O-acetylated imparting important physiochemical properties to the polymers. The position and degree of O-acetylation is genetically determined and varies between organisms, cell types, and developmental stages. Despite the importance of wall polysaccharide O-acetylation, only recently progress has been made to elucidate the molecular mechanism of O-acetylation. In plants, three protein families are involved in the transfer of the acetyl substituents to the various polysaccharides. In other organisms, this mechanism seems to be conserved, although the number of required components varies. In this review, we provide an update on the latest advances on plant polysaccharide O-acetylation and related information from other wall polysaccharide O-acetylating organisms such as bacteria and fungi. The biotechnological impact of understanding wall polysaccharide O-acetylation ranges from the design of novel drugs against human pathogenic bacteria to the development of improved lignocellulosic feedstocks for biofuel production.The extracellular matrix of plants, algae, bacteria, fungi, and some archaea consist of a semipermeable composite containing polysaccharides. Many of these polysaccharides are  The biomass of plants contains considerable amounts of esterified acetate. For example, poplar wood contains 5% of its weight as acetate , while cO-linked acetate on many wall polysaccharides (references below), and to a minor extent on the polyphenol lignin . One of these protein families is the TRICHOME-BIREFRINGENCE-LIKE (TBL) protein family comprising 46 members in the model species Arabidopsis thaliana. Members of the TBL family have been shown to participate in the O-acetylation of specific wall polymers. Loss-of-function of the Arabidopsis ALTERED XYLOGLUCAN 4 (AXY4/TBL27) gene results in a complete lack of O-acetyl substituents on the hemicellulose XyG without affecting the acetylation status of the other wall polymers (AXY4-like (AXY4L/TBL22) – appears to have the same function but specifically in seeds, indicating that AXY4 and AXY4L are XyG-specific acetyltransferases, although the biochemical activity of both proteins remains to be experimentally demonstrated.The identification of plant mutants affected in the polymers . Its partbl29/eskimo1 mutant that was shown to reduce xylan O-acetylation by 46% in the stem (O-acetyl groups to β-(1→4) xylooligosaccharides in vitro thus confirming its role as a xylan O-acetyltransferase . OsXOAT1-7 are able to complement the defects in xylan O-acetylation of the Arabidopsis esk1/tbl29 mutant  (TRICHOME-BIREFRINGENCE-LIKE proteins contain three characteristic protein signatures (igure 1) . A N-terigure 1) .O-acetylation is represented by ALTERED XYLOGLUCAN 9 . Arabidopsis mutants affected in AXY9 expression show a strong reduction in total wall O-acetylation in stems and leaf tissues  suggesting that it could also be an O-acetyltransferase although if this protein harbors any enzyme activity has yet to be determined.A second family of proteins involved in polysaccharide  tissues . Interesproteins . The AXYO-ACETYLATION (RWA) is the third group of proteins involved in plant polysaccharide O-acetylation . The Arabidopsis genome contains four RWA proteins required for O-acetylation of both pectic and non-pectic polysaccharides including xyloglucan, xylan, and mannan. Quadruple rwa mutant plants exhibit a 63% reduction in total wall O-acetylation (RWA genes found in hybrid aspen (Populus tremula x tremuloides) results in reduced wood xylan and xyloglucan O-acetylation, suggesting that RWA function is conserved among plant species . Although no experimental evidence has been reported yet, the existence of intermediary acetyl donor(s) is a likely option  polymer. This heteropolymer is the main component of the bacterial wall, and consists of alternating N-acetylglucosaminyl--N-acetylmuramoyl residues cross-linked with stem peptides. PG O-acetylation can occur in 20–70% of the MurNAc residues, depending on the species and growth conditions and provides protection against lytic enzymes such as lysozyme . Hence, Gram-positive bacteria seem to be simultaneously translocating the acetyl groups from a cytoplasmic source and O-acetylating the N-acetylmuramoyl residues in the extracellular PG polysaccharide using a single bimodular protein. Several OatA homologs have been identified and characterized but only recently the crystal structure of the C-terminal domain of OatA has been resolved and point mutations in the DxxH and GDS motifs demonstrated that these amino acids are essential for catalyzing O-acetylation of PG in Streptococcus pneumoniae and Staphylococcus aureus . Although its activity has not been determined experimentally, the human HsCasD1 protein, showing high similarity and structure to CnCas1p, has been demonstrated to be essential and sufficient for O-acetylation of sialic acids, a family of nine-carbon monosaccharides typically found capping the glycan chains attached to cell surface glycoproteins and glycolipids in mammals including humans . A multitransmembrane protein might translocate the acetyl moieties from a cytoplasmic source into the periplasm, where one or more plasma membrane-anchored proteins containing a SGNH/GDSL motif facing the periplasm might transfer the acetyl-moiety to the polysaccharide . This two-component mechanism involves the coordinated expression of multiple components arranged in operons. A model was originally proposed based on the O-acetylation of alginate, a linear exopolysaccharide consisting of 1-4-linked L-mannuronyl and D-glucuronyl residues present in Pseudomonas aeruginosa    . In thisin vitro . Accordiigure 1) . In thesO-acetylating systems, proteins such as OatA, RWA2 or AlgI share very limited sequence homology with PatA. For example, SaOatA and NgPatA share only 15.1% sequence identity and 23.6% similarity. A similar situation occurs when comparing the O-acetyltransferase domain of plant TBL or Gram-positive bacterial OatA proteins with the Gram-negative AlgX or PatB proteins. For example, there is only 15.4% identity and 18.3% similarity between the globular domain of SaOatA and HgPatB . On the one hand, a bimodal OatA homolog has been characterized exhibiting a mechanism as described above, involving the simultaneous translocation of acetyl groups and PG O-acetylation . As plants contain multiple wall polymers an expansion and increased diversification of the TBL protein family might have become necessary. Interestingly, although plant RWA proteins belong to the same sugar acyltransferase superfamily containing 10 transmembrane domains as bacterial OatAs, CnCas1p, and HsCasD1, they lack the globular O-acetyltransferase domain, indicating that plants need the additional involvement of other components such as members of the TBL family and/or AXY9 in order to O-acetylate their wall polysaccharides. Accordingly, the globular domain of OatA or CnCas1p proteins contains the GDS and DxxH motifs similar to plant TBLs and AXY9 hinting their analogous functions. A similar separate, multiple component mechanism was also developed by Gram-negative bacteria in order to O-acetylate extracellular polysaccharides, albeit likely arising through convergent evolution. The development of a multiple component system in these bacteria could reflect again a more complex wall with a variety of extracellular O-acetylated polysaccharides. In these bacteria, an increased diversification of the O-acetyltransferases is also observed .Gram-positive bacteria, fungi, and mammals developed a one component machinery to O-acetylation of plant wall polysaccharides can be found in vascular plants but also in pteridophytes and bryophytes, including hornworts, mosses, and liverworts . A sequence comparison of nine representative embryophytic species showed that AXY9, TBL29, and RWA2 proteins seems to be highly conserved in dicots , monocots (Oryza sativa) and gymnosperms (Pinus radiata) sharing identities higher than 50% and similarities around 75% with the Arabidopsis representatives. Primitive plants such as Equisetum hyemale, liverworts (Marchantia polymorpha), hornworts (Phaeoceros carolinianus) and mosses (Physcomitrella patens) also contain highly conserved sequences sharing identity and similarity values around 40% and 60%, respectively.All three families of proteins involved in Volvox aureus or Nephroselmis pyriformis) and Charophyta  divisions. However, algae seem not to encode proteins with sequence similarity to AXY9 or TBL29. Since algal RWA orthologs do not contain a GxxH and/or GSD domain required for polysaccharide O-acetylation algae might harbor additional, hitherto unidentified proteins that would be necessary for O-acetylation to occur. These results indicate that RWA proteins emerged earlier than AXY9 and the TBLs and suggest that green algae may also use a polysaccharide O-acetylation system based on RWA. The walls of several Chlorophyta and Charophyta species have been reported to contain plant-type wall polysaccharides such as xylan, mannans or XyG -like phenotypes of Arabidopsis axy4 and axy4L knockout mutants demonstrate. Reinforcing this notion, a natural Ty-0 Arabidopsis accession displays an almost complete lack of XyG O-acetylation without detrimental plant morphological and developmental side-effects when grown in its native environment in the Highlands of Scotland  the glucan-backbone of XyG is partially O-acetylated  loss of function increases stem diameter and activates the development of larger tracheary elements. As a consequence, kak mutations are able to recover almost completely from tbl29/esk1-associated dwarfism without affecting wall acetate content. Although KAK has been described previously as an endoreduplication repressor affecting trichome morphology, the mechanism how it regulates vascular development is not known  synthesis in tbl29/esk1 plants  is able to completely suppress both developmental defects and increased freezing tolerance without affecting the reduced acetate content  to wall polysaccharides instead of sugars (C5-C6) could have evolved as a more energetically favorable strategy as described above for XyG. Other TBL proteins than TBL29/ESK1 participate in the regiospecific O-acetylation of xylan glucuronic acid (methyl-GlcA) residues by xylan glucuronosyltransferases termed GUX. Together, these decorations have been found to be important for xylan-cellulose binding . Actuallitutions . In a tb fibrils . Intrigurestored . GUX1 isof xylan , suggestO-acetylation of pectic polysaccharides has also been associated with plant signaling processes. rwa2 mutant alleles show increased resistance to the necrotrophic fungal pathogen Botrytis cinerea accompanied by leaf surface defects including trichome collapse, enhanced leave permeability and altered cuticle formation  remain to be discovered, albeit it is likely that members of the TBL family are involved. The identification and characterization of such proteins is not only needed to understand the wall O-acetylation mechanism of particular wall polysaccharides, but also to gain insights into the function of the O-acetyl substituent on this polymer. Fourth, the recent advent of identifying the genes responsible for polysaccharide O-acetylation and their genetic manipulation in vivo lead to the discovery of intriguing function of this substituent. However, at this stage the phenotypic results are rather descriptive and additional research in the future is required to ascertain causal relationships as well as mechanistic insights into polymer interactions, cellular sensing and responses.There are still open questions regarding the mechanism of polysaccharide proteins . Recent transfer . The exiMP and VR designed and wrote the 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.


