Cancer biology relies on intrinsic and extrinsic deregulated pathways, involving a plethora of intra-cellular and extra-cellular components. Tyrosine kinases are frequently deregulated genes, whose aberrant expression is often caused by major cytogenetic events . The resulting tyrosine kinase fusions (TKFs) prompt the activation of oncogenic pathways, determining the biological and clinical features of the associated tumors. First reported half a century ago, oncogenic TKFs are now found in a large series of hematologic and solid tumors. The molecular basis of TKFs has been thoroughly investigated and tailored therapies against recurrent TKFs have recently been developed. This review illustrates the biology of oncogenic TKFs and their role in solid as well as hematological malignancies. We also address the therapeutic implications of TKFs and the many open issues concerning their clinical impact. The discovery of Philadelphia chromosome (Ph) in Chronic Myeloid Leukemia (CML) , the subSince these findings, many studies have investigated cell transformation via Tyrosine Kinase Fusions (TKFs). A recent appraisal of the literature shows that ~1000 independent studies have been published describing the oncogenic properties of TKFs. Recurrent kinase chimeras are detected in ~2-3% of human tumors and this frequency increases when non-recurrent fusions 4-5%) or cancer-associated abnormal karyotypes 5-7%) are considered % or canc, 7. All % are conThe widespread occurrence of TKFs in human cancers, their ability to synergistically fire multiple signaling pathways and their overall pivotal role in clinical oncology justify the many recent efforts to identify new anti-TKF target therapies. Of note, similar TKF-related pathways have been reported in different tumors; this constitutes the rationale for the use of similar therapies in tumors bearing the same molecular derangements. This approach is well represented by Ph-like leukemias, whose transcriptomes closely resemble those of conventional Ph-positive ALL, even in the absence of BCR-ABL translocations , 9. ThisIn this review, the role of TKFs in both hematologic and solid neoplasms will be discussed. Special attention will be devoted to the many open issues concerning the treatment of TKF-bearing neoplasms.Tyrosine kinases are the most common kinases and catalyze the transfer of a phosphate group from ATP to tyrosine residues. On a structural level, TK display a variety of modules dictating their cellular localization, function stability and response to therapy . The enzTKD are highly conserved modules, frequently composed by a N-terminal ATP-binding region and by a regulatory C-terminal domain. Upon activation, a cascade of events occurs, including ATP consumption, kinase dimerization, conformational changes, trans-autophosphorylation and oligo-complex formation. TK regulation is disrupted in oncogenic TKFs, which display constitutive dimerization due to loss of the inhibitory domains and forced oligodimerization of partner peptides . Both inGene fusions typically replace the TK promoter; therefore TKF expression becomes ectopically regulated by the promoter of the partner gene. Partner genes contribute to the oncogenic potential in a number of ways. In most instances, the partner N-terminus region provides dimerization domains, which recruit molecular adaptors and lead to the constitutive trans-phosphorylation and activation of the kinase. TKF partners can also directly activate oncogenic pathways, as observed in the TFG-NTRK1 and TRAF1-ALK fusions, independently activating the NF-kB pathway . We and RET , 2814, 2d tumors , which cTo date, fusions involving 14 different TKs have been described in solid tumors  or GTF2IRD1 (thyroid carcinoma) have also been reported.To date, fusions involving 14 different TKs have been described in solid tumors , and in clinically unmet patients  analyses have highlighted new patterns of translocations, defining specific subgroups of common solid tumors  exposure to toxic agents (probably the major initiating event); (ii) exposure to ionizing radiation (e.g. in papillary thyroid carcinoma); (iii) oxidative stress and chemotherapeutic agents (e.g. topoisomerase inhibitors).The oncogenic role of TKFs has prompted huge efforts to improve their detection in cancer patients to better guide patient-directed treatment strategies .Affinity purification coupled with mass spectrometry (AP-MS) is a powerful tool to characterize aberrant gene products and protein-protein interactions (PPIs). AP-MS has been successfully used to discover novel TKFs in NSCLC (ALK and ROS1)  and rareThe introduction of NGS-based approaches has rapidly enriched the overall knowledge of TKF, giving a better representation of their distribution in human cancers. In hematologic malignancies, NGS studies of Ph-like ALL have detected TKFs in >90% of patients . TechnicClinically, TKF in human hematological cancers can be detected by an array of techniques. Genomic/conventional cytogenetics (CG) or FISH are commonly used for the detection of BCR-ALB translocations. Negative samples (<5% of cases) frequently carry variant translocations or cryptic rearrangements, which can occasionally be detected by FISH. Both approaches are also used to monitor drug responses and disease evolution. FISH and RT-PCR have shown higher sensitivity at diagnosis and better MRD detection than CG. In particular, qRT-PCR has become the gold standard for the diagnosis of MRD . FuseFISResistance to TKi treatments is currently detected by RT-PCR coupled with DNA Sanger sequencing, but new methods have been developed. Massive parallel sequencing is rapidly replacing the above reported strategies, as it can also identify subclonal populations and characterize tumor clonal heterogeneity . AnalogoALK fusions or ALK protein expression in NSCLC. Similar strategies were proposed to assess ROS1 and RET fusions [RET-rearrangements has been used to enroll patients in NSLCL clinical trials . It is conceivable that screening approaches to detect simultaneously multiple fusions will be implemented in the clinical arena [In solid tumors, TKFs are clinically identified by several techniques, some of which are performed as companion tests to select the best TKi agent. In 2013, the USA and China's FDA approved FISH- and IHC-based diagnostic tests to identify  fusions . Alternaal arena .Since different TKFs can be present in distinct cancer subsets, multi-targeted platforms have emerged as powerful tools to screen diagnostic samples. As multi-gene screening methods are entering into routine diagnostic procedures, new technical and therapeutic questions need to be addressed. Indeed, the greater efficacy of TKi compared to conventional treatments needs to be established for naïve patients carrying targetable fusions. Similarly, patients treated with multiple rounds of chemotherapeutic regimens need to be evaluated for the compliance to selective TKi, as limited data are available to justify their usage.ALK or ROS-1 gene rearrangements [The discovery of recurrent tumor-associated TK translocations has brought new strategies for the treatment of hematologic and non-hematologic tumors. In the last decades, TKi have revolutionized the treatment of CML, Ph-positive B-ALL  and NSCLngements , 90.BCR-ABL1 gene rearrangements are reported in all cases of CML and in about 30% of adult B-ALL. The fusion gene usually results from the t translocation, but 5-10% of cases bear variants and/or cryptic translocations [m the t9;2. The most promising therapeutic agents are the conventional TKi, Ponatinib, and the BCR-ABL1 switch control inhibitor, DCC-2036 [et al. have recently re-proposed the utility of Axitinib, an anti-VEGFR molecule approved for renal cell carcinoma, to counteract the resistance phenotype of T315I gatekeeper CML. Indeed, Axitinib tightly binds the T315I mutated kinase and inhibits leukemic cell growth, showing a better toxicity profile than other new-generation drugs, such as Ponatinib [In CML patients, resistance to TKi can result from two major mechanisms: (i) occurrence of TKi-resistant BCR-ABL1 mutations; (ii) enhancement of BCR-ABL1-independent pro-survival pathways. BCR-ABL1-dependent resistance has been overcome by third generation TKi that also target the most common ABL1 mutations  has reported complete clinical remission in 93% of adults with active Ph-positive ALL, with a 5-year overall survival (OS) of 43%. Notably, allogeneic stem cell transplant (ASCT) after Imatinib and HyperCVAD improved median OS only in patients with residual molecular disease. The role of Dasatinib and Nilotinib for the treatment of pediatric and adult Ph-positive ALL is still under evaluation, but preliminary results indicate their safety and efficacy when used with high dose chemotherapy and/or ASCT [These data provide the rationale for the use of multi-modal regimens for the treatment of Ph-positive ALL, based on a combination of intensive chemotherapy and TKi. Very encouraging results have been first obtained in pediatric trials  , 105 and/or ASCT . Encoura/or ASCT . Finally/or ASCT .NTRK1 and FGFR1/2/3 have been reported  and PROFILE 1014 (phase III) clinical trials. Of note, in such tumors, Crizotinib displayed even greater efficacy than standard chemotherapy . SimilarDespite these outstanding results, TKi often lose their efficacy due to the development of treatment-related resistance . Much liAs for target mutations, different genetic alterations associated with resistance have been described for ALK-positive NSCLC: i) L1169M within the “gatekeeper” domain (the most frequent); ii) L1152R, associated with EGFR signaling activation; iii) G1202R and S1206Y within the ATP-binding pocket, leading to a decreased Crizotinib affinity  and TyroDifferent strategies have been proposed to overcome TKi acquired resistance, including the development of next-generation drugs, such as the ALK-inhibitors LDK378-Ceritinib and AP26113-Brigatinib (for ALK-positive NSCLC resistant to Crizotinib), Lecitinib (for ALK-positive NSCLC carrying the L1169M mutation) and PF06463922  . AdditioA final major roadblock concerning TKi is the poor response of central nervous system (CNS) metastases to therapy. This drawback may be overcome by different strategies: (i) combined therapies with drugs favoring TKi CNS accumulation, as recently attempted through the co-administration of Vandetanib and Everolimus  ; and . These analyses have to be performed by a pool of medical centers following certified programs, with consequent huge health costs.in vitro preclinical tests on patient-derived cell lines or PDTX may be introduced  should be performed together with genomic analyses. As an alternative, d Figure .in vitro tests with patient-derived tissue samples may allow personalized pharmacologic interventions without the limits related to the use of immortalized cell lines or other tumor models. Patient-derived cancer models are indeed pivotal for drug discovery, for the characterization of cancer biology and for the assessment of personalized therapies [Of note, herapies -139. Sevherapies , 140-142in vitro primary cell lines will allow for the rapid testing of drug libraries, with subsequent validation in more complex models (e.g. PDTX). It is also desirable that new clinical trials will include parallel co-clinical mouse PDTX-trials, which will establish the efficacy of such models. The molecular stratification of these models will finally provide new cancer classifiers with possible diagnostic/prognostic implications  lead to phenotypes closely resembling those of TKF-driven neoplasms. This is, for instance, the case of subsets of ALK-negative ALCL, carrying JAK1 and STAT3 activating mutations, which induce transcriptional signatures akin to those of ALK-positive ALCL . MoreoveIn conclusion, it is desirable that the academic community promotes dedicated programs on TKi and TKF-bearing cancers, also encouraging the support of pharmaceutical companies and the distribution of compounds under compassionate use. This approach, together with the creation of open-access clinical databases, will greatly contribute to the understanding of cancer-related TKFs and to a better management of (TKF-positive and TKF-negative) cancer patients.


