In particular, the treatment with the cocrystal MetH2++•2DCA− induced a synergistic apoptotic cell death coupled to a marked down-modulation of the anti-apoptotic Mcl-1 protein. Taken together, our data emphasize that innovative compounds based on Metformin-DCA combination merit to be further evaluated as chemotherapeutic agents for the treatment of B-CLL.Metformin and the mitochondrial targeting dichloroacetate (DCA) have recently received attention due to their ability to inhibit anaerobic glycolysis, which renders most cancer cells resistant to apoptosis induction. We observed that Metformin alone exhibited a dose-dependent anti-leukemic activity in both B leukemic cell lines and primary B-chronic lymphocytic leukemia (B-CLL) patients' cells and its anti-leukemic activity was enhanced when used in combination with DCA. In order to overcome the problems of poor bioavailability and cellular uptake, which limit DCA efficacy, we have designed and synthetized cocrystals consisting of Metformin and DCA (Met-DCA) at different stoichiometric ratios. Of note, the MetH Most cancers are characterized by enhanced glycolytic flux for ATP production, enhanced glucose to lactate conversion and reduced mitochondrial oxidative phosphorylation, even under aerobic conditions -3. Interin vitro studies have documented the antiproliferative, anti-invasive, and antimetastatic effects of Metformin in multiple cancer cell types  from the structures determined by single-crystal X-ray diffraction. The crystal data of compounds Met-DCA 1:1 and Met-DCA 1:2 were collected at room temperature using a Nonius Kappa CCD diffractometer with graphite monochromated Mo-Kα radiation . , 62.For Western blotting analysis, cells were lysed as previously described , 64. ProThe results were evaluated by using analysis of variance with subsequent comparisons by Student's t-test and with the Mann-Whitney rank-sum test. Statistical significance was defined as p<0.05. In order to investigate the effect of Metformin plus DCA combination, leukemic cells were treated with serial doses of Metformin or DCA, individually or in combination using a constant ratio (Metformin:DCA). Results were analyzed with the method of Chou and Talalay  to deter


House dust mites (HDM) represent one of the most important inducers for respiratory allergies worldwide. The aim of this study was to investigate the IgE- and IgG-reactivity profiles of HDM-allergic individuals suffering only from allergic rhinitis or from allergic asthma.This study included sera from clinically well characterized asthmatic (n=105) and non-asthmatic (n=53) HDM-allergic patients. IgE- and IgG-reactivity to seven HDM-allergens  were measured using a customized allergen microarray .HDM-allergic individuals suffering from asthma showed striking differences regarding their IgE reactivity profiles compared to the non-asthmatic group. First, the frequency of IgE reactivity to the tested HDM allergens was up to 3-fold higher in the asthmatic than in the non-asthmatic group. Seventy percent of the asthmatics reacted with 3 to 6 of the tested allergens compared to only 45% in the non-asthmatic group. Furthermore, IgE-levels to nDer p 1, rDer p 2, rDer p 5 and rDer p 23 were significantly higher in the asthmatic group. In contrast, HDM-allergic asthma patients showed a lower IgG-binding frequency to the seven HDM allergens than HDM-allergic patients without asthma.The IgE and IgG reactivity profiles to HDM allergens differ considerably in patients with mild  and severe  respiratory symptoms due to HDM allergy.


In addition, we also analysed the effect of a novel nano-stroma incorporating XAV939 (XAV), a potent inhibitor of the developmentally important Wnt–β-catenin signalling pathway, to investigate whether it could also promote the survival and differentiation of human fetal dopaminergic precursors; we found that the numbers of both tyrosine-hydroxylase-positive neurons (a marker of dopaminergic neurons) and total neurons were increased. This is the first demonstration that LIF-nano-stroma and XAV-nano-stroma each have pro-survival effects on human dopaminergic neurons, with potential value for target-specific modulation of neurogenic fate in cell-based therapies for PD.The endogenous reparative capacity of the adult human brain is low, and chronic neurodegenerative disorders of the central nervous system represent one of the greatest areas of unmet clinical need in the developing world. Novel therapeutic strategies to treat them include: (i) growth factor delivery to boost endogenous repair and (ii) replacement cell therapy, including replacing dopaminergic neurons to treat Parkinson’s disease (PD). However, these approaches are restricted not only by rapid degradation of growth factors, but also by the limited availability of cells for transplant and the poor survival of implanted cells that lack the necessary stromal support. We therefore hypothesised that provision of a transient artificial stroma for paracrine delivery of pro-survival factors could overcome both of these issues. Using leukaemia inhibitory factor (LIF) – a proneural, reparative cytokine – formulated as target-specific poly(lactic-co-glycolic acid) (PLGA) nano-particles (LIF-nano-stroma), we discovered that attachment of LIF-nano-stroma to freshly isolated fetal dopaminergic cells improved their survival fourfold: furthermore,  In vivo, PLGA slowly degrades by hydrolysis to lactic acid and glycolic acid, monomeric derivatives that feed into metabolic pathways for eventual release as carbon dioxide and water. PLGA allows for the creation of ‘designer’ particles for delivery; these particles can be modified for specific targeting as well as the type of cargo (which can be multiple) and the release kinetics of that cargo. Other approaches that have used synthetic biomaterials to mimic the extracellular microenvironment include bioengineered fibrillar matrices  is the second commonest neurodegenerative disorder of the central nervous system (CNS). In PD, the disease process critically involves the nigrostriatal dopaminergic (DA) neurons, resulting in their loss and subsequent decreased dopamine levels in the dorsal striatum. The concept of replacing lost DA neurons with healthy cells of the same lineage underpins one major clinical approach that uses allografts of human fetal ventral mesencephalic (hfVM) tissue. This strategy has shown significant long-term benefits in some patients with PD  is the second commonest neurodegenerative disorder. In PD, the disease process critically involves nigrostriatal dopaminergic (DA) neurons, resulting in their loss and decreased dopamine levels in the dorsal striatum. For individuals affected by PD, the development of tremor, rigidity and cognitive decline are compounded by an increased risk of early dementia and postural instability. The concept of replacing lost DA neurons with healthy cells of the same lineage underpins one current clinical approach that uses allografts of human fetal ventral mesencephalic (hfVM) tissue. However, many fetuses are required to graft a single individual, owing to the loss of harvested DA neurons denuded of their natural stromal microenvironment.Resultsex vivo. Moreover, human fetal DA neurons pre-coated with LIF-nano-stroma outnumbered cells pre-coated with empty-nano 12 weeks after transplantation into the striatum of nude rats. Notably, DA cells showed the greatest benefit from LIF-nano-stroma pre-coating. The authors also developed a nano-stromal construct using the small molecule XAV939 (XAV) as cargo. XAV is a potent inhibitor of the Wnt–β-catenin signalling pathway, which is involved in neural development. XAV-nano-stroma retained bioactivity and promoted the survival of hfVM ex vivo, including that of DA precursors.In this study, the authors pioneer the use of nanotechnology to model a surrogate ‘nano-stroma’ engineered to deliver proneural growth factors to targeted cells. They report that LIF-nano-stroma, a prototype biodegradable poly(lactic-co-glycolic acid) (PLGA)-based nanoparticle matrix (PLGA is FDA approved for therapeutic devices) loaded with leukaemia inhibitory factor  provided fourfold increased survival when targeted to DA neurons maintained Implications and future directionsThese findings provide the first demonstration that LIF-nano-stroma and XAV-nano-stroma both have pro-survival effects on human DA neurons. Moreover, they show that, to work, the nanoparticles must be attached physically to their target cells and must include a cargo – simple attachment of empty nano-stroma was not beneficial. More generally, these findings have implications for the application of nanotechnology to healthcare. Nano-medicine is a rapidly expanding field, and modelling the outcome of treatment is crucial for its progress. Notably, these findings highlight the potential of using surrogate nano-stromal niches to modulate the fate-determination pathways of precursor cells towards a desired phenotype. This approach might in future be used to support the translation of cell therapies derived from embryonic stem cells or induced pluripotent cells for neurodegenerative disease into clinical practice.in vivo, we previously developed LIF formulated as PLGA-nanoparticles (LIF-nano) and demonstrated that LIF-nano is superior to soluble LIF in being able to provide a stable source of low-dose LIF, mimicking paracrine delivery (in vivo for primary hfVM cells transplanted into the striatum of nude rats.Leukaemia inhibitory factor (LIF) is an IL-6 family member; LIF-specific activity is endowed by the LIF-specific receptor subunit gp190. LIF is attractive as a therapeutic agent in cell therapy for PD, being both proneural and reparative  as well delivery , with acdelivery . Likewisdelivery  as well delivery . TherefoIn addition to LIF-nano, we generated and tested a range of other novel nano-therapeutics as stromal candidates, including the small molecule XAV939 (XAV). XAV is a potent inhibitor of tankyrase , and thiPLGA formulation of the neurogenic factors was based on the successful LIF-nano construct with proven bioactivity in guiding T lymphocyte lineage differentiation . In the +) cells co-express gp190 and gp130. in vitro (DIV), with co-staining for TH plus gp190, and TH plus gp130. Unexpectedly, gp190 staining was nuclear. This subcellular stain is seen in the Human Protein Atlas (HPA) database  cells, known to include DA precursors, benefit from stromal support provided by LIF-nano? Because LIF signalling requires the heterodimeric receptor consisting of gp190 (LIF-specific subunit) and gp130 , we needed to confirm that the tyrosine-hydroxylase-positive  cells express GFRα-1 . Because serotoninergic -positive astrocytes  demonstr+ cells as a function of total cell counts, then counting the number of primary neurites per TH+ cell and measuring the longest neurite, the effect of signalling through TrkB was recorded as detailed in Because soluble BDNF has been reported to support DA cells  and incrThe BDNF and 7,8-DHF experiments established that different types of cell surface receptor are accessible to nanotherapeutic formulations, and that different types of molecule acting on the same cell surface receptor retain potency in nano-formulation. Of note, the cost of 7,8-DHF is some thousand-fold less than recombinant BDNF – an important consideration in the context of potential therapeutic application.When preparing donated primary human fetal tissue for cell transplantation into a recipient PD patient, loss of cells – already a scarce resource – is substantial throughout the procedure from harvest to grafting. This means that many fetuses are required to graft a single patient and there is a need to store the fetal tissues at +4°C in hibernation media for several days to allow for the accrual of sufficient