Knowledge about the health effects of e-cigarette use (or vaping) among past and current combustible cigarette users is limited. Several studies have assessed vaping-related adverse events (AEs) and beneficial health effects, however, most studies focused on AEs in general and examined only a few physiological changes that vapers experience. This study aims to explore self-reported AEs and perceived health changes due to e-cigarette use among Hungarian adult e-cigarette-only users (former smokers who switched completely to e-cigarette use) and dual users (smokers who use e-cigarettes and combustible tobacco cigarettes concomitantly).A cross-sectional, web-based survey of 1042 adult Hungarian e-cigarette users was conducted in 2015. Participants reported AEs and changes in physiological functions since they switched from smoking to e-cigarette use or while dually using e-cigarettes and combustible cigarettes. Confirmatory factor analysis with covariates was applied to explain perceived health changes due to e-cigarette-only use and dual use.p < 0.001). Experiencing health improvements were significantly more likely among e-cigarette-only users than for dual users for all surveyed physiological functions. E-cigarette-only users reported larger effects of vaping on sensory, physical functioning, and mental health factors compared to dual users. Self-reported changes in sensory and physical functioning were significantly higher among individuals using e-cigarettes more than a year and people who were past heavy smokers (smoked ≥20 cigarettes per day). Gender was related to sensory improvement only; males reported greater improvement than females.Dual users (17.6%) were significantly more likely to report AEs of vaping than e-cigarette-only users  contains supplementary material, which is available to authorized users. Electronic cigarettes (e-cigarettes) are rapidly evolving products with a wide range of design and engineering variations. The basic function of all e-cigarettes is to generate a heated aerosol typically containing nicotine, that is inhaled through a mouthpiece , 2. SomeThere is an ongoing debate on the potential harms or benefits of e-cigarettes in the public health community. Advocates of e-cigarettes emphasize the harm reduction potential of these products as substitutes for combustible tobacco among persons trying to quit or reducing the amount of cigarette smoking . However, dual use will undermine the potential harm reduction due to the known short- and long-term health effects of smoking . On the Despite the debate and growing perceptions that e-cigarettes are harmful, experimentation with e-cigarettes is increasing in the European Union . In HungCurrent knowledge about both self-reported and objectively measured health effects of e-cigarette use among past combustible cigarette users is limited. Among the scant literature, most health effects were measured by self-report rather than objectively measured. In recent years, several studies have assessed vaping-related adverse events (AEs) and beneficial health effects. However, most studies focused on AEs in general and consistently examined only a few physiological changes that e-cigarette users experience. Of studies indicating no conflicts of interest regarding support from e-cigarette manufacturers or lobby, 84.2% of Dutch vapers reported health improvements since e-cigarette use initiation , while 8This study aims to explore self-reported health effects of vaping among Hungarian adult e-cigarette-only users (former smokers who switched completely to e-cigarettes) and dual users (smokers who use e-cigarettes and combustible tobacco cigarettes concomitantly). Our objectives were (1) to describe characteristics of Hungarian adult e-cigarette-only and dual users, (2) to assess self-reported AEs and changes in physiological functions after e-cigarette use initiation among e-cigarette-only and dual users, and (3) to explain perceived health changes due to e-cigarette use.www.ecigiforum.com, www.ecigiblog.blog.hu, www.ecigaretta.eu) and an e-cigarette webshop (www.sbcig.com) inviting website visitors to participate. After reading the description of the study, participants consented to participate by voluntarily answering the survey questions. A lottery-based incentive was offered after two months to increase participation. 800 participants completed the survey anonymously without the incentive, and 784 completed the survey after the incentive was introduced, indicating their e-mail address to participate in the lottery. The study was approved by the Institutional Review Board of Semmelweis University, Budapest, Hungary.A cross-sectional online survey was conducted among adult (age 18+) Hungarian e-cigarette users in September–December, 2015. The convenience sample was obtained by posting the survey on Hungarian e-cigarette forum websites (n = 4); had never smoked (n = 22); did not respond whether they used e-cigarette and/or combustible cigarette (n = 63); and responded inconsistently to questions assessing e-cigarette-only and dual use (n = 40). Since we did not have access to respondents’ internet protocol (IP) address to exclude multiple responses from the same participants, we searched for duplicate cases (n = 413) using all socio-demographic variables . We applied an a priori decision rule that only the first case of potential duplicate respondents was included in the final analytical sample. As a result, 1042 unique respondents who ever smoked and were current e-cigarette users  were included in the study.Of the 1584 initial respondents, we excluded the followings: those who were < 18-year-old (The questionnaire consisted of seven parts: 1) socio-demographic characteristics of respondents, (2) e-cigarette use, (3) perceived harm of e-cigarette use, (4) combustible cigarette use, (5) oral hygiene, (6) vaping-related adverse events, and (7) changes in physiological functions , and education .E-cigarette-only versus dual use was assessed by a question “Do you use e-cigarette or combustible cigarette?” . Only persons who were e-cigarette-only users and dual users were included in the study.Past combustible cigarette use was measured by the number of tobacco cigarettes smoked per day before initiating e-cigarette use. Response options were categorized into: ≤10 CPD – light smoker, 11–19 CPD – moderate smoker, ≥20 CPD – heavy smoker.Current e-cigarette use characteristics variables included in this study were (1) time since respondent started using e-cigarettes , (2) frequency of e-cigarette use per day , and (3) nicotine concentration of the e-liquid (0 mg/ml – 18 mg/ml or more) (Table udy were  time sinVaping-related adverse events were assessed in a question where participants could indicate (yes/no) if they had experienced any of 14 AEs . ExplorCompared to dual users 17.6%), e-cigarette-only users (82.4%) were more likely to be male and started using e-cigarettes more than two years prior to the survey . The majority of respondents reported ≤2 AEs (94.0%). The most common AE was cough among dual users and sore/dry mouth and throat among e-cigarette-only users  followed by a confirmatory factor analysis (CFA) to estimate the measurement model of improvement in health. We supported three main factors of physiological functions Fig. . The detOur study found that e-cigarette-only users report more perceived improvement in health and less adverse health effects of vaping than dual users. The majority of e-cigarette-only users reported better respiratory and sensory functions and indicated only minor AEs since they started using e-cigarettes. However, dual users were probably under-represented while satisfied e-cigarette-only users were over-represented, therefore, our results may overestimate perceived benefits of vaping , 23. FurImproved olfactory and gustatory functions, both sensory improvements, were frequently reported benefits of vaping in both independent and sponsored studies , 17, 23.Improved breathing, general physical status and stamina were correlated with past combustible cigarette smoking habits and e-cigarette use duration. The most commonly reported improvement among these physiological functions was breathing in our whole sample, consistent with studies of Dutch vapers, and a large international sample of a sponsored study involving more than 19,000 e-cigarette users where almost 90% of respondents reported better breathing due to vaping , 17, 23.E-cigarette-only users experienced significantly greater improvements in mental health. They reported greater improvements in their mood and quality of sleep compared to a large international study  and a smPositive changes in memory were reported by a minority of our sample similarly to another study . A previSexual performance has rarely been investigated as it pertains to e-cigarette use. Approximately one third of e-cigarette-only users in our sample agreed that their sexual performance improved since they initiated vaping, similarly to an international study which found 28.9% improvement rate . This asAlmost two-thirds of dual users and more than half of e-cigarette-only users did not report improved appetite consistent with a few other studies detecting greater improvement in appetite among switchers compared to dual users , 23. It In our analysis, we also identified three main dimensions of perceived health improvements attributed to e-cigarette use. Sensory improvement included improved smell and taste. The improvement in physical functioning encompassed improved breathing, physical well-being and improved stamina. Finally, the mental health improvement factor incorporated the improved appetite, sexual life, mood, memory and sleeping. We also noted that dual users reported much lower degree of improvement in all three dimensions than e-cigarette-only users. This result highlights that greater perceived health improvements are related with the complete cessation of regular cigarette use. Longer duration of e-cigarette use was associated with higher score on sensory improvement and physical functioning. Interestingly, intensity of smoking before e-cigarette use was also associated with greater improvement in sensory and physical functioning dimensions suggesting that heavy smokers compared to light smokers may gain much more from switching to e-cigarette from combustible cigarette. Furthermore, our study provides a useful self-report tool to measure perceived health effects due to vaping for future studies.Regarding AEs, mouth and throat, respiratory and neurological symptoms were the most frequently reported by a minority of Hungarian vapers parallel with other studies indicating similar side-effect patterns , 23, 27.Among AEs, cough was more frequently reported especially by dual users than breathing difficulties. However, cough was less commonly mentioned in our sample compared to previous studies reporting frequencies between 12.8–69.0% , 20, 28.This study provides more insights to patterns of perceived beneficial and adverse health effects of vaping, however, limitations exist. First, self-reported data are prone to recall and social desirability bias, particularly past tobacco smoking habits and experiencing AEs and physiological changes. Second, individuals with more positive perceptions and experiences of vaping may have been more motivated to participate in the survey leading to respondent bias. Third, the cross-sectional design and convenience sample limit causal inference. Fourth, we cannot separate the impact of positive expectancies for vaping or a placebo effect from improvements due to reducing combustible cigarette use or quitting smoking. Furthermore, we cannot exclude also the possibilities of a general response tendency toward the improvement in functions in self-report due to cognitive dissonance or other self-servicing biases. The possibility of these effects is reflected in relatively high correlations between factors of perceived health