In vitro experiments, the elevated sirtuin1 (SIRT1) activity and protein expression through the cAMP–CREB pathway was observed after ghrelin and ghrelin potentiator treatment in ghrelin receptor 1a-expressing cells and human umbilical vein endothelial cells. Furthermore, rikkunshito increased hypothalamic SIRT1 activity and SIRT1 protein expression of the heart in the all three mouse models of aging. Pericarditis, myocardial calcification and atrophy of myocardial and muscle fiber were improved by treatment with rikkunshito. Ghrelin signaling may represent one of the mechanisms activated by CR, and potentiating ghrelin signaling may be useful to extend health and lifespan.Caloric restriction (CR) is known to retard aging and delay functional decline as well as the onset of diseases in most organisms. Ghrelin is secreted from the stomach in response to CR and regulates energy metabolism. We hypothesized that in CR ghrelin has a role in protecting aging-related diseases. We examined the physiological mechanisms underlying the ghrelin system during the aging process in three mouse strains with different genetic and biochemical backgrounds as animal models of accelerated or normal human aging. The elevated plasma ghrelin concentration was observed in both klotho-deficient and senescence-accelerated mouse prone/8 (SAMP8) mice. Ghrelin treatment failed to stimulate appetite and prolong survival in klotho-deficient mice, suggesting the existence of ghrelin resistance in the process of aging. However, ghrelin antagonist hastened death and ghrelin signaling potentiators rikkunshito and atractylodin ameliorated several age-related diseases with decreased microglial activation in the brain and prolonged survival in klotho-deficient, SAMP8 and aged ICR mice.  Caloric restriction (CR) is known to retard aging and delay functional decline as well as the onset of diseases in most organisms.2 The beneficial effects of CR are considered to be mediated by several signaling pathways, including sirtuin (SIRT), insulin-like growth factor-1 (IGF-1)/insulin, adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin. Hypothalamic SIRT1 is required for normal response to CR3 and regulates energy balance, which is implicated in aging/longevity.5 However, key regulated hormone activated by CR or precise mechanisms involved remain uncertain.The increased lifespan of world population illustrates the success of modern medicine; however, the risk of developing many diseases increases exponentially with old age.6 Ghrelin receptors  are expressed in several organs, including the brain and heart.7 Ghrelin has much broader physiological functions as an orexigen, including stimulation of growth hormone (GH) secretion,8 gastrointestinal motility9 and cardiac contraction,10 inhibition of energy metabolism6 and insulin secretion12 and inhibition of inflammation13 and apoptosis. Orexigenic effect of ghrelin is mediated by activated AMPK in neuropeptide Y neurons in the hypothalamic arcuate nucleus.16 AMPK enhances SIRT1 activity by increasing cellular nicotinamide adenine dinucleotide (NAD+) levels and regulates energy expenditure.17 Therefore, we hypothesized that in CR ghrelin has a role in protecting aging-related diseases through a mediation of SIRT1.Ghrelin is an acylated hormone that is secreted mainly from stomach in response to fasting and CR.22 Of the 43 major chemical compounds contained in rikkunshito, atractylodin shows a marked increase in ghrelin/GHS-R-binding activity and ghrelin-induced cytosolic calcium ion (Ca2+) concentration in GHS-R-expressing cells through an allosteric mechanism. Atractylodin is a major ingredient detected in the plasma of rikkunshito-treated healthy volunteers.23The traditional Japanese Kampo medicine rikkunshito is able to potentiate ghrelin signaling by stimulating ghrelin secretion via serotonin 2b/2c receptor antagonism and by enhancing GHS-R activity.In this study, we examined the impact of ghrelin signaling on the survival of three mouse strains with different genetic and biochemical backgrounds. These animal models mimic accelerated or normal human aging. These models are useful in studying the common protective mechanisms regulating many age-related diseases. We show here that the elevated plasma acyl ghrelin concentration was observed in homozygous klotho mutant  and senescence-accelerated mouse prone/8 (SAMP8) mice. The treatment with ghrelin antagonist (D-Lys3)-GHRP-6 hastened death, whereas the ghrelin signaling potentiators rikkunshito and atractylodin, but not ghrelin, prolonged survival in klotho-deficient mice. Rikkunshito extended the survival of another pathological model of both SAMP8 and aged ICR mice, a model of normal aging. These results suggest the importance of potentiating endogenous ghrelin signaling or attenuation of ghrelin resistance in the extension of lifespan in animal models of human aging. Several age-related diseases were also ameliorated in all rodent models, including improved cardiac involvement in both klotho-deficient and SAMP8 mice and improved learning in aged ICR mice. The effects on survival were mostly independent of the orexigenic activity and interactions with insulin and IGF-1 signaling. As a protective mechanism against the aging process, ghrelin signaling was considered to be associated with increased SIRT1 activity and decreased microglial activation in the brain.Klotho-deficient, SAMP8 and ICR mice as models of aging and GHS-R knockout mice were used. All experimental procedures were performed according to the ‘Guidelines for the Care and Use of Laboratory Animals' approved by each Laboratory Animal Committee. The detailed description of the materials and methods is provided in 24 We examined the potential role of ghrelin signaling in klotho-deficient mice. We found that 5-week-old klotho-deficient mice showed an increase in acyl ghrelin, desacyl ghrelin, GH and corticosterone under fed or fasted conditions, while a decrease in acyl ghrelin/desacyl ghrelin (A/D) ratio suggestive of cachectic state, IGF-1, insulin and glucose were observed in klotho-deficient mice  in both klotho-deficient and age-matched wild-type mice  failed to prolong survival, but ghrelin antagonist (D-Lys3)-GHRP-6  decreased the median survival without changing body weight in klotho-deficient mice  and atractylodin  administration significantly increased median survival  did not affect 24-h food intake  mice; however, it was significantly increased by the daily rikkunshito (1%) administration  prolonged the median survival , with noNo significant change of plasma concentrations of acyl ghrelin was observed in 26-month-old aged ICR mice compared with 4-month-old young ICR mice. Eight-month treatment with rikkunshito did not affect plasma acyl ghrelin concentration and SIRT1 protein expression in the heart but increased SIRT 1 activity in the hypothalamus of aged ICR mice .Twelve-week-old GHS-R knockout, heterozygous and wild-type C57BL/6 mice were treated with rikkunshito (1%) containing chow or control chow for 4 weeks. Hypothalamic SIRT1 activity decreased in rikkunshito-treated GHS-R knockout mice .We examined the cellular effects of ghrelin signaling on SIRT1 activity in GHS-R1a-expressing human embryonic kidney (HEK) 293 (293-GHS-R) cells. Ghrelin and rikkunshito increased SIRT1 activity in 293-GHS-R cells , the eff27 GPCRs coupled to Gαq/11 activate phospholipase C, which opens the endoplasmic inositol trisphosphate-gated Ca2+ channel and activates protein kinase C through production of diacylglycerol. On the other hand, GPCRs coupled to Gαs activate adenylyl cyclase, produce cyclic adenosine monophosphate (cAMP) and activate protein kinase A (PKA) and other downstream effectors. In this study, ghrelin elicited an increase in intracellular Ca2+ concentration in 293-GHS-R cells (2+ in 293-GHS-R cells and mock-transfected (293-Mock) cells. However, it potentiated the ghrelin-induced Ca2+ flux in 293-GHS-R cells  that initiates multiple intracellular signaling cascades.-R cells . Rikkuns-R cells , as we p-R cells . Gastric-R cells .28 In the present study, ghrelin increased cAMP response element (CRE) reporter activity and phosphorylated CREB in 293-GHS-R cells and the effect was augmented by rikkunshito , which express both ghrelin and GHS-R1a.RT1 ref.  and AMPKctylodin . These ectylodin .33 AMPK enhances SIRT1 activity by increasing cellular NAD+ levels, resulting in deacetylation and modulation of the activity of downstream SIRT1 targets.17 Activation of exchange protein directly activated by cAMP (Epac1), a cAMP effector protein, increases intracellular Ca2+ levels and activates the CamKKβ–AMPK pathway.34 Rikkunshito elevated phosphorylated AMPK levels, which was inhibited by the treatment with (D-Lys3)-GHRP-6  acts upstream of AMPK in mammalian cells.)-GHRP-6 , suggestmpound C , suggestGhrelin in combination with rikkunshito inhibited the hydrogen peroxide-induced apoptosis of 293-GHS-R cells, a model of the oxidative cell death .35 To examine the possible involvement of SIRT1 in amelioration of microglial inflammation, rikkunshito was orally administered to klotho-deficient mice, an animal model of progeria, and microglial pathological activation in the dissected brains was morphologically analyzed. Rikkunshito significantly reduced the number of pathologically activated microglia with amoeboid morphology, which was predominantly observed in the brains of non-administered klotho-deficient mice (Previous studies demonstrated that the activation of SIRT1 pathway ameliorated microglia-mediated inflammation through its inhibitory effect on receptor activator of nuclear factor-κB.ent mice . In the ent mice  and agedent mice .We found that ghrelin antagonist (D-Lys3)-GHRP-6 hastened death, whereas the ghrelin signaling potentiators rikkunshito and atractylodin ameliorated several age-related diseases and prolonged survival in three mouse models of accelerated or normal human aging. These findings suggest that the elevated endogenous ghrelin signaling has an important role in preventing aging-related premature death.37 Increased hypothalamic inflammatory cytokines observed in these mice may underlie the resistance to appetite-stimulating effects of ghrelin, the situation similar to cachexia associated with cancer and other diseases.25 In SAMP8 mice only, rikkunshito improved in part the adaptive feeding and body weight response, suggesting that the effects on survival were mostly independent of the orexigenic activity.Plasma acyl ghrelin concentrations were increased in both klotho-deficient and SAMP8 mice. It is most likely due to the anorexia and decreased body weight, because weight loss is a potent stimulus to ghrelin secretion in humans and animals.41 In this study of pathological aging, ghrelin increased GH but not IGF-1 in klotho-deficient mice and the GH–IGF-1 axis appeared not to downregulate in SAMP8 mice. Despite the studies indicating the importance of adequate