fetal material for transplantation. If the yield of DA cells per donated hfVM could be increased, the benefits would be great because fewer donors would be required per patient. Accordingly, we went on to ask, can the nano-stromal approach protect and improve the yield of healthy human DA cells under conditions used for transplantation?Here, in addition to LIF-nano, we included nano-stromal cargos to target delivery of BDNF, GDNF and XAV, reasoning firstly that BDNF and GDNF are already known to be pro-survival for DA neurons , and secin vitro experiments, we first asked, does the type of growth medium (PM versus DM) influence the response of hfVM cells to nano-stromal delivery of cargo? Responses to the tested nano-stromal types were unaffected by PM versus DM, and + cells  neuronal cell numbers (left panel), total TH+ cell numbers (centre panel) and percent TH+ cells (right panel). Only XAV-nano had a selective effect, in that there was a greater increase in total neuronal cell numbers (sixfold) but the percentage TH+ cells decreased when compared with empty-nano controls  is used to prepare hfVM cells for clinical transplantation, whereas use of differentiation medium (DM) is required for the in vitro, we next wanted to know, does coating of hfVM cells with a nano-stroma increase TH cell survival following transplantation into the adult rat brain. Using the xenograft model outlined in + cells co-stained for HuNu in a graft pretreated with LIF-nano. + cells in the LIF-nano-treated group compared with empty-nano controls, but this failed to reach statistical significance . The stereology included recording HuNu+ cell distance from the needle tract, where there was evidence for increased migration in the LIF-nano-treated group  the need for a cargo to be delivered: simple nano-stromal cross-linking of cell surface antigen by targeting antibody did not constitute a pro-survival signal in the absence of cargo; and (2) the need to physically attach the stromal nanoparticles to the target cell: unbound cargo-carrying nanoparticles failed to have an effect.ex vivo treatment of therapeutic cells prior to grafting. Notably, we found no adverse effects following treatment of primary embryonic VM cells: indeed the more vulnerable DA population showed preferential survival following nanotherapy targeted to GFRα-1, or Thy-1. The technology platform used to prepare the nano-therapeutic particles is the same as that of BIND Therapeutics (http://www.bindtherapeutics.com), where clinical efficacy and safety are already established: notably, BIND technology is being used in ongoing clinical trials for non-small cell lung cancer, and for prostate cancer.The versatility of using nano-spheres to create a local stroma enables cells to be treated individually, a major advantage for Our experiments showed that a pro-survival effect can be achieved by nano-stromal delivery of either a biologic or a small molecule, and also when subcellular targets differ. Of the small molecules tested, 7,8-DHF acts at the plasma membrane as an agonist of TrkB; in contrast, XAV acts intracellularly, inhibiting tankyrase enzyme activity. Such versatility of specifically targeted cargos provides potential advantages when considering, for example, a multiplicity of agents to create a favourable niche during transplantation of neural precursor cells. Here, our finding of apparent stromal-derived interactions when XAV-nano is combined with LIF-nano is of interest: firstly, recent work has shown that XAV induces cytoplasmic retention of β-catenin/Axin2 ; and, se+ cells was detectable in rat E14 VM at 2 DIV, as evidenced by cells expressing cleaved caspase-3, before overt cell death within the TH+ population. This suggests that DA cells are especially vulnerable to stress at an early stage of differentiation. In vivo, the sustained benefit over 12 weeks gained by a single ex vivo treatment of primary hfVM cells demonstrates long-lasting effects, including early integration within the host tissue. These preliminary findings using hfVM support the use of LIF-nano in cell-based therapies, with the added value of tolerogenic properties. Furthermore, conceptually, future developments would include tailored nano-stromal support to provide a surrogate, transient, cell-specific stromal niche: for example, nano-stromal combinations using early delivery of factors linked to embryonic development such as LIF and/or XAV to promote precursor cells transiently, followed by delayed release of BDNF-nano or GDNF-nano, to guide lineage differentiation.A further point of note was the finding that the pro-survival effect of LIF-nano on THImproved survival of DA neurons in cell-based transplantation therapies for PD is needed for this therapy to become a clinical reality. Here, using the highly relevant, stringent model of primary DA cell survival and maturation, we demonstrate that nanoparticles used to create a nano-stroma designed to deliver developmental growth factors, or small molecules, to single cells provides a simple and effective means to support DA cell survival, including primary hfVM cells.Cargos of either recombinant growth factor or small molecule included recombinant murine LIF  or human LIF ; recombinant human BDNF and human GDNF (R&D Systems); 7,8-DHF (Sigma); and XAV939 (Selleckchem). Cargo was encapsulated in avidin-coated PLGA nanoparticles using a modified version of a previously described water/oil/water double emulsion technique . BrieflyCulture medium was either for proliferation (PM), consisting of Neurobasal A (Invitrogen), 2% B-27 supplement, 100 U/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin B, 2 mM L-glutamine, 20 ng/ml epidermal growth factor and 20 ng/ml fibroblast growth factor-2, or for differentiation (DM), which was made up of Dulbecco’s Minimal Essential Medium, 10% fetal calf serum, 2% B-27, 100 U/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B.2 air. Subsequent medium exchanges involved 50% of the medium being removed and replaced but without nanoparticles. Cultures were fixed for analyses of the total βIII-tubulin+  and TH+  cell numbers as well as serotoninergic cells  and glia  cells: apoptosis was assessed using caspase-3 release using standard techniques . To measure the time course of any therapeutic effects, replicate cultures were analysed after 1, 2, 3, 4 and 5 DIV.VM cultures from time-mated Sprague-Dawley rats at E14 followed the protocol described previously . After ain vitro experiments, nano-stroma-coated hfVM cells were cultured in differentiation medium for 4 days before fixation and staining for HuNu and TH as previously described  and processed for culture as outlined in escribed .For transplantation, unbound nanoparticles were removed by centrifugation prior to transplanting into the left striatum of male Nu-Nu rats aged 12 weeks as described previously . Specifiin vitro experimental treatment was repeated three times using different donor sources. Within each experiment, five replicate counts/coverslip were made with four coverslips/therapy analysed by ANOVA to give standard error of the mean (s.e.m.).Immunocytochemistry of human cells grown on coverslips followed standard procedures. Primary antibodies were diluted in blocking solution and applied overnight at 4°C using rabbit anti-TH  and mouse anti-βIII tubulin  followed by secondary antibodies for 2 hours at 4°C: antibodies were from Millipore Molecular Probes, including anti-rabbit IgG  and anti-mouse IgG . Cells were counterstained with DAPI (1 μg/ml). Immunohistochemistry of grafted brains used 40-μm coronal sections collected sequentially into 24-well plates. Sections were taken at regular intervals  and stained for HuNu  and TH  using standard protocols. Similarly, a further series of sections were stained for human-specific tau. Each t-test. *P<0.05; **P<0.01; ***P<0.001. Further statistical information is provided in the figure legends.Data are presented as mean ± s.e.m. Multiple comparisons in the same data set were analysed by a one-way ANOVA; pairwise multiple comparison procedures were used for post-hoc analysis. Single comparisons to controls were made using a two-tailed Student’s


DMD gene. The DMD gene, spanning over 2.4 megabases along the short arm of the X chromosome (Xp21.2), is the largest genetic locus known in the human genome. The size of DMD, combined with the complexity of the DMD phenotype and the extent of the affected tissues, begs for the development of novel, ideally complementary, therapeutic approaches. Genome editing based on the delivery of sequence-specific programmable nucleases into dystrophin-defective cells has recently enriched the portfolio of potential therapies under investigation. Experiments involving different programmable nuclease platforms and target cell types have established that the application of genome-editing principles to the targeted manipulation of defective DMD loci can result in the rescue of dystrophin protein synthesis in gene-edited cells. Looking towards translation into the clinic, these proof-of-principle experiments have been swiftly followed by the conversion of well-established viral vector systems into delivery agents for DMD editing. These gene-editing tools consist of zinc-finger nucleases (ZFNs), engineered homing endoculeases (HEs), transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases (RGNs) based on clustered, regularly interspaced, short palindromic repeats (CRISPR)–Cas9 systems. Here, we succinctly review these fast-paced developments and technologies, highlighting their relative merits and potential bottlenecks, when used as part of in vivo and ex vivo gene-editing strategies.Duchenne muscular dystrophy (DMD) is a genetic disorder caused by mutations in the dystrophin-encoding  DMD gene [DMD locus, with 66 % nested within a major, recombination-prone, hotspot region spanning exons 45 through 55 [Duchenne muscular dystrophy (DMD) is a lethal X-linked genetic disorder (affecting approximately 1 in 5000 boys) [DMD gene  which leDMD gene . AlthougDMD gene . These dIn muscle cells, the long rod-shaped dystrophin protein anchors the intracellular cytoskeleton to the extracellular matrix via a large glycoprotein complex embedded in the plasma membrane called the dystrophin-associated glycoprotein complex (DGC). This structural link is fundamental for proper cellular signaling and structural integrity. Indeed, in the absence of dystrophin, a relentless degenerative process is initiated that consists of the substitution of muscle mass by dysfunctional fibrotic and fat tissues . As timeThe complexity of DMD, combined with the extent of affected tissue, demands the development of different, ideally complementary, therapeutic approaches. The goal of pursuing parallel approaches is to target different aspects and stages of the disease and hence maximize the length and quality of patients’ lives. Towards this end, various candidate therapies are currently under intense investigation , 5, 6. TStreptococcus pyogenes Cas9 and its smaller orthologue Staphylococcus aureus Cas9, respectively) [More recently, genome-editing strategies based on sequence-specific programmable nucleases have been proposed as another group of therapies for DMD –10. Progctively) , 24. Henctively) –13.Fig. trans-acting and cis-acting genomic elements [Regardless of the DNA cutting system that is selected, the repair of the ensuing DSBs by different endogenous cellular DNA repair processes can yield specific genome editing outcomes. For example, the engagement of homologous recombination (HR) and non-homologous end joining (NHEJ) mechanisms can result in targeted exogenous DNA additions and endogenous DNA deletions, respectively –13. The elements –13. By oDMD editing based on targeted addition of “exon patches” corresponding to missing