Orius in pest control is widely spread, particularly in Mediterranean Europe. Commercial mass rearing of predatory insects is costly, and research efforts have concentrated on diet manipulation and selective breeding to reduce costs and improve efficacy. The characterisation and contribution of microbial symbionts to Orius sp. fitness, behaviour, and potential impact on human health has been neglected. This paper provides the first genome sequence level description of the predominant culturable facultative bacterial symbionts associated with five Orius species  from several geographical locations. Two types of symbionts were broadly classified as members of the genera Serratia and Leucobacter, while a third constitutes a new genus within the Erwiniaceae. These symbionts were found to colonise all the insect specimens tested, which evidenced an ancestral symbiotic association between these bacteria and the genus Orius. Pangenome analyses of the Serratia sp. isolates offered clues linking Type VI secretion system effector–immunity proteins from the Tai4 sub-family to the symbiotic lifestyle.Pest control in agriculture employs diverse strategies, among which the use of predatory insects has steadily increased. The use of several species within the genus  The food needs of an ever-increasing human population must be matched by intensification of crop production, albeit using sustainable approaches to ensure the preservation of ecosystems, biodiversity, and environmental quality. Pest control is a key element in food production and with thousands of described crops pests , a multitude of pest management strategies have been developed throughout human history to minimise damage and reduce crop losses. Although chemical pesticides were considered at one point a “silver bullet” for pest control, unwanted side effects for both human and environmental health have prompted the search for alternative approaches .Orius (Wolff), most of them with a substantial market presence since the 1990s. Of those, the generalist predator Orius laevigatus in particular is currently used in over 20 countries as a pest control agent (Integrated pest management (IPM) strategies have evolved to address the need for a systems approach combining crop protection strategies with the monitoring of agricultural pests and their natural enemies. The use of insect predators as biological control agents has ancient origins, and their use has gathered popularity within the context of IPM programmes as a more environmentally safe and economically viable pest management method. Several types of biological control are in use  – with the latter consisting of mass rearing of indigenous or exotic predators on a semi-industrial scale. Indeed, augmentative biological control is now a successful commercial activity (>$200 million) with more than 200 predatory species (mostly Arthropoda) produced and sold by numerous suppliers to be used in field and greenhouse grown crops. Among the predators used, the family Anthocoridae (Hemiptera: Heteroptera) is well represented by several species within the genus ol agent .Orius, most commonly distributed in the Palearctic, but also described in the Nearctic and Neotropic realms  currently the only known exception; feeding primarily on pollen of Ecballium elaterium . O. sauteri (Poppius) and O. strigicollis (Poppius), among others, have been used in Japan as biological control agents , O. laevigatus (Fieber), Orius albidipennis (Reuter), Orius niger (Wolff), Orius minutus (L.), Orius horvathi (Reuter), and Orius laticollis (Reuter) do occur  or horizontally transmitted. The nature of the symbiont–host relationship defines two categories of symbiosis: primary or obligate symbionts and facultative or secondary ones. Primary symbionts are essential for host survival and share a long evolutionary history with the host, as evidenced by the 270 million years association between Sulcia muelleri and Auchenorrhyncha , symbionts that confer protection to parasitoids (Hamiltonella defensa in aphids), or fungal infection , and to environmental stress (Serratia symbiotica in aphids). The latter putatively provides evidence of the early stages in the transition from facultative to primary symbiosis, as it likely complements essential functions lost by the primary symbiont Buchnera in the aphid Cinara cedri  being published. Of those studies, Wolbachia infection was detected using multilocus sequence typing (MLST) profiling in multiple populations of five Orius species  from Japan. Only two predominant Wolbachia strain types were detected in the study, which prompted the conclusion that both vertical transmission from a common ancestor and horizontal transfer account for the low diversity of Wolbachia lineages observed  was used as a refuge; frozen and sterilised E. kuehniella eggs were used as a prey for nutrition; surfaced sterilised pods of Phaseolus vulgaris  were provided for egg laying; and a water vial sealed with cotton wool as a source of moisture. Containers were maintained at 25–26°C, in 70–80% relative humidity and 16L:8D daylight cycle. Maintenance and multiplication of these populations were undertaken every 2 or 3 days to collect Orius eggs, launching a new age cohort of insects and avoiding cannibalism from adults to nymphs. Moreover, Ephestia eggs and a fresh bean pod piece were added each time, water vials were replaced as necessary. Four commercial O. laevigatus specimens were purchased from Syngenta Bioline®, Koppert (THRIPOR-L), BioBest (Orius-System), and AgroBio (Oricontrol). O. pallidicornis specimens were collected in the field (Southeast Spain) and processed immediately or preserved in 70% ethanol. Taxonomic classification of insect specimens was confirmed by amplifying COI by PCR from total insect DNA using primers CoxILCO1490F and CoxIHCO2198R  at the Crop Protection laboratory (Universidad Politécnica de Cartagena). These procedures were carried out in previously sterilised, 1 L plastic containers with tight fitting lids containing air vents covered in philtre paper. Sterilised buckwheat husk  was performed on the selected colony morphotypes and the resultant amplicon sequenced to confirm taxonomic identity Primers used here were U1 and U1R  were surface sterilised twice with 70% ethanol, washed in sterile water three times, and crushed using sterile micropestles in liquid Nutrient Broth. The homogenate was diluted in Nutrient broth and plated on Nutrient Agar plates using aseptic techniques, followed by incubation at 28°C until colonies were visible (2–3 days).  and U1R .® Blood & Tissue Kit (insect protocol) from Qiagen. To increase yield, specimens were incubated in Proteinase K overnight. Total bacterial DNA was isolated from overnight Nutrient Broth cultures using a similar kit applying the corresponding protocol (Qiagen). Total genomic DNA was quality controlled by electrophoresis and spectrophotometry. Bacterial genome sequencing was performed on an Illumina MiSeq platform. Sequence ready libraries were constructed using the Illumina Nextera XT library preparation procedure. Raw reads were subjected to quality and adapter filtering using the Trim Galore wrapper tool specifying the Illumina and Nextera adapter strings to be trimmed  phylogeny. Briefly, COI sequences were amplified using primers CoxILCO1490F and CoxIHCO2198R. Resultant amplicons were precipitated overnight and sequenced in both directions using the original PCR primers. Consensus COI sequences were constructed by alignment of both forward and reverse sequences. Orthologous COI sequences from other Orius species were retrieved from GenBank and also added to the dataset. ML phylogenetic reconstruction was performed in MEGA using the Tamura-Nei model and uniform rates among sites. Robustness of the phylogeny was assessed with 1000 bootstrap pseudoreplicates.Phylogenetic reconstruction of Orius sp. was performed using a multilocus amino acid phylogeny. Briefly, ortholog retrieval, alignment, and concatenation of 400 ubiquitous and phylogenetically informative protein sequences were performed in PhyloPhlAn  using GGDC 2 genomes .Serratia sp. SCBI as a reference. Predicted Genomic Islands (GIs) sequences were clustered using CD-Hit-est2 using an 80% identity threshold in order to identify representative GIs . The rest of the isolates (24) belonged to Enterobacteriales . Thirteen Orius-derived isolates distributed to a tight small clade together with 10 Serratia species within the “Serratia SCBI complex,” while two isolates grouped with the reference strain S. marcescens DB11 in a smaller clade (DB11 complex) within the major “S. marcescens complex.” Further genome–genome distance calculation (GGDC) analysis of the genome sequences present in the “Serratia SCBI clade,” using digital DNA:DNA hybridisation confirmed that all genomes in this clade belong to the same species and will be referred to as Serratia SCBI-like isolates (data not shown). However, this analysis also showed that this sequence similarity is not sufficient to consider the Orius-derived isolates the same sub-species as those within the SCBI clade , the accuracy of the S. marcescens taxonomic classification in some genomes seems questionable. Therefore, genome distance comparisons using as query S. marcescens WWW4 to all available fully assembled genomes of bacteria classified as S. marcescens in the NCBI nucleotide database were conducted, revealing in some cases  genome sequence distances that disprove their taxonomic classification as the same species (data not shown).Since some of the organisms grouping within the SCBI complex are classified as Erwinia-like and Leucobacter-like isolates were processed in a similar manner using PhyloPhlan. From the former, only one isolate (OLMDLW33) grouped within the Erwinia/Pantoea sensu stricta, forming a small clade with the F. occidentalis symbiont BFo1 . Surprisingly, the remaining seven “Erwinia-like” isolates disputed the 16S rRNA gene-based classification and distributed to a novel monophyletic clade within the Erwiniaceae . The value of this distribution was confirmed by GGDC analyses, as it showed that our isolates were too distant from any of the available Erwinia, Pantoea, or Tatumella species genomes; probably constituting a new genus yet to be properly classified. Further attempts with GGDC analyses failed to identify a close relative in publicly available genomes, and from this point these isolates are referred to as Erwiniaceae-like.The genome sequences from the ont BFo1 . This diLeucobacter-like isolates formed a strongly supported novel monophyletic clade within the Microbacteriaceae, as part of the Leucobacter clade . Despite this distribution and the 16S rRNA gene sequence similarity, genome distance comparisons estimated by GGDC revealed that none of the isolates were similar enough to be considered the same species as the Leucobacter sp. genomes available at NCBI databases. These genomes are referred to as Leucobacter sp. Orius throughout the paper. Further exploration identified one additional Leucobacter sp. genome sequence, namely Leucobacter sp. AEAR, which was assembled from the raw genome sequences of the nematodes Caenorhabditis angaria and Caenorhabditis remanei, although never isolated and cultured  that suggest different sub-species. Indeed, while GGDC produced a high probability score  confirming that these genomes belong to the same species, the probability of them being the same sub-species (33.31%) falls well below the defined threshold (>79%).The cultured . When coO. pallidicornis) insect species tested were lab-reared, total insect DNA was also extracted from specimens collected in the field or acquired from commercial sources, but not propagated in the lab. Genome-specific