levels of circulating GH and IGF-1 for healthy aging, the role of these anabolic hormones in the genesis of aging phenotypes and in the extension of lifespan remains highly controversial.42Ghrelin, GHS and rikkunshito were reported to improve the GH–IGF-1 axis decline in aged animals and humans.44 In particular, hypothalamus has a programmatic role in the development of whole-body aging via immune-neuroendocrine integration.45 Enhanced ghrelin signaling improved the levels of neuronal genes involved in inflammation, apoptosis, DNA repair and migration in the hypothalamus of klotho-deficient mice. These findings are consistent with previous reports in which the beneficial effect of CR on age-related hypothalamic gene changes46 and the neuroprotective effect of ghrelin47 were observed. Cancer and dementia are the most debilitating conditions associated with aging. Rikkunshito facilitated memory consolidation of passive avoidance learning in aged ICR mice. The improved ghrelin signaling appears to protect against several age-associated neurodegenerative changes, including the pathology of brain and heart, as in CR.Neurodegenerative disorders are one of the most potent risk factors for any age-related diseases. CR delays brain senescence and prevents neurodegeneration.48 Klotho attenuates cellular apoptosis and senescence in vascular cells.49 CR attenuates oxidative stress and improves endothelial function in the aorta of aged F344 rats.50 Recently, ghrelin have been demonstrated to have beneficial effects in the cardiovascular system through a combination of direct and indirect actions.10 Ghrelin improves cardiac function and survival in heart failure using a mouse model of inherited dilated cardiomyopathy.51 Ghrelin inhibits sympathetic nervous system activity and stimulates GH secretion, which may help treat cardiovascular diseases and cardiac cachexia.52 Histological analysis in this study revealed that pericarditis and calcification and focal atrophy of cardiac muscle developed in SAMP8 or aged ICR mice were improved by the treatment with rikkunshito. Calcification was also significantly attenuated by rikkunshito in klotho-deficient mice, suggesting a reason for the better prognosis. Ghrelin was reported to attenuate myocardial calcification induced by nicotine and vitamin D3 via an increase and decrease in osteopontin and endothelin-1 expression, respectively, in the myocardium.53 The mechanism through which leukemia, the main cause of death in SAMP8 mice, was inhibited by treatment with rikkunshito remains clarified in relation to ghrelin.Cardiovascular disease is one of the leading causes of death and disability in the aged society. Klotho gene polymorphisms in humans are associated with an altered risk for coronary artery disease.Changes in muscle mass during aging influence both lifespan and healthspan. The present study demonstrated improved atrophy of myocardial and muscle fiber by treatment with rikkunshito. These findings suggest ghrelin signaling improves age-related sarcopenia and could contribute to prolonged survival in mouse models. Rikkunshito administration recovered the decreased locomotor activity in SAMP8 mice. This result may also be mediated by improvement of muscle atrophy. Ghrelin and rikkunshito decreased the sympathetic nerve activity to brown adipose tissues. This finding suggests the reduced basal energy expenditure.Ghrelin and rikkunshito increased SIRT1 activity and protein expression in 293-GHS-R cells and HUVECs through the GHS-R–cAMP–CREB pathway. GHS-Rs are expressed in several organs, including the hypothalamus and heart. Rikkunshito increased hypothalamic SIRT1 activity and SIRT1 protein expression in the heart in all three mouse models of aging. However, hypothalamic SIRT1 activity significantly decreased in rikkunshito-treated GHS-R knockout mice. These results suggest that SIRT1 activity in aged mice is mediated by ghrelin signaling.35 Recent studies demonstrated that gut and intestine influenced the microglial behavior in the brain and determines the prognosis of some neurological disorders, such as multiple sclerosis; however, the pathophysiology has not been appreciated well.54 Our findings indicate that ghrelin signaling activates SIRT1, and treatment for ghrelin resistance may exert a protective effect against brain and other organ/tissue pathologies during the process of aging through the SIRT1 pathway. These outcomes may be independent of the orexigenic activity of ghrelin55 (Present findings that administration of rikkunshito to SAMP8, klotho-deficient and physiologically senescent mice resulted in the amelioration of microglial pathological activation are consistent with the previous report in which SIRT1 diminished the activation of nuclear factor-κB in microglia and the toxicity of amyloid β.hrelin55 .57 although these results are still somewhat controversial. Many studies have shown that CR results in reduced oxidative stress, which is considered to be a key mechanism underlying the aging process.58 These CR effects are mediated by several signaling pathways, including SIRT, IGF-1/insulin, AMPK and mammalian target of rapamycin. These pathways may interact and may all have important roles in mediating different aspects of the response.59 Rapamycin has been shown to extend both the mean and maximum lifespan, and the development of newer, safer antiaging therapies based on this analog are expected.60CR has been established to extend the lifespan and improve the health in several species. A long-term CR study in rhesus monkeys demonstrated that CR lowered the incidence of aging-related deaths and delayed the onset of age-associated pathologies, such as diabetes, cancer, cardiovascular disease and brain atrophy,The present study demonstrates that the key regulated hormones and mechanisms activated by CR should include ghrelin–GHS-R signaling pathways. The potentiation of ghrelin signaling may be an additional approach for the improvement of both healthspan and lifespan in modern aging societies.


The current yellow fever outbreak in Brazil prompted widespread yellow fever virus (YFV) vaccination campaigns, imposing a responsibility to distinguish between vaccine- and wild-type YFV-associated disease. We developed novel multiplex real-time reverse transcription PCRs that differentiate between vaccine and American wild-type YFV. We validated these highly specific and sensitive assays in an outbreak setting. Flavivirus within the family Flaviviridae (Yellow fever virus (YFV) is a mosquitoborne member of the genus In December 2016, Brazil reported the country’s largest yellow fever (YF) outbreak in decades. Through May 31, 2017, a total of 3,240 suspected cases were reported, including 435 deaths  can occur (We followed 2 rationales for real-time RT-PCR design. First, a small number of oligonucleotides per assay can be beneficial in resource-limited settings. Therefore, we designed 5 different single-target assays using primers capable of simultaneously amplifying vaccine and wild-type YFV strains. However, these criteria restricted the ability to design optimal oligonucleotides. Therefore, we designed 2 additional dual-target assays that target 2 separate genomic regions in which vaccine and wild-type strains differ sufficiently from one another , panel AWe selected the 2 most sensitive single- and dual-target assays on the basis of preliminary experiments using full viral RNA of wild-type and vaccine strains  Figure 26 copies/reaction. Assay specificity was assessed using a set of 39 high-titer flavivirus cell culture isolates (The 95% lower limit of detection of the single- and dual-target assays ranged from 4.0 to 8.8 RNA copies/reaction for vaccine and wild-type YFV strains  Figure 3<1,000 copies/reaction upon the presence of 100–500-fold higher concentrations of the heterologous target.Hypothetically, near-simultaneous infection with wild-type YFV and vaccination may occur in the outbreak setting in Brazil. In the case of co-occurrence of vaccine and wild-type YFV within a single sample, 1 target may occur in relatively higher concentrations than, and thus outcompete amplification of, the other target, resulting in an incomplete test result. We observed no target competition with the dual-target assay even in the presence of high concentrations of the heterologous RNA , panel A1–106 copies/mL of either vaccine or wild-type YFV. Three replicates of each spiked specimen were purified individually and tested by using our PCRs and a YFV reference assay (>1,000 copies/mL in all replicates irrespective of the clinical matrix (<100 copies/mL was unreliable in all 3 assays. As exemplified before for Zika virus, final RNA copy numbers in eluates used for RT-PCR will depend on the RNA extraction protocol, illustrating that even assays with analytical sensitivity in the single-copy range may not correctly detect weakly positive clinical specimens (Commonly used clinical specimens for YFV diagnostics may contain substances that can interfere with PCR (l matrix , panel BClinical specimens may differ from spiked materials, and assay performance needs to be assessed in an outbreak context. Therefore, we compared the new assays to the reference assay , panel CThe new PCRs we describe enable YFV detection with diagnostic sensitivity. The dual-target assay was superior to the single-target assay in sensitivity and robustness to target competition. However, the single-target assay may be advantageous in resource-limited settings and may be more convenient for multiplex usage in combination with assays targeting co-circulating arboviruses, such as chikungunya, Zika, and dengue viruses. Beyond rapid test results, the real-time RT-PCR–based protocols provide considerably higher sensitivity than protocols aiming at generating longer PCR amplicons necessary for strain discrimination by nucleotide sequencing, enabling conclusive results even when virus concentrations in specimens are low or when these materials are available only in limited quantity. Of note, PCR-based YFV detection is most reliable 5–7 days after symptom onset, during the viremic phase. Reliable YFV surveillance should thus include serologic methods. However, serologic tests used for virologic diagnostics cannot discriminate between vaccination and wild-type YFV infection.Of note, our novel assays are limited to vaccine and American YFV wild-type strains. West African wild-type stains would be detected by our YFV vaccine assays due to the close genetic relatedness between these strains, but our assays are not suitable to detect the genetically diverse Eastern and Central African wild-type strains. If needed, one could extend our assays by an additional primer/probe combination targeting the Eastern and Central African genotypes.Recently, the first attenuated live dengue virus vaccine was approved in several countries, including Brazil (Additional information about yellow fever virus surveillance, Brazil, 2017.


