Tyrosine kinase inhibitors (TKIs)-treatments bring significant benefit for patients harboring epidermal growth factor receptor (EGFR) mutations, especially for those with lung cancer. Unfortunately, the majority of these patients ultimately develop to the acquired resistance after a period of treatment. Two central mechanisms are involved in the resistant process: EGFR secondary mutations and bypass signaling activations. In an EGFR-dependent manner, acquired mutations, such as T790 M, interferes the interaction between TKIs and the kinase domain of EGFR. While in an EGFR-independent manner, dysregulation of other receptor tyrosine kinases (RTKs) or abnormal activation of downstream compounds both have compensatory functions against the inhibition of EGFR through triggering phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signaling axes. Nowadays, many clinical trials aiming to overcome and prevent TKIs resistance in various cancers are ongoing or completed. EGFR-TKIs in accompany with the targeted agents for resistance-related factors afford a promising first-line strategy to further clinical application. EGFR is a transmembrane glycoprotein belonging to the ErbB family of RTKs which includes ErbB-1 (EGFR), ErbB-2 (HER2/neu), ErbB-3 (HER3), and ErbB-4 (HER4) . ConsequTo attenuate the effects that EGFR pathways take on cancers, EGFR TKIs that bind the tyrosine kinase domain of EGFR specifically and inhibit its activity are widely administrated for clinical application. For instance, erlotinib and gefitinib  are used to treat patients with EGFR-mutant NSCLC and show significant efficacy . NeverthRTKs are a kind of receptor for various growth factors, cytokines, and hormones. RTKs have a similar molecular structure: an extracellular ligand-binding region, a single hydrophobic transmembrane domain, and a cytoplasmic protein tyrosine kinase region plus additional carboxy terminal and juxtamembrane regulatory regions . The RTKTwo primary downstream signaling pathways of EGFR are the PI3K/Akt/PTEN/mTOR and the RAS/RAF/MEK/ERK Fig. . PhosphoMET, belonging to the RTKs family, is amplified and relevant to the TKIs resistance in EGFR-dependent cancers, especially in lung cancer. In a gefitinib-sensitive lung cancer cell line HCC827, focal amplification of MET was found to stimulate ErbB3 phosphorylation which in turn activated downstream PI3K/Akt signaling axis compensating the inhibitory effect of gefitinib on EGFR . On the Referring to the mechanisms of MET amplification in TKI-resistant tumors, it was acknowledged that MET amplification was pre-existed at low frequencies in untreated HCC827 cells and NSCLC patients (approximately 4%) , and undDual targeting of EGFR and MET may provide an effective approach to prevent the development of MET-amplified EGFR TKI–resistant tumors . CurrentHGF, known as the ligand of MET, is primarily produced by lung cancer cells  and stroHGF is not spontaneously secreted at a detectable level in two gefitinib-sensitive lung adenocarcinoma cell lines (PC-9 and HCC827 cells) . By pretInterestingly, HGF facilitated MET amplification both in vitro and in vivo through upregulating pre-existing MET-amplified clones , 37. TheIn recent years, there are some inconsistent views concerning the influence of ErbB2 dysregulation on the susceptibility of tumor cells to EGFR-TKIs in NSCLC –40. TradIt was elucidated that the resistances to EGFR- or ErbB2-TKIs during the treatment of several malignancies were initiated by ErbB3 –50. ErbBErbB3-induced drug resistance is primarily mediated by three methods. At first, as mentioned above, MET amplification was known to endow ErbB3 signaling with persistent activation and contribute to the resistance to gefitinib in lung cancer cell lines . BesidesActivation of IGF1R is another mechanism conferring the acquired resistance against gefitinib to EGFR-amplified and EGFR-mutant cancer cell lines . And theIn gefitinib-resistant A431 squamous cancer cells, sustained PI3K signaling in the presence of gefitinib was a result of IGF1R-induced signal transduction . ConcurrAlbeit above preclinical researches showed the potent correlation between the IGF1R activation and TKIs resistance, there was insufficient study focusing on this trend in clinical patients. It has been reported that the high frequency of IGF1R (39–84%) was detected in patients with various cancers –67, howeAXL, a subfamily member of RTKs, is correlated with cell survival, proliferation, metastasis, and phagocytosis , 69. ThePTEN, acting as a tumor inhibitor, negatively regulates the PI3K/Akt signaling cascade by converting PIP-3 back to PIP-2 , 78. TheMechanically, the transcription factor, EGR1, is responsible to the abnormal expression of PTEN. By a nuclear translocation manner, EGR1 played a positive role in regulating PTEN expression . HoweverPIK3CA gene encodes the catalytic subunit of PI3K and has occasionally mutation in lung cancer [PIK3CA mutation which led to sustained PI3K/Akt signaling conferred the resistance of EGFR-mutant HCC827 cells to gefitinib [PIK3CA mutations in 5% EGFR-mutant patients with acquired resistance to EGFR-TKIs [PIK3CA mutations.Mutational activation of the downstream signaling components, such as PI3K/Akt or MEK/ERK, which was independent on the EGFR was identified as a novel mechanism of TKIs resistance , 85. PIKg cancer . In a viefitinib . WhereafGFR-TKIs . CombiniBRAF, known as a member of RAS signaling pathway genes, was reported to be involved in pro-mitogenic activity and acquired resistance to EGFR TKIs in lung cancer and colorectal cancer through activating the MAPK signaling axis [BRAF mutations were generally existed in malignant melanoma (30%–40%), whereas it only accounted for approximately 1% of NSCLC [BRAF mutations resulted in negative results (poor prognosis) and provided cognition about mechanisms of acquired resistance to EGFR-TKIs in lung cancer [Additionally, ing axis , 88. BRAof NSCLC . Neverthg cancer .NCT02151981) showed that AZD9291 significantly improved objective response rate (ORR) and PFS in T790 M-mutant NSCLC patients who had disease progression on first-line EGFR-TKIs [Nowadays, the third generation EGFR-TKIs, including osimertinib, rociletinib (CO-1686), HM61713 (BI 1482694), ASP8273, EGF816, and PF-06747775, were widely introduced to replace the first generation EGFR-TKIs to overcome the status of drug resistance –92. A reGFR-TKIs . SubsequMoreover, bypass tracts including amplifications of other tyrosine kinases or abnormal activation of downstream compound also mediated the resistance to third-generation TKIs. HER2 and MET amplifications led to poor response to CO-1686 and were detected in patients who had disease progression on CO-1686 or osimertinib treatment , 95. BesCurrently, the mechanism study on the resistance to EGFR-TKIs has attracted broad attention. There are two major ways involving the initiation and development of resistance to TKI. One is the secondary mutations of EGFR which alter the drug target site of EGFR so that prevent effective interaction with TKIs , 98. AnoBased on the recognition of above resistant mechanisms, new clinical trials covering phase I-IV are emerging to provide therapeutic interventions adapting for patients with refractory or recurring cancers by inhibiting the alternative pathways –101 (Tab