PCR amplification detected the presence of Orius-derived Serratia SCBI-like strains in insect specimens from where Serratia-like isolates were not recovered by culturing techniques; and also from field collected or non-lab reared specimens . The Serratia isolates belonging to the DB11 complex (OLAL2 and OMLW3) were not detected in any of the insect DNA samples tested (not shown), and therefore it was concluded that there was insufficient evidence to catalogue these isolates as true symbionts, and were not further analysed.Representative genome sequences from the distinct isolates described above were used to design genome-specific PCR primers . In contrast, the Erwiniaceae-like organism was only detected in lab-reared O. albidipennis and not detected in some commercial specimens , questioning the symbiotic association of these isolates with Orius sp. This finding, together with the limited number of genome sequences from the same species, led to the decision of not performing further analyses on these strains until their symbiotic nature is confirmed by future studies.Attempts to detect the presence of the Serratia SCBI-like genomes was predicted in Island Viewer using Serratia SCBI as reference genome. Overall 87 GIs were predicted, with sizes ranging from 50 to 262 Kb (Supplementary Table O. laevigatus-derived isolates displayed a comparable number of GIs (ranging from 5 to 9), with the exception of OLFL2 (4) and OLJL1 (3). Overall there is poor correlation between genome size and GI number  and O. pallidicornis -derived isolates possess, respectively, a large number of genome-specific GIs . Interestingly, the distribution of these insect hosts based on presence/absence of representative GIs in genomes of corresponding symbionts is reminiscent of the COI-based phylogenetic relationship of O. niger and O. pallidicornis, as proposed earlier. The lack of available reference genomes for the Erwiniaceae-like and Leucobacter sp. Orius prevented the prediction of GIs in the genomes of these isolates.The GI sequences were clustered into representative GIs  to identify those shared across genomes from genome-specific GIs  as part of the assemblies. In order to ensure a meaningful comparison, before undertaking pangenome analysis the sequence of the Serratia sp. SCBI plasmid (SCBI_Pl) was included as part of this organism’s genome sequence.The absence in databases of fully assembled reference genomes from the same species as the Serratia SCBI complex” reveals that the pangenome contains a total of 8165 genes with some 43.1% of genes shared across all isolates – yielding a core genome of 3518 genes. The accessory genome can be sub-divided into soft core genes (95% ≤ strains < 99%), shell genes (15% ≤ strains < 95%), or cloud genes (0% ≤ strains < 15%) of which there are 515, 1160, and 2972 genes, respectively.Comparative analysis of the gene content between the 21 genomes within the “Serratia SCBI complex is unlikely to significantly alter the estimated core genome size; on the other hand, the complete pangenome is likely to increase in size as additional members of the SCBI complex are sequenced , indicating an open pangenome for species within this complex. A total of 279 accessory genes were found to be strictly associated with the Orius facultative symbiosis trait, occurring in all 13 Orius-derived isolates but not in any of the other strains from the SCBI complex .Sequencing of additional members of this Orius symbionts’ accessory genome (referred as Orius accessory genes) revealed a high number of hypothetical proteins, transcription factors, bacteriophage proteins, mobile elements, and seemingly plasmid-related genes  was used as query in BLASTN to search available Serratia sequences, disclosing that these plasmid sequences possess higher sequence homology to plasmids PSM22 (S. marcescens strain B-6493) and PWN146p1 (S. marcescens PWN146) than to SCBI_P1. This explains why some plasmid genes are included within the Orius accessory genes. S. marcescens strain B-6493 is a human pathogen and S. marcescens PWN146 was isolated from the nematode Bursaphelenchus xylophilus, and both are different species from those in the SCBI complex as confirmed by GGDC. With a lack of replicon segregation within available Serratia species draught genome assemblies, it is difficult to expand on plasmid sequence comparisons at this stage to define a plasmid genealogy for the genus, but our findings are certainly indicative of plasmid exchange and sequence plasticity across the Serratia genus and worthy of future studies.Annotation of the predicted Orius accessory genome a Type VI secretion system (TSS6) associated protein was identified, and annotated as an immunity protein belonging to the Tai4 protein family. Considering the implication of TSS6 in bacterial virulence and inter-bacterial competition, the TSS6-related genes in the genomes analysed were scrutinised. The Orius-derived Serratia genomes possess two TSS6 genetic loci, with most of the genes in these loci contained within the SCBI core genome, with the exception of the aforementioned Tai4-like encoding gene identified as part of the Orius accessory genome. The TSS6 locus encoding this protein was further analysed; revealing the presence of a well-conserved TSS6 gene cluster. It is worth noting that the PGAP annotation pipeline failed to predict some of the ORFs in this locus that were otherwise identified by the PROKKA annotation pipeline, thus these annotations were used to map the genes. In addition to the conserved core TSS6 genes, several genes encoding for effector and immunity cognate partners from the Tae4/Tai4 subfamily were identified. Tae4-like proteins are bacterial cell wall targeting amidases, whose activity is neutralised by cognate Tai4-like immunity proteins to prevent self-killing. This effector–immunity complex have been characterised in Salmonella enterica Typhimurium, Enterobacter cloacae, and S. marcescens DB10 (SmDB). The latter possesses two effector–immunity loci within the same TSS6 cluster, namely the Tae4-like Ssp1 and Ssp2, and the Tai4-like immunity proteins Rap1a, Rap1b, Rap2a, and Rap2b .Interestingly, within the nd Rap2b . The TaiOrius-derived Serratia genome (OLFL2) were used as query to interrogate the Ssp–Rap loci in related genomes, in order to assert the degree of sequence conservation across orthologs. Comparison of this genetic locus in the available genomes of the “SCBI-complex” and S. marcescens DB11 (same species as DB10) revealed striking differences. In contrast to the locus composition in SmDB, the Orius-derived Serratia sp. encodes only one effector protein Ssp2, more similar to SmDB Ssp2 (45% identity) than SmDB Ssp1 (30%), and three Rap-like immunity proteins. The latter were annotated as Rap1b, Rap2a, and Rap2b based on sequence similarity to the corresponding SmDB orthologs. This locus composition is conserved across the Orius-derived Serratia sp. and partially in S. ureilytica.The Ssp-like and Rap-like sequences from a single representative Figure 6), mostly in terms of number of Ssp-like and Rap-like encoding genes. Interestingly, most of the “SCBI-complex” genomes are devoid of Ssp1 and Rap1a coding sequences, except for two strains; while Serratia sp. SCBI and S. marcescens 90-166 lack a gene encoding for an Ssp effector protein. An additional interesting feature is the occurrence of two copies of rap2a in five genomes of the “SCBI-complex,” with one copy displaying close sequence homology (>80% identity) to its corresponding ortholog in OLFL2, while the other is only marginally similar (∼30% sequence identity). The occurrence of this trait in five genomes strongly suggests that the extra rap2a copy in those genomes may have originated from a gene duplication event, and probably constitutes a new sub-family within the Rap-like proteins.The locus composition and architecture differs across the rest of the “SCBI-complex” genomes . Similarly, Rap2b is well conserved (>95% identity) in the Orius-derived isolates, Serratia sp. SCBI, and S. marcescens FDAARGOS 62, but its sequence conservation decreases below the threshold in the rest of the genomes composing the SCBI complex. Rap1b is only encoded by the Orius-derived isolates and S. ureilytica, but absent from the rest of the genomes in the SCBI clade . The above considerations highlight the fact that relaxing the stringency of the conditions used to generate the Orius accessory genome, allowing for the occurrence of sequences in all 13 Orius-derived isolates and one or two extra genomes from the SCBI complex, would have resulted in the inclusion of additional Ssp and Rap sequences as part of the Orius accessory genome.Close examination of the sequence identity across the range of sequences examined demonstrated that, except for B. aphidicola and Wolbachia sp. The accessibility of next generation sequencing platforms has impacted the way in which insect–bacteria symbiosis is studied, with a plethora of reports describing symbionts at the genome level, across a wide range of insect species. However, most studies make use of single laboratory propagated populations or specimens obtained from single field collections, rather than attempting to generate population wide profiles not limited at a particular host species, but expanding onto related ones to provide characterisation of symbiotic associations at host genus level.The studies of microbial symbionts associated to insect hosts have been biased towards the characterisation of primary or obligate symbionts in insect species prioritised by their role as vectors for human pathogens or agricultural pests. Similarly, the nutritional benefits of the symbiotic association or the manipulation of host’s reproduction have defined paradigms in the experimental strategies used to explore symbiosis in insects; as demonstrated by the knowledge accumulated in relation to O. laevigatus populations originating in Spain, Italy, and Greece, together with populations of the related species O. pallidicornis, O. niger, O. albidipennis, and O. majusculus. Molecular phylogeny based on COI sequences revealed a close evolutionary relationship between O. niger and O. pallidicornis, indicating that O. pallidicornis can be considered a cryptic species within O. niger. The existence of O. niger cryptic species has been proposed based on COI haplotype diversity  failed to identify genomes similar enough to be classified as the same species, which further supports the proposal of them being new species in need of taxonomic classification. However, the lack of available genome sequences from the same species as our isolates prevented further genome comparison studies. In the absence of reference genomes belonging to these species, attempting pangenome studies would entail comparing to genomes from different, albeit related species, which would be procedurally incorrect to draw species-related information. Our observations provide the foundations for future studies exploring the symbiotic association of these bacterial species with other Orius species or additional insect genera.Isolation of culturable bacteria from whole insect macerates, followed by whole-genome sequencing and assembly, revealed three predominant species colonising the Serratia sp. based on 16S rRNA gene sequence similarity, and indeed MLSA analysis grouped these isolates within the Serratia sp. SCBI complex. Further refinement based on GGDC confirmed the MLSA results, that the Orius-associated Serratia sp. are sufficiently similar to genomes of the Serratia sp. SCBI complex to be considered the same species. However, none of the genomes within this complex is similar enough to be considered the same species as other S. marcescens (of which there are over 