemicals, which is structure-or function-nondestructive and much more straightforward and milder than those by chemical conjugation or indirect encapsulation with conventional fluorogenic labels.

Platelet-derived microvesicles deliver miR-30e and promote VSMC apoptosis after balloon injury

During vascular remodeling after intimal injury,circulating platelets are activated by exposed collagen and release platelet-derived microvesicles(PMVs),which may contribute to the injury-induced apoptosis of vascular smooth muscle cells(VSMCs).However,the mechanisms in this process are still unclear.Using a rat balloon injury model,platelet adhesion at the injured site was detected,and promoted medial VSMC apoptosis was revealed with dT-mediated dUTP nick-end labeling(TUNEL)in vivo.VSMC apoptosis was promoted by coincubation with PMVs in vitro.Transcriptomics analysis indicated the abound expression of the microRNA-30(miR-30)family in platelets,and the expression of the miR-30 family in platelets and PMVs was confirmed with qPCR.Ingenuity Pathway Analysis(IPA)software identified transcription factor 2(RUNX2)as a pivotal molecule linking miR-30 and cellular apoptosis.In vitro,PMVs delivered miR-30e,an important member of the miR-30 family,into VSMCs and increased cell apoptosis.A dual-luciferase assay was adopted to verify the interaction of miR-30e with RUNX2,and fluorescence in situ hybridization(FISH)proved the cytoplasmic localization of miR-30e in VSMCs.The apoptotic effect on VSMCs was further confirmed by using miR-30e mimics and RUNX2 siRNA.Furthermore,RUNX2 siRNA altered the expression of its downstream genes,Tnfrsf11b,Smad6 and Mmp13,which may participate in apoptosis,as suggested by IPA.Our results indicated that PMVs play important roles in the interactions between circulating elements and VSMCs by delivering miR-30e to VSMCs and then promoting apoptosis.PMVs and potential intercellular messages may be novel therapeutic targets for preventing pathological vascular remodeling after intimal injury.

Microvesicles:the functional mediators in sorafenib resistance

Overcoming drug resistance in cancer therapies remains challenging,and the tumor microenvironment plays an important part in it.Microvesicles(MVs)are functional natural carriers of cellular information,participate in intercellular communication,and dynamically regulate the tumor microenvironment.They contribute to drug resistance by transferring functional molecules between cells.Conversely,due to their specific cell or tissue targeting ability,MVs are considered as carriers for therapeutic molecules to reverse drug resistance.Thus,in this mini-review,we aim to highlight the crucial role of MVs in cell-to-cell communication and therefore their diverse impact mainly on liver cancer progression and treatment.In addition,we summarize the possible mechanisms for sorafenib resistance(one of the main hurdles in hepatocellular carcinoma treatments)and recent advances in using MVs to reverse sorafenib resistance in liver cancer therapies.Identifying the functional role of MVs in cancer therapy might provide a new aspect for developing precise novel therapeutics in the future.

Adipocyte-derived microvesicles from obese mice induce M1 macrophage phenotype through secreted miR-155

The pro-inflammatory profile of M1 macrophage accumulation in adipose tissue is a central event leading to the metabolic complications of obesity.However,the mechanisms by which M1 macrophages are enriched in adipose tissue during weight gain remain incompletely understood.Here,we investigated the effects of adipocyte-derived microvesicles(ADM)on modulating macrophage phenotype in mice and explored the involved molecular signalling pathways.We found that,compared with ADM from lean mice(SD ADM),ADM from obese mice(HFD ADM)significantly enhanced M1 marker expression.The quantitative RTPCR assay demonstrated that miR-155 was upregulated in both HFD ADM and HFD ADM-treated macrophages.By depleting miR-155 expression in HFD ADM and increasing miR-155 level in SD ADM,we further illustrated that miR-155 in ADM-induced M1 macrophage polarization.Functionally,in contrast to SD ADM,HFD ADM significantly decreased the protein level of SOCS1,a proven miR-155 target,leading to activation of STAT1,and suppression of STAT6 signalling;these effects were reversed by silencing miR-155 in HFD ADM.Furthermore,the supernatant of bone marrow-derived macrophages pre-stimulated with miR-155-bearing ADM interfered with insulin signalling and insulin-induced glucose uptake in adipocytes.Collectively,these results provide the first evidence that M1 macrophage polarization can be mediated by miR-155-bearing ADM,which reciprocally regulates insulin signalling and glucose uptake in adipocytes.Our study reveals a novel mechanism through which obesity induces an imbalance in the M1-to-M2 macrophage ratio in adipose tissue,thus causing chronic inflammation and local insulin resistance.