The RV volume is measured by tracing the endocardial border on a stack of SSFP cine images, with most approaches including RV trabeculations in the blood pool. Semi-automated, threshold-based software  allows for volume measurements that exclude trabeculations from the RV with the aim of improved accuracy of the true blood pool volume. Threshold-based analysis has yielded significantly different RV volumes and ejection fraction in the adult population. This study compares the impact of threshold-based analysis of RV volumes in children and adults following ToF repair.Tetralogy of Fallot (ToF) repair often involves placing a transannular patch to augment the right ventricular outflow tract, resulting in right heart dilation and failure over time. Right ventricular (RV) volume measurement by cardiac magnetic resonance imaging (CMR) is the gold standard to guide timing of pulmonary valve replacement in this population - often at an indexed right ventricular end-diastolic volume (RVEDVi) > 160 ml/mThirty-nine patients with repaired ToF underwent clinically indicated CMR including twenty-one pediatric patients ≤ 18 years. Indexed RV end-diastolic and end-systolic volume measurements (RVEDVi and RVESVi) were repeated for independent comparison using the standard contour method and the threshold-based method . Similarly, the pediatric RVESVi using the contour method was 73 ml/m2, and 51 ml/m2 using threshold-based analysis . The pediatric RVEF was 50 percent using the contour method and 56 percent using threshold-based analysis . In the pediatric subgroup, seven patients had an RVEDVi greater than 160 ml/m2 using the contour method, while none had an RVEDVi greater than 160 ml/m2 using the threshold-based method. The adult subgroup had similar significant differences in RVEDVi, RVESVi, and RVEF - consistent with previously reported data. In the adult group, six patients had an RVEDVi greater than 160 ml/m2 using the contour method, while only one had an RVEDVi greater than 160 ml/m2 using the threshold method.In the pediatric age group, the mean RVEDVi using the contour method was 144 ml/mThreshold-based and contour-based analysis of RV volumes using CMR data yields differing RV volume measurements in both children and adults with repaired ToF. This analysis demonstrates that care must be taken prior to incorporating alternative endocardial contour techniques into clinical decision-making. Incorporating these techniques into clinical practice may help refine indications and timing of pulmonary valve replacement by accounting for RV trabeculations, which was previously difficult using traditional contour methods.


P<0.05). Post-transplant BAFF levels showed significant association with pre-transplant sensitization, but did not show association with anti-HLA antibodies and positive donor-specific antibodies at the time of biopsy. We did not find any association between post-transplant BAFF levels and allograft biopsy results, Banff scores and microvascular inflammation scores. In conclusion, pre-transplant BAFF levels are associated with pre-transplant sensitization and are useful in predicting allograft rejection. But post-transplant BAFF levels measured at the time of indication biopsy are not associated with the appearance of de novo HLA-DSA, allograft rejection, biopsy findings and other allograft outcomes.It is well known that pre-transplant B cell activating factor (BAFF) levels are associated with the development of de novo anti-HLA antibodies and antibody mediated rejection post-transplant. However, the clinical significance of BAFF values at allograft rejection has not been determined. In this study, we investigated the clinical significance of pre-transplant BAFF level as well as post-transplant BAFF levels measured when indication biopsy was done. We checked for anti-HLA antibodies in 115 kidney transplant recipients who required allograft biopsy due to an increase in serum creatinine. With the same serum specimen, we measured BAFF levels, and in 78 of these patients, pre-transplant BAFF and anti-HLA antibody levels were detected as well. Patients in each group were divided into tertiles according to BAFF levels. We investigated the relationship between BAFF levels and the occurrence of anti-HLA antibodies. Pre-transplant BAFF levels showed significant association with pre-transplant sensitization, and also with early rejection (Tertile 3, 26.9% vs. Tertile 1, 11.5%;  The role of B-cells in kidney transplantation, especially in acute and chronic antibody-mediated rejection (AMR) and transplantation tolerance, has been repeatedly emphasized in recent studies ,2. BesidB-cell activating factor (BAFF) is a cytokine belonging to the tumor necrosis factor family that plays a role in the survival, proliferation and differentiation of B-cells ,5. SeverHowever, the clinical significance of monitoring of post-transplant BAFF levels has not yet been determined. Therefore in this study, we measured both pre-transplant and post-transplant BAFF serum levels in kidney transplant recipients, with the intention to investigate the significance of pre and post-transplant BAFF levels in predicting clinical outcomes such as acute rejection and allograft failure.We analyzed a hundred and thirty cases of serum taken from 115 patients at the time of indication biopsy performed between January, 2010 and December, 2014 at Seoul St. Mary’s Hospital. Pre-transplant serum taken just before initiation of any immune suppressants or desensitization therapy such as rituximab or plasmapheresis was available in 78 of these cases. We measured serum BAFF level in the 130 post-transplant serums and also in the 78 pre-transplant serums and investigated the clinical significance of both. This study received approval of the Institutional Review Board of Seoul St. Mary’s Hospital (KC13TNMI0701) and all patients signed an informed consent before inclusion.In our center, indication biopsy was done in patients with an increase in serum creatinine of 20% above baseline value. The biopsy was performed as previously described , using aHLA antibodies were detected using Lifecodes LSA Class I and Class II kits  or LABScreen Single Antigen , according to the manufacturer’s instructions. Fluorescence was analyzed using the Quick-Type User’s Manual Research Use Only program, version 2.4, of the LABScan100 flow cytometer . Normalized median fluorescence intensity (MFI) greater than 1,000 were considered positive. HLA typing was done by DNA molecular typing in all donors and patients using sequence-specific oligonucleotide probes (SSOP) with LIFECODES HLA SSO typing kits . HLA-DSA was defined as the anti-HLA antibodies of the recipient corresponding with HLA types of the donor.Serum BAFF levels were measured using serum that had been stored at -70°C. Commercially available ELISA kit  was used according to the manufacturer’s recommendations and results were expressed in pg/mL.Correlation of serum BAFF levels with pre-transplant sensitization status such as PRA levels, and detection of HLA-DSA were observed, as well as the development of biopsy proven acute rejection (antibody-mediated and T-cell mediated), and allograft survival post-transplant. Patients in both the pre-transplant (n = 78) and post-transplant groups (n = 130) were also divided into tertile groups  according to BAFF levels, and clinical parameters or outcomes were compared across the 3 BAFF tertiles. Detailed pathologic findings based on the Banff classification were compared among tertiles in the post-transplant BAFF measured group. In 78 patients in whom both pre- and post-transplant BAFF level were measured, we calculated the change of BAFF level after KT in comparison with before KT (delta BAFF) in each patient and observed the correlation of these values with the above mentioned parameters. These patients were also divided into three tertiles according to delta BAFF value and clinical parameters or outcomes were compared.Results are described as mean ± standard deviation (SD) or median and range for continuous variables and as percentages for categorical data. Comparisons were made with chi-square test for categorical data and Student’s t-test for continuous variables. Allograft survival was compared between tertiles by Kaplan-Meier analysis with a log-rank test. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software  and the statistical package MedCalc version 15.5 . Results with P values below 0.05 were considered significant.Post-transplant BAFF levels were measured concomitantly with 130 indication biopsies in 115 patients and median post-transplant BAFF level was 99.49 pg/mL (9.63~897.89 pg/mL). In 78 patients in whom pre-transplant serum was available, median pre-transplant BAFF level was 7.48 pg/mL (range of 0.91~ 60.97 pg/mL). Patients were divided into 3 tertile groups according to serum BAFF levels and a comparison of baseline and clinical characteristics among the tertiles were made Tables . There wWe observed the correlation of serum BAFF levels with pre-transplant sensitization status and early acute rejection episodes, defined as rejection within 6 months from KT, and also compared the parameters among the 3 tertile groups. Higher serum BAFF levels before transplantation were significantly associated with pre-transplant sensitization; 50% of patients in tertile 3 showed positive PRA compared with 34.6% of patients in tertile 1 (P = <0.05), 42.3% of patients in tertile 3 showed high PRA levels (defined as PRA levels higher than 50%) compared with 20.8% of patients in tertile 1 (P = <0.05) . Higher Post-transplant BAFF levels showed significant association with pre-transplant sensitization; 46.5% of patients in tertile 3 showed positive PRA, compared with 25.6% of patients in tertile1(P = <0.05) and presence of HLA-DSA was found in 23.3% of patients in tertile 3 while only 5.5% of tertile 1 patients showed positive HLA-DSA before transplantation (P = <0.05) . PRA I lIn 78 patients, in whom both pre and post BAFF level were available, we compared the change of serum BAFF levels before and after transplantation. Serum BAFF levels showed significant increase at post-transplant compared to pre-transplant (P<0.05) . Median In this study, we sought to analyze the clinical significance of measuring pre-transplant and post-transplant serum BAFF levels in kidney transplant recipients We found that although pre-transplant BAFF was associated with pre-sensitization and allograft rejection as in previous studies ,13, postTo determine the clinical significance of serum BAFF levels measured before and after transplantation, we chose to observe the correlation of BAFF levels with clinical parameters, and then to divide each group of patients into tertiles according to serum BAFF levels as there has been no reference range yet that has been agreed upon, and levels vary enormously from study to study ,16,17. OHowever, in the present study, we were more interested in determining the clinical significance of measuring post-transplant BAFF levels, as this aspect has not been thoroughly investigated yet. We measured post-transplant serum BAFF levels in samples taken at the time of indication biopsy in hopes to investigate the relationship between BAFF levels and allograft biopsy results. Interestingly, we found that post-transplant serum BAFF levels also showed significant association with pre-transplant sensitization. The reason is unclear but, it is possible that once humoral immunity has been activated before transplantation, high BAFF levels may persist until the post-transplant period. This may explain the high risk of chronic antibody mediated rejection and allograft dysfunction during long-term follow up especially in patients who were highly sensitized pre-transplant ,20.Contrary to our expectations, no significant associations between post-transplant BAFF levels and allograft biopsy findings regarding rejection, findings based on the Banff classification and allograft survival, were found. Our negative findings may be explained by previous studies of serum BAFF kinetics in renal transplantation and chronic GVHD ,17. Thes2, intravenously) a month before transplantation. B-cell depleting agents such as this have been reported to be associated with significant elevation of serum BAFF levels for more than 1 year after transplantation [P = 0.000)  . However= 0.000) . These rFinally, we performed an additional analysis to investigate the change in BAFF levels between pre and post-transplantation. We also evaluated whether delta BAFF levels were associated with post-transplant clinical outcomes in 78 patients in whom pre and post-transplant serum were available. Delta BAFF levels also showed significant association with pre-transplant sensitization but not with post-transplant DSA or allograft rejection. This non-specific rise in serum BAFF levels immediately after transplantation, may similarly be explained by the activation of various BAFF-producing immune cells due to normal immune responses stimulated by the transplanted allograft .There are a few limitations to this study. As mentioned earlier, we only took post-transplant samples at the time of indication biopsy, therefore we could not assess the point at which serum BAFF levels may have shown correlation with allograft dysfunction or rejection. A longitudinal study with sampling at multiple time points will be needed to determine whether post-transplant serum BAFF is at any time useful in predicting graft outcomes. Secondly, in our study, we were only able to measure serum BAFF levels which represent a fraction of the total BAFF pool. We could additionally analyze the cell-membrane-bound form of BAFF by measuring the BAFF mRNA on peripheral blood mononuclear cells as previously done by Thibault-Espitia et al .In conclusion, this is the first study to examine the clinical significance of both pre and post-transplant serum BAFF levels in adult kidney transplant recipients. Pre-transplant BAFF levels may be useful in predicting allograft rejection, but post-transplant BAFF levels measured at the time of renal dysfunction failed to show significant correlation with allograft outcomes.S1 Fig(A) Correlation of pre-transplant BAFF levels with DSA titer. Note that no significant correlation was observed. BAFF, B cell activating factor; KT, kidney transplant; DSA, donor specific antibody; MFI, median fluorescence intensity.(TIF)Click here for additional data file.S2 FigComparison of delta BAFF levels with (A) pre-transplant PRA I, (B) PRA II, (C) presence of pre-transplant HLA-DSA, and (D) post-transplant acute rejection showed weak association with pre-transplant PRA I but did not show significant association with PRA II, prevalence of pre-transplant HLA-DSA or allograft biopsy findings. BAFF, B cell activating factor; PRA, panel reactive antibody; DSA, donor specific antibody; TCMR, T cell mediated rejection; AAMR, acute antibody mediated rejection.(TIF)Click here for additional data file.S3 Fig(A) Comparison of post-transplant BAFF levels in patients who underwent Rituximab desensitization therapy and those who did not showed that serum BAFF levels were significantly higher in the group that underwent Rituximab desensitization therapy. Comparison of (B) pre-transplant sensitization and (C) prevalence of post-transplant HLA-DSA among post-transplant BAFF tertiles in the subgroup analysis showed that post-transplant BAFF levels in the subgroup of patients excluding those who underwent desensitization therapy were also significantly associated with pre-transplant sensitization but not with the prevalence of post-transplant anti-HLA antibody and HLA-DSA. BAFF, B cell activating factor; PRA, panel reactive antibody; HLA-DSA, anti-HLA donor specific antibody.(TIF)Click here for additional data file.