300 available genome sequences). Similarly, the SCBI complex member S. ureilytica has been shown to be distinct enough from S. marcescens by DNA–DNA hybridisation , further supporting the observation that Leucobacter sp. Orius are hosted by the insects tested.The inability to detect the Leucobacter sp. have been isolated from several insects hosts like the non-biting midges Chironomid sp. . Based on this evidence, a facultative symbiotic association between Leucobacter sp. Orius and its insect host is therefore feasible. Once more genomes from this species become available, pangenome analyses will lead to the identification of genes and gene networks required for the facultative symbiotic lifestyle.Various omid sp. , the scaa oblita , and AnoSerratia sp. isolates from Orius sp. hosts. Despite the close genome sequence similarity across the isolates, the characterisation of genomic islands permitted their segregation into putative independent strains, suggesting that despite their common origin independent lineages are emerging within their respective host populations. Indeed, GI-based hierarchical clustering of these genomes closely resembles the host phylogenetic relationship. The close sequence conservation, together with the association of these Serratia strains to various Orius species, suggests a host–symbiont association established in the last common ancestor to present day European Orius species, albeit not reflected by genome size reduction. An alternative explanation is to interpret this facultative symbiosis as a consequence of the routine acquisition of this Serratia species from environmental sources. The failure to isolate other Serratia species from the Orius sp. specimens tested adds weight to the interpretation that this Orius–Serratia association is an ancient symbiotic event. However, expanding this study to Orius species from distant geographical locations and archived specimens is needed to determine the extent of the symbiotic relationship.The availability of same species genome sequences allowed the extensive analysis of the Serratia species possesses the ability to colonise a variety of niches and are both successful mutualistic symbionts and opportunistic pathogens. In particular their ability to persist in symbiosis with insect-associated nematodes has been demonstrated  in Orius-associated Serratia, without the presence of cognate Ssp-like effector, suggests that a trait has evolved to expand the resistance range against effector proteins secreted by invading bacteria, as suggested for other bacterial species , plant associated , and human pathogens . The latter in particular is extremely relevant in lieu of the use of Orius species as pest control agents in crops destined for human consumption, and at least one reported case of targeted biting of humans by Orius majusculus (Serratia sp. Orius isolates are a distinct sub-species from those shown to be pathogenic to humans, and therefore expressing concerns over the safety of their insect hosts in IPM applications is premature. Nevertheless, regulatory agencies monitoring the use of biological control should engage in discussion with the wider academic community to explore the feasibility of implementing microbiome characterisation as part of the standard quality control measures governing the use of insects as pest control agents.The results described earlier demonstrate the existence of a facultative symbiotic association between European jusculus . The genXC performed molecular microbiology tasks and genome sequence analyses. MH performed genome sequencing, genome assembly, pangenome analyses, contributed to experimental design, and manuscript preparation. PB, JM, and VB collected and propagated insect populations, performed taxonomic classification, and contributed to molecular biology tasks and manuscript preparation. PF performed molecular phylogeny analyses, contributed to experimental design, and manuscript preparation. PD contributed to manuscript preparation. RD supervised the project, designed experiments, performed genome sequence and molecular phylogeny analyses, and drafted the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Non-muscle invasive bladder cancers are a heterogeneous group of cancers whose spectrum includes low grade Ta lesions and high-grade T1 lesions. Accurate staging and grading during initial evaluation and TUR ensures appropriate treatment and prevents the risk of understaging. TUR should be ideally performed under spinal anesthesia, with a continuous flow video resectoscope to maintain a stable bladder capacity, and a video monitor. The entire bladder must be visualized, with both 30- and 70-degree lenses, and all abnormal areas must be resected, with separate biopsies from each tumor's base. Repeat TUR is recommended for all high grade tumors and T1 tumors, especially if muscle was not present in the initial specimen. Immediate instillation of single dose chemotherapy agents following TUR is highly recommended to reduce the risk of tumor recurrences.


Tumor development and tumor progression is not only determined by the corresponding tumor cells but also by the tumor microenvironment. This includes an orchestrated network of interacting cell types ) via the extracellular matrix and soluble factors such as cytokines, chemokines, growth factors and various metabolites. Cell populations of the tumor microenvironment can interact directly and indirectly with cancer cells by mutually altering properties and functions of the involved partners. Particularly, mesenchymal stroma/stem cells (MSC) play an important role during carcinogenesis exhibiting different types of intercellular communication. Accordingly, this work focusses on diverse mechanisms of interaction between MSC and cancer cells. Moreover, some functional changes and consequences for both cell types are summarized which can eventually result in the establishment of a carcinoma stem cell niche (CSCN) or the generation of new tumor cell populations by MSC-tumor cell fusion. Solid tumors can be regarded as a complex organ with tumor cells and a variety of differentially organized cell types, establishing a certain immune status, contributing to blood vessel formation and neovascularization, and building an extracellular matrix which enables the associated cell populations to communicate within this tumor microenvironment (TME) , 2. For + T cells and CD4 TH1 T cells mainly exhibit anti-cancer effects since strong infiltration of tumor tissue by these cell types is associated with favorable prognosis in many solid tumors including breast, ovarian, cervical, lung and colorectal cancer [H2 and Treg cells have been correlated with poor clinical outcome in several cancer types [Predominant cell types within the TME are represented by immune cells, fibroblasts, pericytes, endothelial cells, mesenchymal stroma/stem cells and sometimes adipocytes . Immune l cancer . In conter types . Likewiser types , 7. Moreer types , 8. Tumoer types . For inser types .In perivascular niches of tumor blood vessels pericytes are predominantly present and can associate with vascular endothelial cells by contributing to the formation of blood capillaries . In contFurther cell populations are represented by stromal cells or fibroblasts within the TME. The so-called cancer- or carcinoma-associated fibroblasts (CAFs) which are derived from various precursors like aberrant mesenchymal stroma/stem cells (MSC) or endothelial cells secrete a plethora of growth factors, cytokines, chemokines, structural protein components, and metabolites that communicate with tumor cells and promote oncogenesis by activating cell proliferation, tumor angiogenesis and invasive properties . Thus, aLikewise, MSC are one of the key players within the TME and can either inhibit or promote tumor cell growth by distinct types of cellular interaction . ReductiMSC are recruited to tumor sites and can be activated by certain stimuli such as TGF-β1 to develop a CAF-like phenotype .In addition to a variety of different cell populations in the tumor microenvironment, the extracellular matrix (ECM) also plays an important role in the regulation of tumor development und progression. The ECM does not only provide a structural scaffold for the tumor stroma with fibrous proteins such as elastin, collagen and fibronectin, and proteoglycans like chondroitin sulfate and hyaluronic acid, but in addition, ECM is abundant source of soluble factors including growth factors, angiogenic factors, cytokines and chemokines. This dynamic and complex network contributes to the intercellular cross-talk with cancer cells. During tumor development the ECM is usually dysregulated, remodeled and appears disorganized , 18. ColTaken together, the tumor microenvironment contains a variety of initially non-malignant cell types  which develop tumor-associated functionalities together with soluble factors and ECM components that all communicate with cancer cells thereby inhibiting and promoting tumorigenesis. Nonetheless, it is important to strengthen that the TME is a dynamic and heterogeneous environment whose total composition varies between tumors and patients. However, the tumor stroma exhibits common features of these distinct cell types which may serve as interesting therapeutic targets .MSC are multipotent cells that preferentially reside in perivascular niches of nearly all human tissues and organs like bone marrow, adipose tissue, heart or lung and neonatal tissues including placenta, amniotic membranes or umbilical cord –24.Apart from various sources and heterogeneous populations, MSC exhibit certain common properties ranging from the expression of surface markers  to the differentiation along the adipogenic, chondrogenic and osteogenic lineage . Their fIn addition to the MSC heterogeneity, subpopulations are characterized with altered proliferative capacity and aging properties  which maMSC are recruited to sites of injury to support tissue repair, stem cell homeostasis and immunomodulation. Similar functions are displayed by MSC during tumor development, whereby permanently proliferating and invasively growing tumor cells create an inflammatory microenvironment displaying a certain kind of “wound that never heals” . TherebyDifferent types of cross-talk between MSC and cancer cells both directly and indirectly are illustrated in Figs. Indirect communication proceeds via secretion of soluble molecules  that might function as substrates for specific receptors of neighboring cells to activate intracellular signaling pathways. Activated MSC produce and secrete a large variety of bioactive molecules suggesting MSC as Medicinal Signaling Cells . MSC-medVarious metabolites within the TME released and exchanged by the different interacting cell populations strongly affect the progress of malignancy and contribute to alter tumor cell properties such as motility, survival and self-renewal. These effects are relayed in part by altered energy turnover and consumption whereby lactate, glutamine and keton bodies contribute to a functional change of cancer cells toward an OXPHOS addicted phenotype .Besides the secretion of cytokines and chemokines into the tumor stroma, several metabolites such as prostaglandins or indoleamine 2,3-dioxygenase (IDO) represent further released soluble factors stimulating cells in a paracrine manner and contributing to the interaction network of the TME , 42.Previous studies observed that cyclic adenosine monophosphate (cAMP) can inhibit DNA damage-induced p53 accumulation via activation of protein kinase A (PKA) resulting in increased survival of lymphoblastic leukemia cells , 44. In A close metabolic coupling of MSC was demonstrated with osteosarcoma cells whereby tumor