Antibody-drug conjugates (ADCs) have become a promising class of antitumor agents with four conjugates being approved by regulatory agencies for treating cancer patients. To improve the conventional conjugations that are currently applied to generate these heterogeneous products, various site-specific approaches have been developed. These methods couple cytotoxins or chemotherapeutic drugs to specifically defined sites in antibody molecules including cysteine, glutamine, unnatural amino acids, short peptide tags, and glycans. The ADCs produced showed high homogeneity, increased therapeutic index, and strong antitumor activities in vitro and in vivo. Moreover, there are recent trends in using these next generation technologies beyond the cytotoxin-conjugated ADC. These site-specific conjugations have been applied for the generation of many different immunoconjugates including bispecific Fab or small molecule–antibody conjugates, immunosuppressive antibodies, and antibody–antibiotic conjugates. Thus, it is likely that additional technologies and related site-specific conjugates will emerge in the near future, with various chemicals or small molecular weight proteins in addition to cytotoxin for better treatment of many challenging diseases. Therapeutic antibodies have become a major class of biologics in treating many challenging diseases, including cancer. Due to the fact that they have high specificity and affinity, the monoclonal antibodies have less off-target side-effects as compared to small molecular weight compounds. Currently, many monoclonal antibodies are under accelerated clinical developments . It was As one of the targeted therapies or the “magic bullet” proposed by Paul Ehrlich more than a century ago , the conThus, this unique format would allow the use of certain chemotherapeutic drugs which are too potent or toxic to be applied systemically. Since these cytotoxins need to be delivered into the tumor cells targeting intracellular microtubules or DNA, the chemotherapeutic drugs carrying antibodies should be efficiently internalized into lysosome once binding to the antigen on the tumor surface . Within All these ADCs were prepared using conventional conjugation methods that couple antibodies through either surface-exposed lysines (~70 to 90) or cysteines from interchain disulfides (8 in IgG1) after partial reduction. These lysine and cysteine methods generate heterogeneous products with varied numbers of drugs coupled across several possible sites although the average drug-to-antibody ratio (DAR) appears around 4 to 6, creating significant challenges for process consistency and product characterization ,24,25. IIn contrast to the conventional conjugations to the lysines or cysteines, which are abundant in an antibody, the site-specific conjugations couple the antibodies with cytotoxins through the unique and defined sites based on antibody engineering. There are four major categories of methods based on the conjugation sites in the antibody molecules, including specific amino acids, unnatural amino acids, short peptide tags, or glycans .Several native or engineered amino acids, including cysteines and glutamines, are selected as the sites for conjugation.The unpaired cysteine-mediated conjugation was the first described site-specific ADC as THIOMAB or TDC . A cysteMoreover, cysteine insertion was also described in antibody before and after selected sites in either IgG HC or LC, including LC-V205, HC-A114, and HC-S239 . There wIn addition to conjugation through unpaired cysteine, thiol bridge methods were developed to conjugate a bifunctional drug-linker to both cysteines from each of interchain disulfide, instead of only one cysteine in the conventional cysteine conjugation, after partial reduction and re-oxidation ,32. ThusThe site-specific conjugation through glutamine was also reported ,34. InstThe site-specific conjugation through unpaired cysteine is relatively simple and scalable. The drug coupling was done without the need of special reagents. The method has been applied to prepare multiple site-specific ADCs for preclinical or clinical developments. As described above, the ADCs prepared through site-specific cysteines showed two-fold stronger in vivo antitumor activities and were better tolerated than the conventional conjugates. However, the stability of conjugate generated through unpaired cysteine varied dependent on where the cysteine was introduced in an antibody molecule . It appeMethanococcus jannaschii was expressed in the cells to site-specifically incorporate p-acetylphenylalanine (pAcF) in response to an amber nonsense codon engineered in the antibodies. The keto group of pAcF was then selectively coupled to a drug-linker with aminooxy functionality. The pAcF-containing anti-HER2 was produced from Chinese hamster ovary (CHO) cells co-expressing antibody with an amber codon at heavy chain (HC-A121) and an orthogonal amber suppressor tRNA/aaRS pair. The pAcF residue was coupled with a non-cleavable auristatin linker, which is a potent tubulin inhibitor, through oxime chemistry with DAR at ~2. The anti-HER2 ADC showed strong in vitro and in vivo antitumor activities. The method was further optimized, and a stable CHO cell line was generated to express orthogonal amber suppressor tRNA/aaRS pair ..60].Methanococcus jannaschii was co-expressed to site-specifically incorporate pAcF at defined sites in each of two Fab fragments in response to an amber nonsense codon. The unnatural amino acids was incorporated into LC-S202 of Fab fragment of anti-HER2 and subsequently conjugated with PEG linker containing azide, while pAcF was incorporated in HC-K138 of Fab fragment of anti-CD3 before conjugated with PEG linker containing BCN. Both Fab fragments were subsequently coupled using copper-free click chemistry. In an in vitro cytotoxicity assay, the bispecific antibody Fab conjugate efficiently recruited T cells from human peripheral blood mononuclear cells (PBMCs) to kill the target tumor cells at picomolar concentration. Moreover, the same site-specific antibody conjugation approach was applied to prepare bispecific antibody Fab conjugates against both CD3 and C-type lectin-like molecule-1 (CLL1) as well as both CD3 and CD33 [The antibody fragments have been coupled using site-specific method as bispecific antibody Fab conjugates. In a study, bispecific antibody Fab conjugate was generated using genetically encoded unnatural amino acids with orthogonal chemical reactivity as described above for cytotoxin containing ADC . A tRNA/and CD33 . CLL1 anand CD33 . As descBesides the bispecific antibody Fab conjugation, chemically programmed bispecific antibody conjugation is another strategy for site-specifically preparing a bispecific antibody. Kim et al. reported the generation of a bispecific small molecule-antibody conjugate for targeting prostate cancer . They inStaphylococcus aureus that can establish infection even in the presence of antibiotics vancomycin. They prepared an AAC in which an anti-S. aureus antibody was conjugated to a highly efficacious antibiotics rifalogue that is activated only after being released in lysosome. A cysteine residue was engineered at the V205 position of ant-bacteria antibody light chain and the thiol containing antibody was conjugated to MC-vc-PAB-rifalogue. The AAC is superior to vancomycin for the treatment of bacteremia in vivo.Lehar et al. reported generation of novel antibody-antibiotic conjugate (AAC) using THIOMAB approach . They foA site-specific antibody-polymer conjugation (APC) approach was also reported . An unna50 at nM range, but had no significant effect in hepatocytes, indicating that payload delivery was CD11a-mediated. This approach represents a fundamentally different strategy that uses tissue targeting to overcome the limitations of LXR agonists for potential use in treating