Optic neuritis (ON) in patients with anti-myelin oligodendrocyte glycoprotein (MOG)-IgG antibodies has been associated with a better clinical outcome than anti-aquaporin 4 (AQP4)- IgG ON. Average retinal nerve fiber layer thickness (RNFL) correlates with visual outcome after ON.The aim of this study was to examine whether anti-MOG-IgG ON is associated with better average RNFL compared to anti-AQP4-IgG ON, and whether this corresponds with a better visual outcome.A retrospective study was done in a consecutive cohort of patients following anti-AQP4-IgG and anti-MOG-IgG ON. A generalized estimating equation (GEE) models analysis was used to compare average RNFL outcomes in ON eyes of patients with MOG-IgG to AQP4-IgG-positive patients, after adjusting for the number of ON events. The final mean visual field defect and visual acuity were compared between ON eyes of MOG-IgG and AQP4-IgG-positive patients. A correlation between average RNFL and visual function was performed in all study eyes.Sixteen patients were analyzed; ten AQP4-IgG-positive and six MOG-IgG-positive. The six patients with MOG-IgG had ten ON events with disc edema, five of which were bilateral. In the AQP4-IgG-positive ON events, 1/10 patients had disc edema. Final average RNFL was significantly better in eyes following MOG-IgG-ON (75.33μm), compared to 63.63μm in AQP4-IgG-ON, after adjusting for the number of ON attacks . Mean visual field defects were significantly smaller  among MOG-IgG positive ON eyes compared to AQP-IgG positive ON eyes, but last visual acuity did not differ between the groups . Among all eyes, average RNFL positively correlated with mean visual field defect  and negatively correlated with final visual acuity .Following ON, RNFL is better preserved in eyes of patients with MOG-IgG antibodies compared to those with AQP4-IgG antibodies, correlating with better visual outcomes. Optic neuritis (ON) is a common inflammation of the optic nerve associated with numerous autoimmune conditions, including multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), chronic relapsing autoimmune optic neuritis (CRION) and autoimmune optic neuritis (AON). NMOSD iCertain clinical and radiological features are suggestive of MOG-IgG positive ON, such as bilateral ON, disc edema and a predilection for the retrobulbar portion of the optic nerve,5. ON inAverage retinal nerve fiber layer thickness (RNFL) correlates with visual outcome after ON. The aimFor this retrospective cohort study we identified patients from the database of our neurophthalmology-neuroimmunology team from 2003–2015. The study was approved by the institutional review board at the Rabin Medical Center.We conducted the following selection process: We included all patients following AQP4-positive NMOSD-ON and MOG-positive ON seen between 2003–2015. NMOSD was defined according to the Wingerchuk et al. 2015 criteria. Serum aPatients of both groups were admitted for acute-phase treatment during acute episodes of ON. An individualized approach was used, with high-dose intravenous methylprednisolone given to all patients during acute attacks and with the optional addition of plasmapheresis or intravenous immunoglobulins (IVIg) when needed. After the acute episode, continued care, maintenance therapy, and follow-up were provided in a step-wise escalation approach, based on individual response. Patients who elected to receive maintenance therapy were treated with rituximab, azathioprine, daily low-dose corticosteroids, IVIg, methotrexate, cyclophosphamide or mycophenolate mofetil. Maintenance treatment choice was based on a combination of patients’ preference, side effects, insurance coverage, and the perceived clinical course.® 4000–2713  was used to measure the average RNFL. Scans were obtained in adherence with the APOSTEL 9-point recommendations and the OSCAR-IB quality criteria [Spectral Domain (SD-HD) Cirrus OCTcriteria ,12. The The following longitudinal clinical data was recorded for each patient: age, gender, inaugural event at presentation, length of follow-up (months), number of ON attacks, whether a bilateral ON attack occurred, presence of disc edema during ON, the worst visual acuity during the most severe ON attack , final visual acuity at last follow-up, final mean visual field defect on Humphrey perimeter (in decibels (dB)), 30–2 spot size 3, (mean deviation) for each eye, the final average RNFL measurement for each eye on OCT and the ICD-10/WHO Visual Disability scale for each patient at last follow-up.The generalized estimating equation (GEE) models with a Gamma distribution and log link function, using SPSS for IBM (2013) for Windows, Version 22.0. Armonk, NY: IBM Corp were used to compare the final average RNFL (as a dependent variable) between ON eyes of MOG-IgG and AQP4-IgG-positive patients, with the number of ON events as covariate. The absolute mean visual field defect (dB) and the last visual acuity (logMAR) was compared in AQP4-positive ON eyes and in MOG-positive ON eyes using GEE models. Among all study eyes, the average RNFL was correlated with mean visual field defect and with visual acuity. GEE was performed using both eyes of patients for bilateral cases controlling for within subject inter-e ye correlations.We identified 12 patients amongst 18 with AQP4-IgG positive NMOSD who had ON, two of whom were excluded due to insufficient follow-up. We identified six patients following ON with MOG-IgG antibodies. Longitudinal data of at least 12 months following the last event was complete for all six patients with MOG-IgG ON and for ten of the 12 AQP4-IgG positive NMOSD who had ON . Only one of the six patients with MOG-IgG positive antibodies met the criteria for NMOSD.  All AQP4-IgG positive patients met the revised 2015 criteria for NMOSD(2). Demographic data, inaugural event and length of follow-up for each patient are detailed in Sixteen eyes of ten AQP4-IgG positive NMOSD patients experienced 35 ON episodes , and nine eyes of six MOG-IgG positive patients had 10 ON events . Disc edema was observed in all MOG-IgG positive ON attacks and in only one patient with AQP4-IgG antibodies. The final WHO visual disability score was normal in 4/6 MOG-IgG positive patients and mildly impaired in another 2/6. Amongst the AQP4-IgG positive patients, the final WHO visual disability score was normal in only one case. https://dx.doi.org/10.6084/m9.figshare.4300178.v1.RNFL thickness was significantly lower in ON eyes of AQP4-IgG positive patients . The meaAmong ON eyes, MOG-IgG was significantly associated with higher average RNFL compared to eyes of AQP-IgG positive patients, even after adjusting for number of ON attacks .The mean visual field defects were significantly smaller among ON eyes of MOG-IgG positive patients , compared to ON eyes of AQP-IgG positive patients , , but last visual acuity did not differ significantly between the two groups .Among all eyes, average RNFL values positively correlated with mean visual field defect  and negatively correlated with final visual acuity .In this cohort study of ON outcomes; average RNFL was better preserved in eyes of MOG-IgG positive patients compared to those with AQP4-IgG positive antibodies, even after adjusting for the number of ON attacks. The improved anatomical outcome in MOG-IgG positive patients closely correlated with a better visual acuity and visual field outcome.Favorable visual outcome  and relaIn addition to the different antibody targets; MOG-IgG optic neuropathy may be more sensitive to treatment because it involves secondary immune processes. An animal model of AQP4-IgG infused into the CSF has been shown that AQP-IgG is directly pathogenic. This moA limitation of this study is the difference in disease duration between the two groups. This difference is a direct result of the fact that the first reports of MOG-associated disease  began alThe results of the largest study on MOG-IgG ON to date,21, publDespite the caution needed in interpreting analyses from this small cohort, our finding are consistent with prior reports ,22. WhetThe results of this study also highlight the fact that visual acuity alone is a poor indicator of residual deficit after ON. There was an insignificant difference in visual acuity outcome between ON eyes of MOG-IgG and AQP-IgG positive patients. However, both the mean RNFL and the mean visual field defect were better indicators of residual deficit after ON . We did We recognize the inherent statistical flaws stemming from the separate-eyed nature of our analysis, leading to a possible overstatement of statistical estimates . MOG-posIn conclusion, RNFL is better preserved in eyes following ON of patients with MOG-IgG antibodies compared to those with AQP4-IgG antibodies, correlating with visual outcome. Recognizing MOG-antibody positive associated disorders is not only an issue of nomenclature, but of potential clinical importance.