cell-induced oxidative stress in MSC was associated with higher levels of lactate and lactate efflux receptors. Consequently, osteosarcoma cells increased expression of lactate influx receptors whereby lactate secreted by MSC and incorporated by osteosarcoma cells elevated ATP production and increased migratory capacity of the cancer cells . In geneAnother indirect interplay between MSC and cancer cells is represented by the exchange of microparticles including exosomes and microvesicles. Whereas exosomes are defined as small homogeneous membrane particles of endocytic origin ranging in size from 40 to 100 nm, microvesicles are directly shed from the plasma membrane into the extracellular environment representing a larger and heterogeneous population with 50 to 1000 nm in diameter . AlthougPrevious results demonstrated that MSC-derived exosomes can modulate the function of tumor cells by induction of MMP-2 and ecto-5’-nucleotidase activity resulting in a more complex tumor microenvironment with higher tumor heterogeneity , 50. AltAnti-tumor effects have been also observed with microvesicles derived from human umbilical cord Wharton’s jelly MSC which inhibit bladder tumor cell growth via cell cycle arrest and induction of apoptosis, both in vitro and in vivo .Although functional mechanisms for these controversial observations of tumor promoting versus tumor inhibiting roles of MSC-derived microvesicles and exosomes remain to be elucidated, it appears conceivable that the cargo of these microparticles which depends on the activation status and state of development of the originating MSC is primarily responsible for the type of action on tumor cells.Notch signaling plays an important role in fundamental processes such as support of tissue repair or regulation of various immune cell functions . ActivatPrevious experiments indicated a functional involvement of the notch pathway during the interaction between MSC and breast cancer cells . Inhibit2+-ions are transported through gap junctions [Gap junctions connect adjacent cells for intercellular, direct communication called gap junctional intercellular communication (GJIC) which can regulate cell growth and differentiation or maintain tissue homeostasis. One gap junction channel is composed of two hemi-channels from each interacting cell. One hemi-channel is formed by 6 connexin protein subunits and each connexin in turn features four transmembrane domains. Typically small molecules and second messenger such as cAMP and Caunctions .Acquisition of CD90 by breast cancer cells after co-culture with MSC is associated with GJIC signaling since carbenoxolone, a gap junction inhibitor, reduces MSC-mediated CD90 expression of breast cancer cells .Nanotubes represent thin, dynamic cytoplasmic protrusions which connect two cells enabling the exchange of a variety of biological cargo ranging from organelles such as mitochondria to small molecules including calcium ions and glycoproteins over longer distances .Besides exchange of cargo between tumor cells themselves , cancer A further mechanism for direct cross-talk between MSC and cancer cells is displayed by trogocytosis. This type of direct cellular interaction was firstly described between immune cells for the transfer of surface molecules from antigen-presenting cells to lymphocytes as an active mechanism . LikewisThe closest/strongest and most complex interaction between MSC and cancer cells is the formation of fusion or hybrid cells which also represents a very rare event whereby underlying molecular mechanisms are still not fully understood. Co-culture of MSC with cancer cells like breast or ovarian cancer at certain conditions in vitro can lead to the development of hybrid cells by fusion of the two parental cell lines . PotentiGFP and red-fluorescing MDA-MB-231cherry. In such a co-culture system with MSC and cancer cells, yellow-fluorescing hybrid cells are spontaneously formed within 3 to 6 days whereby the cell size and shape varies depending on the culture conditions and the contribution of parental cell populations [Nevertheless, hybrid cells from human breast cancer and breast epithelial cells are well characterized  and sponulations .Fig. 3FoBesides the labeling of both parental cell lines with fluorescent lentiviral vectors, another system has been applied to clearly identify hybrid cells in a co-culture. The so-called bimolecular fluorescence complementation involves a lentiviral transduction of both parental cells as well whereby the eGFP gene is separated into two non-fluorescent halves and each cell type is transfected with one of these halves. Consequently, only a fusion between a mesenchymal stem cell and a cancer cell can bring these two different halves together resulting in a functional GFP fluorescence after expression that is detectable via fluorescence microscopy or flow cytometry .GFP and MDA-MB-231cherry is displayed in Fig. Fused hybrid cells generated in a labeled co-culture can be isolated by FACS and subsequent single cell cloning . A succeWhereas knowledge about tumor cell fusion remains scarce, certain characteristics of properties and functions are available about MSC-cancer hybrid cells.Formation of hybrid populations was reported in various studies between MSC and breast cancer as well as ovarian cancer cells , 36, 38.A variety of important questions concerning tumor hybrid cells remain to be elucidated with respect to the existence, frequency and mechanisms of formation in vivo as well as to the role of these fused cell types in tumorigenesis and initiation of metastasis. At least breast and gastric cancer hybrids provide evidence for enhanced tumorigenic and metastatic properties , 69. NonDuring the multistep procedure of tumorigenesis, intercellular communication of diverse cell types within the TME contributes to the malignancy of primary tumor cells as well as their metastatic ability . In co-cIn ovarian cancer cells, MSC can induce further surface markers besides expression of CD90 such as functional CD73 and CD105. Thus, primary small cell hypercalcemic ovarian carcinoma cells (SCCOHT-1) acquired the capability to metabolize cyclic adenosine 3’, 5’-monophosphate (cAMP) underlining the bidirectional molecular exchange between tumor cells and MSC .DSP gene, the cell-cell adhesion-related genes MPZL2 and SCEL, and the calcium-dependent cell-cell adhesion glycoprotein gene CDH1 are all up-regulated in MSC after co-culture with ovarian cancer cells. Conversely, a variety of cytokine mRNAs such as CSF3, IL1A, CCL20, LIF, TNF, CXCL1, CXCL2, CXCL3, and CXCL12 are down-regulated in MSC in the presence of ovarian cancer cells [Moreover, up-regulated mitotic spindle-associated factors (MZT2A) and epithelial mitogens (EPGN) suggested the promotion of proliferative capacity in ovarian cancer cells. In parallel, a down-modulation of transcription factors like TAL1, transcripts of the basic helix-loop-helix family FOS and FOSB, HES1 and HES5 are also related to the promotion of cancer development. In addition, growth factors of the bone morphogenetic protein family were acquired by ovarian cancer cells in the presence of MSC , wherebyer cells .Furthermore, undetectable expression of the epithelial cell adhesion molecule (EpCAM) in normal MSC significantly increased both, at the mRNA and protein level after co-culture with different ovarian cancer cell lines such as SK-OV-3 or NIH:OVCAR-3 . TogethePrevious work presented evidence for tumor-initiating cells (TIC) in mammary carcinoma also termed cancer stem cells (CSC) with increased expression of mesenchymal characteristics including vimentin, fibronectin and N-cadherin instead of E-cadherin . MoreoveFollowing continuous mutual interaction within the TME, cytokines and particularly IL1 released by tumor cells can stimulate arachidonic acid metabolism and subsequent PGE2 production in MSC. Vice versa, released cytokines and PGE2 together can induce β-catenin signaling in the neoplastic cells which contributes to the development of more immature stem cell-like properties .During these interactions characteristics of a mesenchymal phenotype are progressively acquired by the cancer cells –38 whichConsequently, the newly arising cancer cell populations after EMT and interaction display an enhanced phenotypic plasticity including metastatic potential and altered responsiveness/resistance to therapeutic approaches.Conversion of epithelial cells into a mesenchymal phenotype termed as EMT is a prerequisite in physiological processes during early stages of embryonic development including gastrulation and myogenesis . HoweverEMT has been implicated in several tumors including breast, ovarian and colon cancer –91. In pRecently, it has been demonstrated that colon cancer cells increased expression of EMT-TF such as Zeb1/2, Slug, Snail and Twist which was paralleled by a downregulation of E-cadherin expression. Moreover, these colon cancer cells acquired the expression of stemness genes including Oct4 and Sox2 after co-culture with adipose tissue-derived MSC. Additionally, the morphology of colon cancer cells was altered to an elongated, fibroblast-like cell shape underlying the conversion to a mesenchymal phenotype. Vice versa, colon cancer cells were able to induce secretion of cytokines  and metastasis-related factors  in MSC via activation of Wnt signaling which in turn resulted in activation of Wnt pathways in colon cancer cells. Of interest, inhibition of Wnt signaling reduced the invasiveness and tumorigenicity of cancer cells both in vitro and in vivo .Besides these direct interactions between MSC and colon cancer cells leading to induction of EMT and higher invasiveness, indirect interactions also resulted in EMT induction. Adipose tissue-derived MSC can alter cell confluence and migration of SKBR3 breast cancer cells, increase mammosphere formation, induce EMT, and alter tumor cell morphology . These fNasopharyngeal carcinoma (NPC) cells exhibited a higher proliferation and migration capacity after uptake of MSC-derived exosomes. Moreover, EMT markers were significantly altered after uptake of exosomes including down-modulation of E-cadherin and upregulation of vimentin and N-cadherin. This indirect communication between MSC-derived exosomes and cancer cells induced EMT, promoted tumor growth in vitro and in vivo and metastasis .Further studies substantiate the occurrence of direct and indirect MSC-cancer cell interactions leading to induction of EMT, thereby altering the cell pattern and morphology of cancer cells to a mesenchymal phenotype which facilitates metastasis to distant tumor sites , 97–99.Different types of intercellular communication both, indirect and/or direct between MSC and tumor cells  involve mutual functional alterations whereby the tumor cells acquire certain mesenchymal properties. Depending on the type and the extent of cellular interaction, even completely new tumor cell populations can be formed in the rare event of a MSC and tumor cell fusion. In addition, MSC which are recruited to the invasive tumor sites to initiate regenerative potential are progressively altered into an aberrant MSC phenotype to functionally support tumor cell survival. Therefore, tumor-associated aberrant MSC are involved in tumor cell protection and consequently, contribute to certain effects of chemotherapeutic resistance either directly by expression of protective extracellular matrix proteins as a drug barrier and/or indirectly by promoting EMT of tumor cells and participating in a carcinoma stem cell niche. Such development includes the interplay with various other tumor-associated cell populations and restructure of the ECM, furthermore highlighting the tumor microenvironment as potential therapeutic anti-tumor target.