atherosclerosis.The site-specific antibody conjugation has been applied for preparing immunoconjugates against other diseases such as autoimmune disease and atherosclerosis. A highly potent phosphodiesterase 4 (PDE4) inhibitor, GSK256066, was site-specifically coupled to a human anti-CD11a through unnatural amino acid, pAcF, introduced at HC-A122 with DAR at ~2 . PDE4 isAs next generation technologies, the site-specific antibody conjugations are likely to be applied to different therapeutic areas for preparing homogeneous immunoconjugates. The combination of therapeutic antibodies with wide varieties of small molecular weight proteins or drugs would potentially expand the current treatment options for many challenging diseases. Although it is unknown which methods are better than others, each category of these methods may have their unique advantages related to the in vivo and in vitro properties of the conjugates.Site-specific antibody drug conjugation technologies have been developed by coupling the cytotoxin to engineered specific amino acids, unnatural amino acids, short peptide tags, and N297 glycans. As next generation methods, these approaches generated homogeneous ADCs with a high therapeutic index as compared to the conventional conjugations. Some of the site-specific ADCs even showed better antitumor efficacy in vivo than the ADCs prepared using conventional methods. Moreover, there are trends in applying these site-specific antibody conjugations to other therapeutic areas or diagnosis. All those studies have provided promising results that suggest the usefulness of these next generation methods in the coupling of small proteins, small molecular weight compounds, DNAs, and RNAs. It is not surprising that these new technologies will lead to important therapeutic platforms for many unmet medical needs.


Fungi have adapted to diverse habitats and ecological niches, including the complex plant systems. Success of the pathogenic or symbiotic fungi in colonizing the plant tissue depends on their ability to modulate the host defense signaling . StrategUstilago maydis secretes the chorismate mutase (Cmu1) into the invaded host cells to interfere with the plant SA pathway during disease development in maize [U. maydis also produces a salicylate hydroxylase, Shy1, which degrades the host-derived SA and helps in biotrophic invasion [SHY1 expression is activated in the presence of SA. Thus, it appears that U. maydis has a robust strategy to suppress SA-mediated plant immunity by blocking the synthesis of fresh SA while degrading the existing hormonal pool in the host. While SHY1 orthologs have been reported only in a few fungal genera, chorismate mutase is found in various plant-associated microbes, and its secretion is likely to be a common strategy for modulating the host defense response.Fungal pathogens either establish a biotrophic relationship, in which the host plant is kept alive, or are necrotrophic and rampantly kill the invaded host cells to feed on the dead material. Hemibiotrophs begin their invasive lifestyle as biotrophs and, once established, switch to necrotrophy. During host invasion and colonization, fungal pathogens typically target phytohormones such as salicylic acid (SA), jasmonic acid (JA), or ethylene (ET), which are primarily involved in the host defense response, or modulate growth hormones like indole-3-acetic acid (IAA), abscisic acid (ABA), cytokinin (CK), or gibberellin (GA), which can also regulate immune signaling in plants . For insin maize . Thus, tinvasion . InteresPuccinia graminis f. sp. tritici, expresses a tryptophan 2-monooxygenase (Pgt-IaaM) specifically in the specialized invaginating structure called the haustorium, leading to excess accumulation of host-derived IAA during establishment of the pathogenic interaction in wheat [Another biotrophic fungus, in wheat .Botrytis cinerea induces the accumulation of a conjugated form of plant IAA (IAA-Aspartate or IAA-Asp) to promote disease development in Arabidopsis thaliana. Preliminary studies indicate that the host-derived IAA-Asp supports in planta invasive growth by regulating the transcription of virulence genes in the fungal pathogen [B. cinerea also secretes an exopolysaccharide as an elicitor of the host SA pathway to eventually suppress the JA-mediated signaling during invasion of tomato plants [Several necrotrophic fungi utilize various low-molecular-weight phytotoxic metabolites to influence the accumulation of defense-related plant hormones or employ their own phytohormone-mimics to suppress the plant immunity and/or aid in disease progression . The necpathogen . IAA-Aspo plants .Sclerotinia sclerotiorum has the ability to degrade host-derived SA during necrotrophic growth [Hyaloperonospora arabidopsidis attenuates the phytohormone signaling pathways via the RxL44 effector protein, containing the highly conserved arginine (R), a random amino acid (x), leucine (L), and another arginine (R) sequence motif, that targets the host mediator complex subunit MED19a, which is a positive regulator of SA-triggered immunity in A. thaliana [Intriguingly, c growth . Howeverthaliana . These fMagnaporthe oryzae produces and secretes an analog of a phytohormone to modulate host immunity [M. oryzae converts intrinsically produced as well as host-derived JA into 12-hydroxyjasmonic acid (12OH-JA) during establishment of the blast disease in rice ([M. oryzae accumulates methyl JA (MeJA), which strongly induces the defense response in rice plants  [in planta growth in the CKS1 mutant due to a precocious induction of the defense response in the invaded rice leaves. It was found that the fungus-derived CKs were likely involved in dampening the host immunity and in mobilizing host nutrients at the site of invasion [M. oryzae has the ability to produce ABA, too. A recent study showed that the endogenously produced ABA helps M. oryzae in proper pathogenic development and host invasion, likely via the suppression of plant immunity [In addition to JA, 1 (CKS1) . The inainvasion . M. oryzimmunity .M. oryzae and rice [These studies highlight the chemical arms race that has shaped the interaction between and rice , 16 in cand rice –21.Laccaria bicolor interacts with PtJaz6, which is a negative regulator of JA-induced gene expression in poplar trees [L. bicolor and if 12OH-JA, the aforementioned metabolite from M. oryzae, targets a Jaz-like repressor of JA signaling in rice.Just like the pathogenic counterparts, the symbiotic fungi, too, secrete effectors and/or host-mimic molecules to modulate the plant defense machinery to establish mutually beneficial associations . The secar trees . Thus, MGlomus intraradices suppresses ET signaling by secreting SP7, which directly targets the ET-responsive transcription factor ERF19 [Tuber melanosporum and T. borchii produce endogenous ET, which likely manipulates the hormonal signaling in the host and thus induces morphological changes in the roots [Similarly, 2 separate studies suggest that down-regulation of the ET pathway by the arbuscular mycorrhizal (AM) fungus is crucial for the establishment of symbiotic associations in the roots –26. Indeor ERF19 . Intriguhe roots . It is pThe emerging areas of metabogenomics would further help in revealing the secrets of the chemical communication between plant hosts and fungi. Overall, there is a great potential for new and/or as-yet-uncharacterized chemical molecules at the fungus-host interface, for improving the growth potential and inducing disease resistance in plants, and for agribiotech applications in precision agriculture.