improvement. Sixth, like prior research, we did not compare perceived health impacts of switching to e-cigarettes to complete tobacco use abstinence which would be the optimal strategy to reduce tobacco-induced harm. We also do not have data on the long-term stability of the perceived benefits. Seventh, perceived improvement in health is influenced by several factors that we may did not measure which can explain the relatively low explained variance of health improvement factors. Further research should investigate those factors that can influence how e-cigarette users perceive and report health benefits from their product use. Finally, this study similar to others, is based on a convenience sample of users, therefore the generalization of results is limited, however having a representative sample of e-cigarette users is difficult to define, and rarely applied in e-cigarette research. Furthermore, the pattern of e-cigarette use is continuously changing which may also limit the generalizability of our results.In conclusion, our study supports that the majority of e-cigarette-only users report more perceived beneficial changes in physiological functions while less AEs than dual users. However, perceived short-term benefits of e-cigarette use may reinforce users despite the uncertainty of long-term health consequences. Further research needs to determine the role of placebo effects and expectancies on objectively measured health improvements associated with switching from combustible cigarettes to e-cigarettes. Health professionals should provide balanced information regarding the possible short- and long-term health effects of e-cigarettes during consultations with patients. E-cigarettes are not without negative health consequences as numerous minor and some major adverse events have been reported previously and in our study . FurtherAdditional file 1:Survey questionnaire. An English language translation of the survey used to study the perceived health effects of vaping among Hungarian adult e-cigarette-only and dual users. (XLSX 14 kb)Additional file 2:Psychometric analysis. Exploratory factor analysis followed by a confirmatory factor analysis to estimate the measurement model of improvement in health. (DOCX 17 kb)


Mechanistically, Gal-3 enhances actin assembly and reduces Erk 1/2 activation, leading to early OLG branching. Gal-3 later induces Akt activation and increases MBP expression, promoting gelsolin release and actin disassembly and thus regulating OLG final differentiation. Altogether, findings indicate that Gal-3 mediates the glial crosstalk driving OLG differentiation and (re)myelination and may be regarded as a target in the design of future therapies for a variety of demyelinating diseases.Galectin-3  is the only chimeric protein in the galectin family. Gal-3 structure comprises unusual tandem repeats of proline and glycine-rich short stretches bound to a carbohydrate-recognition domain (CRD). The present review summarizes Gal-3 functions in the extracellular and intracellular space, its regulation and its internalization and secretion, with a focus on the current knowledge of Gal-3 role in central nervous system (CNS) health and disease, particularly oligodendrocyte (OLG) differentiation, myelination and remyelination in experimental models of multiple sclerosis (MS). During myelination, microglia-expressed Gal-3 promotes OLG differentiation by binding glycoconjugates present only on the cell surface of OLG precursor cells (OPC). During remyelination, microglia-expressed Gal-3 favors an M2 microglial phenotype, hence fostering myelin debris phagocytosis through TREM-2b phagocytic receptor and OLG differentiation. Gal-3 is necessary for myelin integrity and function, as evidenced by myelin ultrastructural and behavioral studies from  N-acetyllactosamine and, despite lacking specific receptors, form multivalent complexes with cell surface glycoconjugates containing suitable oligosaccharides and trigger intracellular signals to regulate cell survival and differentiation  constitute a 15-member family of β-galactoside-binding lectins which recognize ntiation . Among Gntiation . In contIn this context, the present review firstly summarizes Gal-3 structure, location in cells and tissues, extracellular and intracellular space functions, regulation, internalization and secretion. The manuscript then focuses on the current knowledge of Gal-3 role in central nervous system (CNS) health and disease, particularly regarding oligodendrocyte (OLG) differentiation, myelination and remyelination, and its potential as a target in the design of future therapies for a variety of demyelinating diseases.6 and Ser12, important for nuclear localization and oligomerization; (b) a proline and glycine-rich collagen-like sequence susceptible to metalloprotease (MMP) cleavage; and (c) a COOH terminal domain containing the CRD and the NWGR anti-death motif, highly conserved within the BH1 domain of the Bcl-2 protein family  and bind to β-galactoside-containing glycoconjugates. Gals are classified into three groups on the basis of their structural architecture: proto, chimera and tandem types, with Gal-3 being the only representative of the chimeric type . Gal-3 hn family . In the tosamine .in vivo by astrocytes in the subventricular zone (SVZ), being indispensable for cytoarchitecture maintenance but dispensable for apoptosis and proliferation . It is often incorporated to the ECM to modulate cell adhesion or interact with plasmatic membrane receptors by binding to carbohydrate moieties in an autocrine or paracrine fashion to form membrane lattices and trigger intracellular events. Gal-3 impact on cell adhesion and migration depends on its multivalent capacity, as it functions as a nexus between ECM and membrane receptors and can thus inhibit or potentiate these cell properties. Gal-3 binds laminin, hensin, elastin, collagen IV, tenascin-C and -R, and crosslinks with integrins, the major cell adhesion receptor modulators .Having introduced the main features of Gal-3, the present review will now focus on the current knowledge of Gal-3 role in CNS myelination, remyelination and OLG differentiation  and mature  OLG isolated from primary rat cultures, with higher levels in mature ones . Recent LGALS3-/- microglia, promotes OLG differentiation, suggesting that microglia express the Gal-3 necessary to induce OLG maturation. In addition, using neurosphere cultures, our group has also proven Gal-3 to promote cell commitment toward the OLG lineage , 10 (P10), 15 (P15), and 20 (P20) using transgenic mice expressing the enhanced green fluorescent protein (EGFP) driven by the promoter of oligodendroglial protein CNPase (CNP–EGFP). Gal-3 expression showed substantial changes during white matter development, with high expression levels at P5 and a reduction upon myelin development. In agreement, high levels of Gal-3 were found at P5 in microglial cells localized in the CC and cingulum, fairly close to CNPase+ cells. Interestingly, confocal microscopy showed some CNPase+ cells with maturen OLG-like morphology to colocalize with Gal-3. On the other hand, Gal-3 was detected at low levels in astrocytes at P10 and P15, and at high levels in the SVZ at all ages evaluated. This work also revealed a critical role for Gal-3 during OLG myelination, as reflected by the morphological changes observed in the myelin of LGALS3-/- mice. These animals presented hypomyelination in the striatum and CC, as revealed by electron microscopic morphometric analysis. These myelin defects were deeper in 8-week-old relative to 4-week-old mice. Also, myelin showed lesser integrity and abnormal compaction compared to WT mice. Furthermore, LGALS3-/- mice exhibited substantial alterations in behavior with diminished levels of innate anxiety than their WT littermates  signaling , advanced glycation end-products (AGE) are a source of inflammation and oxidative injury. Gal-3 acts as an AGE receptor and leads them to lysosome degradation and removal . In lineOligodendroglial injury leads to demyelination, which is followed by a regenerative response consisting in the formation of new myelin sheaths –a process called remyelination . This prSera from patients with secondary progressive MS has been recently shown to present auto-antibodies against Gal-3, which may be responsible for blood brain barrier progressive damage . These aLGALS3 gene upregulation only in primary progressive MS CFS-treated OPC and establishes a link between Gal-3 upregulation and increased OPC branching. Gal-3 upregulation has also been observed in post-mortem human brain tissues with primary progressive MS.Further work has described the effect of cerebrospinal fluid (CSF) from patients with primary progressive MS or relapsing remitting MS in the morphology and transcriptional response of treated OPC . The resTaken together, these findings indicate that Gal-3 is a positive target for OLG differentiation in human MS tissue and drives the downregulation of ICAM-1 in BMEC, mediating a protective effect on the blood–brain barrier. However, the presence of anti-Gal-3 auto-antibodies is responsible for blood brain barrier damage, a negative effect which might be counteracted through neutralizating therapy.Current evidence has proposed exosomes as possible biomarkers and therapeutic agents in CNS disease. Most importantly, as therapeutic agents, exosomes display some advantages, as they can deliver cargo to other cells, rapidly pass the brain–blood barrier and render low immunogenicity . As desc-/-LGALS3 mice display clearly diminished CNS macrophage infiltration during EAE, which leads to lesser disease severity , in which mice are immunized with myelin oligodendroglial glycoprotein. EAE constitutes the most widely exploited model and is especially useful to study the autoimmunity aspects in MS pathology. However, numerous therapeutic agents displaying beneficial effects in this model have proven poorly or not beneficial at all in the treatment of MS. severity . These fseverity .Virus-induced demyelination models support the hypothesis that some environmental factors, such as viral infections, are involved in MS and may be actually triggering the disease. MS-induced inflammation may decrease SVZ cell proliferation and thus hinder repair. Gal-3 expression increases in active human MS lesions , in periAnother increasingly used and more recently described demyelination model consists in the administration of cuprizone (CPZ) as part of the diet of young adult mice, which produces massive demyelination through pathogenic T cell-independent mechanisms, with different areas specifically affected in the CNS such as the CC . CPZ-indPrevious studies have described four different lesion subtypes in MS: pattern-1 and -2 lesions are thought to be autoimmune response-mediated, while pattern-3 and -4 lesions are regarded as primary oligodendrogliapathy . The firLGALS3-/- microglia is usually deficient  expression and TREM-2b upregulation during CPZ-induced demyelination, unlike CPZ-treated LGALS3-/- mice, which display more numerous microglia with activated caspase-3. These findings support Gal-3 as a modulator of the microglial response to favor the onset of remyelination and OLG differentiation , or Nasu–Hakola disease , which pLGALS3-/- mice submitted to CPZ-induced demyelination exhibit higher microglial proliferation and apoptosis, as evidenced by their increased activation of caspase-3, which may respond to the well-established Gal-3 anti-apoptotic role  could be involved in MS pathology, as its neutralization with anti-CCL2 significantly reduces EAE relapsing severity . Our resand CCL2 . Taken tlination , and micin vitro myelination through glial cell communication and therefore a target in the design of future therapies for a variety of demyelinating diseases.LT designed the figures. LT and LP wrote and designed the manuscript. LP supported manuscript submission.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.