Acute bioprosthetic valve thrombosis can occur after surgery and sometimes cause hemodynamic instability and cardiogenic shock. Risk factors for bioprosthetic valve thrombosis are hypercoagulability, atrial fibrillation, atrial dilatation, low cardiac function, and lack of anticoagulation therapy. The authors present a case of severe mitral stenosis due to bioprosthetic valve thrombus. The patient was diagnosed with dilated-phase hypertrophic cardiomyopathy and underwent mitral valve replacement. He required venoarterial extracorporeal membrane oxygenation (VA-ECMO) due to extremely low cardiac output and was scheduled for left ventricular assist device (LVAD) implantation. Transesophageal echocardiographic examination before LVAD implantation revealed severe mitral stenosis due to bioprosthetic mitral valve thrombus, which was not detected by transthoracic echocardiography in the intensive care unit and contributed to the low cardiac function. The thrombus was removed through an unscheduled left atriotomy before LVAD implantation. The possibility of bioprosthetic valve thrombosis must be considered when the patient is dependent on VA-ECMO support. Early transesophageal echocardiographic examination of the bioprosthetic valve may be helpful and contribute to surgical decision-making. Bioprosthetic valve thrombosis is not an uncommon complication and can occur several years after surgery . Acute t2); he was registered as a candidate for transplantation before MVR. He was weaned off cardiopulmonary bypass (CPB) with intra-aortic balloon pumping support and was transferred to the intensive care unit (ICU). In the ICU, heparin therapy was started after achieving hemostasis and controlled to prolong the activated partial thromboplastin time to approximately twice that of the control value over days. On postoperative day (POD) 2, he experienced atrial fibrillation and ventricular tachycardia storm and required venoarterial extracorporeal membrane oxygenation (VA-ECMO) support. He was dependent on VA-ECMO (pump speed of 3300 revolutions/min with a blood flow rate of 4.2 L/min) because of extremely low cardiac output. Transthoracic echocardiography (TTE) examination in the ICU revealed pericardial hematoma, decreased cardiac contractility, and poor opening of the aortic valve, but it could not point out the abnormality of the bioprosthetic mitral valve due to poor echocardiographic images. His clinical status did not improve, and he was scheduled for surgical treatment of pericardial hematoma and DuraHeart  LVAD implantation as bridge to transplantation to prevent multi-organ dysfunction on POD 9. Transesophageal echocardiography (TEE) before surgical incision revealed right ventricular collapse. Our initial analysis indicated that the hematoma was a part of the cause of the persistent hemodynamic instability  with symptomatic functional mitral regurgitation underwent on-pump beating-heart mitral valve replacement (MVR) with a 27-mm Epic stented tissue valve . He had a history of dilated-phase hypertrophic cardiomyopathy, with a decreased left ventricular ejection fraction of 16% and a severely dilated left atrium (LA)  and estimates of mitral valve areas using either continuous or pulsed-wave Doppler.Risk factors for prosthetic mitral valve thrombosis have been reported as atrial fibrillation, left atrial dilatation, low cardiac output syndrome, hypercoagulability such as heparin-induced thrombocytopenia, and lack of anticoagulation , 3, 5, 6The possibility of bioprosthetic valve thrombosis must be considered when the patient is completely dependent on VA-ECMO support. A closed aortic valve is a sign of blood stagnation in the left heart and of the possibility of thrombosis. Early TEE monitoring of the bioprosthetic valve would be helpful for intraoperative surgical decision-making and contribute to better patient outcomes.


Tumor occurrence, progression and metastasis depend on the crosstalk between tumor cells and stromal cells and on extrinsic factors outside the tumor microenvironment. Exosomal microRNA (miRNA) not only is involved in communications within the tumor microenvironment but also mediates communications between the extrinsic environment and tumor microenvironment. However, most reviews have been limited to the role of endogenous exosomal miRNA in remodeling the tumor microenvironment. Hence, we herein review the role of endogenous exosomal miRNA in mediating intercellular crosstalk within the tumor microenvironment, inducing the formation of the premetastatic niche. To place our vision outside the microenvironment, we also summarize for the first time the most recent studies regarding how exogenous miRNA derived from milk, plants and microbes influences the tumor microenvironment. Furthermore, to improve the value of exosomal miRNA in cancer research and clinical applications, we also provide some novel ideas for future research based on the comprehensive role of exosomal miRNA in remodeling the tumor microenvironment. Researchers are gaining an improved understanding of tumor evolution, and the role of the tumor microenvironment in tumor occurrence and development has accordingly attracted increasing attention. The tumor microenvironment is composed of various types of cells, including tumor cells and a variety of stromal cells, such as endothelial cells, immune cells, fibroblasts, adipocytes and mesenchymal stem cells. The occurrence, development and metastasis of a tumor depend not only on its own characteristics but also on crosstalk between tumor cells and stromal cells in the tumor microenvironment . In normmicroRNA (miRNA) comprises a class of noncoding RNA of 21–24 nucleotides . A maturBecause exosomes can be detected in almost all bodily fluids and because these vesicles are easily absorbed by recipient cells due to their nanoscale structures, exosomes may be valuable for early diagnosis, curative effect assessment, prognostic analysis and drug target development . This rePubMed was mainly used to search for related articles published using the keyword “exosome,” “miRNA” and “cancer.” Then, screened articles were used as references for this review. Additional keywords, such as “exogenous,” “virus” and “bacteria,” were also used.Various cell-derived endogenous exosomes function via the paracrine or autocrine signaling pathway. The endogenous exosomal miRNA-regulated network connects many types of cells in the microenvironment that can act as the donor or recipient of exosomal miRNA. Through intercellular transfer of exosomal miRNA, tumor parenchymal cells can “educate” stromal cells, and stromal cells in turn regulate the proliferation, apoptosis, migration and invasiveness of tumor cells. A mutual loop may also form between tumor parenchymal and stromal cells.Tumor cells modulate the biological behavior of nearby cells via paracrine and autocrine signaling. Exosomes released from breast cancer MCF-7 cells can be ingested by other MCF-7 cells and promote tumor proliferation in the recipient cells by downregulating expression of the pro-apoptotic protein Bax. In turn, shikonin (SK) inhibits proliferation of MCF-7 breast cancer cells by suppressing exosome release, thus exerting an anticancer effect .Different malignant tumor cells can affect the invasion and migration of tumor cells by releasing exosomal miRNA. Malignant MDA-MB-231 breast cancer cells contain a large abundance of miR-10b, which can be released to the extracellular environment via exosomes and transferred to nonmalignant HMLE breast cells to suppress expression of homeobox D10 (HOXD10) and Kruppel-like factor 4 (KLF4), thereby promoting HMLE cell invasion and migration .Tumors comprise a class of highly heterogeneous diseases. Indeed, a specific tumor is composed of groups of clonal cells that have different growth rates and different sensitivities to chemotherapy and radiotherapy. Thus, tumor cells with different malignancies have different abilities to metastasize and invade. An overall understanding of the heterogeneity of tumor cells and communication among heterogeneous tumor cells via miRNA transfer may help guide clinical treatments.A Cy3-labeled miR-21 mimic harbored by the human esophageal cancer EC9706 cells were found to be transferred to untransfected EC9706 cells via exosomes, and this exosomal miR-21 directly suppressed its target, programmed cell death protein 4 (PDCD4), and activated the JNK signaling pathway to promote migration and invasion of the recipient cells . SimilarIt is well known that tumor cells have a high demand for energy and that a suitable blood supply can help tumor cells acquire oxygen and nutrients and remove tumor cell metabolites, which are closely related to the function of endothelial cells in the tumor microenvironment. The interaction between tumor parenchymal cells and endothelial cells mediated by exosomal miRNA can affect tumor progression.In vitro, human leukemia K562 cells secrete a large number of miR-17/92 clusters  into the culture supernatant. These miRNAs can then be transferred to endothelial cells via exosomes, promoting angiogenesis and endothelial cell migration . Umezu eIn addition, endothelial cell-derived exosomal miRNA also regulates the biological behavior of tumor parenchymal cells. By adding vascular endothelial growth factor (VEGF) and basic fibroblast growth factor to mimic the tumor microenvironment in the culture medium of endothelial cells, exosome release and the level of miR-503 in exosomes was reduced, resulting in decreased transfer of endothelial cell-derived exosomal miR-503 to the tumor cells. Decreased miR-503 resulted in upregulation of cyclin D2 and cyclin D3 to promote tumor cell proliferation. However, an elevated plasma level of miR-503 was observed after neoadjuvant chemotherapy, which may be explained in part by enhanced endothelial cell secretion. This process may augment the efficacy of chemotherapy drugs .The immune microenvironment of the host is closely related to the occurrence and development of tumors. The large numbers of infiltrating immune cells in