By using a gus gene and an hpRNAi cassette targeted for OsPDS as reporters, we demonstrated that soaking transgenic seeds with estradiol solution could induce highly efficient site-specific recombination in germinating embryos, resulting in constitutive and high-level expression of target gene or RNAi cassette in intact rice plants from induced seeds. The strategy reported here thereby provides a useful gene activation approach for effectively regulating gene expression in rice.Inducible gene expression has emerged as a powerful tool for plant functional genomics. The estrogen receptor-based, chemical-inducible system XVE has been used in many plant species, but the limited systemic movement of inducer β-estradiol in transgenic rice plants has prohibited a wide use of the XVE system in this important food crop. Here, we constructed an improved chemical-regulated, site-specific recombination system by employing the XVE transactivator in combination with a Cre/ Inducible gene expression systems are powerful tools for basic research in plant functional genomics, and for plant biotechnology applications such as the deletion of selectable marker genes in transgenic plants . Over thArabidopsis demonstrated that the target gene expression controlled by this system was highly induced and tightly regulated upon estradiol induction  is one of the world’s most important staple food crops and has been established as a model organism for the functional genomics of monocot plants. The availability of highly accurate genomic sequences of both indica  gene showed that soaking transgenic seeds with estradiol solution could activate highly efficient site-specific recombination in germinating embryos, resulting in constitutive activation of the target gene or RNAi cassette in intact rice plants. Our method thereby provides a useful gene activation approach for functional genomics in rice.In the present study, based on detailed investigation of XVE-controlled gene expression patterns in calli, detached leaf pieces, and intact plantlets, we proposed that limited systemic movement of estradiol might be the key reason for low inducibility in intact rice plants. To meet the need for high-level target gene expression in intact rice plants, we further constructed an improved XVE-controlled, site-specific recombination system and optimized the induction procedure for rice seeds. Experiments based on a reporter Plant expression vectors pUX-GUS, pXCL-GUS, pXCLF-GUS, and pXCLF-PDSi were constructed following standard molecular manipulation procedures , except O. sativa cv. Nipponbare. PEG-mediated protoplast transfection was carried out by following previously described procedures  analysis of transgene copy number were used for further analyses  was dissolved to 20 mM in dimethyl sulfoxide (DMSO), and stored at -20°C as stock solution. The stock solution was added to sterilized water, or solid medium at a final concentration of 20 μM immediately before use. The same volume of DMSO was added to the negative control solution or medium without estradiol. For induction treatment, rice transgenic calli were directly cultured on N6 medium supplemented with estradiol, and rice detached leaf pieces or rice seeds were submerged in estradiol water solution. In whole-plantlet treatment, seedlings germinated on 1/2 MS medium plates were transferred to small tubes or flasks, and plantlet roots were submerged in estradiol water solution.D-glucuronide  as substrate, and the histochemical staining was performed using X-Gluc  as substrate.GUS assays were performed as described by RBE4  and was subsequently treated with RNase-free DNase I  to remove DNA contamination. Reverse transcription was carried out using 2.5 μg of DNase I-treated total RNA with the RevertAid First Strand cDNA Synthesis Kit  according to the manufacturer’s instructions. The qPCR was performed on an ABI 7500 real-time PCR system  using SYBR Premix Ex Taq  according to the manufacturer’s instructions.All primers used for PCR, qPCR, and qRT-PCR were listed in the Supplementary Table Southern blot analysis was performed following standard procedures . Rice gepUX-GUS  using a gus gene as a reporter, because the GUS system has been proved to be a reliable and extremely sensitive reporter allowing both quantitative and histochemical assessment of gene activity in plants . In transfected rice protoplasts without treatment with estradiol, GUS expression was detected at a very low level after 24 h incubation, and remained at a similar low level after 36 or 48 h incubation .We generated the estrogen-inducible expression construct n plants . The pUX1 in which a gus gene is driven by a constitutive CaMV 35S promoter . By contrast, almost no GUS staining was observed in X-GUS calli without treatment with estradiol during the culture period .To further analyze XVE-controlled gene expression in stably integrated transgenic rice tissues, pUX-GUS and a control construct pCAMBIA1305.2promoter , were th0 X-GUS lines were obtained. Small pieces of transgenic plant leaves were cut and exposed to 20 μM estradiol for 24 h, and the GUS activity was determined by histochemical staining. From 17 independent lines, two lines showed no GUS staining in detached leaf pieces either treated or not treated with estradiol, and one line showed weak GUS staining in both induced and uninduced leaf pieces. For the remaining 14 lines, while no GUS staining was detected in leaf pieces without treatment, clear GUS staining was observed in estradiol treated-leaf pieces . Taken together, these results demonstrated that the XVE system could stringently and efficiently regulate gene expression in rice cells and tissues.Transgenic calli were further cultured to regenerate rice plants, and a total of 17 independent T1 generation, Southern blotting and qPCR analysis of transgene copy number  were treated with estradiol, and GUS staining could be observed in induced leaf tissues but not in uninduced tissues, similar to that of T0 generation plants . These results suggested that XVE-controlled GUS expression pattern could be inherited stably over generations.The transgenic X-GUS, and 35S-GUS rice plants were grown to produce progeny of advanced generation for further analysis. Among the 14 screened X-GUS lines, #5 and #11 were examined to confer a strong estradiol-inducible GUS expression pattern in detached leaf pieces, and contain a single copy of T-DNA insertion based on genetic segregation analysis of T5 homozygous lines were cultured in liquid solution via a root absorption manner for 2 days. The cultured seedlings were subjected to histochemical staining. While no GUS expression was observed in plantlets of X-GUS-#5 or X-GUS-#11 cultured in liquid medium without estradiol, strong GUS staining was observed in the roots of X-GUS-#5 and X-GUS-#11 cultured in estradiol solution . However, when compared with 35S-GUS plants, X-GUS plantlets showed only weak GUS staining in leaf tissues . A fluorometric assay was also performed to detect GUS expression level in transgenic plantlets. Three-leaf-stage seedlings of transgenic plants were cultured in liquid solution for different induction periods , and the roots, shoots, and leaves of the cultured seedlings were collected and evaluated for GUS activity. With 20 μM estradiol induction for 2 days, GUS expression was induced in X-GUS plantlets. In the roots of X-GUS-#5 and X-GUS-#11, GUS level was about two times higher than or similar to that of 35S-GUS plants . However, GUS level in the leaves of X-GUS-#5 and X-GUS-#11 was much lower than that of 35S-GUS plants , similar to previous report that XVE-controlled GFP was expressed at a low level in the leaves of transgenic seedlings cultured on the inductive medium .To examine the estrogen-induced target gene expression patterns in intact rice plantlets, 4-day-old seedlings of Te medium . FluoromArabidopsis, rice plants are larger in size, and have a relatively longer life cycle. The difficulty in inducing target genes to be expressed at a high level in the leaves of intact plantlets, and the relatively short induction duration, have limited the use of the XVE system in rice. We reasoned that a system permitting constitutive gene expression after chemical induction would be more feasible for rice. We thus designed an alternative approach by employing the XVE system coupled with a site-specific recombination cassette. A basic construct pXCL-GUS, in which a XVE-controlled Cre/loxP-mediated recombination would lead to fusion of a maize (Zea mays) ubiquitin-1 promoter , was developed initially. However, rice transformation experiments showed that pXCL-GUS gave only very low transformation efficiency , in which the original nos promoter for the selectable marker gene hpt was replaced with a CaMV 35S promoter, was constructed. In pXCLF-GUS, the two directly oriented loxP sequences were also replaced with two loxP-FRT fusion sequences which were previously reported to have enhanced excision efficiency mediated by Cre recombinase . By histochemical staining assay, about 12 T1 independent lines were detected to display induced GUS expression upon seed-soaking treatment. In LF-GUS lines, GUS expression was detected in the embryos of LF-GUS transgenic seeds soaked in the inductive solution, but not in seeds soaked in the solution without estradiol . The LF-GUS seedlings developed from induced seeds displayed a constitutive expression pattern of GUS, whereas no GUS or only very faint GUS spots were observed in uninduced LF-GUS seedlings .The uninduced rice calli transformed with pXCLF-GUS (named LF-GUS) were cultured on media for regeneration, and a total of 18 independent T2 generation of four LF-GUS homozygous lines, #15, #21, #22, and #42 were subjected to more detailed analysis. In mature flowering plants developed from estradiol-induced LF-GUS seeds, constitutive GUS expression was detected in root, stem, leaf, and floret tissues of LF-GUS plants by histochemical staining . Consistent with results observed in T1 seedlings, almost no GUS staining or only very faint GUS spots were observed in tissues of LF-GUS plants developed from uninduced seeds . Fluorometric assays confirmed that GUS was highly expressed in all tested root, stem, and leaf tissues in the induced mature LF-GUS plants . While the level of GUS activity in root, stem, or leaf tissues of LF-GUS-#15 plants were comparable to that of 35S-GUS-#14 plants, GUS expression level of LF-GUS-#21, #22, or #42 plants was about 1.5–2 times higher than that of 35S-GUS-#14 plants . In uninduced LF-GUS plants, only a low GUS level similar to that of non-transgenic plants was detected . No GUS expression was detected in tissues of mature plants of X-GUS line #11 developed from seeds germinated in liquid solution with or without inducer , indicating that estradiol induction in seeds did not persist to later times.The TpUbi-loxP-FRT-XVE, and LF-P3/LF-P4 to the joint region of Cre-int-Tnos-loxP-FRT-gus of the non-recombinant T-DNA , the expected 1019 and 679 bp fragments were detected in the tissues of uninduced LF-GUS plants, respectively . No LF-P1/LF-P2 or LF-P3/LF-P4 fragments were detected in the tissues of induced LF-GUS plants except for line #42 showing a faint band, indicating highly efficient DNA recombination in induced plants. By contrast, PCR amplifications using primers LF-P1/LF-P4 generated a 626 bp fragment consistent with reconstitution of the pUbi-loxP-FRT-gus transcription unit  in induced LF-GUS plants, except that a faint band was detected in uninduced line # 22 , indicating no DNA or almost no DNA recombination in uninduced plants. The PCR products of LF-P1/LF-P4 fragments amplified from induced plants of the four LF-GUS plants were sequenced. And sequencing results from all four lines were identical, showing that the maize ubiquitin-1 promoter was contiguous with one copy of loxP-FRT sequence, followed by the gus sequence . DNA isolated from LF-GUS-#15 and LF-GUS-#21 plants was double cut with XhoI and SpeI , and Southern blot analysis was performed using a 3′-end fragment of the maize ubiquitin-1 promoter as a probe. As showed in Figure 6E, an expected 4.2 kb band was detected in uninduced LF-GUS-#15 and LF-GUS-#21. By contrast, a 1.4-kb band was detected in induced