Considerable progress has been made in cancer therapeutics recently with targeted strategies that are efficacious and less toxic. Immunotherapy and chimeric antigen receptor (CAR) T-cells are increasingly being evaluated in a variety of tumors in the relapsed/refractory as well as frontline disease settings, predominantly in hematologic malignancies (HM). Despite impressive outcomes in select patients, there remains significant heterogeneity in clinical response to CAR T-cells. The gut microbiome has emerged as one of the key host factors that could potentially be modulated to enhance responses to immunotherapy. Several recent human studies receiving immunotherapy showed a significantly superior response and survival in patients with the more diverse gut microbiome. Currently, it is unknown if gut microbiota modulates anti-tumor responses to CAR T-cells. Based on molecular and immunological understanding, we hypothesize that strategically manipulating gut microbiota may enhance responses to CAR T-cells. In this review, we further discuss resistance mechanisms to CAR T-cells in HM, potential approaches to overcome resistance by harnessing gut microbiota and other related novel strategies. Tremendous advances have been made in cancer therapeutics recently with the advent of targeted immunotherapeutic approaches that are efficacious with potentially less toxicity than the standard-of-care chemotherapy. Both immunotherapy, via programmed cell death 1 (PD-1) receptor, programmed cell death ligand 1 (PD-L1) or cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitors, and chimeric antigen receptor (CAR) T-cells have impacted the natural history of both solid and hematological malignancies (HM). These are increasingly being studied in a variety of tumors in patients with relapsed/refractory (R/R) malignancy . CAR T-cThe role of the gut microbiota in health and disease, its bidirectional negotiation with the systemic immunity and the existing evidence of the critical role of the gut microbiome in immune checkpoint inhibition (ICI) responses and survival are summarized in Abid MB, 2019 .The results of pre-clinical and clinical studies thus far demonstrate an impact of the gut microbiota on ICI responses, both positive and negative. Predominantly observational, the existing evidence shows that diversity and compositional differences of the gut microbiome may influence response to ICI treatment  and outcome , 11. TheCAR T-cells are autologous T-cells re-directed towards a tumor-specific antigen and are a successful modality for patients with refractory B-cell hematological malignancies. The intracytoplasmic and transmembrane portions of a CAR T-cell contain signaling domains that are involved in T-cell activation and durability, such as CD3ζ, CD28, 4-1BB, ICOS, and OX40. A characteristic second-generation CAR connects an antigen-binding domain, typically single-chain variable fragments (scFv) derived from a monoclonal antibody, to the T-cell signaling domain (CD3ζ: CD3 zeta-chain) coupled to the intracellular signaling domain of a costimulatory molecule such as CD28 or 4-1BB. Higher-generation CAR T-cell constructs may contain multiple signaling domains. These components, including the engineered receptor protein and signaling domains, then facilitate activation of CAR T-cells and render durability, thus allowing execution of the effector functions of a CAR T-cell independent of MHC restrictions , 17, 18.While there is no evidence on the impact of gut microbiome manipulation on CAR T-cell responses as of this writing, we hypothesize that gut microbiota modulation carries potential for enhancing CAR T-cell responses: based on pre-clinical and growing clinical evidence elucidating escape mechanisms in CAR T-cell non-responders, clinical evidence of enhancement of ICI responses with gut microbiota manipulation, and shared immunological features between CAR T-cells and ICI. We discuss below factors that impact CAR T-cell responses and known resistance mechanisms to CAR T-cell therapy. We further highlight potential immunologic associations based on which we postulate that the gut microbiota manipulation may be harnessed to homogenize CAR T-cell response.Bacteroides spp. enhanced ACT function in an IL-12-dependent manner. This finding was further reiterated by a French study by Routy et al. involving 249 patients with advanced NSCLC, RCC and urothelial carcinoma receiving ICIs with anti-PD1 and anti-PDL1. They showed that patients who received antibiotics in the peri-ICI blockage period had shorter survival and metagenomic analysis revealed that Akkermansia muciniphila was enriched in responders. This effect was mediated by an increment in IL-12 and a concomitant decrease in Tregs in the TME  into synthetic biology, increasingly sophisticated and specific CAR T-cells are being designed for clinical usage that will possess a knockout of a multiplex of inhibitory proteins –46. SomeFor instance, Qasim et al.  successfSeveral clinical trials are currently underway analyzing the efficacy of CRISPR/Cas9-mediated PD-1 knocked out CAR T-cells, both in HM and solid tumors . Although hypothetical currently, growing clinical evidence of gut microbiota manipulation to enhance responses to ICI may render a viable alternative to genome editing-based knockout of inhibitory molecules. This hypothesis is based on the shared immunological impact of gut microbiota and genome editing.T-cells redirected for universal cytokine-mediated killing (TRUCKs) are the fourth generation of CAR T-cells that are designed to engage allied immune effector cells by cytokine production within the TME. This is particularly efficacious in solid tumors as these employ complex immune evasion strategies in the tumor bed and localized delivery of cytotoxic cytokines via TRUCKs averts systemic toxicity.Chmielewski et al.  first deIn a separate study, Tanoue et al.  recentlyTaken together, the gut microbiome may also hold potential to achieve similar results as those obtained by such sophisticated TRUCKs: specific gut taxa have also shown the capability to differentially activate effectors T-cells—Th17, CD8+ T-cells and Tregs—and induce cytokines, in early, pre-clinical studies.Clostridioides difficile infection (CDI) when given concurrently with systemic antibiotics. Specifically, in a Phase IIb clinical trial, SYN-004 (ribaxamase), a novel β-lactamase engineered to degrade higher-generation cephalosporins and penicillins, was demonstrated to be effective in curtailing AMR and CDI in patients treated with intravenous (IV) ceftriaxone for a lower respiratory infection  are associated with good response and superior survival whereas the phylum Proteobacteria (e.g. Enterobacteriaceae) is associated with poor response and inferior survival. Akkermansia muciniphila belonging to the phylum Verrucomicrobia is associated with a boost in response and potentially superior survival. The impact of Bacteroidetes and Bifidobacteria are dependent upon the species involved and will need to be tailored during studies. For instance, Bifidobacteria longum has been shown to boost response in ICIs whereas Bifidobacteria bifidum was shown to be immunosuppressive. In order to make responses to CAR T-cells more homogeneous, predictable and durable, human studies will be needed. These studies may be performed in the following ways:There is growing pre-clinical evidence that delineates differential roles of the gut microbiota and allows their stratification with potentially stimulatory and inhibitory properties towards engineered CAR T-cells. Albeit the results from clinical studies in the ICI setting do not uniformly stratify “favorable” vs “unfavorable” gut microbial taxa, certain conclusions may yet be drawn (Table Clostridioides difficile infections) that include hematological patients who received a variety of cancer treatments at different treatment time-points. These stool samples, if available for CAR T-cell patients, may be ideal to study to delineate “responders” vs “non-responders,” in a retrospective setting. Taxonomic profiling using 16S ribosomal RNA (16S) gene sequencing and whole-genome shotgun sequencing (WGS) would further stratify the bacterial taxa involved in each of the two cohorts of CAR T-cell patients. The influence of behavior, lifestyle, dietary habits, obesity, co-morbidities, recent use of antibiotics or probiotics may further be analyzed, to study potential association with response and survival.Large, academic medical centers typically treat refractory cancer patients with CAR T-cells and the information is stored in robust databases. The centers may also have ongoing stool studies  microbiome profile from baseline (via 16S profiling), the persistence of CAR T-cells, changes in immune cell populations and/or cytokines in the TME , response to treatment as well as objective survival rates. Radiological imaging, tumor biopsies, and blood samples will need to be collected at matched treatment time-points and responders vs non-responders can be defined based on the established response assessment criteria for the underlying hematological illnesses.It is noteworthy that WGS is superior to the 16S amplicon-based method for bacteriome sequencing as the former renders enhanced detection sensitivity, specificity and increased detection of diversity , 58. AltThe microbiome studies have not expanded to CAR T-cell or other types of novel immune-engaging therapy yet and the earliest evidence of the role of antibiotics in ACT setting has been conflicting. The only one available thus far is a single-center observational study performed by van den Brink and colleagues. The study included 25 patients who received CAR T-cells with varying conditioning regimens and showed that responders had distinctive baseline microbiota composition as compared to non-responders. Interestingly, non-responders also did not experience toxicity and the microbiota in patients from both cohorts belonged to the Firmicutes phylum. The small study corroborates our hypothesis discussed above and hints towards a possible association between baseline gut taxa and response to CAR T-cells . AdditioThe findings discussed above illustrate that controlled manipulation of the gut microbiome could potentially be a rather inexpensive strategy to substantially enhance responses to posh and engineered therapeutic modalities such as immunotherapy and CAR T-cells in the future. Further large-scale, prospective studies will delineate its role in a myriad of engineered cancer therapeutics.Dysbiosis can increase the representation of deleterious microbiota that produces harmful metabolites and antigens, leading to maladaptive immune responses. Strategically averting gut dysbiosis, preventing the loss of diversity during treatment, and maintaining desirable taxa are needed to augment response to cancer treatment. Although there are suggestions that certain genera may hold a uniformly positive effect on responses, there are no definite or optimal consortia of bacterial taxa established yet that could be used in trials as a ‘probiotic’. Randomized studies are being planned to study differential responses and survival in melanoma patients receiving immunotherapy with FMT from long-term immunotherapy responders and an oral pill containing an exclusive assortment of microbes that mimic key taxa, explored thus far, in collaboration with the industry.Meanwhile, we recommend against using commercially available probiotics specifically during cancer treatment . These aImmunotherapy is relatively safer than conventional treatment and prolongs survival in a variety of tumors in the relapsed/refractory disease settings.CD19-directed CAR T-cells are FDA-approved for patients with R/R ALL and DLBCL and benefit patients with otherwise poor outcomes.Despite impressive outcomes in select patients, there remains significant heterogeneity in clinical responses to both immunotherapy and CAR T-cells.The diversity and composition of the gut microbiome influences response to immunotherapy, according to recent evidence.The role of the gut microbiome in ACT or CAR T-cell setting has not been explored.Pre-clinical and growing clinical evidence elucidating escape mechanisms in CAR T-cell non-responders.Clinical evidence of enhancement of immunotherapy responses with gut microbiota manipulation.Shared immunological features between CAR T-cells and other types of immunotherapy.Early clinical evidence of the potential role of IL-6/STAT3 signature in superior CAR T-cell responses.Early data on the suppressive role of Tregs on ACTs.We hypothesize that the gut microbiome modulation carries the potential for enhancing CAR T-cell responses and this theory is based on:Broad-spectrum antibiotics during immunotherapy and CAR T-cells may lead to dysbiosis and inferior responses and survival.Although we are aware of certain taxa that carry ‘favorable’ versus ‘unfavorable’ impact on immunotherapy responses, a definite, rationally designed conglomerate of live bacteria is yet to be established for use in prospective trials.Retrospective studies analyzing the association between gut microbial diversity/composition and CAR T-cell responses.Retrospective studies analyzing any potential association between broad- versus narrow-spectrum antibiotics and CAR T-cell responses.Retrospective studies analyzing the potential association between behavior, lifestyle, dietary habits, obesity, co-morbidities, and probiotics and CAR T-cell responses.Delineate the relationship between the gut microbiome and cytokine and immune cell infiltration within the TME.Establish a clear relationship between the gut microbiome and tumor genome, if one exists.Metabolomic profiling to identify microbial metabolites as ‘biomarkers’ of response.Multicenter, prospective, randomized trials analyzing the impact of diet, non-absorbable oligosaccharides contained in potato starch, FMT from immunotherapy and CAR T-cell responders, or a rationally designed probiotic-containing consortia of live bacteria that mimic favorable taxa.