tumor tissues are among the important components of the tumor microenvironment . The immDendritic cells are professional antigen-presenting cells that efficiently ingest, process and present antigens, playing an important role in the initiation, regulation and maintenance of the immune response . CompareAt the same time, exosomal miRNA secreted by immune cells can be transferred to tumor cells. Hu et al. found that the level and release of exosomal miR-7 was increased after stimulating macrophages with TNF-like weak inducer of apoptosis (TWEAK). Exosomes can also be transferred to epithelial ovarian cancer (EOC) cells, and miR-7 can inhibit the EGFR/AKT/ERK1/2 signaling pathway to block the metastasis of EOC . SimilarA mutual loop mediated by exosomal miRNA may also form between tumor cells and immune cells to promote tumor development. Exosomal miR-21 released by neuroblastoma cells (NBLs) can be ingested by mononuclear cells to elicit NF-κB signaling pathway and can induce mononuclear cells to prevalently differentiate into M2 macrophages. miR-155 in turn released by mononuclear cells can be taken up by neuroblastoma cells, reducing expression of the telomerase inhibitor telomeric repeat-binding factor 1 (TERF1), enhancing the activity of telomerase and promoting drug resistance in tumor cells .Fibroblasts are the major cellular components of the tissue matrix and have the ability to synthesize and secrete extracellular matrix (ECM) proteins and regulate tumor angiogenesis, epithelial–mesenchymal transition (EMT), and tumor metastasis . Normal Pang et al. found that NFs can be transformed into CAFs or cells with a CAF phenotype by co-culturing NFs with pancreatic cancer cells or by treating NFs with pancreatic cancer cell-derived microvesicles (MVs). One possible mechanism for this transformation is that pancreatic cancer cells are able to secrete miR-155-containing exosomes, which can be received by NFs and convert them into CAFs via inhibition of tumor protein p53-inducible nuclear protein 1 (TP53INP1), a target protein of miR-155 . AnotherCancer-associated fibroblasts can help pancreatic cancer cells develop drug resistance to gemcitabine, the main chemotherapeutic drug for pancreatic ductal adenocarcinoma. Gemcitabine treatment not only promotes the release of CAF exosomes but also increases expression of the chemotherapy resistance factor snail and its target miR-146a in exosomes. After exosomes are absorbed by pancreatic cancer epithelial cells, the levels of the transcription factor snail and miR-146a are increased, promoting the resistance of these tumor cells to gemcitabine .As a common social phenomenon worldwide, obesity is known to be associated with certain chronic diseases, but it is also one of the major risk factors for cancer, and it may indicate a bad prognosis in several types of cancer . AdipocyFor instance, Au Yeung and other researchers found that tumor-associated adipocytes express a high abundance of miR-21, which can be transferred to ovarian cancer cells via exosomes to promote their motility and invasiveness. Exosomal miR-21 can directly downregulate the expression of apoptotic protease activating factor 1 (APAF1), a tumor drug resistance and apoptosis-associated protein that promotes drug resistance and aggressiveness in ovarian cancer cells . Mouse pRegardless, the effect of tumor-secreted exosomes on adipocytes has not been reported to date. We look forward to more studies exploring this topic to contribute to our understanding of the crosstalk between tumor cells and adipocytes.Mesenchymal stem cells (MSCs) are collectively derived from the mesoderm and have the ability to migrate to specific tissues, a process also known as homing. MSCs show a double-edged sword effect on tumor development: MSCs can be transferred to the tumor site to promote tumorigenesis and tumor metastasis, but they also inhibit tumor growth. This dual effect may be related to the source of MSCs, the type of tumor cells and the distribution of cytokines in the microenvironment . SimilarMesenchymal stem cells take up tumor cell-derived exosomal miRNA to promote cell-type transformation. Chronic lymphocytic leukemia cell-derived exosomes can be transferred to endothelial cells and MSCs within the tumor microenvironment, and exosomal miRNA and protein can induce an inflammatory phenotype in receiving cells that is similar to the inflammatory CAF phenotype. Therefore, these stromal cells acquire an improved ability to proliferate, migrate, and secrete inflammatory cytokines, generating a more suitable microenvironment for cancer progression .Mesenchymal stem cell-secreted exosomes are ingested by tumor cells and have a complex effect on tumor evolution. As an example, MSC-derived exosomal miR-16 reduces VEGF expression in tumor cells, and as a consequence, tumor angiogenesis is inhibited, which is the first piece of evidence that MSC-derived exosomal miRNA can remodel the tumor microenvironment . In addiIn summary Exosomal miRNA works as an intercellular “messenger” to convert the tumor microenvironment into a pro-tumorigenic environment. Donor cells can selectively package and release specific miRNAs to induce changes in biological activities in recipient cells. On the other hand, several interventions, such as chemical agents, can modify the quantity of exosomes released to exert their antitumor effect.Recent statistical analyses have shown that more than 90% of cancer patients die of tumor metastasis . Metasta6β4 specifically bind to lung-resident fibroblasts and epithelial cells, directing tumor cell metastasis to the lung, and exosomal integrins αvβ5 bind to Kupffer cells, favoring liver tropism, while brain-tropic primary tumor-derived exosomal integrins β3 bind brain-resident endothelial cells, inducing brain metastasis  ligand 2 (CCL2) secretion. IBA1or cells .Tumor cells may enter into a temporary dormant stage after migrating to a new environment. Ono et al. established such a model, and after co-culturing mouse bone marrow-infiltrated breast cancer (BM2) cells with human bone marrow mesenchymal stem cells (BM-MSCs), they found that expression of myristoylated alanine-rich C-kinase substrate(MARCKS), a target gene of miR-23b that is related to cell invasion and cell cycling, was significantly reduced in BM2 cells via transfer of BM-MSC-derived exosomal miR-23b. Thus, breast cancer cells in a metastatic microenvironment exhibit decreased proliferation and migration, with entry into a dormant phase and concomitant reduced sensitivity to many chemotherapeutic agents .Through distal transfer, tumor cell-derived exosomal miRNA educates cells in the premetastatic niche, while tumor cells can also be regulated by stromal cell-released exosomal miRNA in the metastatic microenvironment. In other words, exosomal miRNA orchestrates a fertile “soil” for the “seed” to strike root and sprout or provides protective mechanisms against harmful conditions.Previous studies have shown that miRNAs not only are synthesized endogenously but also can be acquired from food and other exogenous routes, such as milk, herbs and microbial infection .There are large amounts of exosomal miRNAs in bovine milk. These miRNAs are protected from RNase digestion through encapsulation in vesicles and can remain stable under acidic conditions that mimic the gastric intestinal environment . Some stIn addition, viral infection is one of the etiological factors for the occurrence of tumors because insertion of a viral genome into a host’s genome may activate proto-oncogenes or inactivate tumor-suppressor genes, destabilizing the host genome . ThroughAll the instances mentioned above suggest that mammalian cells can ingest exogenous exosomal miRNA that regulates target genes. Predictably, exogenous exosomal miRNA can influence intercellular communication to reprogram the tumor microenvironment. Microbial infection can increase the level of exosomal miRNA in the plasma to influence the growth and angiogenesis of tumors in a mouse model . HoweverAs an important molecule for intercellular communication, an increasing number of studies are focusing on exosomal miRNA-mediated crosstalk among different types of cells in the tumor microenvironment. It is well established that the tumor and its surroundings secrete exosomes that convert the normal tissue microenvironment into a pro-tumorigenic environment through bilateral miRNA exchange. However, the mechanisms by which exosomal miRNA reprograms the tumor microenvironment are not fully understood.As an intercellular signaling molecule, miRNA can exist in many forms: freely, in complex with other proteins or encapsulated in exosome . This fimiRNA and other exosome components, such as various protein, mRNA, and DNA, have an important role in tumor progression . HoweverExosomes can be released into the blood, urine, saliva, cerebrospinal fluid, synovial fluid, pleural effusion and ascites . Therefohttp://www.exocarta.org), Vesiclepedia (http://www.microvesicles.org) and EVpedia (http://evpedia.info) (http://cancergenome.nih.gov/), Arrayexpress (http://www.ebi.ac.uk/arrayexpress/) and other platforms can also be used to download and apply initial data from exosomal miRNA research  . TCGA (hresearch . These pIn addition, due to the insufficiency of pathologic technology for exosome study, it is difficult to quantitate and locate exosomal miRNA in clinical tissue specimens, and accordingly, most of the literature thus far is related to cellular investigation rather than examination of clinical tumor specimens.In conclusion, the remodeling of the tumor microenvironment is dynamic. Whether constructing a better microenvironment for tumor survival or preparing a suitable metastatic microenvironment, endogenous exosomal miRNA-mediated intercellular crosstalk cross is an important component of the regulation process. However, some evidence has also shown show that exogenous miRNA may also play a pivotal role in remodeling the tumor microenvironment, and thus it is a valuable field that merits further exploration in the future.