or disrupted coding sequences might become ideal therapeutic options as they result in the synthesis of full-length dystrophin [DMD-repairing exon patches with engineered meganucleases [DMD editing approaches under investigation are based on inducing NHEJ to disrupt or delete specific sequences [DMD transcripts, despite being shorter than the full-length DMD transcript, often yield functional dystrophins [DMD alleles by targeting: (1) splicing sites for inducing DNA-borne exon skipping; (2) exonic sequences for resetting reading frames and “overwriting” downstream premature stop codons; and (3) flanking intronic sequences for directly excising mutations through the use of pairs of programmable nucleases (multiplexing) [strophin , 25. Proucleases  can indeequences –10. Thesequences , 27, whitrophins –30. Indetrophins –30. Therplexing) .Table 1CDMD correction approaches based on targeted multi-exon deletions do not give rise to indel-derived epitopes and are applicable to a wider range of DMD-causing genotypes, with de novo-generated intronic junctions leading to predictable in-frame mRNA templates [In contrast to those triggering single-exon deletions, the emplates , 32. Howemplates . These iDMD-editing concepts will require improved methods for delivering large and complex molecular tools into target cells, as well as increasing the efficiency, specificity, and fidelity of the ensuing DNA modifications [DMD-editing agents in both patient-derived cells and mouse models of DMD [The clinical application of ications . Similarications , viral vications ). Indeedications –37  dystrophic phenotype that is due to a nonsense mutation located in exon 23 of the Dmd gene; historically, this has been the principal animal model for investigating DMD-targeted therapies and certain pathophysiological aspects of the disease [S. pyogenes Cas9 or sgRNAs that targeted sequences flanking Dmd exons 21 through 23 were co-injected into the gastrocnemius muscles of newborn Dmdmdx mice [The  disease . In one mdx mice . At 3 weA notorious characteristic of prototypic serotype-5 AdVs is their immunogenicity and, although they can be made without viral genes , 44, capDmd exon 23. These rAAV particles consist of rAAV DNA from serotype 2 packaged in capsids from AAV serotype 8 (rAAV-8) [S. pyogenes Cas9 [S. aureus Cas9 [Dmdmdx mice. Nelson and colleagues detected abundant dystrophin protein synthesis 8 weeks after co-injecting a mixture of rAAV-8 particles encoding S. aureus Cas9 and cognate sgRNAs into tibialis anterior muscles [Three other studies investigated the in vivo delivery of RGN components  by capsid-pseudotyped rAAVs for inducing the in-frame deletion of (rAAV-8)  or serot(rAAV-8) , 41, who(rAAV-8) , 51. Paines Cas9  or the seus Cas9 , 41 were muscles . Importa muscles , Nelson  muscles .S. pyogenes RGN complexes into striated muscle tissues of newborn Dmdmdx mice [Dmdmdx mice subjected to direct intramuscular injections with the engineered viral vector contained approximately 8 and 26 % of dystrophin-positive fibers at 3 and 6 weeks post-administration, respectively [Instead of rAAV-8, Long and colleagues used rAAV-9 to introduce mdx mice . Dystropmdx mice . Consistmdx mice , a time-ectively .S. aureus Cas9 and sgRNAs to the tibialis anterior muscle of dystrophin-defective Dmdmdx mice [Dmd exon 23 deletion experiments [Dmd exon 23 excision in cardiac and skeletal muscle tissues ranging from 3 to 18 %, as determined by RT-PCR, depending on the muscle groups analyzed [Dmd-editing rAAV-9 particles were also administered intramuscularly or systemically to Pax7-ZsGreen Dmdmdx mice whose satellite cells are marked by green fluorescence. Subsequently, after isolating, expanding, and inducing myogenic differentiation of the Pax7-ZsGreen-positive cells, the authors reported in-frame Dmd exon 23 deletions in myotubes derived from these cells [Dmdmdx mice showed that, in addition to its other functions, dystrophin has a transient but critical regulatory role in activated Pax7-positive satellite cells, which further supports the therapeutic relevance of this cell population. In particular, the 427-kilodalton dystrophin isoform is expressed at very high levels in these cells, where it governs asymmetric cell division, a process that is indispensable for maintaining the stem cell pool and for generating committed Myf5-positive myoblast progenitors for muscle repair [DMD editing, as this strategy is expected to restore proper regulation of dystrophin synthesis.In the third study, Tabebordbar and coworkers used rAAV-9 pairs for delivering mdx mice . Similareriments , 40, admeriments . In addianalyzed . Importase cells . The popse cells . The “stse cells , 55. Rece repair . Among oe repair , 57. If e repair . Such diDMD-repairing agents, thus warranting further research. Future developments should include assuring the transient presence of programmable nucleases in post-mitotic tissues, preclinical testing in large outbred animal models [Taken together, these findings demonstrate that rAAV delivery of RGN complexes can result in the structural improvement of treated striated tissues and also lead to the partial rescue of specific muscle functions in dystrophic mice. Although dystrophin synthesis was detected at 6 months after a single injection in one experiment , no longl models , and idel models .DMD editing, the synthesis of programmable nucleases should be restricted not only spatially but also temporally.The administration of rAAVs to some human individuals resulted in clinical endpoints that had not been predicted on the basis of the available preclinical data. These findings are simultaneously sobering and illuminating. An example is provided by the elimination of transduced hepatocytes in patients with hemophilia B, which was due to the development of a dose-dependent T-cell response to capsid epitopes from an rAAV-2-encoding human factor IX . This tyDMD editing strategies to generate genetically corrected human cells with myoregenerative capacity for autologous transplantation can also be envisaged  and, in doing so, enable the in vitro survival and self-renewal of bona fide satellite cells [Despite these encouraging developments, the hurdles towards the clinical application of ex vivo DMD cell therapies remain numerous and complex. Preeminent examples of such hurdles include achieving sufficient numbers of undifferentiated cells in vitro, as well as robust cell engraftment, migration, and differentiation of the transplanted graphs in vivo. Ideally, the transplanted cells should also be capable of homing to damaged tissue after systemic administration and should dedifferentiate or transdifferentiate  into satellite cells Table . Therefotrophies , 79 but trophies . Howevertrophies . Importate cells . The devIn addition to that of skeletal muscle, cardiac muscle impairment is a key component of DMD that also needs to be tackled in future therapies. Despite intense research on the isolation and characterization of stem and progenitor cells for the repair of damaged heart tissue , so far there is no evidence for a significant functional improvement of the myocardium through the cell-autonomous differentiation of the transplanted cells into mature, electrically coupled cardiomyocytes , 90.DMD alleles and yielded permanent and regulated dystrophin synthesis. In this work, we exploited targeted NHEJ-mediated correction of DMD-causing intragenic deletions by reading-frame resetting, DNA-borne exon skipping, and in-frame excision of single or multiple exons [DMD editing reagents and strategies in panels of human myogenic cells harboring the various DMD mutations, which are not represented in the currently available animal models. In addition, the well-defined in vitro conditions permit the straightforward monitoring of intended as well as unwarranted or potentially deleterious interactions between the gene-editing reagents and the human genome  deepening our knowledge about the origins and biology of the various cell therapy candidates and their interaction(s) with their respective niches; (2) gathering all possible information on the fate and behavior of transplanted cells from ongoing and future cell therapy trials; (3) moving forward with gene replacement approaches involving stable transduction of recombinant constructs; and (4) testing different gene-editing reagents and strategies for developing autologous cell transplantation approaches. Regarding the latter research avenue, it will be crucial to efficiently introduce different gene-editing tools into human muscle progenitor cells and non-muscle cells with myogenic capacity. AdVs outperform rAAVs in ex vivo settings owing to their higher functional vector particle titers, larger packaging capacity (up to 37 kb), and faster kinetics of transgene expression , 52. Ourle exons . The resle exons . Bypassi deepeninDMD repair purposes is expanding the range of genetic therapy options for tackling DMD. In this context, the coopting of viral vector systems as carriers of programmable nucleases is set to have an important role in the path to DNA-targeted DMD therapies and, along the way, in defining the best strategies and optimizing the corresponding reagents. In view of the complexity of the DMD phenotype and the extent of the affected tissues, it is sensible to consider that future DMD therapies will profit from integrating complementary approaches. For example, the simultaneous treatment of skeletal and cardiac tissues from patients with DMD might be approached by combining ex vivo and in vivo gene-editing strategies, respectively. Such schemes can potentially address the skeletal and heart components of DMD while circumventing the current lack of cell entities capable of differentiating into functional cardiomyocytes. Regardless of the particular therapy or combination of therapies ultimately selected, there is widespread agreement that they should preferably be applied as early as possible so that most striated musculature is still in place and the degeneration process can be halted or, ideally, reversed in the treated muscle groups. Finally, the insights gained from these DMD-targeted research efforts will probably also be useful for devising advanced genetic therapies for addressing other neuromuscular disorders for which, at present, there are no therapeutic options available.The application of genome-editing principles for