Recombinant viruses are novel therapeutic agents that can be utilized for treatment of various diseases, including cancers. Recombinant viruses can be engineered to express foreign transgenes and have a broad tropism allowing gene expression in a wide range of host cells. They can be selected or designed for specific therapeutic goals; for example, recombinant viruses could be used to stimulate host immune response against tumor-specific antigens and therefore overcome the ability of the tumor to evade the host’s immune surveillance. Alternatively, recombinant viruses could express immunomodulatory genes which stimulate an anti-cancer immune response. Oncolytic viruses can replicate specifically in tumor cells and induce toxic effects leading to cell lysis and apoptosis. However, each of these approaches face certain difficulties that must be resolved to achieve maximum therapeutic efficacy. In this review we discuss actively developing approaches for cancer therapy based on recombinant viruses, problems that need to be overcome, and possible prospects for further development of recombinant virus based therapy. Cancers are the second most common cause of death worldwide. According to statistical data, the number of new cases increases every year despite significant improvements in existing therapy and the emergence of new innovative therapeutic approaches . SurgeryRecently, gene and cell therapies were introduced as having significant potential for cancer treatment. Gene therapy implies correction or modulation of the expression of a gene of interest in the host. It can be used to achieve both increased expression of immunomodulating or oncosuppressor genes as well as to inhibit oncogene expression leading to tumor regression . Cell-meCell-free therapy is an alternative to cell-based therapy, which is based on the use of extracellular vesicles (exosomes and microvesicles) derived from native or genetically/chemically modified cells. The cell-free approach was developed in an attempt to circumvent the limitations of cell-based therapy. Extracellular vesicles (EVs) retain properties of the donor cells, however, more importantly, they can carry donor cell cargo or externally loaded content . HoweverGenetic modification of cells using recombinant viruses can promote high levels of transgene expression in a wide range of host cells . With diRecombinant viruses can be used to genetically modify cells, triggering the expression of tumor-specific antigens. These viruses can also be used to directly infect antigen-presenting cells to boost anti-cancer immune responses . The enhTumor-specific antigens used for therapeutic vaccines should induce strong immune responses and be expressed only in tumor cells. Target specificity can result from antigens generated by somatic point mutations  or integSeveral viruses have been used in attempts to develop optimal strategies for transgene delivery for a range of cancers. These include, retroviruses, lentiviruses (including HIV-1) , adenovi+ and CD8+ mediated immune response against heterologous antigens, are the most widely used viruses for anti-tumor vaccine production [Poxviruses, which are able to induce both CD4oduction . Poxviruoduction ,32,33.MVA has been used to generate a number of vaccines carrying specific antigens overexpressed in tumor cells including 5T4 , mucin 1IL-2) gene, was shown to be safe in Phase I clinical trials. The Phase II clinical trial showed significant activation of MUC1 CD8+ specific immune response in more than half of the patients with PSA [+ and CD8+ immune responses [For example, TG4010 vaccine, which is an MVA encoding the MUC1 antigen as well as the human interleukin 2 , and lymphocyte function-associated antigen 3 (LFA-3 or CD58), was used to treat patients with CEA-expressing cancers in combination with IFN-α-2b. Administration of IFN-α-2b led to significant increase in overall survival (OS) compared to the vaccine alone . PreclinMultiple clinical trials have demonstrated the efficacy of combined therapy where various viral vaccines were used together with chemotherapy or radiotherapy. For example, TG4010 vaccination enhanced the effect of cisplatin and gemcitabine in a Phase IIB clinical trial in patients with non-small-cell lung cancer . HoweverIn addition to the ability of tumors to evade immune surveillance, the natural ability of the host’s immune system to neutralize viral vaccines can limit the efficacy of viral vaccines, because booster doses do not increase antigen-specific immune responses . AnotherThe idea of stimulating the host anti-tumor immune response by injecting viruses encoding tumor-specific antigens has been tested in large number of in vitro and in vivo studies, as well as clinical trials. However, patients, participating in these clinical trials, were already at the advanced stages of tumor development, when standard therapy was not effective. At these stages the immune system becomes significantly suppressed by tumor , leadingThe large number of ongoing clinical trials see  using viThe idea of using autologous ex vivo modified immune cells inspired new ideas in the field of antitumor vaccines. This approach has proven more successful and, currently, FDA has approved three immune cell-based vaccines to stimulate antitumor immunity. KYMRIAH (tisagenlecleucel) and YESCARTA (axicabtagene ciloleucel) are patient’s T-cells genetically modified using lentiviral or retroviral vectors, respectively, encoding anti-CD19 chimeric antigen receptor (CAR). YESCARTA was FDA approved in October 2017 for relapsed-refractory lymphoma (NCT02348216). KYMRIAH was approved for diffuse large B-cell lymphoma (DLBCL), TFL and high-grade B-cell lymphoma treatment after at least two lines of therapy on 1 May 2018 (NCT02445248). The third antitumor therapeutic vaccine PROVENGE (sipuleucel-T) was approved in 2010 for the treatment of prostate cancer. This vaccine is not subjected to direct genetic modification, instead, autologous antigen presenting cells (APCs) are cultivated in presence of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and prostatic acid phosphatase (PAP), an antigen expressed in prostate cancer tissue . All appP53 gene has been used to produce a DC-vaccine to promote anti-p53-specific immunity . Three active dose-escalation trials of MSCs are using viral infection to express TRAIL (NCT03298763), IL-12 (NCT02530047) or IFN-β genes (NCT02079324). The number of clinical trials with viral vectors for cell-based vaccine production is shown in The use of CAR T-cell approaches for immunotherapy is gaining momentum as evidenced by the number of active clinical trials. Few clinical trials have used virus-engineered DC-based vaccines to investigate the use of DCs, genetically modified to express various tumor-associated antigens, to induce antigen-specific immune responses. For example, adenovirus carrying the O-sulfate-modified heparan sulfate receptors, while HSV-2-based oncolytic viruses bind to HVEM, nectin-1 and nectin-2 receptors [The genetic modification of viral genomes, which was the basis for the development of viral vaccines, has become the framework for developing another approach for cancer therapy using oncolytic viruses. Oncolytic viruses are able to preferentially infect tumor cells directly, in contrast to viral vaccines, which are mainly used to modify the immune cells . Most vieceptors .γ34.5 gene, encoding infected cell protein 34.5 (ICP34.5), which is necessary for HSV-1 infection of host cells. In addition, a disabling lacZ insertion in UL39 that encodes the large subunit of viral ribonucleotide reductase (RR), inactivates RR. This increases virus specificity for tumor cells  and thus enhances virus safety for non-malignant cells and tissues [γ34.5 and α47 genes (the latter encoding ICP47 protein), and human GM-CSF gene insertion, which increases the immunogenicity of the virus [γ34.5 and α47 genes, there is an insertion of lacZ gene into the icp6 gene site leading to RR inactivation [HSV-1-based viruses are the most commonly used viruses in oncolytic therapy. To date, there are three generations of these viruses, among them second and third generations have the most potent anti-cancer activity. The second-generation HSV-based virus G207 carries one mutation at both loci of  tissues . T-Vec (he virus . The thitivation .tk) gene is replaced with human GM-CSF and β-galactosidase  genes, is also undergoing clinical trials [e1a gene is commonly deleted in oncolytic adenoviruses [Vaccinia virus-based oncolytic virus JX-594 , in which the thymidine kinase  (NCT01584284). An increase in median OS after administration of a large dose of JX-594 also indicates the effectiveness of therapy with oncolytic viruses (NCT00554372). A high success rate was achieved in clinical trials of T-Vec oncolytic virus. In a randomized Phase III trial (NCT00769704), patients with unresectable stage IIIB-IV melanoma were treated with T-Vec or subcutaneous human GM-CSF injection. The median OS in the human GM-CSF arm was 18.9 months, while in the T-Vec arm it was 23.3 months. The overall response rate in the arms was 5.7% and 26.4% of patients, respectively . This da+ T-cell response. For example, administration of T-Vec to patients with metastatic melanoma increases the number of melanoma associated antigens recognized by T-cells (MART-1) specific T-cells as compared to controls [In addition to the oncolytic effect, viruses cause local inflammation, which manifests with increased infiltration of immune cells into the tumor , local rcontrols . A phasecontrols . Interescontrols .Oncolytic virus infection can trigger immune responses which suppress viral replication. The immune system produces neutralizing antibodies  and mobiMost of the adverse effects associated with oncolytic vaccination are flu-like symptoms, such as fever/chills, rigors, nausea or vomiting ,75. AnemOncolytic virus-based therapy is also combined with chemotherapeutic drugs and human monoclonal antibodies. Potentially, combination of various treatment strategies could improve antitumor responses. For example, in a Phase Ib trial, T-Vec in combination with ipilimumab was used for melanoma treatment. Objective response rate in the combination arm was 39%, while in ipilimumab arm OR was 18%   gene induced apoptosis in SGC-7901 cells in vitro by secreting bioactive sTRAIL protein [PTEN) gene, which is an oncosuppressor, and LY294002, the selective phosphatidylinositol 3-kinase (PI3K) inhibitor, led to significant induction of apoptosis in U251 glioma cells in vitro and in U251 subcutaneous glioblastoma xenograft model in vivo [REIC/Dkk-3 gene suppressed the cell growth of human prostate adenocarcinoma LNCaP cells in vitro by inhibiting CD147 expression [Recombinant viruses can also be used to deliver tumor suppressor genes and apoptosis-inducing genes. For instance, lentivirus carrying human soluble tumor necrosis factor-related apoptosis-inducing ligand ( protein . In turn protein . Interes protein . Combine in vivo . Adenovipression . Thus, vCancer therapy using recombinant viruses shows great promise and is currently being extensively investigated. The most studied approach includes using viral vectors as vaccines carrying tumor-specific antigens. These vaccines are aimed at stimulating the host’s immune system to suppress tumors and enhance T-cell-specific immune responses. Combination of these vaccines with immunomodulating agents is important to achieve the highest antitumor effect. It appears that the search for a successful combination of tumor antigen-bearing vaccines and other therapeutic agents will be the key to successful cancer treatment in a greater number of patients and cancer types.Another breakthrough in virus-mediated cancer therapy is the use of oncolytic viruses, which have a direct cytotoxic effect on tumor cells, and additionally stimulate the immune response by releasing tumor-specific antigens and inducing local inflammation. Genetic modification of oncolytic viruses allows improvements in their specificity and immunogenicity to increase the effectiveness of therapy. As in the case of viral vaccines, combination with other therapeutic agents allows researchers to find the most effective approach for cancer treatment. This includes an FDA-approved oncolytic virus T-Vec which is successfully used to treat patients with melanoma.In view of the fact that cancers are complex diseases that can arise as a result of various mutations, the approaches for the treatment of different cancers will vary. Most likely, the combination of various methods of treatment, including virus-mediated therapy, will lead to the most promising results in the fight against cancer.