There are limited data about clinical outcomes of biodegradable polymer biolimus-eluting BioMatrix stents (BP-BES) and durable polymer everolimus-eluting Xience stents (DP-EES) in real world practice. We sought to compare the clinical outcomes of BP-BES and DP-EES in real world cohorts of patients undergoing percutaneous coronary intervention. A prospective multicenter registry enrolled 999 patients treated with BP-BES and 1,000 patients treated with DP-EES. The primary outcome was target lesion failure, defined as a composite of cardiac death, target vessel-related myocardial infarction, or target lesion revascularization. Definite or probable stent thrombosis was also compared in total and propensity score-matched cohorts. The median follow-up duration was 24 months, and mean age was 65 years . Patients receiving BP-BES had a lower prevalence of acute coronary syndrome, prior myocardial infarction, multi-vessel disease, bifurcation lesions, and left anterior descending artery lesions than those receiving DP-EES. After propensity score matching (692 pairs), target lesion failure occurred in 22 patients receiving BP-BES and in 25 patients receiving DP-EES . The risk of definite or probable stent thrombosis did not differ between the 2 groups . The results were consistent across various subgroups. In the propensity score-matched analysis of real world cohorts, BP-BES showed similar clinical outcomes compared to DP-EES. We need to investigate about whether differences in clinical outcome emerge during long-term follow-up. Drug-eluting stents (DES) consisting of a metal platform and a polymer coating with the release of antiproliferative drugs had reduced restenosis compared with bare metal stents ,2. HowevA total of 1,999 patients who underwent percutaneous coronary intervention (PCI) with BP-BES  or DP-EES  at the 16 coronary intervention centers in Korea between July 2010 and June 2012 were enrolled in the prospective multicenter registry . All patAll interventions were performed according to current practice guideline. All patients received loading doses of aspirin (300 mg) and clopidogrel (300–600 mg) before PCI unless antiplatelet medications had previously been prescribed. Unfractionated heparin was administered during PCI in order to achieve an activated clotting time of 250–300 seconds. The choice of DES type and duration of dual antiplatelet therapy was at the operator’s discretion.The primary outcome was target lesion failure (TLF) after stent implantation, defined as a composite of cardiac death, target vessel-related myocardial infarction, or target lesion revascularization. Individual components of the primary outcome, all-cause death, myocardial infarction, and definite or probable stent thrombosis were compared as the secondary outcome. All deaths were considered cardiac unless a definite non-cardiac cause could be established. Myocardial infarction was defined as elevated cardiac enzymes  greater than the upper limit of the normal value that occurred alongside ischemic symptoms or electrocardiography findings indicative of ischemia unrelated to the index procedure. Target lesion revascularization was defined as revascularization within stent or within 5mm border of stent deployment. Definite or probable stent thrombosis was assessed according to the definition of the Academic Research Consortium (9). Basal clinical and angiographic characteristics and all follow-up data were collected prospectively into a dedicated PCI registry, and missed additional clinical information was obtained by the review of medical record or telephone encounter.Continuous variables were expressed as mean ± SD and compared using an independent t-test or the Mann-Whitney test. Categorical variables were summarized as numbers with percentages and compared using chi-square test or Fisher’s exact test. Time-to-event hazard curves were presented with Kaplan-Meier estimates and were compared using a log-rank test. To balance the patients for various clinical and angiographic characteristics, we used the propensity score matching method in a pairwise manner. The propensity score, which represents the probability of the use of BP-BES, was estimated without regard to outcome using multiple logistic regression analysis . The paiAmong a total of 1,999 patients , 999 patients  underwent PCI using BP-BES and 1,000 patients  underwent PCI using DP-EES. Mean age was 65 years . Baseline clinical and angiographic characteristics of patients were described in The median follow-up was 24 months  in the BP-BES group and 24 months  in the DP-EES group. The cumulative outcomes are summarized in The definite or probable stent thrombosis occurred in 4 patients treated with BP-BES and in 4 patients treated with DP-EES  trial compared with durable polymer sirolimus-eluting stents . EspeciaConcerning efficacy, the cumulative rate of TLF defined as a composite of cardiac death, target vessel-related myocardial infarction, or target lesion revascularization was similar between groups in our propensity score-matched population. This result was coincident with those of randomized trials compared with BP-BES and DP-EES, including COMPARE II and NEXT ,11. BP-BThe present study could not avoid certain limitations associated with its observational nature. The use of BP-BES and DP-EES was at the discretion of the physician. Several confounding factors may have affected the results of our study. For example, DP-EES was used more frequently in the complex situation, possibly due to better deliverability of DP-EES . AlthougThe target lesion failure and stent thrombosis of BP-BES were similar to that of DP-EES in this propensity score-matched analysis of an observational registry.S1 Dataset(CSV)Click here for additional data file.