Here, we show that Midline (Mid), an evolutionarily conserved transcription factor, expresses in the wing disc of Drosophila and plays a vital role in wing development. Loss or knock down of mid in the wing disc induced hyper-expression of wingless (wg) and yielded cocked and non-flat wings. Over-expression of mid in the wing disc markedly repressed the expression of wg, DE-Cadherin (DE-Cad) and armadillo (arm), and resulted in a small and blistered wing. In addition, a reduction in the dose of mid enhanced phenotypes of a gain-of-function mutant of hedgehog (hh). We also observed repression of hh upon overexpression of mid in the wing disc. Taken together, we propose that Mid regulates wing development by repressing wg and hh in Drosophila.Wingless (Wg) and Hedgehog (Hh) signaling pathways are key players in animal development. However, regulation of the expression of  The development of multicellular organisms relies on participation and coordination of several signaling pathways that contribute to regulation of organ growth and pattern. Aberrant activity of these pathways can lead to many human diseases including cancers123Drosophila wing disc, as a gradient morphogen, Wg protein is secreted from the producing cells and acts at long range to activate target gene expression in surrounding cellswg in cells along the dorsal-ventral (DV) boundary1113151617wg is activated by Dachs and repressed by Expanded and Hinge11920The evolutionarily conserved Wg signaling pathway plays a crucial role in growth and pattern formation during development5671Drosophila wing disc, Hh regulates cell proliferation and cell fate specification2530decapentaplegic (dpp), ptc and en32hh is activated by Dlp and repressed by Ihog and Hyd34The Hh signaling pathway is evolutionarily conserved and plays an essential role in embryonic development and adult tissue patterning2223125In the nucleus, several common regulators are shared by more than one pathways, such as: Groucho (Gro), functions as a corepressor in the downstream of the N, Wnt, Hh, Dpp and EGFR pathways36Drosophila homolog of Tbx20 in vertebrates. As an evolutionarily conserved T-box family transcription factor, Mid plays a vital role in cell fate specification and tissue morphogenesis during a series of developmental processes4041424344mid and its paralog H15 are directly activated by Tinman (Tin) and required for the formation and specification of cardioblast464748mid and H15 function as selector genes to specify ventral fate in the leg discMid is the In this study, we analyzed the function of Mid in the wing disc and its role during wing development. Our study reveals a novel role for Mid as an important regulator of the Wg and Hh signaling pathways.To study the role of Mid in wing development, we first analyzed the expression pattern of Mid in the wing imaginal disc using immunostaining with anti-Mid antibody. In wild-type third instar larvae, Mid ubiquitously expressed in the nucleus marked by TOTO-3 in both peripodial epithelium (PE)  and the mid function. Considering the lethality of mid null homozygotes during late embryogenesis (mid1 and mid2), we expressed mid RNAi using MS1096-GAL4 or sd-GAL4 to knock down mid in pouch and margin cells of the wing disc. The expression patterns of MS1094-GAL4 and sd-GAL4 are schematically illustrated in mid resulted in defect wings -GAL4, we observed a wider space between vein 3 and vein 4  gradient of DE-Cad and normal cell shape , suggesting that Mid represses Wg, at least partly, in an indirect manner. Therefore, Mid possibly attenuates Wg through two mechanisms, direct and indirect. Further studies are necessary to dissect the detailed mechanisms.What is the underlying mechanism of the Mid-mediated repression on Wg? Mid could directly repress hh mRNA was enhanced in the mid mutant. Secondly, using anti-Hh antibody and hh-lacZ reporter, we observed reduced expression of Hh upon overexpression of mid in the wing disc. Thirdly, mid reduction can enhance the wing phenotype of hhMrt, a gain-of-function allele of hh. Lastly, we observed decrease in the expression of a hh target dpp in response to overexpression of mid. Overexpression of mid reduced the expression of hh-lacZ in a non-cell autonomous manner. Therefore, the repression of hh by Mid is not direct but mediated through an unknown mechanism.We also demonstrated that Mid acts as a negative regulator of the Hh pathway  from foumid function. Knock down of mid in the wing disc induced hyper-expression of Wg and its downstream DE-Cad, and yielded the cocked and non-flat wings. The hyper-expression of DE-Cad would lead to abnormally strong cell-cell adhesion and epithelial misfoldingMS1096-gal4 driver is mainly expressed in the dorsal compartment -GAL4, hhMrt, UAS-mid, UAS-wg, UAS-GFP, UAS-wg-RNAi, hh-lacZ were originally obtained from Bloomington Stock Center. sd-GAL4 was obtained from Drosophila Genetic Resource Center. UAS-mid-RNAi was originally obtained from NIG-Fly. All fly stocks were grown at 25 °C. Mutant clonal analyses were done with hs-FLP; FRT42D, Ubi-GFP/CyO and FRT42D, mid1/CyO. FLP-out clonal analyses were done with hs-FLP; UAS-mid/CyO and act > y + > gal4;UAS-GFP.Following stocks were used: The antibodies were used at the following dilutions: rabbit anti-Mid 1:500050, mousImages were acquired under Leica TCS SP5 confocal microscope, Olympus DP2-BSW microscope and Olympus cellSens, processed using Adobe Photoshop CS6.mid heterozygote or mutant embryos were identified using CyOact-GFP balancer chromosome. Total RNA was extracted separately for each of the heterozygous and homozygous mid mutant embryos using the QIAGEN RNeasy isolation kit. The probes labeling and hybridization were performed as described previouslywww.ncbi.nlm.nih.gov/geo) under accession number GSE73802.The TM II 1st strand cDNA synthesis kit (TaKaRa #6210A) was used for cDNA synthesis. qPCR was performed in a 20 ul reaction containing 2 pmol of relevant primers . Prime Script primers  in Bio-RHow to cite this article: Fu, C.-L. et al. The T-box transcription factor Midline regulates wing development by repressing wingless and hedgehog in Drosophila. Sci. Rep.6, 27981; doi: 10.1038/srep27981 (2016).


The central nervous system (CNS) possesses powerful local and global immunosuppressive capabilities that modulate unwanted inflammatory reactions in nervous tissue. These same immune-modulatory mechanisms are also co-opted by malignant brain tumors and pose a formidable challenge to brain tumor immunotherapy. Routes by which malignant gliomas coordinate immunosuppression include the mechanical and functional barriers of the CNS; immunosuppressive cytokines and catabolites; immune checkpoint molecules; tumor-infiltrating immune cells; and suppressor immune cells. The challenges to overcoming tumor-induced immunosuppression, however, are not unique to the brain, and several analogous immunosuppressive mechanisms also exist for primary tumors outside of the CNS. Ultimately, the immune responses in the CNS are linked and complementary to immune processes in the periphery, and advances in tumor immunotherapy in peripheral sites may therefore illuminate novel approaches to brain tumor immunotherapy, and vice versa. Contrary to common perceptions of central nervous system (CNS) “immune privilege,” the brain can in fact elicit vigorous immune-stimulatory as well as immunosuppressive responses, the determinants of which are highly contextual. Understanding the determinants and mechanisms of both the stimulatory and suppressive responses may help elucidate novel immune-based strategies for brain tumor immunotherapy. In this review, we will discuss routes of glioma-mediated immunosuppression, including mechanical and functional barriers of the CNS, immunosuppressive cytokines, immune checkpoint molecules, tumor-infiltrating immune cells, and suppressor immune cells Table . In addiThe notion of “immune privilege” has long been ascribed to tissues wherein the immunological responsiveness is ostensibly blunted or modified . Early eIn recent decades, the consensus view of the CNS immune environment has shifted from one in which the blood–brain barrier (BBB) serves as a static barrier against the exchange of cells and soluble molecules into one in which egress and entry are dynamically regulated, often by mechanisms observed in other organ systems. During inflammation, immune cells migrate into the CNS parenchyma following dynamic gradients of chemotactic cues, including IFN-γ inducible cytokines , 124, α In vivo tracer studies have demonstrated that CNS antigens drain via cerebrospinal fluid across the cribiform plate and into the nasal sub-mucosa , B lymphocytes, and vascular pericytes may provide other cellular sources for CNS tumor antigen presentation. TAMs, which infiltrate gliomas in large numbers and possess cross-presentation capabilities , are thoin vitro . To our in vitro . Given tin vitro , it is pfms-like tyrosine kinase 3 ligand (Ad-Flt3L), which were used to kill tumor cells and recruit APCs to the microenvironment, Candolfi et al. showed that treatment with Ad-TK + Ad-Flt3L produced long-term survivors in 60% of WT mice but 0% in B-cell depleted mice β7 integrin, the latter of which was found to be important for T-cell retention within the brain (E(CD103)β7 integrin compared to fewer than 5% of CD8+ T cells in peripheral blood (E(CD103)β7-expressing CD8+ T cells in human glioma tissue also co-expressed granzyme B (E(CD103)β7 expression in non-CNS sites (E(CD103)β7 expression on T cells within gliomas  tumor sizes and increased T-cell infiltration at 14 days post-implantation compared to mice with IL-10− tumors . Additio− tumors .More recently, Vleeschouwer et al. reported that persistent and elevated IL-10 production by T-cells was in fact required for T-cell suppression of glioma growth following stimulation with tumor lysate-loaded dendritic cells . EctopicWhile IL-10 also plays a duplicitous role in tumor suppression and progression at tissues outside of the CNS, its biological actions in peripheral sites also differ in several important ways. IL-10 mRNA and protein have been isolated from a variety of human tumors, including ovarian , breast At the same time, other peripheral tumors, similar to gliomas, may also rely upon TAMs to produce the majority of IL-10. HPV-16 associated carcinomas, for example, have been shown to recruit TAMs, which produce the majority of IL-10 . WhetherIn certain peripheral tumors, IL-10 also appears to have a particularly strong stimulatory effect on NK cells . In a muin vitro data suggest that the pro-proliferative effects of IL-10 in malignant gliomas may outweigh the inhibitory effects  techniques was shown to prevent NKG2D down-regulation on NK cells and enhance MICA expression on glioma cells  , 246. IDIndolamine 2,3-dioxygenase 1 is not expressed in the brain under normal physiological conditions. IDO mRNA, however, is substantially elevated in human glioma tissues and correlates negatively with overall survival , 61. SimRecent data also indicate that glioma cells, rather than TAMs, microglia, and DCs, directly produce the majority of the IDO , which iAlthough tumors both in the brain and the body can exploit IDO-mediated immunosuppression to overcome host anti-tumor immune responses, molecular inhibition of IDO activity has produced different responses in different organs, which may reflect unique tissue-specific factors.In a murine breast tumor model, 1-methyl-tryptophan (1-MT), a widely studied inhibitor of IDO, failed to inhibit tumor growth ; howeverInterestingly, administering 1-MT with anti-CTLA-4 and anti-PD-L1 monoclonal antibodies produced a 100% long-term survival rate in glioma-bearing mice , an imprLastly, differential patterns of IDO expression among tumor types may also impact therapeutic efficacy of IDO modulation. Recent tissue analysis of 15 human tumor types showed that IDO expression was largely restricted to tumor cells, myeloid-lineage cells, and endothelial cells . The disRegulatory T lymphocytes (Tregs) are a highly diverse and plastic subset of CD4+ immunosuppressive helper T cells that play an essential role in promoting immunological tolerance , 253. AsCD4+ Tregs comprise approximately 5–10% of circulating CD4+ T cell population, and, based on developmental origin, Tregs are classified as either thymus-derived natural Tregs (nTregs) or peripherally induced “adaptive” Tregs (iTregs) . SubsetsnTregs develop in the thymus from CD4+ single-positive thymocytes via antigen presentation by thymic epithelial cells . nTregs By comparison, iTregs, which encompass several distinct CD4+ T cell types , differeUltimately, whether Tregs are thymically or peripherally derived, Tregs are capable of wholesale suppression of innate and adaptive effector immune cell function . From a Greater numbers of glioma-associated Tregs has been associated with higher tumor grade , and levin vitro migration studies have demonstrated the ability of glioma-derived CCL22 to induce Treg chemotaxis  have been identified, including monocyte-derived tumor-associated macrophages (TAMs); angiogenic monocytes; immature, immunosuppressive myelomonocytic cells known as MDSCs; tumor-associated neutrophils (TANs); as well as microglia within the CNS . Their eMicroglia and monocyte-derived macrophages  together account for the majority of glioma-associated myeloid cells , 159. MiSimilar to monocyte-derived macrophages, both microglia and TAMs can embody pro-inflammatory (M1) as well as immunosuppressive (M2) phenotypes depending on environmental cues , 297. Inhigh/CD45low and TAMs expressing CD11bhigh/CD45high, but the reliability of these markers in practice remains controversial , which, for example, could trigger apoptosis in activated T cells by decreasing Bcl-2 expression , while me arrest –328. Adde arrest . In a stin vitro .In gliomas, the majority of circulating MDSCs appear to be predominately granulocytic . InteresMyeloid-derived suppressor cell-targeted immunotherapy is an area of active research. For example, various murine glioma models have shown that depletion of MDSCs, either via COX-2 inhibition , antibodImmunosuppressive myeloid cells are not unique to CNS tumors and are equally important facilitators of tumor growth and invasion in peripheral sites as well. Higher density of tumor-associated macrophages (TAMs) has been associated with poorer prognosis in several human cancers, including breast, prostate, bladder, colorectal, and gastric cancers . IncreasAlthough the relative distribution of microglia and TAMs in gliomas has yet to be fully characterized (see above discussion), an intriguing observation that the majority of tumor-infiltrating mononuclear phagocytes in murine gliomas may represent monocyte-derived macrophages rather than native microglia suggests that monocyte-derived macrophages may play a significant role in coordinating glioma growth . Immunotin vitro data from Rodrigues et al. revealed that direct contact between monocytes and glioma cells was needed to induce an MDSC-like phenotype in monocytes , a co-inhibitory molecule expressed on activated T-cells and Treg cells. Ipililumab, an mAb directed against CTLA-4, was the first therapy to procure survival benefits for patients with metastatic melanoma , providiNumerous immune checkpoint molecules have subsequently been identified  and holdProgrammed cell death protein-1 ligand  is a trans-membrane glycoprotein of the B7 family of co-stimulatory molecules with potent immune-regulatory properties , 342. UnAside from endothelial cells of the BBB, PD-L1 is usually not expressed in the CNS –347, andIn addition to glioma cells, TAMCs provide another source of PD-L1. Microglia are known to upregulate PD-L1 expression under inflammatory states , and micNotably, in the first study that extensively characterized the role of neurons in the GBM microenvironment in inhibiting tumor growth, Liu et al. reported that high levels of neuronal PD-L1 expression in tumor-adjacent brain tissue (TABT) corresponded favorably with overall patient survival, and low TABT PD-L1 expression and high GBM PD-L1 expression portended poorer patient survival . MechaniThe biological pathways implicated in enhancing PD-L1 expression in gliomas are equally important to the development of immunoresistance in tumors outside of the CNS. Expression of PD-L1 on colorectal carcinomas, for example, has also been linked to loss of PTEN tumor suppressor . SimilarAt the same time, other malignancies also utilize distinct signaling pathways to enhance PD-L1 expression, requiring that targeted molecular suppression of PD-L1 be tailored toward each site based on tumor-specific biology. For example, MyD88/TRAF6 and MEK/ERK pathways enhance PD-L1 expression in multiple myeloma , while cTargeting the PD-L1/PD-1 axis by indirectly modulating PD-L1-bearing TAMs may also prove to be relevant strategy for CNS and non-CNS tumors alike , 110. HeLastly, the astonishing discovery that neuronal expression of PD-L1 in the tumor microenvironment protects against gliomagenesis  may alsoBeyond PD-L1, effector immune cells express myriad checkpoint molecules that also contribute tumor immunoresistance both in brain and body. Targeting cytotoxic T-lymphocyte antigen 4 (CTLA-4), which modulates early stages of T-lymphocyte activation, has proved useful in reversing immunoresistance in gliomas, as it has in non-CNS tumors –355. In A major challenge for the field of brain tumor immunology lies in elucidating key determinants and constituents of the pro-inflammatory and anti-inflammatory responses in the CNS such that they might be augmented for therapeutic gain. Contrary to the historical view of the CNS as immunologically sequestered from the rest of the body, the immune responses in the CNS are linked and complementary to immune processes in the periphery. The phenotype of the immune response often hinges upon cytokines and cellular mediators that exert highly pleiotropic and sometimes paradoxical actions depending on the specific tumor and environmental context. In evaluating these processes, however, it will be helpful to recognize that routes by which the CNS coordinates immune modulation are not without precedent: analogous immunological mechanisms exist at sites outside of the CNS, and advances in tumor immunotherapy in peripheral sites may therefore illuminate novel approaches to brain tumor immunotherapy, and vice versa . TherefoThe 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.


Default-mode network (DMN) functional connectivity and its task-dependent down-regulation have attracted a lot of attention in the field of neuroscience. Nevertheless, the exact underlying mechanisms of DMN functional connectivity, or more specifically, the blood oxygen level-dependent (BOLD) signal, are still not completely understood. To investigate more directly the association between local glucose consumption, local glutamatergic neurotransmission and DMN functional connectivity during rest, the present study combined for the first time 2-Deoxy-2-[18F]fluoroglucose positron emission tomography (FDG-PET), proton magnetic resonance spectroscopy (1H-MRS), and resting-state functional magnetic resonance imaging (rs-fMRI). Seed-based correlation analyses, using a key region of the DMN i.e. the dorsal posterior cingulate cortex as seed, revealed overall striking spatial similarities between fluctuations in FDG-uptake and the BOLD signal. More specifically, a conjunction analysis across both modalities showed that DMN areas as the inferior parietal lobe, angular gyrus, precuneus, middle and medial frontal gyrus were positively correlated with the dorsal posterior cingulate cortex. Furthermore, we could demonstrate that local glucose consumption in the medial frontal gyrus, posterior cingulate cortex and left angular gyrus was associated with functional connectivity within the DMN. We did not find a relationship between glutamatergic neurotransmission and functional connectivity. In line with very recent findings, our results provide further evidence for a close association between local metabolic activity and functional connectivity and enable further insights towards a better understanding of the underlying mechanisms of the BOLD signal.


Objectives: Individuals with chronic traumatic brain injury (TBI) often showdetrimental deficits in higher order cognitive functions requiring coordination ofmultiple brain networks. Although assessing TBI-related deficits in higher order cognitionin the context of network dysfunction is promising, few studies have systematicallyinvestigated altered interactions among multiple networks in chronic TBI.Method: We characterized disrupted resting-state functional connectivity ofthe default mode network (DMN), dorsal attention network (DAN), and frontoparietal controlnetwork (FPCN) whose interactions are required for internally and externally focusedgoal-directed cognition in chronic TBI. Specifically, we compared the network interactionsof 40 chronic TBI individuals (8 years post-injury on average) with those of 17 healthyindividuals matched for gender, age, and years of education. Results: Thenetwork-based statistic (NBS) on DMN-DAN-FPCN connectivity of these groups revealedstatistically significant  reductions in within-DMN,within-FPCN, DMN-DAN, and DMN-FPCN connectivity of the TBI group over healthy controls.Importantly, such disruptions occurred prominently in between-network connectivity.Subsequent analyses further exhibited the disrupted connectivity patterns of the chronicTBI group occurring preferentially in long-range and inter-hemispheric connectivity ofDMN-DAN-FPCN. Most importantly, graph-theoretic analysis demonstrated relative reductionsin global, local and cost efficiency (p<.05) as a consequence ofthe network disruption patterns in the TBI group. Conclusion: Our findingssuggest that assessing multiple networks-of-interest simultaneously will allow us tobetter understand deficits in goal-directed cognition and other higher order cognitivephenomena in chronic TBI. Future research will be needed to better understand thebehavioral consequences related to these network disruptions.  A traumatic brain injury (TBI) is induced by external force, leading to abnormalities inbrain structure and function. Individuals with chronic TBI (6-months or more post-injury)often have difficulties in cognitive functions such as attention, memory and executivefunctions, which are critical to daily life tasks. One of the most relevant mechanisms ofsuch deficits following TBI is diffuse axonal injury  studies in TBI have provided valuableinformation for a better understanding of TBI, most of these previous studies have focusedon investigating connectivity alterations within a single-network or patterns ofconnectivity for a single-brain-region with rest of the brain. For example, TBI-relateddisruptions of functional connectivity within the default mode network (DMN), which is“de-activated” when subjects engage in tasks .Goal-directed cognition is key in everyday life as it represents an ability to coordinatethoughts and actions to achieve goals while adjusting these goals in the context of changingtask demands. Individuals with chronic TBI often exhibit difficulties in goal-directedcognition  TBI screening form  forthe participants, using the Beck Depression Inventory-II  and PTSD Check List Stressor-specific . Note that we did not acquire PCL-S scores from the healthy controlparticipants, as PCL-S requires reporting on a specific traumatic event that participantexperiences. The healthy individuals, by definition, did not experience traumatic eventsto be reported on for the PCL-S assessment.We administered neuropsychological tests at the time of MRI scanning to characterizeparticipants’ cognition in a variety of domains. These tests included similarities, matrixreasoning, and full scale intelligent quotient-2 (FSIQ-2) from the Wechsler AbbreviatedScale of Intelligence (WASI) for estimated current IQ /echo time(TE)=2000/30 ms; flipangle (FA)=80°; field of view (FOV)=22.0×22.0 cm; matrix=64×64; 37 slices, 4.0 mm thick].Total number of rsfMRI runs was different across the participants because, at the earlystage of our study, we observed that the QA procedures with only one rsfMRI run yieldedhigh rates of participant exclusion. Thus, we additionally acquired two rsfMRI runs forthe remainder of the data collection . During rsfMRIacquisition, the participants were asked to remain still with their eyes closed. ForrsfMRI alignment, we obtained one high resolution T1-weighted image of thewhole brain  for each participant, using the same head coil.The participants underwent MRI scanning on a Philips Achieva 3 Tesla (T) scanner  at the Advanced Imaging Research Center. In each imagingsession, one or two 416-s runs of rsfMRI were acquired using a standard 32-channel headcoil with Thttp://afni.nimh.nih.gov/pub/dist/doc/program_help/afni_proc.py.html) from AFNI, normalization to whole brain mode of 1000, band-pass filtering (0.009<f <0.08 Hz), and linear regression.RsfMRI data were preprocessed with standard methods using a modified version of a shellscript generated by afni_proc.py  six parameters for the rigid body head motion acquired fromthe motion correction of 1.8 to prevent potential motion artifacts  analysis with covariates of within-group-centered BDI-IIscores on each of the connectivity matrix elements due to statistically significantgroup differences in BDI-II scores (p<10−4), introducing co-linearity into the designmatrix.To obtain a connectivity matrix for each of the subjects, we first defined nodes as 43regions  affiliated with the DMN, DAN, and FPCN  with correction for multiple comparisons at p<.05 usingnetwork-based statistic . Atotal of 10,000 permutations were generated to estimate the null distribution of maximalcomponent size. NBS-corrected group differences in connectivity were visualized onanatomical space using BrainNet Viewer within- versus between-network connections.Statistically significant group differences in connectivity were identified atK, an average of suparthreshold correlation coefficients.Thresholding based on K allowed us to account for the potential effectsof group differences in overall functional connectivity strengths on group comparisonsof the subsequent network measures 0≤K≤1. When a correlationcoefficient for thresholding increases, the network becomes more sparse, yielding lowcost K. When a correlation coefficient for thresholding decreases, thenetwork becomes denser, yielding high cost K.Weighted and undirected networks were constructed for graph theoretic analyses. Wedefined an edge as the Pearson correlation coefficient for time-series from a pair ofthe nodes. The correlation coefficients controlled for the BDI-II scores bywithin-group-centered BDI-II score covariates were then thresholded by the network cost,K, starting from 0.01 in step size of 0.01. Note that, in thisthresholding procedure, only positive correlation coefficients were considered for thenetwork construction since the meaning of negative correlations in rsfMRI is unclear atthe present time (see the Discussion section for relevant limitations). For eachsubject, networks with higher K were obtained until the smallestpositive correlation coefficient was included. Since the maximum K thatallows only positive correlation coefficients was different across subjects, theavailable number of subjects for group comparisons of the network measures decreased aswe applied higher K  across subjects. Thus, we limited the ranges of K forgroup analyses of the network measures up to K=0.23 at which wereliably performed group analyses (N≥5 per group).As numerical values and topological properties of network measures vary with thesethreshold levels, we performed group analyses of the network measures as a function ofi and j, dij, was defined:luv is connection length between nodes u and i andj and wuv is a network weight between nodes u and v. Ifnodes i and j are disconnected, With the connection weight matrices, we obtained the global efficiency .Local efficiency, α=0.05 to assess the normality of distributions ofeach group’s demographics and each network measure. Age did not pass the Shapiro-Wilk normality test. Thus, theMann-Whitney U test was used to compare age between the groups. Twosample t tests were used to compare other demographics between thegroups. The Fisher’s exact test was used to compare the gender distributions between thegroups. Group comparisons on the neuropsychological test scores were carried out using theGLM with age, years of education, and within-group-centered BDI-II score covariates.Within-group-centered BDI-II score covariates were included to assess group differences inneuropsychological measures at average BDI-II scores of each respective group  the effects of demographics  on our results, (2) “overall” neuropsychological deficits inthe TBI group, (3) effects of estimated initial injury severity, (4) effects of depressivesymptoms in the TBI group, (5) effects of a mixture of civilians and veterans in the TBIgroup, (6) group differences in whole-brain volumes, (7) sustained DAI in the TBI group,(8) effects of an initial threshold level on NBS, (9) discrepancies in our results whenusing partial-correlations, (10) effects of GSR, and (11) effects of short distance nodes.estimated initial injury severity of the TBI participants was primarilymild, but the types of injury were diverse. Although the TBI participants were notclinically diagnosed with depression or PTSD (per inclusion criteria),the TBI group was higher on depressive symptom severity than the controls(p<.05). The TBI group had mild depressive symptoms on average, asmeasured by the BDI-II manual .The TBI participants were in the long-term chronic phase of TBI (approximately 8 yearspost-injury time on average). There were no statistically significant differences in age,education, or gender between the groups . There whttp://www.ptsd.va.gov/professional/assessment/adult-sr/ptsd-checklist.asp). Thepresence of depressive and PTSD-related symptoms of these TBI participants was notsurprising as comorbid psychiatric disorders or symptoms are common in chronic TBI  on the word reading condition of the color-wordinterference test and on the satisfaction with life scale. More TBI individuals thanexpected by chance  showed deficits in matrix reasoning, digitspan forward and backward, all sub-domains of color-word interference, card sorting, andtrail making, category switching  of verbal fluency, delayed recall, andsatisfaction with life scale.There were no statistically significant group differences in estimated current IQ betweenthe groups (Table 3). The TBI group did not show statistically significant differencesbetween premorbid and current IQ. The TBI group showed significantly lower performance local efficiencyfor K=0.15, 0.22 at p<.05 andK=0.16, 0.17 at p<.1  occurred in three regions and noticeable group differences in the regional global efficiency(p<.1) occurred in eight regions . As for the regional local efficiency, statisticallysignificant group differences (p<.05) occurred in 9 regions andnoticeable group differences (p<.1) occurred in other 13 regions, r-MFG (BA9),l-rlPFC, and r-rlPFC).Assessments of the regional global and local efficiency for  regions . Of the Our motion analysis  revealedvs. 1/40expected, p=.0007) had abnormally poor neuropsychological performancerelative to the controls in more than two neuropsychological measures ,indicating overall deficits in neuropsychological measures for the TBI group. Groupcomparisons of global and local efficiency in the TBI sub-group with neuropsychologicaldeficits (N=26) versus the TBI sub-group without anyneuropsychological deficits (N=14) did not show statisticallysignificant group differences .More TBI participants than would be expected by chance . There were also nosystematic effects of estimated initial injury severity on our NBS findings .There were no systematic effects of estimated We found no systematic effects of comorbid depressive symptoms on our findings on NBS or network efficiency measures .There were no systematic effects of military status (civilian or veteran with TBI) onour NBS findings  or network efficiency measures .There were no statistically significant group differences in whole-brain volumes .The TBI group had relatively reduced fractional anisotropy in the anterior thalamicradiation, corpus callosum, corticospinal tract, forceps major and minor, inferiorfronto-occipital fasciculus, and superior and inferior longitudinal fasciculi,confirming sustained DAIs among the TBI individuals .NBS analyses with two additional threshold levels  replicated thepatterns of relatively reduced connectivity of the TBI group in Consistent with the full correlation analysis results, group comparisons of directconnectivity using partial correlation coefficients revealed relative reductions inconnectivity for the TBI group . There were a few exceptionsshowing relatively increased connectivity . Again, NBS analyseswith partial correlations allowed us to demonstrate disrupted patterns of the TBI group consistent with the patterns in K.Analyses of the global and local efficiency without GSR essentially replicated the results after GSR in Retaining connectivity strengths between short distance nodes did not markedly alterdisruption patterns of long-range connections .We characterized disrupted resting-state functional connectivity of DMN-DAN-FPCN in chronicTBI comparing to healthy individuals. We confirmed that there were disruptions in chronicTBI individuals not only within-network connectivity but also between-network connectivity–3. Of thThis study demonstrates the advantages of combining both complex network andhypothesis-driven approaches over either simple network or whole-brain-wide data-drivenapproaches only in identifying network disruptions in chronic TBI relevant to specificdeficits. As such, comprehensive assessments of multiple networks-of-interest allowed usto identify markedly disrupted between-network connectivity of chronic TBI individuals–3. TheseOn the other hand, data-driven complex network approaches for TBI individuals at thewhole-brain level . Prior literature indicated an antagonisticrelationship in DMN-DAN, supported by observed anti-correlation between the DMN and DANin Fox et al.  and KellWe observed markedly reduced functional connectivity in the TBI individuals relative tothe control participants; however, there is a body of literature that has reportedelevated functional connectivity in TBI  and methodologies (whole-brain connectivity) inNakamura et al.  and short-term chronic (6months–2 years post-injury) stages of TBI. However, at the long-term chronic stage of TBI(>2 years post-injury), initial injury severity often plays lesscritical role in characterizing TBI individuals at the time of study .Nonetheless, care should be taken in interpreting our findings with regard to TBI severityas initial injury severity was retrospectively estimated.In our case, there were no systematic effects of estimated Second, a portion of the TBI sample showed comorbid depressive symptoms, which are commonin chronic TBI populations. Although comorbid psychiatric symptoms are common in chronicTBI and the presence of depressive symptoms did not alter our findings , care should be taken due to (1) potential bias in self-reported BDI-II scoresdriven by frequent impairments in self-awareness among TBI individuals  and (2) relatively small sample size of the TBIsub-groups.Third, the behavioral consequences of the phenomena reported in this study are as yetunclear. To identify behavioral consequences of disrupted between-network connectivity inchronic TBI, task-based fMRI brain activation studies are of interest.Fourth, it is unknown that how disrupted resting-state functional connectivity amongDMN-DAN-FPCN in TBI could influence “reconfiguration” of functional connectivity inspecific tasks.Fifth, we excluded negative correlations in our network analyses due to ongoing debatesabout the meaning of negative correlations after GSR (Anderson et al., Our future works include an assessment of relationship between disrupted networks andbehavioral consequences in this cohort and efforts to address the other concerns discussedabove. Furthermore, we will identify if and how disrupted connectivity among DMN-DAN-FPCNin chronic TBI could be reorganized following rehabilitation.In conclusion, we demonstrated pronounced disruptions in DMN-DAN-FPCN connectivity. Ourfindings suggest that the three networks should be analyzed together to better understanddeficits in goal-directed cognition and other higher order cognitive functions in chronicTBI. Further studies are required to explain the behavioral consequences of the phenomenareported in this study.