Meningiomas are the most common benign intracranial tumors accounting for up to 30% of non-glial tumors of the central nervous system (CNS); on neuroimaging studies, they usually appear as a lobular, extra-axial mass with well-circumscribed margins mostly located in the parasagittal aspect of the cerebral convexity. On magnetic resonance imaging (MRI) of the brain, meningiomas are typically isointense to hypointense relative to grey matter in the T1-weighted sequence and isointense to slight hyperintense relative to grey matter on the T2-weighted sequence with avid homogeneous enhancement after contrast administration. A thin linear enhancement along the dura infiltrating away from the lesion, known as the dural tail sign, was once thought to be a pathognomonic feature of meningiomas, but this non-specific sign can also be seen in other meningioma-like lesions. Several benign and malignant pathologies may mimic some of the neuroimaging characteristics of meningiomas; among them dural metastases of lymphomas. When approaching a patient with suspected meningioma, close attention to the neuroimaging features may help distinguish them from meningioma-like lesions. Here we present the case of a woman with CNS involvement of non-Hodgkin lymphoma that presented with a dural mass resembling the neuroimaging characteristics of a meningioma.  Secondary central nervous system (CNS) involvement of aggressive systemic non-Hodgkin lymphomas occurs in less than 5% of diffuse large B-cell lymphomas (DLBCL) cases -2. At thA previously healthy 77-year-old woman, with a one-month history of headache and left arm weakness presented to the emergency department with nausea, vomiting, and abdominal pain for the past three days. On admission, she was alert; the neurological examination was relevant for left upper motor neuron facial paralysis and mild ipsilateral arm weakness  scale 4/5). An abdominal examination revealed a round palpable mass in the right lower quadrant. Blood workup, including a full blood count, serum electrolytes, lactate dehydrogenase, liver, and kidney function tests, were all within the normal range. Testing for human immunodeficiency virus (HIV) and hepatitis C antibodies were negative. An abdominal computed tomography (CT) showed a contrast-enhancing ill-defined appendicular mass Figure . Three dAs part of the seizure investigation, an electroencephalogram (EEG) was performed; relevant findings included an encephalopathic rhythm within the delta-theta range without epileptiform activity. MRI of the brain showed an extra-axial right frontotemporal dural mass with heterogeneous gadolinium enhancement and perilesional edema Figures -1D.Treatment with levetiracetam, midazolam, and dexamethasone for brain edema was started. Under the suspicion of meningioma, she underwent surgical resection of the dural mass without any complications; also colonoscopic biopsies of the appendicular mass were taken. Histologic analysis of both masses showed diffuse infiltration of large lymphoid cells; in immunohistochemistry, the large lymphoid cells were positive for cluster of differentiation 20 (CD20), B-cell leukemia/lymphoma 2 (BCL-2), BCL-6, and multiple myeloma oncogene 1 (MUM 1); and negative for CD3, CD10, and C-MYC, findings consistent with non-germinal center DLBCL (Figure Secondary CNS lymphoma may present as a dural-based lesion mimicking meningiomas . On MRI,When approaching a patient with a suspected meningioma, close attention to imaging features may help distinguish them from meningioma-like lesions. The distinction between meningioma and meningioma-like lesions is essential due to distinct changes in therapy depending on tissue-based pathology.


Depending on the type of DC-therapy used, long-term clinical efficacy upon DC-therapy remains restricted to a proportion of patients, likely due to lack of immunogenicity of tumor cells, presence of a stromal compartment, and the suppressive tumor microenvironment (TME), thereby leading to the development of resistance. In order to circumvent tumor-induced suppressive mechanisms and unleash the full potential of DC-therapy, considerable efforts have been made to combine DC-therapy with chemotherapy, radiotherapy or with checkpoint inhibitors. These combination strategies could enhance tumor immunogenicity, stimulate endogenous DCs following immunogenic cell death, improve infiltration of cytotoxic T lymphocytes (CTLs) or specifically deplete immunosuppressive cells in the TME, such as regulatory T-cells and myeloid-derived suppressor cells. In this review, different strategies of combining DC-therapy with immunomodulatory treatments will be discussed. These strategies and insights will improve and guide DC-based combination immunotherapies with the aim of further improving patient prognosis and care.Dendritic cells (DCs) are antigen-presenting cells (APCs) that are essential for the activation of immune responses. In various malignancies, these immunostimulatory properties are exploited by DC-therapy, aiming at the induction of effective anti-tumor immunity by vaccination with  Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) and mediate a critical role in the interface between the innate and adaptive immune system. DCs can be subdivided in different subsets including conventional DCs (cDCs) and plasmacytoid DCs (pDCs) that arise in the bone marrow and reside in peripheral tissues in an immature state. In addition, monocytes are able to differentiate into monocyte-derived DCs (moDCs) upon inflammatory conditions –4. Activex vivo-activated DCs loaded with tumor antigens may circumvent suppressive tumor-derived signals, thereby inducing effective anti-tumor immunity upon vaccination. For the past two decades, DC-therapy has shown to be safe, well-tolerated and capable of inducing anti-tumor immunity  and interleukin (IL) 4 will lead to differentiation into immature moDCs , although to date the superior route of administration is still not established. Also the percentages of DCs that migrate successfully toward the lymph nodes is limited, with up to 4% of injected DCs reaching the lymph node after intradermal injection and 0–56% reaching the lymph node after intranodal injection . The migex vivo antigen-loaded DCs will be discussed. A summary of the main characteristics of the studies is presented in Table Chemotherapeutics are traditionally designed to eradicate and eliminate malignant cells to lower tumor burden. However, more recent insights indicate that chemotherapy also has off-target immunological effects depending on the type of chemotherapy, such as immunogenic cell death (ICD) of tumor cells, thereby enabling the induction of anti-tumor immunity . ICD stiCyclophosphamide is an alkylating agent that has tumoricidal effects, thereby reducing tumor burden . In addiThe alkylating agent temozolomide (TMZ) induces lymphoablation upon high doses whereas at low doses it primarily targets Tregs Figure  49). As. As49). +CTL numbers in responders, the concurrent combination did not result in decreased MDSC and Treg numbers in responders vs. non-responders  combined with bortezomib and dexamethasone improved CD4+/CD8+ T-cell ratios compared to baseline and treatment with chemotherapy alone , a protein used to assess T-cell responses initiated by DC-therapy . As this as well . An effepy alone . Specifipy alone . In addipy alone . Howeverpy alone , 71. TheEver since radiotherapy was found to affect non-radiated tumor lesions in a process called the abscopal effect, the immunomodulatory effects of this therapy have been more thoroughly appreciated. As radiotherapy induces ICD, one primary effect is the release of DAMPs and tumor-derived antigens, thereby initiating the activation and migration of DCs to the LN where DCs subsequently cross-present these antigens to T-cells and induce systemic anti-tumor immune responses Figure  76–80).–80.76–80In cancer, tumor cells and immune cells often overexpress co-inhibitory molecules, such as PD-1/PD-L1 and CTLA-4, which suppress anti-tumor immunity. Checkpoint inhibitors targeting these co-inhibitory molecules improve existing anti-tumor immunity when administered as monotherapy , 100. Ad+ T-cells was decreased. In addition, cytotoxicity of CTLs improved when PD-1 was inhibited in a co-culture of tumor cells and T-cells isolated from mice treated with DC/myeloma fusions , IL-2, IL-5, and IL-12 and promoted cytolysis of tumor cells in vitro , Boehirnger Ingelheim (inst). Patents, Royalties, Other Intellectual Property: Patent: Tumor cell lysate for dendritic cell loading (Inst), SNP analyses for immunotherapy (Inst). The remaining 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.


Microtubules (MTs) play fundamental roles in cellular functions including cell division, cell shape, and intracellular transport. Abnormal MT regulation has been linked to age-related disorders and diseases, and MTs often serve as targets for disease therapies. MT regulation is particularly important for neurons, since their polarized morphologies dictate heavy reliance on MT function for their maintenance. MT regulation is involved on several levels in neuronal function and maintenance of neuronal structure, and also appears to be a general downstream indicator and effector in age-dependent neurodegeneration . The nerC. elegans and delayed age-associated changes in neuronal integrity, such as axon blebbing and branching as well as mislocalization of synaptic vesicles to dendritic structures within touch sensory neurons [C. elegans. The efa-6 loss-of-function mutants also maintained greater touch sensitivity and motor function during aging compared to wild-type worms. These differences in neuronal integrity and functional ability were time/aging-dependent with no significant differences between the efa-6 loss-of-function mutants and wild-type worms at day 1 of adulthood but significant differences between the groups by days 9 and 10 of adulthood [We have recently reported that mutations in MT regulators can affect lifespan in a DAF-16 dependent manner , increas neurons . The eff neurons  as well ptrn-1 and hdac-6 were also studied [ptrn-1 and hdac-6 mutants were similar to the lifespan changes seen with ptl-1 and efa-6 mutants respectively, providing more support that a preference for microtubule stabilization over destabilization within neurons promotes increased mean lifespan. Further, DAF-16/FOXO, the downstream transcription factor in the insulin/insulin-like growth factor signaling (IIS) pathway, is necessary for the increased mean lifespan seen in both efa-6 and hdac-6 mutants. To understand how the MT regulators contribute to animal lifespan, we examined the transcription profiles in the mutants with RNA-seq. Differentially expressed genes in MT regulator mutants were genes for lipid metabolism and lipid transport respectively. Consistent with the gene expression changes, altered fat storage was observed in the microtubule regulator mutants, suggesting that these changes may also contribute to the changes in lifespan. Taken together, these observations lend support to the concept that MT status impact neuronal function, which can influence lipid metabolism in other tissues and organismal longevity  by which DAF-16 influences these microtubule regulators during normal aging as well as alterations that may occur in models of disease states in which altered microtubule dynamics are known to play a role.