LF-GUS-#15 and LF-GUS-#21 plants, confirming the recombination events in LF-GUS plants.PCR analysis was performed on genomic DNA isolated from root, stem, and leaf tissues to investigate the recombination events in LF-GUS plants. Using specific primers LF-P1/LF-P2 targeted to the joint region of Figure 1), which contained an hpRNAi cassette targeted to a rice phytoene desaturase (OsPDS) gene, was developed and introduced into rice by Agrobacterium-mediated transformation. A total of 14 independent T0 plants (named LF-PDSi) were obtained, and no uninduced plants showed detectable albino phenotype. In the T1 generation, eight lines of induced transgenic plants showed a clear albino phenotype , whereas all transgenic lines developed from seeds geminated in liquid solution without estradiol grew similar to non-transgenic control plants. Real-time RT-PCR analysis showed that the endogenous OsPDS mRNA level was greatly reduced in the induced plants . Using the same primers LF-P1, LF-P2, and LF-P3 used for detecting LF-GUS plants, and a specific primer LF-P5 targeted to the hpRNAi cassette of the OsPDS fragment , PCR analysis detected LF-P1/LF-P2, LF-P3/LF-P5 non-recombinant fragments only in uninduced plants, except for induced line #26 showing faint amplified bands . By contrast, PCR analysis detected a 583 bp LF-P1/LF-P5 recombinant fragment only in induced LF-PDSi plants . Moreover, sequencing results of the LF-P1/LF-P5 amplified products from induced LF-PDSi plants indicated the accurate reconstitution of the recombinant cassette of pUbi-loxP-FRT-hpRNAi (OsPDS) , confirming again that the XVE system is highly inducible and tightly regulated in rice cells. However, in intact plantlets induced with estradiol via root immersion, while the roots of the plantlets displayed high inducibility of GUS, the stems and the leaves expressed a much lower level of GUS. XVE was driven by a maize ubiquitin-1 promoter , which gives constitutive and high-level expression of transgenes in rice  also suggested that estradiol uptake and XVE level should be sufficient for activating target gene induction. Therefore, the most likely reason for low inducibility of target gene expression in the leaves of intact plantlets could be that systemic movement of estradiol was limited within the rice plant.By using the firefly luciferase system as a reporter, n 2 days . Similar in rice . In addi in rice . The facpER8 . Transformation experiments indicated that this modification greatly enhanced the rice transformation efficiency of the constructs , indicating that uptake of estradiol by rice seeds at early stages of germination could efficiently induce Cre/loxP-FRT-mediated DNA excision in rice embryos. Consistent with this, rice seedlings and matured plants developed from induced seeds displayed a constitutive GUS expression pattern , and the GUS expression level in the roots, stems, or leaves were higher or at least similar to that of transgenic rice plants expressing GUS under control of a 35S promoter . This property of the XCLF system would allow target genes to be expressed at the desired high level for functional characterization. In the present study, the XCLF system was also tested for knocking down target gene expression. We tested the XCLF system using an hpRNAi cassette for the OsPDS gene. The clear albino phenotype and the reduction of OsPDS mRNA of induced LF-PDSi plants indicated that the system could be applied in knocking down endogenous gene expression in rice with high efficiency.To meet the need for chemical-regulated, high-level expression of target gene in intact rice plants, we have designed an alternative approach by employing the XVE system coupled with a site-specific recombination cassette. In the backbone of the original XVE constructs pER8 , the selpromoter . In the loxP system was not efficiently induced in the germline cells in rice. In the present study, we detected highly efficient induced DNA excision in germinating rice embryos. Molecular analyses of induced rice plants displaying strong recombinant phenotypes showed that complete or almost complete DNA excision occurred in the tested plants . The possible reason for high efficiency of induced DNA recombination in our experiments could be the efficient uptake of estradiol in germinating embryos by the soaking treatment of rice seeds. Unlike previous work, where transgenic seeds were germinated on solid inductive media , therefore providing a useful tool for functional genomics and biotechnology applications such as the deletion of selectable marker genes in rice.The chemical-induced, seed-soaking activation procedure based on the XCLF system appears to be effective in regulating gene expression in intact rice plants (MF434112 (pUX-GUS), MF434111 (pXCL-GUS), MF434113 (pXCLF-GUS), and MF434110 (pXCLF-PDSi).The sequence data from this article can be found in the GenBank database, under accession numbers ZJC, G-LW, FW, and SC conceived and designed the experiments. ZJC, QC, CH, XG, ZQC, YL, TH, and MB performed the experiments. ZJC, QC, ZW, FW, and SC analyzed the data. GL and ZW contributed reagents and tools. ZJC and SC wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A newer source of MSCs derived from human pluripotent stem cells (PSC) can offer the ‘best-of-both-worlds’ scenario, abrogating the concern of teratoma formation while preserving PSC proliferative capacity. PSC-derived MSCs (PSC-MSCs) also represent MSCs at the earliest developmental stage, and we found that these MSCs harbor stronger neuro-differentiation capacity than post-natal MSCs. PSC-MSCs express higher levels of neural stem cell (NSC)-related genes and transcription factors than adult bone marrow MSCs at baseline, and rapidly differentiate into neural-like cells when cultured in either standard neurogenic differentiation medium (NDM) or when the cytoskeletal modulator RhoA kinase (ROCK) is inhibited. Interestingly, when NDM is combined with ROCK inhibition, PSC-MSCs undergo further commitment, acquiring characteristics of post-mitotic neurons including nuclear condensation, extensive dendritic growth, and neuron-restricted marker expression including NeuN, β-III-tubulin and Doublecortin. Our data demonstrates that PSC-MSCs have potent capacity to undergo neural differentiation and also implicate the important role of the cytoskeleton in neural lineage commitment.Mesenchymal stem cells (MSCs) are paraxial mesodermal progenitors with potent immunomodulatory properties. Reports also indicate that MSCs can undergo neural-like differentiation, offering hope for use in neurodegenerative diseases. However,  The discovery of a stem cell population in the adult brain has opened the possibility of using such tissue-specific stem cells for therapy in neuro-degenerative and ischemic diseases, including Parkinson's disease and stroke . As exciex vivo expansion to reach the volumes necessary for therapeutic use, and this can result in replicative senescence and functional decline [A number of studies have shown that mesenchymal stem cells (MSCs) harbor neural differentiation potential and have therapeutic relevance for treatment of related diseases . Multili decline . To over decline –10. The  decline . Moreove decline . Being d decline .Traditionally, induction of neural differentiation has involved the use of biochemical factors in serum-free conditions . Recent Nestin, an intermediate filament, and Musashi, a RNA-binding protein, which are neural stem cell (NSC)-associated genes [Nestin and Musashi than adult BM-MSCs , iPS-MSCs-1 (25.9-fold), and iPS-MSC-2 (30.0-fold) compared to BM-MSCs. We then performed neural differentiation using standard neurogenic differentiation medium (NDM), which consist of serum-free medium with addition of retinoic acid (RA) [Nestin, Musashi, and MAP2 (microtubule-associated protein 2)—a critical protein involved in neural differentiation [To assess the neurogenic potential of human PSC-MSCs, including human ESCs-derived MSCs (ES-MSCs) and iPS-derived MSCs (iPS-MSCs), we performed real-time PCR to detect baseline expression of ed genes . We founs Figure . ES-MSCscid (RA) . After cntiation —in all MCytoskeletal rearrangement in post-natal MSCs has been shown to direct neural lineage commitment through RhoA kinase (ROCK), one of the most important mediators in cytoskeletal dynamics , 16. We MAP2, which is a marker of NRPs [Given the strong baseline expression of NSC-related genes and response to cytoskeletal manipulation, we were interested whether PSC-MSCs could further undergo differentiation into more committed neural lineage cells. NSCs are known to further differentiate into committed cell types: neuron-restricted progenitors (NRPs) and glial-restricted progenitors (GRPs) , 24. To  of NRPs , was alsNRPs are positive for expression of β-III-tubulin, Doublecortin and NeuN, but negative for NSC markers such as Nestin , 27. β-Iin vitro expansion for therapeutic use [in vivo disease models for future clinical applications.Increasing evidence for MSC plasticity has brought hope for use of these versatile somatic stem cells for a multitude of clinical indications . Howeverutic use , 31. Humutic use , 32. Addutic use , 11. Ourin vitro studies in which ROCK inhibition of PSC-MSCs in SF conditions—rather than with serum in complete medium—results acquisition of a more committed neural phenotype that is more profound than that induced with RA in SF medium. Our findings in this study are in line with other in vitro studies, in which inhibition of ROCK promotes neurite outgrowth in rat embryo hippocampal neurons and neuronal differentiation in mouse NSCs and ESCs [in vitro modeling of human PSC neural differentiation has also recently been shown in human iPSCs [in vitro recapitulation of in vivo information.Cytoskeletal rearrangement has increasingly been shown to be a critical component during neural development. The proteins of RhoA family are known to be involved in neuronal cytoplasmic extension and growth of dendritic processes seen during the development of the central nervous system , 17, 33.and ESCs , 39. Thean iPSCs . Moreovean iPSCs . These sm-Numb mRNA translation and activate Notch signaling [While iPSCs and direct reprograming of fibroblasts can generate NRPs or other neural cells, these two cellular sources are not without its problems. For iPSCs, unless complete differentiation of all cells is achieved, teratoma formation is always a concern; the use of PSC-MSCs abrogates this possibility. On the other hand, direct reprograming of fibroblasts to neural cells is highly inefficient as not to be clinically relevant currently . In compignaling . This reignaling . FurtherPSC-MSCs were derived and characterized as previously reported . ES-MSCs2) and treated with 0.5 μM retinoic acid  for 48 hours in serum-free medium [PSC-MSCs and BM-MSCs were characterized for trilineage paraxial mesodermal differentiation capacity as previously described , 50. Fore medium . The ROCe medium .IF staining was performed as previously reported . Briefly® Green Master Mix qPCR Kit on the ABI 7500 Real-Time PCR System (both from Applied Biosystems-Thermo Fisher Scientific). Relative gene expression levels were analyzed as indicated by the manufacturer. Primers used are shown in Table Real-time PCR was performed as previously reported . Total Rt test. ANOVA was used to analyze the significance between multiple samples. A value of p < 0.05 was defined as statistical significance.All data were expressed as mean ± SEM. Statistical significance between two samples was analyzed by the Student's PSC-MSCs show potent capacity to differentiate into a committed neural phenotype with high expression of NSC genes at baseline. Cytoskeleton rearrangement through inhibition of the ROCK-myosin II axis in a neural differentiating environment can strongly induce PSC-MSCs to acquire a committed neural cell phenotype, with reduction of nuclear size and upregulation of late neural lineage-specific markers including β-III-tubulin, Doublecortin, and nuclear NeuN. Our data gives support to the use of PSC-MSCs as a possible candidate progenitor cell in therapeutic application towards neuro-degenerative and related diseases, as well as highlight the importance of the cytoskeleton in neural differentiation.