The PD-A patients exhibited altered putamen FC with cortical and subcortical regions. The PD-A patients showed enhanced putamen FC with the caudatum, which correlated with increased emotional processing during anxiety. Decreased putamen FC with the orbitofrontal gyrus and cerebellum also correlated with increased anxiety in Parkinson's disease. Our findings demonstrate that anxiety disorder in Parkinson's disease is associated with abnormal putamen FC networks, especially with caudatum, orbitofrontal gyrus and cerebellum.In this study, we used resting state-functional magnetic resonance imaging (rs-fMRI) to explore altered putamen functional connectivity (FC) in Parkinson's disease patients with anxiety disorder. We divided 65 Parkinson's disease patients into anxiety (PD-A;  Anxiety syndrome is more prevalent in Parkinson's disease (PD) patients (6%-55%)  than in In recent years, neuroimaging technology has been widely used to investigate functional and structural alterations of motor and non-motor symptoms in PD brains , 5. PosiResting state-functional MRI (rs-fMRI) measures the resting blood oxygen level-dependent signal to determine spontaneous brain activity between functionally linked brain regions at the level of neural networks. FC indicates inter-regional temporal patterns of the blood oxygen level-dependent signal . RecentlThe aim of this study was to use rs-fMRI to analyze differences in the putamen FC patterns among 3 study subject groups:, namely, PD patients with anxiety (PD-A), PD patients without anxiety (PD-NA) and healthy controls.Sixty-five right-handed PD patients were recruited from the outpatient clinic of our hospital and diagnosed with idiopathic PD according to the UK Parkinson's Disease Society Brain Bank criteria . The foln = 18) and PD-NA (n = 45) groups based on the cut off score of 12 on the Hamilton Anxiety Rating Scale (HAMA) score  fluorodeoxyglucose PET (FDG-PET) showed that anxiety symptoms in PD were inversely associated with DAT availability in the caudatum or decreased caudate metabolism [Our results show increased putamen FC with caudate in PD-A relative to PD-NA patients. Although striatal dysfunction is typically related to motor impairment in PD , it is itabolism . Therefoet al. showed the role of medial orbitofrontal gyrus in conditioned fear response based on various animal studies and human neuroimaging analysis [We also observed reduced bilateral putamen FC with orbitofrontal gyrus in PD-A compared to PD-NA patients. Previous studies in non-PD patients demonstrated that orbitofrontal gyrus as the site for emotion processing. Milad analysis . Patientanalysis . Moreoveanalysis . This suanalysis . Topograanalysis  and the analysis . Based oThere was also reduced FC between cerebellum and putamen in PD-A relative to PD-NA subjects, thereby negatively correlating with anxiety severity. Clinical, experimental and neuroimaging studies indicate that the cerebellum is involved in neural processes beyond the motor domain , 43. ForThis study has several limitations. Firstly, our sample size was relatively small and therefore may not have detected exact neural substrates of PD-related anxiety disorder. Secondly, anxiety and depression overlap in PD patients with similar symptoms and pathophysiology , 47. HenIn conclusion, our study showed that anxiety in PD was associated with altered FC network between putamen and other regions of the brain, especially caudatum, orbitofrontal gyrus and cerebellum.


We aimed at characterizing the transcription profiles of immunological receptors associated with thebiology of mesenchymal stromal cells (MSCs).RAGE) receptor,C-type lectin receptors , leukotriene B4 (LTB4) receptors(BLT1 and BLT2) and cysteinyl leukotrienes (CysLTs) receptors (CYSLTR1 and CYSLTR2) in distinct populationsof MSCs grown under basic or inflammatory conditions. In this experimental study, quantitative real time-polymerase chain reaction (qRT-PCR) was performed to establish the transcription profiles of advanced glycation end-products ( MSCs derived from adipose tissue (AT), foreskin (FSK), Wharton’s jelly (WJ) and bone marrow (BM)exhibited significantly different transcription levels for these genes. Interestingly, these transcription profilessubstantially changed following exposure of MSCs to inflammatory signals.Collectively, for the first time, our data highlights that MSCs depending on their tissue-source, presentseveral relevant receptors potentially involved in the regulation of inflammatory and immunological responses.Understanding the roles of these receptors within MSCs immunobiology will incontestably improve the efficiency ofutilization of MSCs during cell-based therapies. Mesenchymal stromal cells (MSCs) are multipotent progenitor cells characterized by fibroblast-like shape, high self-renewal capacity and multilineage differentiation potential. Initially isolated from the bone marrow (BM), MSCs have been successfully derived from several other tissues including adipose tissue (AT), Wharton’sjelly (WJ) of the umbilical cord and foreskin (FSK) , 2. Besin vivo effects of MSCs have yielded conflicting results  which are associated with danger signals. Furthermore, inflammation greatly influences the transcription patterns of these receptors. Understanding the roles of these receptors, being considered as inducers, sensors, and mediators of the inflammatory processes , 13, wiThe present experimental study was conducted in accordance with the Declaration of Helsinki (1964) and was approved by the local Ethics Committee of the “Institut Jules Bordet” (Belgium). All samples were obtained from healthy donors who gave informed written consent before initiation of the study.2. After 48 hours, non-adherent cells were removed upon changing the medium. When sub-confluency (80-90%) was achieved, adherent cells were harvested by TrypLE Select solution (Lonza Belgium) and then expanded at a lower density . In order to assess the impact of an inflammatory environment, cells were cultivated under both basic and inflammatory conditions as previously described  and IFN-γ (1000 U/ml or 50 ng/ml) .AT-MSCs, BM-MSCs, FSK-MSCs and WJ-MSCs were isolated from seven independent healthy donors as previously described , 14. Briescribed . BrieflyGAPDH was used as a housekeeping gene. ABI Prism 7900 HT system  was used to perform the amplification reactions. In all cases, dissociation curves were generated and the specificity of the PCR reactions was checked. The comparative ΔΔCt method was followed for data analysis. All qPCR reactions were performed in triplicates. To evaluate the fold change, data were normalized against the GAPDH genes to obtain the ΔCt and calibrated using the geometric mean of the GAPDH ΔCt to generate the ΔΔCt. Fold changes were then calculated as fold change=2-ΔΔCt. The sequences of the used primers are indicated in Table 1. Total RNA was extracted using TriPure Isolation Reagent according to the manufacturer’s guidelines . Reverse transcription reaction was applied for 1 mg RNA using