Downregulation of Sty leads to upregulation of MAPK signaling, thereby promoting the growth of synaptic boutons and tracheal branches. Thus, glia-derived miR-274 might be deemed a “gliotransmitter” to mediate communication with neurons and tracheal cells. The modulation of tracheal branches by glial-derived miR-274 is also crucial for fine-tuning larval behavior in response to hypoxia.Our study provides significant advances in the understanding of circulating exosomal microRNA (miRNA) in animals. Circulating exosomal miRNAs mediate communication among tissues and organs. In glia, mature miR-274 is produced and secreted to the circulating hemolymph to target the recipient cells, neurons, and tracheal cells. We also identified the target gene  sty 3′ untranslated region, thereby enhancing MAPK signaling and promoting cell growth. miR-274 expressed in glia of an mir-274 null mutant is released as an exosomal cargo in the circulating hemolymph, and such glial-specific expression resets normal levels of Sty and MAPK signaling and modulates target cell growth. mir-274 mutant larvae are hypersensitive to hypoxia, which is suppressed by miR-274 expression in glia or by increasing tracheal branches. Thus, glia-derived miR-274 coordinates growth of synaptic boutons and tracheal branches to modulate larval hypoxia responses.Secreted exosomal microRNAs (miRNAs) mediate interorgan/tissue communications by modulating target gene expression, thereby regulating developmental and physiological functions. However, the source, route, and function in target cells have not been formally established for specific miRNAs. Here, we show that glial miR-274 non-cell-autonomously modulates the growth of synaptic boutons and tracheal branches. Whereas the precursor form of miR-274 is expressed in glia, the mature form of miR-274 distributes broadly, including in synaptic boutons, muscle cells, and tracheal cells. Mature miR-274 is secreted from glia to the circulating hemolymph as an exosomal cargo, a process requiring ESCRT components in exosome biogenesis and Rab11 and Syx1A in exosome release. We further show that miR-274 can function in the neurons or tracheal cells to modulate the growth of synaptic boutons and tracheal branches, respectively. Also, miR-274 uptake into the target cells by AP-2–dependent mechanisms modulates target cell growth. In the target cells, miR-274 down-regulates Sprouty (Sty) through a targeting sequence at the  Cells communicate at multiple levels during development, from short to long range, between the same or different types of cells, and between different tissues/organs in the body. Long-range communication requires transport of signals, leading to coordinated growth and differentiation in multicellular organisms. Several mechanisms for transporting long-range signals from source to target cells have been identified, including transport by extracellular vesicles (EVs) , 2. ThesSOX-4 mRNA translation  that functionally inhibit protein expression in the target or recipient cells . In animnslation . Exosomapression . Adipocyic cells . In Drosl growth . miRNAs et cells . HoweverDrosophila , we showed that the miR-274 precursor was expressed in glia and the mature form was ubiquitously detected. Consistently, miR-274 was required in glia for synaptic and tracheal growth. Glial expression of miR-274 could be detected in the hemolymph of the larval circulatory system. Indeed, miR-274 was secreted as an exosomal cargo as shown by genetic analysis and biochemical fractionation. miR-274 targets the sprouty (sty) 3′ untranslated region (UTR) to down-regulate Sty expression, leading to enhancement of MAPK signaling and target cell growth. Intriguingly, the mir-274 mutant with fewer tracheal branches was hypersensitive to hypoxia. Thus, glial miR-274 coordinates the growth of synaptic boutons and tracheal branches and links the developmental role to behavior responses to hypoxia.We explored the coupling of synaptic boutons to tracheal branches at larval By immunostaining synaptic, glial, and tracheal structures, we show that glial processes wrap around incoming motor axons, and envelopment ends before terminal branching at muscle 6/7 B, arrowsmir-274KO/mir-274KO) displayed reduced growth of both synaptic boutons and tracheal branches . While the mir-274KO mutant had reduced tracheal branches, the muscle area was equivalent to wild type, leading to the reduction in the overall density of branch tips . We then quantified the distribution of branch tips in the bouton-surrounding proximal area, which accounts for about a quarter of the total muscle area . In wild type, the relative tracheal density within the proximal area was similar to that in the outer area . In the mir-274KO mutant, however, the relative tracheal density within the proximal area was markedly reduced as compared to that in the outer area . Thus, miR-274 might play a role in recruiting tracheal branches to the synaptic bouton area. Furthermore, the reduction in synaptic boutons was not limited to NMJs of muscle 6/7, as we also observed synaptic bouton reduction at NMJs of muscle 4 in the mir-274KO mutant . Likewise, tracheal branching was also compromised in the dorsal region of the mir-274KO mutant . These data suggest that larvae lacking miR-274 fail to develop complete sets of synaptic boutons and tracheal branches.To investigate whether these structures are developmentally coregulated, we screened 51 miRNA-knockout mutants  for defebranches C. Quanti+ larvae F. We als systems C and F. UAS-decoy-mir-274 transgene driven by cell-type-specific GAL4 drivers to inhibit miR-274 functions. Neuronal elav-GAL4, glial repo-GAL4, and tracheal btl-GAL4 were individually crossed to UAS-decoy-mir-274 to analyze phenotypes in synaptic boutons and tracheal branches. Surprisingly, glial depletion caused significant reductions in synaptic boutons and tracheal branches . Whereas the glial processes at NMJs of muscle 6/7 presented a normal morphology in the mir-274KO mutant, the hemolymph–brain barrier  was defective , confirming the findings of a previous study . The reductions in synaptic boutons and tracheal branches are not secondary to the defective HBB, as an intact HBB was retained in glial expression of UAS-decoy-mir-274  while synaptic boutons and tracheal branches were reduced , subperineurial (moody-GAL4), and wrapping (Nrv2-GAL4) glia were used to express decoy-mir-274 for miR-274 trapping. The heterozygous mir-274KO/+ background is more sensitive to decoy-mir-274 trapping in glia , which was used in subtype glia trapping. Trapping miR-274 in pan glia led to significant growth defects , which was used in subtype glia trapping. Trapping miR-274 in subperineurial glia led to significant growth defects , while trapping miR-274 in perineural and wrapping glia had no effect . Thus, subperineurial glia might be the major source of miR-274 in regulating synaptic and tracheal growth.Interestingly, both precursor and mature miR-274 signals were observed in peripheral nerves, suggesting a local peripheral regulation of synaptic boutons and tracheal branches J and K. SI Appendix, Fig. S5A). As miR-274 might merely associate with the exosomes, the isolated exosomes with RNaseA treatment still included miR-274, suggesting that miR274 is likely enclosed inside the exosome . However, the exosomal fraction isolated from the mir-274KO hemolymph did not contain miR-274 . Likewise, TSG101, Rab11, and Syx1A were detected in exosomal fractions of both wild-type and mutant larvae  and efficient suppression of synaptic and tracheal growth . Taken together, these results are consistent with glial expression of miR-274 being secreted into the circulating hemolymph through the exosomal pathway to regulate synaptic and tracheal growth.Cargo-carrying exosomes are assembled through serial actions of the ESCRT complexes, which promote membrane invagination and formation of intraluminal vesicles in MVBs . To suggrepo-GAL4 control, low background signals were present in these structures by glial trapping of miR-274  showed low background FISH signals throughout different tissues, glial expression of miR-274 in the mir-274KO null mutant  displayed strong and punctate signals in synaptic boutons, muscle cells, and tracheal cells . In contrast, tracheal UAS-mir-274 expression in the mir-274KO mutant showed strong miR-274 FISH signals in tracheal cells but not in synaptic boutons and muscle cells . We also failed to detect the FISH signals in both synaptic boutons and tracheal cells upon glial knockdown of Rab11 or Syx1A . Taken together, these results are consistent with the idea that only miR-274 expressed in glia could reach all target cells.To further show that glia-expressed miR-274 could reach synaptic boutons and tracheal branches for function, we first performed the FISH experiment with the mature miR-274 probe. Whereas the FISH signals were detected in synaptic boutons, muscle, and tracheal cells in the  miR-274 A. Also, al cells B. We alsSI Appendix, Fig. S2 C–F), which we suspected might be due to insufficient miR-274 trapping. We therefore expressed the UAS-decoy-mir-274 transgene in the mir-274 heterozygous mutant that reduces one gene dosage of mir-274. Strikingly, trapping miR-274 in glia in mir-274KO/+ caused stronger reductions in synaptic and tracheal growth than in the wild-type background  or tracheal cells (btl-GAL4), the miR-274 FISH signals were reduced in synaptic boutons or tracheal cells, respectively, suggesting that miR-274 failed to be endocytosed into the target cells . Consistently, specific reduction in growth of synaptic boutons or tracheal branches was observed . Taken together, these results strongly suggest that miR-274 is necessary and sufficient to function in target cells to regulate their growth.Glial expression of miR-274 localized in synaptic boutons and tracheal cells B and res deficit C–F. SecoO mutant G–J. We abranches G and H, branches I and J. achinery . By exprmir-274KO larvae . From the candidate genes, we chose sprouty (sty) for further study since Sty plays a critical role in feedback inhibition of receptor tyrosine kinase (RTK)/MAPK signaling during tracheal branching and synaptic bouton formation  restored dpERK levels in tracheal cells and synaptic boutons to the levels comparable to the control , we found reduced Sty and increased dpERK levels, as compared to those without glia-specific miR-274 rescue  . We also detected reduced levels of dpERK at these 2 sites . These results strongly support that glia-expressed miR-274 reaches target cells to down-regulate Sty expression and to promote growth of synaptic boutons and tracheal branches. We also examined the effects of disrupting exosomal biogenesis, transportation, and release in glia. By repo-GAL4–induced knockdowns of Rab11, Syx1A, TSG101 and Shrb, Sty up-regulation and dpERK down-regulation were detected in synaptic boutons and tracheal cells as well .To further show that glia-derived miR-274 regulates Sty and dpERK levels in neuronal and tracheal cells, we first performed immunostaining in glia-specific miR-274–rescued larvae that had restored synaptic and tracheal growth E and H. po-GAL4) A–C. Simisty mRNA was detected in exosomes isolated from S2 cells . It is possible that sty was down-regulated and packaged in exosomes in glia for secretion. However, we failed to detect sty mRNA in the exosomes isolated from larval hemolymph, suggesting distinct populations of exosomes derived from S2 cells and larval hemolymph . Thus, sty is more likely to be directly down-regulated in target cells. To support this possibility, sty was specifically down-regulated in target cells of the mir-274KO mutant, and the respective phenotype was analyzed. When styRNAi was expressed in neurons by elav-GAL4 , growth of synaptic boutons but not that of tracheal branches was rescued  (The  rescued D and F. styRNAi) E and F. Drosophila trachea is a highly branched network with open ends and air-filled terminal branches that function in gas exchange similarly to mammalian circulatory systems (2), about 20% of control larvae (w1118 and Canton S) escaped away from the food paste within 5 min, and this percentage increased to almost 40% by 10 min and to close to 50% by 15 min (Left). Strikingly, almost 50% of mir-274KO mutants exhibited a strong hypoxia response by escaping away from the food paste by 5 min and about 60% by 10 and 15 min (Left). The differential responses between mir-274KO and control larvae were still significant when we