Hemodialysis arteriovenous synthetic grafts (AVG) provide high volumetric blood flow rates shortly after surgical placement. However, stenosis often develops at the vein-graft anastomosis contributing to thrombosis and early graft failure. Two novel fusion proteins, ANV-6L15 and TAP-ANV, inhibit the tissue factor/factor VIIa coagulation complex and the factor Xa/factor Va complex, respectively. Each inhibitor domain is fused to an annexin V domain that targets the inhibitor activity to sites of vascular injury to locally inhibit thrombosis. This study’s objective was to determine if these antithrombotic proteins are safe and effective in inhibiting AVG stenosis.A bolus of either TAP-ANV or ANV-6L15 fusion protein was administered intravenously immediately prior to surgical placement of a synthetic graft between the external jugular vein and common carotid artery in a porcine model. At surgery, the vein and artery were irrigated with the anti-thrombotic fusion protein. Control animals received intravenous heparin. At 4 weeks, MRI was performed to evaluate graft patency, the pigs were then euthanized and grafts and attached vessels were explanted for histomorphometric assessment of neointimal hyperplasia at the vein-graft anastomosis. Blood was collected at surgery, immediately after surgery and at euthanasia for serum metabolic panels and coagulation chemistries.No acute thrombosis occurred in the control group or in either experimental group. No abnormal serum chemistries, activated clotting times or PT, PTT values were observed after treatment in experimental or control animals. However, at the vein-graft anastomosis, there was no difference between the control and experimental groups in cross-sectional lumen areas, as measured on MRI, and no difference in hyperplasia areas as determined by histomorphometry. These results suggest that local irrigation of TAP-ANV or ANV-6L15 intra-operatively was as effective in inhibiting acute graft thrombosis as intravenous administration of heparin, but failed to inhibit hyperplasia development and stenosis in AVG. Chronic hemodialysis, a life-sustaining treatment for those with end-stage renal disease, requires access to high volumetric blood flow to provide consistent shunting of blood to an extracorporeal circuit for rapid cleansing and return. Native arteriovenous fistulas, where the patient’s vein is anastomosed to the side of an artery, are the preferred means of vascular access because of low rates of infection and thrombosis. However fistulas can require several months before they can be used for hemodialysis and 20–60% never become functional –3. AdditThrombosis in AVG most commonly results from underlying neointimal hyperplasia (NH) where the blood vessel wall thickens such that the lumen narrows, restricting blood flow and promoting thrombosis , 7. AberSystemic antithrombotic therapies have been investigated to inhibit thrombosis in AVG with some success  but are Two novel fusion proteins have been developed that inhibit thrombin generation in a site-directed fashion , 16. EacFig 1for the experimental procedure flow chart. Female Yorkshire cross domestic swine (~30kg)  were initially sedated with intramuscular injection of a Telezol® cocktail , tiletamine/zolazepam 3–5 mg/kg , xylaxine 6–10 mg/kg ) and then anesthesia was maintained with isofluorane gas (1–3%) inhalation. All surgeries were performed under aseptic conditions and all efforts were made to minimize suffering. Prior to experimental treatment, peripheral blood samples were collected and used for point-of-care activated clotting times , serum metabolic panel chemistries and plasma coagulation chemistries (prothrombin times (PT) and activated partial thromboplastin times (aPTT) ). Antithrombotic proteins (TAP-ANV or ANV-6L15), produced as previously described [A well-established porcine model of AVG stenosis was used for this study –21, whicescribed .In all groups, treated and control, enteric-coated aspirin , was administered three days before surgery and continued until euthanasia at four weeks. Clopidogrel  was administered one day prior to surgery and then given at 75 mg/day until euthanasia. Enrofloxacin  was administered intramuscularly on the day of surgery. A transdermal fentanyl (50 μg/pig) patch  was applied and buprenorphine (0.1 mg/kg)  was injected intramuscularly after surgery. Animals were allowed to recover and then monitored for untoward swelling and healing issues at the surgical site, difficulty in eating or drinking, and any signs of infection for four weeks. At four weeks, the animals were sedated, MRI was performed, peripheral blood was drawn and used for point-of-care clotting times, for serum metabolic panel chemistries and plasma coagulation chemistries. The animals were then euthanized by intravenous injection of pentobarbital sodium (80–100 mg/kg) and the graft and attached vascular tissues were obtained for histomorphological analyses.in vitro. For the in vitro studies, TAP-ANV solution was added to Celite ACT tubes prior to addition of whole blood to achieve a concentration of 40 μg/mL.Celite ACT tubes  were pre-warmed in the Hemochron device for 3 min prior to the addition of whole blood. The time-to-clot was determined with the Hemochron device using blood obtained immediately prior to surgery, immediately after surgery and just prior to euthanasia. The ACT was similarly measured in whole blood in which the anti-thrombotic fusion protein was added At 4 weeks after AVG placement, animals were sedated and placed supine in the bore of a 3 Tesla MRI machine . An overlap-decoupled 16-channel radiofrequency (RF) coil mounted on a fiberglass support molded to fit the porcine neck, or two sets of bilateral paired-array RF coils with eight total coil elements, were placed over the surgical area to obtain images. A localization scan was performed using 2D time-of-flight (TOF), with echo time (TE) of 6.08 ms and repetition time (TR) of 25 ms, and slice thickness of 3.5 mm. To visualize the graft and blood vessel lumens, T1-weighted 2D black-blood sequences with high in-plane resolution at the anastomoses were obtained using a turbo-spin echo (TSE) sequence with time-efficient double-inversion preparation  = 9, voxel dimensions of 0.3x0.3 mm, field of view (FOV) of 192x192 mm, slice thickness of 2 mm), was applied . The MR http://www.osirix-viewer.com/downloads.html), was used for MR image analysis. The process of image analysis has been described in detail elsewhere [Fig 2. The minimum lumen area was normalized by dividing by the graft cross-sectional area measured at the same location and multiplying by 100% . This region was selected for measurement since the vein-graft anastomosis region is (i) the area in which the NH develops most frequently and to the greatest degree, and (ii) because the smallest lumen cross-sectional area  would impose the greatest limitation on blood flow. All image analyses were performed by an investigator experienced in this technique but blinded to treatment assignments.OsiriX medical image processing software (Osirix v. 3.6.1), an open-source software  tests were used to assess statistical differences among the three treatment groups for the hyperplasia-to-graft ratio (assessed by histomorphometry) and the minimal open lumen area (assessed by MRI).Initial dose-escalation studies using 5, 20 and 100 μg/mL of the anti-thrombotic fusion proteins were performed to assess safety of the drugs before assessing efficacy. No apparent problems were observed in these initial animals so the efficacy experiments were performed using the 100 μg/mL concentration for both ANV-6L15 and TAP-ANV fusion proteins.A total of 38 animals were used for the efficacy studies: 16 for control (heparin-treated), 12 for ANV-6L15 treatments and 10 for TAP-ANV treatments. Two were excluded due to surgical problems (1 in the control group and 1 in the ANV-6L15 group); four were excluded because of unsatisfactory histology quality . One animal was omitted from the control group due to a cardiovascular abnormality.The ANV-6L15 and TAP-ANV treatments were well-tolerated with no incidence of unusual bleeding during or after surgery similar to heparin-treated animals, and no gross difference in redness or swelling compared to heparin-treated (control) animals. No TAP-ANV- or ANV-6L15-treated animals experienced early clot formation during graft surgery, similar to the heparin-treated animals. The wound healing appeared similar and in all groups and no infections occurred in any group. One animal in the ANV-6L15 treatment group developed a large intraluminal thrombus at the vein-graft anastomosis that was detected at the 4 week MRI and which completely occluded blood flow. Upon histomorphometric analysis, the thrombus consisted mostly of fibrin and red blood cells resembling a “white clot” (data not shown).Fig 4). The activity of anti-thrombotic fusion protein was confirmed in an in vitro experiment in which the TAP-ANV was added directly to normal porcine whole blood and the ACT measured. As shown in Fig 5, the addition of TAP-ANV significantly prolonged the time-to-clot compared to the control in which no drug was added.Point-of-care ACT were performed on whole blood collected immediately before surgery and administration of systemic and local ANV-6L15, TAP-ANV or heparin, and immediately post-operatively, within approximately 1.5 h from the last antithrombotic drug administration. The ACT is a measure of the intrinsic coagulation pathway, with celite used as the surface activator. There was no statistically significant change in whole-blood clotting times between paired pre- and immediately post-surgery blood samples in either anti-thrombotic fusion protein-treated animals or heparin-treated animals . In contrast, the PT values from plasma collected pre-operatively and post-operatively from the TAP-ANV-treated animals were unchanged. No significant difference was observed between pre-operative and immediately post-operative plasma aPTT values in any treatment group (Table 1). Domestic pigs are known to have hypercoagulability compared to human with much shorter average PTT values [Heparin activates anti-thrombin that then inactivates thrombin and other proteases such as factor Xa. The antithrombotic fusion protein ANV-6L15 inactivates the tissue factor/factor VIIa complex (extrinsic pathway), and the fusion protein TAP-ANV inactivates the Xa-Va complex (common pathway). Thus, these drugs may alter the PT and aPTT. As shown in T values . Thus it all values were still within normal ranges for swine. Other assessed serum chemistries were unchanged between pre-operative and 4 week post-operative time points (data not shown).Standard metabolic panel chemistries were assessed in serum collected pre-operatively and 4 weeks post-operatively. Although there were statistically significant differences between pre-operative and 4-week post-operative values for serum alanine transaminase (ALT), serum creatinine and total leukocyte counts (WBC)  area ratios in all three groups are shown in Fig 7. No significant differences in the mean H/G ratio were observed amongst the treatment groups.The NH surface area was normalized against the graft surface area in digitized images of histological cross-sections as described previously in in vitro analyses using the ACT assay, similar to what had been reported previously for both TAP-ANV and ANV-6L15 in tissue factor-initiated clotting assays [Table 1). No effect on PT or aPTT were detectable at 4 weeks after administration as expected, as the in vivo systemic half-life of the proteins is only hours [The anti-thrombotic fusion proteins appeared to be well-tolerated, did not appear to affect wound healing or infection rates, and did not alter hemostatic parameters beyond normal ranges as assessed by point-of-care ACT assays. No peri-operative clotting events occurred in any treatment group, indicating that local treatment with the anti-thrombotic fusion proteins at the time of surgery effectively inhibited acute thrombosis similar to heparin. Significantly prolonged clotting times occurred when TAP-ANV was added directly to blood in ly hours . The preAlthough some studies have reported efficacy of anti-thrombotic therapy on AVG thrombosis –28, a reThe failure of the peri-operative treatment with anti-thrombotic fusion proteins to suppress NH formation and stenosis in the present porcine AVG study stands in sharp contrast with previous studies in other animal models that involved simpler forms of vascular injury, such as balloon angioplasty and interposition vein grafting. Tissue factor pathway inhibitor (TFPI) protein is a Kunitz-type protease inhibitor of the coagulation cascade. Local irrigation with recombinant TFPI or the TFPI gene has been shown in rabbit models to inhibit NH formation in vein bypass grafts  and afteThe anti-thrombotic fusion proteins inhibited NH to the same extent as heparin. Heparin is required to prevent perioperative clotting during the AVG placement surgery in swine. Thus, the effects of heparin on NH development alone cannot be determined, due to the confounding issue of clotting. As the NH development was similar in the group treated with the novel anti-thrombotic fusion proteins and that treated with heparin, this suggests that perioperative inhibition of thrombin and its sequela may be insufficient to significantly inhibit AVG stenosis. Although adverse bleeding events were not observed in any treatment group, the novel fusion proteins may have benefit over heparin in other models or in humans because their activity is targeted to the injured vascular surface which may allow for greater or repeat dosing without causing adverse bleeding.In contrast to wire injury or balloon angioplasty, the AVG experiences continuous insults as the presence of the synthetic graft material stimulates a prolonged, robust foreign-body response that results in the release of inflammatory cytokines and growth factors that promote cell proliferation , 39, 40.We have previously reported significantly decreased NH development in porcine and canine models of AVG stenosis with repeat percutaneous delivery of sirolimus-laden and paclitaxel-laden polymer gels to the perivascular region of AVG , 42. TheIn a pig model of carotid stenosis induced by angioplasty, significantly less NH occurred at 4 weeks after placement of PTFE stent grafts coated with thrombomodulin, an endogenous anticoagulant, compared to uncoated stent grafts . FurtherS1 FileWhole blood clotting times for blood from control, TAP-ANV and ANV-6L15-treated pigs.(XLSX)Click here for additional data file.S2 FilePT and PTT plasma coagulation results from control, TAP-ANV- and ANV-6L15-treated pigs.(XLSX)Click here for additional data file.S3 FileALT, creatinine and WBC results from control, TAP-ANV- and ANV-6L15 treated pigs.(XLSX)Click here for additional data file.S4 FileEffect of anti-thrombotic fusion protein treatment on open lumen area determined by MR imaging.(XLSX)Click here for additional data file.S5 FileEffect of anti-thrombotic fusion protein treatment on hyperplasia-to-graft (H/G) ratios determined by histomorphometry.(XLSX)Click here for additional data file.