qScript cDNA SuperMix . Next, mRNA levels were quantified by real-time PCR using 20 ng of cDNA, SYBR Green PCR Master Mix  and 0.32 mM forward and reverse primers. Presented data correspond to means ± SEM of three independent experiments and statistically significant differences in gene expression between control and treated cells were determined using unpaired Mann-Whitney U test. P<0.05 were considered significant.Unravelling the receptors, particularly those related to cell danger/injury, that are expressed by MSCs will ultimately determine MSCs immunotherapeutic profile and function and consequently enhance their therapeutic value. Accordingly, using quantitative real-time PCR (qRT-PCR) we examined the transcription profiles of several relevant receptors   in diffeCell-based therapy requires a well-defined and identified cellular product. Thus, before any experimental assay, the studied cells were critically characterized according to with International Society for Cellular Therapy (ISCT) criteria. We confirmed that different MSCs used in this study, were compliant with ISCT criteria . Indeed,BLT1 and BLT2 genes were constitutively transcribed in all MSCs, but differences in the mRNA levels were noted. The highest transcription levels were observed in FSK-MSCs. Following inflammation-priming, BLT1 and BLT2 transcription was strongly induced in AT-MSCs , moderately elevated in BM- and FSK-MSCs  butshowed no change in WJ-MSCs  . Under inflammatory conditions, WJ-MSCs and FSK-MSCs did not show significant alterations in CYSLTR1and CYSLTR2 transcription levels  . In casen levels .RAGE was comparable among different MSCs. The highest levels of RAGE mRNA were exhibited by FSK-MSCs followed by AT- and BM-MSCs whereas WJ-MSCs displayed less RAGE mRNA than the others. Intriguingly, inflammation priming specifically and strikingly enhanced RAGE transcription in AT-MSCs (3.5-fold increase), while no significant alterations were observed in the other cell types  and following inflammation-priming, only AT-MSCs demonstrated a huge induction of DECTIN-1 mRNA levels (30-fold increase). To a lesser extent, BM-MSCs showed a slight but significant DECTIN-1 induction (5-fold increase) (CLEC6A (DECTIN-2) was also minimally transcribed in different types of MSCs under basic growth conditions. However, inflammation priming induced a substantial increase in DECTIN-2 mRNA levels in AT- and FSK-MSCs . DECTIN-2 mRNA was moderately increased in BM-MSCs (3-fold increase) but remained unchanged in WJ-MSCs (CLEC4E gene (Mincle) appeared to be minimally transcribed in both AT- and FSK-MSCs but neither in BM- nor WJ-MSCs. Inflammation priminghad no significant impact on the transcription ofMincle in different types of MSCs  . In par WJ-MSCs . Under b of MSCs . RAGE, CLRs, BLT1, BLT2, CYSLTR1 and CYSLTR2 in different types of MSCs  andinvestigated the impact of inflammation on them. While MSCs are non-immunogenic, they exert marked immunomodulatory activities, thus, they have emerged as a promising immunotherapeutic tool , 17. In In such context, inflammation is known to critically modulate the properties of stem cells . Moreov(LTB4) is a highly potent inflammatory mediator acting through two G protein-coupled seven-transmembrane domain receptors (GPCR) known as BLT1 and BLT2   are a family of potent bioactive lipids that have amino acid cysteine in their structure. They act upon binding to target cell-surface GPCRs, CYSLTR1 and CYSLTR2  in regulating MSCs’biology. A previous report indicated that BLT1 and BLT2 play opposite roles during regulation of umbilical cord-derived MSCs proliferation  but have no effect on IL-6 release. By modulating the secretion profile of DCs, LTB4 as well as CysLTs may initiate T helper 2-type immune responses , 25. A Advanced glycosylation end products (AGEs) correspond to modified protein or lipid structures resulting from the non-enzymatic glycation and oxidization reactions following contact with reducing sugars and may generate an inflammatory response . Due to Due to its inflammatory role in innate immunity and ability to recognize a common structural motif exhibited by a set of ligands, RAGE is also considered as a pattern recognition receptor (PRR). Indeed, RAGE may mediate inflammasome activation and subsequent release of pro-inflammatory mediators, thus contributing to the propagation of the innate immune response . RAGE varied considerably among different MSCs tested. In particular, AT-MSCs showed constitutively high amount of RAGE mRNA which subsequently increased following inflammation-priming. In the literature, two animal models have demonstrated a possible role for RAGE in the immunomodulatory potential of MSCs. In fact, it has been shown that BM-MSCs overexpressing RAGE could competitively bind the high mobility group box chromosomal protein 1 (HMGB1), an important pro-inflammatory molecule, thus reducing the subsequent immuno-inflammatory response in a rat model of acute liver failure (ALF)  . RAGE in AT-MSCs optimized their immunoregulatory properties by decreasing their production of pro-inflammatory mediators (such as IL-1ß and IL-6) and increasing the expression of regulatory molecules (such as IL-10 and TGF-ß). These immunological changes inhibited the differentiation of Th1 as well as Th17 cells, and reciprocally induced T regulatory cell expansion  demonstrated that overexpression of xpansion . As we oCLRs constitute a large family of soluble transmembrane proteins that possess one or more C-type lectin-like domains (CTLD). Different members of this family including DECTIN-1, DECTIN-2 and MINCLE, are considered to be PRRs. CLRs are primarily expressed by immune cells but might be found on other cell types. By sensing molecules that are associated with infection (i.e. PAMPs) or tissue damage (i.e. DAMPs), CLRs contribute to the regulation of the inflammatory and immune response . DECTIN-1, DECTIN-2, and MINCLE were noted among different types of MSCs. Moreover, inflammation specifically increased the expression of DECTIN-1 and DECTIN-2 while no evident impact could be concluded for MINCLE. Importantly, the inflammation-induced increase was not general, as DECTIN-1 was enhanced particularly in AT-MSCs and DECTIN-2 was restricted in BM-, AT- and FSK-MSCs. CLRs can activate distinct signalling cascades that trigger the production of certain cytokines which specify the fates of T-cell polarization. Signalling pathways activated by CLRs either directly regulate the production of pro-inflammatory cytokines through MAPK (Mitogen-activated protein kinase) and NFkB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation or indirectly by modulating Toll-like receptor (TLR)-mediated immune complexes (In the present study, we reported that the transcription profile of CLRs within MSCs is closely dependant on their tissue-origins as constitutive differences in the expression levels of omplexes .Interestingly, MSCs display different TLR expression profiles and inflammation was shown to differentially alter these