conducted the assay in 10% O2, suggesting that the mutant larvae exhibited hypersensitivity toward reduced oxygen levels (Middle). However, no significant differences were found when we assayed these 3 genotypes under normoxia, with almost all larvae (>95%) staying in the food source (Right). We performed several control experiments to show that mir-274KO larvae are indeed more responsive to lower oxygen levels. First, mir-274KO mutants still exhibited a significantly different hypoxia response compared to control larvae in a 10-fold–diluted food source, suggesting that the enhanced fleeing behavior of mutant larvae is not due to differences in evaluating nutrition . Second, the hypoxia-induced response is not caused by alterations in locomotion, as we observed comparable crawling lengths between mir-274KO mutant and control larvae . Third, feeding motivation toward nutritious (yeast) or nonnutritious (grape juice) foods, as evaluated by counting mouth hook contractions, were almost identical under both fed and starved conditions . Fourth, we performed the behavior assay in response to high salt that serves as an alternative aversive stimulus .The  systems , 37. Ter systems . We post systems . When exy 15 min A, Left. d 15 min A, Left. n levels A, Middled source A, Right.mir-274KO mutants carrying both repo-GAL4 and UAS-mir-274 transgenes showed a reduced percentage of fleeing larvae in response to hypoxia compared to homozygous mir-274KO mutants carrying either the repo-GAL4 or UAS-mir-274 transgenes  significantly reduced synaptic boutons and tracheal branches . Consistently, trapping miR-274 within glia also induced the hyperactive hypoxia response  that reduced synaptic boutons but not tracheal branches  almost completely suppressed the enhanced hypoxia escape response .We propose that miR-274–modulated synaptic and tracheal growth during development is coupled to physiological demands . Detection of miR-274–carrying exosomes in the hemolymph depends partially on the ESCRT components in exosome biogenesis , supporting that glia-secreted miR-274 is carried by exosomes in the larval circulatory system. Circulating exosomes carry diverse molecules including proteins and RNAs. Although some studies have suggested that extracellular miRNAs might be “cellular by-products” disposed of by apoptotic cells . Interestingly, neuron-expressed miR-274 was also detected in muscle cells , which might be transported by transverse exosomes crossing the synaptic cleft at NMJs, similar to the Wnt/Wg signal carried by Evenness interrupted (Evi)-positive exosomes from pre- to postsynapses (Drosophila retrovirus-like Gag protein Arc1 (dArc1) binds to darc1 mRNA to be sorted into exosomes for transport across synaptic clefts . Differential requirements for ESCRT components have also been observed for blocking Hh-borne exosomes from wing-disk epithelial cells  controlling larval self-righting behavior. Lack of miR-iab4/iab8 or overexpressing the target gene Ultrabithorax in SRNs inhibits the ability of larvae to right themselves , allowing larvae to tolerate hypoxia. Our study highlights a coordinating role for glia in regulating a coupled developmental and physiological process.Recently, miRNAs were also shown to be essential for physiological functions. In emselves . Similaremselves . Here, wSI Appendix) and protocols are available by contacting the corresponding author.All materials  or the hemolymph isolated from 50∼100 larvae. In brief, the S2 cell culture medium or larval extracellular fluid was centrifuged at the series of 300 × g for 5 min, 2,000 × g for 10 min, and 10,000 × g for 30 min to remove large cell debris. Exosomes were then collected following the manufacturer’s instructions for the ExoQuick kit (System Biosciences). For Exosome fractions were isolated from the medium used for culturing Graphpad Prism v6 (Graphpad) was used to perform statistical analyses. All data are expressed as mean ± SEM.SI Appendix, Materials and Methods.See other extended methodological details in Supplementary File


Imaging plays a critical role in the management of the highly complex and widely diverse central nervous system (CNS) malignancies in providing an accurate diagnosis, treatment planning, response assessment, prognosis, and surveillance. Contrast-enhanced magnetic resonance imaging (MRI) is the primary modality for CNS disease management due to its high contrast resolution, reasonable spatial resolution, and relatively low cost and risk. However, defining tumor response to radiation treatment and chemotherapy by contrast-enhanced MRI is often difficult due to various factors that can influence contrast agent distribution and perfusion, such as edema, necrosis, vascular alterations, and inflammation, leading to pseudoprogression and pseudoresponse assessments. Amino acid positron emission tomography (PET) is emerging as the method of resolving such equivocal lesion interpretations. Amino acid radiotracers can more specifically differentiate true tumor boundaries from equivocal lesions based on their specific and active uptake by the highly metabolic cellular component of CNS tumors. These therapy-induced metabolic changes detected by amino acid PET facilitate early treatment response assessments. Integrating amino acid PET in the management of CNS malignancies to complement MRI will significantly improve early therapy response assessment, treatment planning, and clinical trial design. Malignancies of the central nervous system (CNS) account for an estimated 23,000 cases and over 16,000 deaths each year -fluoro-D-glucose (18F-FDG PET) combined with computed tomography has emerged as a standard-of-care imaging modality for the detection of tumors based on their elevated glucose metabolic rate (Warburg effect). The utility of 18F-FDG PET in imaging brain tumors, however, is hindered by the elevated background uptake level of glucose by normal brain tissue generating little discernable contrast. Moreover, immune cell activation within inflammatory responses also exhibits elevated glucose metabolism further obscuring distinction from brain or tumor tissue -l-methionine (11C-MET) is the most well-established and utilized amino acid probe. 11C-MET PET has been shown to be useful in delineating ependymomas, medulloblastoma, and astrocytomas in pediatric patients and can also effectively differentiate between radiation-induced brain tissue injury and tumor recurrence  alpha-Methylaminoisobutyric acid (11C-MeAIB), an amino acid analog, can differentiate between low-grade and high-grade astrocytoma as determined by the tumor to normal brain uptake ratio [11C] methyl-l-tryptophan (11C-AMT) can be used to identify LGGs, even if they are not contrast-enhancing on MRI, and distinguish them from HGGs based on tumor-to-cortex radiotracer uptake [The utility of amino acid PET to differentiate between LGG and HGG based on the level of radiotracer uptake has been assessed in a limited number of studies. Generally, amino acid uptake is higher in grade III/IV gliomas compared to grade I/II ,62,63. [ke ratio . Differer uptake .Despite differential LGG/HGG imaging in many studies, a recent systematic review of amino acid PET of LGGs has revealed a difficulty in interpretation due to inconsistencies in radiotracer uptake and different correlations between uptake ratios and LGG molecular status . RadiotrKinetic PET imaging, however, may offer a better differentiating parameter based on the differing rate of radiotracer uptake by LGG and HGG. Time-activity curves of grade II tumors exhibit a slow, steady increase compared to the rapid uptake of amino acid radiotracers by grade III/IV tumors. Thus, LGG and HGG can be more reliably differentiated based on a dynamic or dual-time-point PET than static or endpoint imaging ,68,69,7018F-FET uptake following radiochemotherapy was associated with overall survival compared to increased or stable uptake [11C-MET PET at eight weeks were predicted to have more favorable prognoses [11C-MET-PET is prognostic of progression-free survival in high-grade glioma patients [As an extension to its capacity to report on the early response to therapy, amino acid PET has proven to be a reliable prognostic tool in predicting overall and progression-free survival. Decreased e uptake ,72. Truerognoses . Determipatients  and predpatients .18F-FET PET combined with anatomic MRI predicted outcome and progression-free survival [Amino acid uptake may also serve as a reliable predictor of survival in patients with LGGs. survival . Kineticsurvival ,77. Detesurvival .18F-FET and 18F-DOPA PET proved useful for biopsy guidance and resection planning in most cerebral glioma cases compared to 18F-FDG [Diagnostic inaccuracies based on image interpretation carry risk and compromise CNS tumor therapy outcomes. Biopsy provides diagnostic accuracy at a higher rate than neuroimaging and is essential to tumor grading and treatment planning. Therefore, image-guided stereotactic biopsy of tumor volumes is crucial to achieving higher diagnostic yields. Delineation of tumor extent and identification of regions of higher grade and density with  18F-FDG ,79,80,81 18F-FDG .11C-MET PET was more diagnostically precise than contrast-enhanced MRI in delineating a broad and continuous tumor mass [Some case studies illustrate the advantages of amino acid PET over MRI in delineating tumor extent, informing on response to therapy, and detecting disease recurrence. Amino acid uptake has also been shown to be consistent with exhibited symptoms and biopsy results. A unique case study involving a 37-year old woman with a “butterfly” glioblastoma exemplifies tumor extent delineation by amino acid PET. MRI findings were equivocal and imprecise, indicating bifocal growth with slight contrast enhancement despite the large extent of the bilateral tumor mass. mor mass .18F-DOPA uptake in the left parietal lobe of a 46-year-old patient, where resection of a glioma had taken place two years earlier. 18F-FDG uptake was not elevated and did not indicate any abnormalities. Recurrence of the tumor in this patient was confirmed by biopsy demonstrating the accuracy of amino acid PET in detecting metabolic tumor volume [18F-FET PET, however, was negative for focal uptake and, indeed, histopathology of the resected tumor revealed necrotic tissue consistent with pseudoprogression. The lesion regressed in follow-up MRI further confirming the diagnosis [Amino acid PET has also proved valuable in detecting recurrence and differentiating it from pseudoprogression as confirmed by biopsy. Detection of recurrence by PET was illustrated by high r volume . Similariagnosis .11C-AMT uptake indicating recurrence. 11C-AMT uptake decreased upon chemotherapy and radiotherapy and correlated with a symptomatic improvement [18F-MET PET was able to detect recurrence of a benign oligodendroglioma in a patient exhibiting recurrent temporal epileptic seizures 15 months following resection. 18F-MET PET uptake was high in the region of the previous tumor. Secondary resection and biopsy confirmed recurrence as predicted by PET [In contrast to MRI, consistency of amino acid uptake with patient symptoms has been demonstrated in a number of case studies. Vision problems exhibited by 16-year old female optic pathway glioma patient correlated with an increase in rovement . Likewisd by PET . In both11C-MET-PET has been shown to be a reliable identifier of true responders to bevacizumab therapy with favorable prognoses [The strength of MRI in providing high anatomical resolution of soft brain tissue coupled with the specificity of amino acid PET in delineating CNS tumors promote combining the modalities to improve diagnostic specificity in the management of CNS malignancies. When combined with MRI, rognoses ,88,89. Frognoses  and relirognoses . The benrognoses .Metabolic changes detected by amino acid PET occur in response to therapy sooner than morphological and structural changes providing an earlier response to therapy assessment and prognosis. Moreover, metabolic uptake of radiolabeled amino acids is specific to the proliferating cellular component of the tumor, occurs independently of the status of the blood-brain barrier, and excludes non-cellular lesions created by necrosis, edema, and inflammation. As summarized in The advantages provided by amino acid PET justify its future implementation as a standard procedure in defining biopsy sites, planning surgical resection and radiotherapy, prognostication, and treatment response assessment ,94. Thes