Microvesicles(MIVs)secreted from adipose-derived stem cells(ADSCs)contain multiple microRNAs and promote the migration and invasion of endothelial cells

Extracellular vesicles(EVs)such as microvesicles(MIVs)play an important role in intercellular communications.MIVs are small membrane vesicles sized 100e1000 nm in diameter that are released by many types of cells,such as mesenchymal stem cells(MSCs),tumor cells and adipose-derived stem cells(ADSC).As EVs can carry out autocrine and paracrine functions by controlling multiple cell processes,it is conceivable that EVs can be used as delivery vehicles for treating several clinical conditions,such as to improve cardiac angiogenesis after myocardial infarction(MI).Here,we seek to investigate whether ADSC-derived MIVs contain microRNAs that regulate angiogenesis and affect cell migration of endothelial cells.We first characterized the ADSC-derived MIVs and found that the MIVs had a size range of 100 e300 nm,and expressed the MIV marker protein Alix.We then analyzed the microRNAs in ADSCs and ADSC-derived MIVs and demonstrated that ADSC-derived MIVs selectively released a panel of microRNAs,several of which were related to angiogenesis,including two members of the let-7 family.Furthermore,we demonstrated that ADSC-derived MIVs promoted the cell migration and invasion of the HUVEC endothelial cells.The PKH26-labeled ADSC-derived MIVs were effectively uptaken into the cytoplasm of HUVEC cells.Collectively,our results demonstrate that the ADSC-derived MIVs can promote migration and invasion abilities of endothelial cells,suggesting pro-angiogenetic potential.Future studies should focus on investigating the roles and mechanisms through which ADSC-derived MIVs regulate angiogenesis.

Autographa Californica Multiple Nucleopolyhedrovirus P48(Ac103) Is Required for the Efficient Formation of Virus-Induced Intranuclear Microvesicles

Our previous study has shown that the Autographa californica multiple nudeopolyhedrovirus(AcMNPV)p48(acl03)gene is essential for the nuclear egress of nucleocapsids and the formation of occlusion-derived virions(ODVs).However,the exact role of p48 in the morphogenesis of ODVs remains unknown.In this study,we demonstrated that p48 was required for the efficient formation of intranuclear microvesicles.To further understand its functional role in intranuclear microvesicle formation,we characterized the distribution of the P48 protein,which was found to be associated with the nucleocapsid and envelope fractions of both budded virions and ODVs.In AcMNPV-infected cells,P48 was predominantly localized to nucleocapsids in the virogenic stroma and the nucleocapsids enveloped in ODVs,with a limited but discernible distribution in the plasma membrane,nuclear envelope,intranuclear microvesicles,and ODV envelope.Furthermore,coimmunoprecipitation assays showed that among the viral proteins required for intranuclear microvesicle formation,F48 associated with Ac93 in the absence of viral infection.

Extracellular vesicles in the diagnosis and treatment of central nervous system diseases

Extracellular vesicles,including exosomes and microvesicles,play a fundamental role in the activity of the nervous system,participating in signal transmission between neurons and providing the interaction of central nervous system with all body systems.In many neurodegenerative diseases,neurons pack toxic substances into vesicles and release them into the extracellular space,which leads to the spread of misfolded neurotoxic proteins.The contents of neuron-derived extracellular vesicles may indicate pathological changes in the central nervous system,and the analysis of extracellular vesicle molecular content contributes to the development of non-invasive methods for the diagnosis of many central nervous system diseases.Extracellular vesicles of neuronal origin can be isolated from various biological fluids due to their ability to cross the blood-brain barrier.Today,the diagnostic potential of almost all toxic proteins involved in nervous system disease pathogenesis,specificallyα-synuclein,tau protein,superoxide dismutase 1,FUS,leucine-rich repeat kinase 2,as well as some synaptic proteins,has been well evidenced.Special attention is paid to extracellular RNAs mostly associated with extracellular vesicles,which are important in the onset and development of many neurodegenerative diseases.Depending on parental cell type,extracellular vesicles may have different therapeutic properties,including neuroprotective,regenerative,and anti-inflammatory.Due to nano size,biosafety,ability to cross the blood-brain barrier,possibility of targeted delivery and the lack of an immune response,extracellular vesicles are a promising vehicle for the delivery of therapeutic substances for the treatment of neurodegenerative diseases and drug delivery to the brain.This review describes modern approaches of diagnosis and treatment of central nervous system diseases using extracellular vesicles.