expression patterns in a manner dependent on MSCs tissue source . Ligand-According to our observations, a role for each of these receptors in modulating MSCs biology, particularly during inflammation, could be thus speculated. As a result, induced signaling pathways of either of the studied receptors within MSCs, can precisely control the expression or modulation of several cytokines that govern the inflammatory and immune response.in vitro. MSCs actively sense their surroundings andaccordingly modulate their biological functions. Thecapacity of MSCs to sense new danger/injury signals such as leukotrienes and/or cysteinyl leukotrienes, AGEs, PAMPs and DAMPs have to be taken into account by characterizing and identifying their receptors. Our results revealed differential transcription profiles of these receptors in a manner dependent on both the MSCs tissue source and the inflammatory status of the surroundings. The environment is an essential parameter triggering the MSCs to adapt either anti-inflammatory or pro-inflammatory phenotype and consequently, determining their therapeutic efficacy. Since these receptors are important for the inflammatory and immune response, further investigation of the signaling pathways and the biological effects that they promote is required to achieve the optimal therapeutic effect of MSCs. Before translating MSCs-based therapy into clinical practice, the environment of the host at the moment of transplantation should be well defined and mimicked


In vivo markers for F-actin organization and dynamics are extensively used to investigate cellular functions of the actin cytoskeleton, which are essential for plant development and pathogen defense. The most widely employed markers are GFP variants fused to F-actin binding domains of mouse talin (GFP-mTn), Arabidopsis fimbrin1 (GFP-FABD2) or yeast Abp140 (Lifeact-GFP). Although numerous reports describing applications of one, or occasionally more, of these markers, are available in the literature, a direct quantitative comparison of the performance of all three markers at different expression levels has been missing. Here, we analyze F-actin organization and growth rate displayed by tobacco pollen tubes expressing YFP-mTn, YFP-FABD2 or Lifeact-YFP at different levels. Results obtained establish that: (1) all markers strongly affect F-actin organization and cell expansion at high expression levels, (2) YFP-mTn and Lifeact-YFP non-invasively label the same F-actin structures  at low expression levels, (3) Lifeact-YFP displays a somewhat lower potential to affect F-actin organization and cell expansion than YFP-mTn, and (4) YFP-FABD2 generally fails to label F-actin structures at the pollen tube tip and affects F-actin organization as well as cell expansion already at lowest expression levels. As pointed out in the discussion, these observations (1) are also meaningful for F-actin labeling in other cell types, which generally respond less sensitively to F-actin perturbation than pollen tubes, (2) help selecting suitable markers for future F-actin labeling experiments, and (3) support the assessment of a substantial amount of published data resulting from such experiments. In plants, the actin cytoskeleton, which is composed of filamentous actin (F-actin), plays key roles in organelle transport , membranExcellent techniques are available to visualize F-actin organization in fixed plant cells either based on electron microscopy, or on fluorescence microscopy after staining with fluorescently labeled actin antibodies or derivatives of phalloidin, a membrane-permeable fungal metabolite that specifically associates with actin filaments . AlthougArabidopsis thaliana fimbrin1 , which was attached via a flexible 5× Glycine-Alanine linker to one of the three different F-actin binding domains introduced above. cDNAs coding for the different fusion proteins were cloned into the same plasmid vector and expressed under the control of the pollen specific Lat52 promoter  with an in vivo F-actin markers: (1) Application of this method results in F-actin marker expression in individual transformed pollen tubes at a wide range of different levels, which can be directly inferred from the brightness of fluorescence emission (in vivo markers to non-invasively label F-actin at variable expression levels. (2) The rate of pollen tube growth is extremely sensitive to perturbation of the actin cytoskeleton  F-actin organization in vegetative tobacco pollen tube cells has been extensively studied using a variety of techniques including electron microscopy  Rank the performance of the three fluorescent fusion proteins tested  as non-invasive F-actin marker based on the following two criteria: (a) ability to reveal as much information as possible about F-actin organization in normally growing tobacco pollen tubes, and (b) potential to affect tobacco pollen tube F-actin organization and growth rate at high expression levels.(2) Enhance our understanding of the organization of the pollen tube actin cytoskeleton through the identification of F-actin structures, which (a) can be non-invasively visualized in normally growing tobacco pollen tubes based on YFP-mTn, YFP-FABD2 or Lifeact-YFP expression, or (b) are only observed when one of these markers is expressed at levels that affect F-action organization and cell expansion.in vivo F-actin imaging also in other types of plant cells.Results obtained not only help deciding, which of the three markers tested should be preferentially employed for non-invasive F-actin labeling in future experiments, they are also important for the assessment of a large amount of data generated using these markers, which has already been reported in the literature : PGGAGAGAGAGALEGT (Lifeact-YFP), SRGAGAGAGAGAG (YFP-mTn) and SRGAGAGAGAGAGK (YFP-FABD2).cDNA sequences were constructed coding for Lifeact-YFP, YFP-mTn and YFP-FABD2 fusion proteins composed of eYFP  fused via a flexible 5× Gly-Ala linker to different F-actin binding domains identical to those reported in the original publications introducing the three F-actin markers: (1) The Lifeact-YFP, YFP-mTn and YFP-FABD2 cDNAs were inserted between the Lat52 promoter  and a NONicotiana tabacum Petit Havana SR1) plants were grown at monthly intervals from seeds germinated in soil and maintained in a growth chamber under the following conditions: 16 h illumination (200–250 μmol m-2s-1) at 24°C and 8 h darkness at 18°C per day, relative humidity 60–65%. Fresh pollen collected from mature plants was used for transient transformation.Tobacco , a N PLAN 5×/0.12 lens and a peltier cooled b/w CCD camera (DFC365 FX), which was run in an 8-bit readout mode (dynamic range of 256 gray levels). All micrographs analyzed in this study were recorded using identical imaging parameters .To acquire low magnification epifluorescence images, 6 h after gene transfer transiently transformed pollen tubes growing on solid culture medium were transferred onto a coverslip by cutting a section of the medium and flipping in upside-down directly