Multipotent mesenchymal stem cells (MSCs) are recruited into tumor microenvironment in response to multiple signals produced by cancer cells. Molecules involved in their homing to tumors are the same inflammatory mediators produced by injured tissues: chemokines, cytokines and growth factors. When MSCs arrive into the tumor microenvironment these are “educated” to have pro-metastatic behaviour. Firstly, they promote cancer immunosuppression modulating both innate and adaptive immune systems. Moreover, tumor associated-MSCs trans-differentiating into cancer-associated fibroblasts can induce epithelial-mesenchymal-transition program in tumor cells. This process determinates a more aggressive phenotype of cancer cells by increasing their motility and invasiveness and favoring their dissemination to distant sites. In addition, MSCs are involved in the formation and modelling of pre-metastatic niches creating a supportive environment for colonization of circulating tumor cells.The development of novel therapeutic approaches targeting the different functions of MSCs in promoting tumor progression as well as the mechanisms underlying their activities could enhance the efficacy of conventional and immune anti-cancer therapies.Furthermore, many studies report the use of MSCs engineered to express different genes or as vehicle to specifically deliver novel drugs to tumors exploiting their strong tropism. Importantly, this approach can enhance local therapeutic efficacy and reduce the risk of systemic side effects. In these last decades, many researches focused on the possible role of mesenchymal stem cells (MSCs) to promote tumor progression by interacting with tumor cells and other stroma cells in the complex network of microenvironment . The mulOnce the MSCs come into contact with the tumor microenvironment (TME) they are “educated” to evolve and differentiate in tumor-associated MSCs (TA-MSCs) and cancer associated fibroblasts (CAFs) –4. Both Many signals are involved in the cross-talk between TA-MSCs and other component of tumor microenvironment. Several chemokines, cytokines, growth factors and others are produced by tumor cells to recruit MSCs from bone marrow and adipose tissue. In turn, TA-MSCs release the same molecules to repress immune surveillance, to induce epithelial-mesenchymal transition (EMT) program and to promote tumor cell migration and invasion. Recently, on this topic we reported that bone-marrow MSCs (BM-MSCs) derived from patients induced the metastatic phenotype of osteosarcoma and hepatocellular carcinoma through stromal derived factor 1 (SDF-1)-C-X-C-Chemokine receptor type 4 (CXCR4) axis and also through the aquaporin 1 (AQP1) membrane channel , 7. FurtAlthough many studies reported the pro-tumorigenic activity of MSCs many others showed their tumor suppressive properties .Here, we mainly focused on the progress made to elucidate the key mechanisms in which MSCs are involved to promote a pro-metastatic phenotype.Nowadays, there are few reports regarding the possibility to target MSCs to hamper cancer progression. However, recently Ramos et al. (2017) reported that the inhibition of Histone deacetylases 8 in MSCs derived from myeloproliferative neoplasms selectively decreases their hematopoietic-supporting ability .Currently, there has been a heightened focus on the homing abilities of MSCs into tumors and their role in promoting tumor progression , 11 but Nakamizo et al. (2005) isolated human MSCs (hMSCs) from the bone marrow and followed their fate labeled them using fluorescent protein. Then, they injected labeled-MSCs into the opposite hemisphere of mouse brain of a orthotopic glioma model and after 14 days observed that the fluorescent hMSCs were exclusively within the brain tumors .Moreover, fluorescent-labeled MSCs were detected in metastatic breast tumors in mice after their systemic administration and monocyte chemotactic protein-1 (MCP-1) produced by tumor cells was involved in their recruitment .Interestingly, it has been reported that human adipose-derived MSCs (AD-MSCs) transduced with a retroviral vector encoding full-length human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) specifically localized into xenografts and mediated tumor cell apoptosis without significant apparent toxicities to normal tissues .Furthermore, Hu et al. (2011) observed that human umbilical blood mononuclear cell (UBMC)-derived mesenchymal stem cells (UBMC-MSCs) transfected with recombinant pIRES2-IL-21-enhancement green fluorescent protein delayed tumor growth and prolonged survival in ovarian-cancer-bearing mice .Tumors are considered the “wounds that never heal” and due to this are in a constant inflammatory status . Indeed,At present, although much research is being carried out in this field, it has not yet been cleared which factor could play the most important role in this mechanism, it would seem that more factors are involved in the mediation of MSC tropism to a specific tumor site. In TME there is a chemotactic gradient consisting of different chemokines: CC-chemokine ligand 2 and 5 , CXC – chemokine ligand 12 and 16 , which are able to recruit MSCs . FurtherMany growth factors such as, platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), transforming growth factor-β (TGF-β) and basic fibroblast growth factor (bFGF) have been found to effectively mediate MSC homing to TME , 26, 27.Doucette et al. (2011) showed that syngeneic MSCs are capable of homing to endogenous gliomas in immunocompetent mice. This model of high-grade glioma was induced by overexpression of PDGF-BB, that previously has been well characterized to be a critical mediator of MSC tropism to tumor sites –30.Recent studies suggest that TGF-β1 is a crucial player in inducing MSC migration towards prostatic carcinoma cells (PC3 DU145) as well as to tumor stroma components . In addi2) were able to secrete high levels of interleukin-6 (IL-6), which served to activate and attract MSCs through Stat3 and MAPK signaling pathways , 8281, 8In particular, in TME where a complex crosstalk between tumor cells and stromal cells takes place, the epithelial tumor cells undergo profound morphological and functional changes trans-differentiating in a mesenchymal-like phenotype with a higher metastatic potential . GeneralHepatocyte growth factor HGF and epidermal growth factor, EGF) [Many EMT inducible factors have been demonstrated to be produced or secreted by MSCs such as cytokines  , chemokior, EGF) , 91 as wor, EGF) , 93.These molecules acting in a paracrine manner are able to orchestrate EMT program. It has been reported the importance of expression and functional activation of a group of EMT-inducing transcription factors  and ZEB2 , 94. TheThe pivotal role of EMT program in cancer progression, has been investigated in several pre-clinical cancer models such as breast , ovarianMost recently, it has been reported that EMT program is also under miRNAs control that may promote mesenchymal or inhibit epithelial gene expression. miRNA-9 which is up-regulated in breast cancer cells, directly targets the E-cadherin-encoding messenger RNA, leading to an increase in cell motility and invasiveness . ConversIn conclusion, EMT process can be drawn as very interesting and complex phenomena that offers a way to move cancer cells from primary tumor to the confined tissues and blood vessels thus facilitating metastatization process . In addiA particular pre-requisite for tumor invasion is represented by modifications of ECM involving the degradation of its components by enzymes as the metalloproteinases 2 and 9 (MMP-2 and MMP-9) –108. It in vitro and in vivo [MSCs are being investigated as cellular vehicles for the delivery of anti-cancer agents on the basis of their pronounced tropism and integration capacity into tumor microenvironment as well as for their immunomodulatory abilities  Table 22. Indeed in vivo . Further in vivo . Many ot in vivo , endosta in vivo , throspo in vivo  and pigm in vivo . Interes in vivo , gliobla in vivo  and panc in vivo . In addi in vivo –130. Ano in vivo , while s in vivo . Importa in vivo . Further in vivo , glioma  in vivo , melanom in vivo  and pros in vivo .MSCs derived from different sources and acting in different tumor contexts can have a pro- or an anti-tumorigenic behavior. Probably, these opposite effects of MSCs can depend on the experimental setting in cell and in the animal models. It seems that among the MSCs which that originate from umbilical cord /cord blood (UC-MSCs) show more anti-cancer properties. Many studies report that UC-MSCs not only suppress tumor progression, but also promote drug sensitivity of different blood cancer cells including Jurkat leukemia cells , K562 erIn the present review, we highlight the multiple activities of TA-MSCs in the TME with particular emphasis on their ability to stimulate cancer immunosuppression, to induce EMT program, to trans-differentiate into CAFs and finally to promote pro-metastatic phenotype Figure .Many mechanisms underlying MSCs role in TME have been elucidated but still further studies are needed to understand completely their involvement in tumor progression.These investigations could provide information for targeting TA-MSCs with therapeutic approaches and eventually for using them as vehicles to deliver specifically in TME anti-cancer agents.