一种细胞间通信的新的信号分子——Microvesicle运输的microRNA

Microvesicle（MV）是机体内的细胞在正常和病理状态下都会分泌的直径在30~1000nm之间的微小囊泡。细胞把特异的生物活性分子如蛋白质、mRNA等包裹到MV中，这些生物活性分子会通过MV被运输到相应的受体细胞以调节受体细胞的生物功能。这种由MV介导的细胞间信息传递在一些生理和病理过程中扮演着十分重要的作用。近期研究表明MV中包含microRNA（miRNA），并且通过MV的运输，这些miRNA会被运送人靶细胞以调控靶细胞相关基因的表达。本文就MV运输的miRNA在细胞间通信的功能做一总结。

Detection of Microvesicle miRNA Expression in ALL Subtypes and Analysis of Their Functional Roles

Microvesicles （MVs） are the heterogeneous mixtures of vesicles. MVs released by leukemia cells constitute an important part of the leukemia microenvironment. MVs might act as important reser- voirs of microRNAs （miRNAs）. It is worth evaluating whether MVs possess some unique miRNA con- tents that are valuable in understanding the pathogenesis. In this study, we investigated the miRNA ex- pression patterns of Nalm-6-derived MVs, Jurkat-derived MVs and normal cell-derived MVs using miRNA microarrays. The potential target genes regulated by differentially expressed miRNAs were also predicted and analyzed. Results demonstrated that 182 miRNAs and 166 miRNAs were differentially expressed in Nalm-6-MVs and Jurkat-MVs, respectively. Many oncogenes, tumor suppressors and sig- nal pathway genes were targeted by these aberrantly expressed miRNAs, which might contribute to the development of B-ALL or T-ALL. Our findings expanded the potential diagnostic markers of ALL and provided useful information for ALL pathogenesis.

Methods for the extraction and RNA profiling of exosomes

AIM:To develop protocols for isolation of exosomes and characterization of their RNA content.METHODS:Exosomes were extracted from He La cell culture media and human blood serum using the Total exosome isolation(from cell culture media)reagent,and Total exosome isolation(from serum)reagent respectively.Identity and purity of the exosomes was confirmed by Nanosight?analysis,electron microscopy,and Western blots for CD63 marker.Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit.Finally,RNA was profiled using Bioanalyzer and quantitative reverse transcriptionpolymerase chain reaction(q RT-PCR)methodology.RESULTS:Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum,with subsequent isolation and analysis of RNA residing within these vesicles.The isolation procedure is completed in a fraction of the time,compared to the current standard protocols utilizing ultracentrifugation,and allows to recover fully intact exosomes in higher yields.Exosomes were found tocontain a very diverse RNA cargo,primarily short sequences 20-200 nt(such as mi RNA and fragments of m RNA),however longer RNA species were detected as well,including full-length 18S and 28S r RNA.CONCLUSION:We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes,followed by isolation of RNA and its analysis by q RT-PCR and other techniques.

Defining lung cancer stem cells exosomal payload of miRNAs in clinical perspective

Since the first publication regarding the existence of stem cells in cancer[cancer stem cells(CSCs)]in 1994,many studies have been published providing in-depth information about their biology and function.This research has paved the way in terms of appreciating the role of CSCs in tumour aggressiveness,progression,recurrence and resistance to cancer therapy.Targeting CSCs for cancer therapy has still not progressed to a sufficient degree,particularly in terms of exploring the mechanism of dynamic interconversion between CSCs and non-CSCs.Besides the CSC scenario,the problem of cancer dissemination has been analyzed indepth with the identification and isolation of microRNAs(miRs),which are now considered to be compelling molecular markers in the diagnosis and prognosis of tumours in general and specifically in patients with non-small cell lung cancer.Paracrine release of miRs via“exosomes”(small membrane vesicles(30-100 nm),the derivation of which lies in the luminal membranes of multi-vesicular bodies)released by fusion with the cell membrane is gaining popularity.Whether exosomes play a significant role in maintaining a dynamic equilibrium state between CSCs and non-CSCs and their mechanism of activity is as yet unknown.Future studies on CSC-related exosomes will provide new perspectives for precision-targeted treatment strategies.

Tritrichomonas foetus:New structures by high-resolution scanning helium ion microscopy

Helium ion scanning microscopy(HIM)is a novel high-resolution scanning microscopy technique that uses helium ions instead of electrons to form images of the highest quality and resolution,providing a sub-nanometer resolution sputter uncoated biological cell.Here,we took advantage of HIM to explore the cell surface of Tritrichomonas foetus,a protist parasite of cattle that provokes hard infection and abortion in cows.We describe thin protrusions,like nanotubes described in other cells,with different sizes(27 nm to 81 nm in thickness)and various lengths(from 73 nm to 2μm),as well bulbous structures either budding from the cell surface or present in the extremities of some protrusions.The flagella also presented these thin protrusions and different protein decoration,similar to those previously described using freeze-fracture techniques.Nanotubes between two cells were also seen,and their role in infection is discussed.The cell surface was also examined and showed several pits indicative of endocytic activity and other types of arrays of particles.These observations were confirmed using Scanning Electron Microscopy(SEM),negative staining,and conventional thin sectioning for observation by transmission electron microscopy.Our findings provide new and relevant information that may contribute to a better understanding of protozoan biology and its interaction with mammalian cells.