There is a growing recognition for the need to expand our evidence base for the clinical effectiveness of diagnostic tests. Many international bodies are calling for diagnostic randomized controlled trials to provide the most rigorous evidence of impact to patient health. Although these so-called test-treatment RCTs are very challenging to undertake due to their methodological complexity, they have not been subjected to a systematic appraisal of their methodological quality. The extent to which these trials may be producing biased results therefore remains unknown. We set out to address this issue by conducting a methodological review of published test-treatment trials to determine how often they implement adequate methods to limit bias and safeguard the validity of results.We ascertained all test-treatment RCTs published 2004–2007, indexed in CENTRAL, including RCTs which randomized patients to diagnostic tests and measured patient outcomes after treatment. Tests used for screening, monitoring or prognosis were excluded. We assessed adequacy of sequence generation, allocation concealment and intention-to-treat, appropriateness of primary analyses, blinding and reporting of power calculations, and extracted study characteristics including the primary outcome.One hundred three trials compared 105 control with 119 experimental interventions, and reported 150 primary outcomes. Randomization and allocation concealment were adequate in 57 and 37% of trials. Blinding was uncommon , as was an adequate intention-to-treat analysis (29%). Overall 101 of 103 trials (98%) were at risk of bias, as judged using standard Cochrane criteria.Test-treatment trials are particularly susceptible to attrition and inadequate primary analyses, lack of blinding and under-powering. These weaknesses pose much greater methodological and practical challenges to conducting reliable RCT evaluations of test-treatment strategies than standard treatment interventions. We suggest a cautious approach that first examines whether a test-treatment intervention can accommodate the methodological safeguards necessary to minimize bias, and highlight that test-treatment RCTs require different methods to ensure reliability than standard treatment trials.http://bmcmedresmethodol.biomedcentral.com/articles/10.1186/s12874-016-0286-0.Please see the companion paper to this article:  Diagnostic tests are an essential component of the clinician’s armory for deciding how best to manage their patients. But how should clinicians identify the ‘best’ test to use for a given indication? In an ideal world, such decisions would be guided by large meta-analyses of rigorous clinical effectiveness studies that summarize how competing tests impact on downstream patient health. In reality, there is a serious paucity of this evidence for most diagnostic procedures in use today –7. AcknoHelicobacter pylori and treating those positive with eradication therapy, would effectively reduce their symptoms when compared to the standard approach of giving acid suppression to all dyspeptic patients ) and allocation concealment methods (k = 0.71 [95%CI 0.5–0.8]), and most disagreements concerned conflicting interpretations of whether meagre descriptions should be judged as ‘unclear’ or ‘inadequate’. Agreement was perfect when judging the presence of patient blinding (k = 1.00), near-perfect for outcome assessor blinding (k = 0.90 [95%CI 0.8–1.0]) and substantial for blinding care-providers (k = 0.65 [95%CI 0.3–1.0]); all discrepancies were due to inaccuracies in data extraction and the three disagreements regarding whether care-providers had been masked owed to the misidentification of whether personnel described as blind were treating physicians performing the experimental or comparator test.This tool was piloted on five randomly selected test-treatment trials by three authors  and modified to improve consistency. One reviewer undertook extraction and quality assessment of all trials (LFR) and a second reviewer (JaD) independently extracted and assessed a 64% convenience sample . Disagreements were discussed to reach consensus judgements. If uncertainties remained after discussion, the data were checked by a third member of the team and resolved by discussion. We assessed agreement using kappa for a core subset of categorisations: agreement was substantial for assessing the adequacy of sequence generation  detailed methods of measurement for the primary outcome with adequate detail to replicate, while neither the measurement method nor timing were described by 17 trials (17%). Incomplete reports most commonly omitted the time at which outcomes were measured . Complete accounts of participant flow, reporting numbers who were eligible, allocated, received interventions, followed-up and analyzed (as recommended by CONSORT ) were prMethods of sequence generation and allocation concealment were adequate in 59 (57%) and 38 (37%) trials Table . Only 2 Few trials reported blinding: 5 (5%) trials reported blinding patients, 4 (4%) blinded care-providers and 22 (21%) blinded outcome assessors. Few trials explicitly stated that blinding was not used. The trials that blinded patients gave both experimental and control tests to all patients (4/5) –35 or coInadequate reporting of primary outcomes made ascertaining the identity of the outcome assessor difficult, hence we often deduced this detail implicitly from the outcome type and descriptions of measurement methods. Blind outcome assessments were achieved using independent expert panels in nine trials (9%), clinicians not involved in patient care in six trials (6%), or by independent research assistants in 7 (7%).Most trials  used objective primary outcomes  and standardized measures of health response , and 27% (16/60) of these trials performed blinded evaluations. Subjective primary outcomes were less frequently blinded .Fifty-nine (57%) studies were incomplete due to the exclusion of participants after randomization 32, 31%) and/or missing outcome data . Missing outcome data were adequately explained in 26 of these trials, 11 provided no description and 25 gave partial accounts that were insufficient to determine the reasons for missing data. The number of participants with missing data on the primary outcome in the 59 trials ranged from 0.1 to 46% of randomized participants  and 25 trials excluded more than 10% of the original study population. The proportion of missing data could not be calculated for four trials due to poor reporting. Attrition differed by more than 5% between arms in 21 comparisons made by 16 trials, and in 18 cases (86%) experimental interventions lost the most participants. Attrition differed by more than 20% in only one trial  imputed all missing values, while three others imputed partial responses but excluded wholly missing records. No trial reported using multiple imputation methods.Although 72 (70%) of trials analyzed paitents according to their allocated inteventions, the first requirement for an intention-to-treat (ITT) analysis; only 30 29%) analyzed patients by their assigned groups and had no missing data or imputed missing data, and so comply with the most rigorous definition of intention-to-treat analysis [% analyzeThe majority of trials performed consistent between-arm comparisons 82/103, 80%), either clearly using the same measurement method across all study arms  or assessing test performance outcomes  for which use of different tests is appropriate . For three trials the outcome was measured in different ways between study arms. For example, a trial of patients with suspected scaphoid fracture compared expedited MRI imaging within 5-days of presentation with standard testing   were judged as unfair due to analysis across improper subgroups; typically this entailed measuring the primary outcome in a single diagnostic or treatment subgroup, such as counting the frequency of antibiotic prescription amongst patients diagnosed with non-pneumonic respiratory tract infection (the study target condition) . Since aMore than half  the studies were inadequate in either their use of consistent outcome assessments, analysis of patients by allocated group or avoidance of inappropriate exclusions; only 19 trials (18%) performed adequate primary analyses that minimized all assessed risks of selection bias.Power calculations were reported in 81 (79%) trials. Nearly all related to a specific outcome parameter  which matched the primary outcome in 72% (59/81). The remainder either did not define a primary outcome , powered on a single variable when the study evaluated multiple primary outcomes , or used a different variable to power the study .Study populations ranged from 20 to 5341 participants in individually-randomized trials, and 145 to 972 participants in cluster RCTs. Trials reporting power calculations had considerably larger study samples  than those omitting this description . Trials using patient primary outcomes had slightly larger median study samples  compared to those using process outcomes .n = 2), or significantly different outcome rates to those expected. None provided details of any stopping rules.Of the 79 trials in which a comparison between target and achieved sample sizes could be made , 11 (14%) failed to reach 75% of their estimated targets, including four that recruited less than 50%. Of the 13 trials with a ≥5% deficit, 5 (38%) reported difficulties in recruiting, 4 (31%) were stopped early due to either routine uptake of the new test-treat strategy  recruited by 225 contemporary (2005–6) parallel-group RCTs [Similar reviews have assessed the methodological quality of treatment intervention trials, and we compare our findings for randomization, allocation concealment and blinding with those of an assimilated analysis of 1973 trials of mainly pharmaceutical interventions . In test. 27–45% , 46) medOn the other hand, our findings are broadly consistent with appraisals of complex intervention trials; ITT analyses were found in 24% (vs. 30%), although power calculations were reported considerably less often than by test-treatment trials (28% vs 79%) . Rates oThe low quality of test-treatment RCTs is partly explained by the suboptimal quality observed across all types of RCT , yet theAs with therapeutic complex intervention trials, the scarcity of blinding in test-treatment RCTs almost certainly reflects the practical and ethical difficulties involved in blinding all trial participants  from multiple elements of lengthy care pathways, which may be invasive and are typified by active patient and clinician participation. For the majority of test-treatment comparisons blinding is likely to be impossible, particularly for clinicians who would need to be masked from the test(s) being used, and possibly also from the test result itself. It is difficult to imagine many clinical situations in which this would be ethical or practicable.Then again, the degree to which not blinding exposes test-treatment trials to bias cannot necessarily be directly inferred from what we know of treatment trials. Diagnostic tests are decision-making tools, hence it is possible that blinding itself can have unintended consequences on the validity of trial results, for example from removing the clinician’s role from decision-making. In one of our cohort, patients with suspected pulmonary embolism (PE) received treatment directed either by results of a standard ventilation-perfusion (V/Q) scan, or by an initial triage test  followed by V/Q scan if PE could not be ruled out . InterprWe also provide empirical confirmation that test-treatment trials are particularly susceptible to attrition and lack of power. High rates of missing outcome data could indicate that test-treatment RCTs are at an increased risk of losing participants after randomization, perhaps due to the practical difficulties of maintaining patient compliance throughout numerous interventions, longer study periods and potentially more intensive follow-up regimes. The lack of blinding in these trials may also drive drop-out rates and missing responses, since patients dissatisfied or disillusioned with their diagnostic allocations may have been less motivated to comply with the trial’s follow-up protocol. This was documented by authors of one trial evaluating the benefits of investigating patients with suspected fractures using an MRI scan in addition to the usual X-ray, who reported that patients randomized to the MRI arm were more likely to return their questionnaires than those who knew they had not received the new technology .The lower sample sizes observed were unexpected since test-treatment sample sizes generally need to be substantially larger than is usual for treatment trials as health effects are diluted by the subgroup of patients who would receive the same diagnosis and treatment by both test-treat strategies under evaluation , 23, 51.Often an RCT may be used to assess whether there are reductions in the proportions of patients undergoing inappropriate additional investigations or interventions. We observed instances where results were computed based on the numbers undergoing additional investigations or interventions, rather than the total numbers randomized. This introduces bias as those receiving additional testing and treatment are determined post-randomization and are not based on the same information in both study arms .Despite these practical challenges, there is no theoretical reason why test-treatment RCTs cannot minimize the risks of attrition bias and type II error to a similar degree as standard treatment RCTs. Ensuring that published trials are internally valid, however, will require current methods of critical appraisal to be adapted to reflect the particular requirements of test-treatment interventions.Our study examines the internal validity of a systematically-derived and unrestricted group of test-treatment RCTs measuring patient outcomes. The cohort comprises a diverse range of test-treatment interventions, conducted across a wide range of clinical settings. We have previously shown  that ourTwo factors may have impacted on the reliability of our estimate of trial quality. We only undertook duplicate data extraction for 64% of our sample, however the perfect or near-perfect agreement we found indicates the likelihood of error is small. Lastly, the quality of trial reporting and conduct has improved over the last 15 years , 54 so iThere is a clear need to improve the conduct and reporting of test-treatment RCTS, and guidance for trialists to resolve these issues is urgently needed. Existing RCT quality checklists, such as those provided by NICE  and CochOur review emphasizes methodological weaknesses intrinsic to the RCT design when used to evaluate test-treatment interventions. Minimizing attrition poses a much greater practical difficulty for test-treatment trialists than those undertaking standard trials, while blinding and adequate powering present additional challenges which in many circumstances may be impossible to overcome. These pose considerable obstacles to successfully completing test-treatment trials, particularly when viewed alongside evidence that test-treatment interventions are difficult to capture and translate into trial protocols , and thaWhen an RCT design is used, our findings highlight that standard RCT methods need to be tailored to suit test-treatment interventions. Since blinding clinicians is unlikely to be feasible, the use of more detailed or rigid protocols may serve to limit systematic differences in care provision that occur beyond those generated by using different diagnostic tests for decision-making. Close accounting of clinical behavior, including test use, decision-making and treatment use, will assist investigators to discern between effects due to genuine divergence in test performance from those that reflect artefacts of study design. The noncomparability of outcome measurements between study arms is another unique challenge to test-treatment RCTs that occurs because the intervention we wish to evaluate is itself designed to categorize patients into subgroups. From a clinical perspective it is intuitive to want to know how patients fare between particular treatment categories in order to gauge the appropriateness of decision-making, however the introduction of a third test to ensure comparability of outcomes in all participants may prove practically difficult. Finally, adequate powering requires trialists to inflate estimates based on standard treatment effects by the projected fraction of participants who would receive a difference in diagnosis between test-treatment interventions , leadingWe maintain that protection against bias in RCT studies is not often feasible, and thus these designs should be mandated with care. First, it is essential to examine whether the test-treatment strategies being compared can accommodate the key methodological safeguards, in particular adequate powering, consistent outcome measurement and outcome blinding. In cases where it is impossible to control for bias, smaller scale observational studies and modelling may prove to be more valid. Urgent research is needed to establish: whether blinding is feasible, the impact that not blinding has on the reliability of trial results, and–importantly–the validity of alternative linked-evidence approaches.