Receptor for advanced glycation end products (RAGE) belongs to a immunoglobulin superfamily of cell surface molecules that could bind to a number of ligands such as advanced glycation end products, high-mobility group protein box-1, S-100 calcium-binding protein, and amyloid-β-protein, inducing a series of signal transduction cascades, and being involved in a variety of cellular function, including inflammation, proliferation, apoptosis, angiogenesis, migration, and fibrosis. RAGE is expressed in hepatic stellate cells and hepatocytes and hepatoma cells. There is accumulating evidence that engagement of RAGE with various ligands elicits oxidative stress generation and subsequently activates the RAGE downstream pathway in the liver, thereby contributing to the development and progression of numerous types of hepatic disorders. These observations suggest that inhibition of the RAGE signaling pathway could be a novel therapeutic target for liver diseases. This article summarizes the pathological role of RAGE in hepatic insulin resistance, steatosis and fibrosis, ischemic and non-ischemic liver injury, and hepatocellular carcinoma growth and metastasis and its therapeutic interventions for these devastating disorders. Receptor for advanced glycation end products (AGEs) (RAGE) is a multiligand cell surface receptor that belongs to the immunoglobulin superfamily -4. RAGE RAGE is found in an oligomeric or preassembled state within the plasma membrane -4. RAGE RAGE activation by various ligands have been reported to increase oxidative stress generation and subsequently evoke inflammatory, proliferative, angiogenic, fibrotic, thrombogenic, and apoptotic reactions in numerous cell types via activation of diverse intracellular signaling pathways such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), Janus kinase-signal transducers and activators of transcription (JAK-STAT), and phosphoinositol 3 kinase -10. RAGEIn the present review, literature searches were undertaken in Medline by the PubMed interface. Non-English language articles were excluded. Key words (RAGE) and (review or liver or hepatic) have been used to select the articles.Hepatic insulin resistanceNon-alcoholic fatty liver (NAFL) is the most common chronic liver disease in the world -16. NAFLWe have previously shown that serum levels of AGEs are elevated under oxidative stress, inflammatory, and/or diabetic conditions and correlated with insulin resistance and decreased adiponectin levels, thereby being one of the useful biomarkers for differentiating NASH from simple steatosis -24. FurtHepatic steatosisHigh AGE-containing methionine choline-deficient (MCD) diet increased AGE contents, lipid peroxidation product, 4-hydroxynonenal levels, and NADPH oxidase-driven superoxide generation in the liver of Sprague Dawley (SD) rats compared to SD rats with MCD diet alone, which were associated with severity of steatohepatitis and hepatic fibrosis in these animals . FurtherHepatic fibrosisHSCs are the main extracellular matrix-producing cells in the liver and thus play a pivotal role in liver fibrosis . Fehrenbde novo synthesis of glutathione, which could lead to the suppression of oxidative stress generation, inflammation, and HSCs activation [Curcumin, a main curcuminoid present in turmeric, a popular Indian spice, inhibited AGE-induced RAGE expression in HSCs by increasing PPARγ activity and stimulating tivation . Curcumitivation ,38. We htivation -44. StimIn normal rats, chronic AGEs administration induced significant increases in α-smooth muscle actin levels, but did not induce fibrosis or biochemical evidence of liver injury . HoweverHepatic ischemia/reperfusion (I/R) injury associated with liver transplantation and hepatic resection is characterized by hepatocyte damage and enhanced inflammatory reactions . AdminisUncoupling protein-2 (UCP2) knockout mice showed higher malondialdehyde levels and reduced glutathione/glutathione disulfide ratios as well as significantly higher hepatic levels of AGEs and RAGE compared with normal mice . GalactoRAGE is up-regulated in liver remnants selectively after massive (85%) versus partial (70%) hepatectomy, principally in mononuclear phagocyte-derived dendritic cells . FurtherRAGE was expressed in human HCC cell line, Hep3B and HepG2 cells, whereas AGEs increased vascular endothelial growth factor (VEGF) expression in these cell types ,60. FurtWe have previously shown that AGE-RAGE interaction-mediated, NADPH oxidase-induced ROS generation stimulates proliferation and tube formation of ECs, the key steps of tumor angiogenesis, through VEGF expression via transcriptional activation of NF-κB and activator protein-1 -66. FurtHCC lines resistant to hypoxia were found to have higher levels of RAGE expression, and RAGE overexpression showed significantly prolonged survival under hypoxia . FurtherKnockdown of RAGE by small interfering RNAs inhibited proliferation of HuH7 cells and induced G1 arrest of this cell type, whereas HMGB1, one of the ligands for RAGE, exerted opposite effects on HuH7 cells . HMGB1-RAs discussed here, there is accumulating evidence that RAGE could play a pathological role in numerous liver diseases via increased oxidative stress generation and inflammatory reactions Figure . RAGE li


The hexapeptide SLIGRL-amide activates protease-activated receptor-2 (PAR-2) and mas-related G protein-coupled receptor C11 (MRGPRC11), both of which are known to be expressed on populations of sensory nerves. SLIGRL-amide has recently been reported to inhibit influenza A (IAV) infection in mice independently of PAR-2 activation, however the explicit roles of MRGPRC11 and sensory nerves in this process are unknown. Thus, the principal aim of this study was to determine whether SLIGRL-amide-induced inhibition of influenza infection is mediated by MRGPRC11 and/or by capsaicin-sensitive sensory nerves.mrgpr-cluster∆−/− mice) or had impaired sensory nerve function (induced by chronic pre-treatment with capsaicin). Complementary mechanistic studies using both in vivo and ex vivo approaches investigated whether the anti-IAV activity of SLIGRL-amide was (1) mimicked by either activators of MRGPRC11 (BAM8-22) or by activators (acute capsaicin) or selected mediators  of sensory nerve function, or (2) suppressed by inhibitors of sensory nerve function (e.g. NK1 receptor antagonists).The inhibitory effect of SLIGRL-amide on IAV infection observed in control mice in vivo was compared to effects produced in mice that did not express MRGPRC11  and CGRP receptor (CGRP8-37) antagonists. Direct stimulation of airway sensory nerves through acute exposure to the TRPV1 activator capsaicin also failed to mimic SLIGRL-amide-induced inhibition of IAV infectivity. The anti-IAV activity of SLIGRL-amide was mimicked by the purinoceptor agonist ATP, a direct activator of mucus secretion from airway epithelial cells. Additionally, both SLIGRL-amide and ATP stimulated mucus secretion and inhibited IAV infectivity in mouse isolated tracheal segments.SLIGRL-amide and BAM8-22 dose-dependently inhibited IAV infection in SLIGRL-amide inhibits IAV infection independently of MRGPRC11 and independently of capsaicin-sensitive, neuropeptide-releasing sensory nerves, and its secretory action on epithelial cells warrants further investigation. Worldwide, influenza A virus (IAV) infections are estimated to cause up to 5 million cases of severe illness, and between 250,000 and 500,000 deaths each year . The pot2) inhibits IAV infection in mice -7,7-diphenyl-4H-isoindol), substance P, CGRP and L-733,060 were obtained from Tocris Bioscience . Sodium pentobarbitone was supplied by Virbac Australia , methoxyflurane by Medical Developments International Ltd  and Schiff reagent by Australian Biostain .InVivoStat software. Differences between groups were considered statistically significant at p <0.05.Unless otherwise stated, grouped data are expressed as mean ± SEM. Statistical comparisons between groups were made using one-way ANOVA with post-hoc testing  using As shown in Fig. Mrgpr-cluster∆−/− mice that do not express MRCPRC11 [Mrgpr-cluster∆−/− and wild-type) with IAV (in the absence of peptides) was associated with a marked increase in total BAL cell number on day 4 post-inoculation  chronic exposure to capsaicin or (2) administration of a neurokinin receptor antagonist.in vivo protocol to induce airway sensory nerve dysfunction was obtained from isometric tension recording experiments  receptors. Consistent with this, NK1 receptor antagonists inhibit substance P-induced effects on microvascular leakage, mucus secretion and bronchomotor tone in the airways (see ). In theex vivo [ex vivo perfused tracheal system preserves tissue viability, architecture and microenvironment of the airways, and enables the study of the cellular mechanisms through which SLIGRL-amide inhibits IAV virus infection. Furthermore, this ex vivo perfusion system can be adapted to airways of other non-murine species, including human airways. In ex vivo studies, the levels of immunoreactive staining for IAV within the airway epithelium is the primary endpoint used to indicate IAV infectivity [in vivo  that induced large relaxation responses in mouse isolated tracheal preparations . These rSensory nerve C fibre function has been reported to influence the response of the host to respiratory tract insults including bacterial endotoxin, mycoplasma and environmental toxicants , 48–50, in vivo and ex vivo via mechanisms that are independent of MRGPRC11 and of capsaicin-sensitive sensory nerves, and also independent of the sensory neuropeptides substance P and CGRP and their receptors. However, it is important to note that vagal bronchopulmonary C-fibers in the mouse are heterogeneous – a substantial fraction of C-fiber afferent nerves in the mouse respiratory system are capsaicin-insensitive [The current study has established that SLIGRL-amide inhibits IAV infectivity ensitive , and thaensitive . Thus, wensitive . Of partSLIGRL-amide and related peptides have been reported to directly stimulate secretory pathways in the airways , 52–54. in vivo and ex vivo approaches provides compelling evidence that the anti-IAV activity of SLIGRL-amide in murine airways occurs independently of the sensory nerve receptor MRGPRC11 and of neuropeptide release from TRPV1-expressing sensory nerves. Preliminary data show that both SLIGRL-amide and ATP release airway secretions and inhibit IAV infection, raising the possibility that SLIGRL-amide inhibits IAV infectivity via the release of antiviral substances from the airway epithelium. Additional release and intervention studies, particularly in human airways, will provide invaluable insight into the molecular target and cellular signalling processes through which SLIGRL-amide inhibits IAV infectivity.The current study using a combination of