Hepatitis E virus (HEV) is a fecal-orally transmitted foodborne viral pathogen that causes acute hepatitis in humans and is responsible for hepatitis E outbreaks worldwide. Since the discovery of HEV as a zoonotic agent, this virus has been isolated from a variety of hosts with an ever-expanding host range. Recently, a subunit HEV vaccine developed for the prevention of human disease was approved in China, but is not yet available to the rest of the world. Meanwhile, notable progress and knowledge has been made and revealed in recent years to better understand HEV biology and infection, including discoveries of quasi-enveloped HEV virions and of a new function of the HEV-ORF3 product. However, the impact of these new findings on the development of a protective vaccine against zoonotic HEV infection requires further discussion. In this review, hallmark characteristics of HEV zoonosis, the history of HEV vaccine development, and recent discoveries in HEV virology are described. Moreover, special attention is focused on quasi-enveloped HEV virions and the potential role of the HEV-ORF3 product as antibody-neutralization target on the surface of quasi-enveloped HEV virions to provide new insights for the future development of improved vaccines against zoonotic HEV infection. Orthohepevirus, family Hepeviridae (Hepatitis E virus (HEV) is a single-stranded, positive-sense RNA virus which belongs to the genus eviridae . HEV vireviridae . In humaeviridae . For a leviridae . Hepatiteviridae . Consequeviridae , cross-seviridae .In recent years, both chronic HEV infection in immunocompromised patients and extrahepatic illnesses caused by HEVs have been documented . These f®) has been approved in China, while all other HEV vaccine programs have been discontinued. However, the most recent HEV discoveries have raised concerns that conventional subunit vaccine designs based on a single protein from a single viral strain might not confer protection against infection by various zoonotic HEV strains. In this review, the historical HEV vaccine pipeline and potential antigenic variation among zoonotic HEV isolates are discussed. In addition, the impact of newly identified quasi-enveloped HEV virions, as well as the potential role of HEV-ORF3 protein in HEV neutralization, is reviewed and perspectives and new insights are discussed.The goal to develop an anti-HEV vaccine can be traced back to soon after the initial discovery of HEV. However, HEV has been less investigated than other hepatic viruses. Consequently, much information has only recently become available to design HEV vaccine, including the discovery of zoonotic infection as well as detection of highly diverse HEV genotypes and newly identified quasi-enveloped HEV particles. Currently, only one HEV vaccine (Hecolincis-reactive elements (CRE) that are essential for viral replication  and Piscihepevirus (only trout HEV isolates) within the family Hepeviridae. It is notable that all four previously known HEV genotypes (1–4) infecting humans are now known to belong to the species Orthohepevirus A  include isolates from various non-human hosts  required for HEV replication. The functional domains within the HEV-ORF1 polyprotein include an RNA capping enzyme domain, a papain-like cysteine protease, a hypervariable region, a macro domain, an RNA helicase domain, and an RNA-dependent RNA polymerase domain (RdRp) . The hypFigure 1). Genetic analysis of ORF2 has demonstrated over 85% similarity among genotypes 1–4 HEV strains that infect humans  were known to be zoonotic except for experimental infections of non-human primates by above hepatic viruses . HoweverThe hallmark of zoonotic HEV research was the identification of swine HEV closely related to the human hepatitis E virus circulating in swine herds in the mid-western United States . SimultaSince the mid-1990s, numerous HEV isolates have been identified from various animal species. The presence of HEV in domestic pig and wild boar populations has been confirmed in both developing and developed countries . MeanwhiStudies have also suggested that humans appear to be at risk of infection from rabbit HEV as well . Other sSubsequently, the identification of swine and rabbit HEV has stimulated further research to investigate the serological prevalence of HEV in other animal species . As a rein vitro models for generation of inactivated vaccine or modified live virus (MLV) vaccines is not reported yet.Cell culture systems have greatly facilitated development of numerous vaccines. However, until now available cell culture systems for HEV have not been robust, creating a major obstacle that has impeded HEV research and vaccine development. One culture system using S10-3 cells, a subclone of Huh7 hepatoma cells, supported improved replication efficiency of genotype 1 HEV strain Sar-55 above that of its Huh7 parental cell line. However, this system relies on the transfection of full-length HEV RNA . AlternaFortunately, the capsid protein of HEV shares over 85% identity with all four major HEV genotypes infecting human or other mammalian hosts. Therefore, ORF2 is a good HEV subunit vaccine candidate  and promE. coli. However, this recombinant vaccine only worked in rhesus macaques immunized with three 100 μg doses in Freund’s adjuvant and was poorly immunogenic in mice when administered with alum adjuvant  as the first recombinant protein HEV vaccine . Prelimiadjuvant . Next, tadjuvant ,b. Afterredicted .50) of homogenous genotype 1 strain, resulting in only one observed case of viral shedding  forms VLPs , 2005c. shedding . Anothershedding . Unlike shedding . By contshedding . It is sshedding . MeanwhiV strain . As a reV strain . TherefoE. coli  which is based on an Indian genotype 1 strain , which was finally approved and commercialized in China, is a truncated protein (aa 368–606 of ORF2) expressed in E. coli  is likely only a subset of potential cases at present.As mentioned above, genotype 1 HEV is restricted to humans and thus has no known animal reservoirs . Therefo®. However, it remains unknown whether or not Hecolin® would provide full protection against all human or animal zoonotic HEV infections and therefore prevent foodborne zoonotic HEV transmission. Indeed, several studies have demonstrated that Hecolin® vaccine could protect rabbits against genotype 4 HEV challenge (Because most reported HEV cases in recent years have been caused by zoonotic HEV (genotypes 3 and 4), for maximum protection an effective HEV vaccine should protect recipients against zoonotic HEV rather than against human HEV only (genotypes 1 and 2) . The highallenge .in vitro (Orthohepevirus B (avian HEV) derived ORF2 peptide (aa 389–399) recognized only human HEV-ORF2 protein (® (p239) and another truncated p179 protein (based on ORF2 of genotype 4 HEV) showed that Hecolin®-induced IgGs produced in mice and humans reacted strongly with genotypes 1 and 2 recombinant ORF2 proteins. However, p179-induced IgGs from mice exhibited stronger reactivity to ORF2 proteins of genotypes 3 and 4 rather than for genotypes 1 and 2 ORF2 products . In this example, neutralizing antibodies to the vaccine strain of MLV were unable to both neutralize virus and did not achieve protection against heterogeneous strains that share homology of as high as 86% with the vaccine strain . Taken tAs mentioned above, HEV has traditionally been classified solely as a non-enveloped virus. The presence or absence of an envelope has traditionally been used to classify viruses. This distinction was based on the sensitivity of enveloped virus infectivity to bile salts exposure, which removes the envelope lipid layer on the outside viral surface and abrogates infectivity . GeneralVirus envelopes confer both advantages and disadvantages. On the one hand, an envelope can facilitate virus release and trafficking out of cells, while release of non-enveloped viruses relies on lysis of infected cells . On the The HAV was the first non-enveloped virus later confirmed to possess an enveloped form derived from the host cell membrane . The bioFigure 2) suggested that quasi-enveloped virions which were unable to bind antibodies against the capsid protein , the C-terminus of pORF3 is not highly conserved  is also highly immunogenic, with most of its epitopes located at the C-terminus . Soon afonserved . Meanwhionserved , which sMore than two decades have passed since the discovery of HEV and the characterization of its genome. In spite of numerous efforts during that time, our understanding of HEV still remains limited. Nevertheless, ongoing research continues to uncover new information regarding its biology and pathogenesis. However, as a further complication, HEV is no longer viewed as a public health concern only in developing countries, but is now an increasing concern in developed countries due to its complex mechanisms of transmission.®, a prophylactic vaccine based solely on the capsid protein of a genotype 1 HEV isolate has been approved in China. However, it is still uncertain whether this vaccine will offer protection against zoonotic HEVs in humans or domestic animals due to new knowledge of cross-species transmission and zoonotic HEV host tropism. Moreover, recently discovered quasi-enveloped HEV particles may partially explain why vaccines targeting capsids have not worked, while also reshaping our understanding of HEV biology and vaccine design. Fortunately, ongoing research is revealing more and more valuable information regarding pORF3 function and is generating optimism that pORF3 could serve as a new target of vaccine design against quasi-enveloped HEV particles.HecolinAcross HEV genotypes, ORF3 sequence homology is lower than for ORF2, especially within the immunogenic domain of approximately 66 aa within the ORF3 C-terminus. It is hoped that epitope scanning and analysis may soon help to identify major neutralizing epitopes against quasi-enveloped HEV virions. Subsequently, if neutralizing epitopes from different HEV genotypes could be identified, full-length pORF3 may not be needed as an immunogen. Instead, a combination of peptides containing neutralizing epitopes from various HEV genotypes could be explored as a novel peptide vaccine. Such a vaccine might serve as a booster shot for the current pORF2-based subunit vaccine to ultimately provide better protection than from the pORF2 vaccine alone. Using these new strategies, it is hopeful that the next generation of HEV vaccines will finally achieve solid cross-protection against zoonotic HEV.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.Mentioning of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.