on the glass surface as previously described . EpifluoFigure 1A): level I , level II , level III  and level IV . To determine the length of analyzed pollen tubes, they were traced with the “freehand line” tool and measured  after image calibration .To broadly classify the level of YFP or YFP-fusion protein expression in brightly fluorescent pollen tubes, and to determine the length of these cells, micrographs were imported into IMAGEJ . Subsequently, the “Image/Adjust/Threshold” function was used to highlight in red color pixels displaying gray levels above an adjustable threshold. Moving this threshold stepwise down from the maximal gray level (256), the brightest pixels (the first ones to turn red) in the image of each pollen tube excluding the pollen grain was identified. It was necessary to exclude the pollen grain from this analysis, because pollen grains often contained parts of the living, transformed protoplast and in this case emitted very bright fluorescence as a consequence of their large diameter. Based on this procedure, transformed pollen tubes were broadly classified into four groups emitting fluorescence at four different levels  and an HCX PL APO 63×/1.20 water immersion lens. Fluorescence was excited by a 514 nm argon laser and imaged in the range from 530 to 600 nm. Line-by-line sequential imaging was employed to quasi-simultaneously record confocal images of YFP-FABD2 labeled F-actin organization and transmitted light reference images . Growth rates of individual pollen tubes were determined after confocal imaging of F-actin organization by measuring the distance between the positions of the apex on two images taken in the same focal plane at a 2 min interval.post hoc Bonferroni–Holm testing (performed using the XL Toolbox NG for Microsoft Excel1). P-values smaller than 0.05 indicate statistically significant differences between two data sets.Microsoft Excel  was employed to compute the average and the 95% confidence interval of individual sets of pollen tube length or growth rate measurements; as well as to assess the significance of differences between such data sets based on one-way ANOVA Figure 1A). On these micrographs, brightly fluorescent pollen tubes strongly expressing transgenes were selected for length measurements. Based on the gray level of the brightest pixels in the image of each of the analyzed pollen tubes (excluding the pollen grain), they were broadly classified into four categories emitting YFP fluorescence at different levels: level I , level II , level III  and level IV  .A direct comparison was performed of effects of high-level expression of either free YFP, or of one of the F-actin markers Lifeact-YFP, YFP-mTn or YFP-FABD2, on the growth of tobacco pollen tubes transiently transformed by particle bombardment. In these experiments, the Lat52 promoter was used to drive transgene expression, which unlike the 35S promoter is highly active in pollen tubes . PresumaFigure 1B). Pollen tubes expressing free YFP were morphologically unaffected and displayed a length within the normal range . By contrast, all three F-actin markers strongly reduced pollen tube growth already at lower expression levels. At expression levels II, III and IV, Lifeact-YFP reduced pollen tube length by 39.7–55.5% as compared to free YFP expressed at level II. YFP-mTn expression at the same levels inhibited pollen tube growth slightly but detectably more strongly than Lifeact-YFP and cause a reduction of pollen tube length by 55.8–68.2%. Interestingly, YFP-FABD2 reduced pollen tube length to the same extent as YFP-mTn at expression level II, but was never found to be expressed at higher levels (levels III or IV). The low maximal expression level of YFP-FABD2 may be due to rapid turn-over, or to cytotoxicity that prevents marker accumulation to higher levels. Although all three F-actin markers strongly reduced pollen tube growth when expressed at high levels , they only induced minor morphological alterations under these conditions, which included moderate tip swelling and a slightly irregular tube diameter .Analysis of the average length of pollen tubes emitting fluorescence at different levels as defined above demonstrated that high-level expression of all marker proteins tested inhibited pollen tube growth in a dose-dependent manner . This analysis further supported data shown in Figure 1B. In general, a decreasing percentage of pollen tubes expressing each marker emitted fluorescence at increasing levels . Furthermore, a slightly larger proportion of Lifeact-YFP expressing pollen tubes, as compared to YFP-mTn expressing pollen tubes, emitted fluorescence at each of the two highest levels (III and IV), supporting the notion that Lifeact-YFP is a somewhat less invasive F-actin marker. Interestingly, YFP also appeared to be less frequently expressed at the highest level IV than Lifeact-YFP. Quantifying fluorescence emission based on maximal gray level is presumably responsible for this observation, as this will result in a moderate underestimation of the expression level of diffusely distributed YFP, as compared to F-actin markers that locally accumulate at cytoskeletal structures. Maximal YFP-FABD2 fluorescence at level II (see above) was only emitted by a small percentage of the pollen tubes expressing this marker, as determined when YFP-FABD2 expressing pollen tubes were included in the analysis, which emitted weaker fluorescence (level I) than any of the Lifeact-YFP and YFP-mTn expressing pollen tubes examined to generate Figure 1B.In addition, the percentage of all YFP, Lifeact-YFP or YFP-mTn expressing pollen tubes analyzed to generate the graph shown in Figures C demonstrate that all three F-actin markers interfere with pollen tube growth when expressed at high levels. Furthermore, they indicate that at high expression levels Lifeact-YFP is somewhat more readily tolerated by living pollen tubes than YFP-mTn, whereas YFP-FABD2 appears to be considerably less stable and/or more cytotoxic than both these two markers.Together, data shown in Figures 4). Analysis of the growth rates of all imaged pollen tubes showed that Lifeact-YFP and YFP-mTn expressing pollen tubes were elongating nearly as rapidly as weakly fluorescent YFP expressing pollen tubes at average rates of more than 5 μm/min , which are within the range of normal growth rates of cultured tobacco pollen tubes reported in the literature .Six to eight hours after gene transfer, F-actin organization was visualized using confocal microscopy in morphologically unaffected pollen tubes , which expressed the different F-actin markers at the lowest detectable levels and emitted the weakest fluorescence among all transformed pollen tubes observed  and YFP-mTn  labeling patterns in normally elongating pollen tubes were nearly indistinguishable and revealed the same F-actin organization as previously reported in the literature , indicating possible direct connections between these F-actin structures. Filamentous F-actin structures in the apical clear zone between the extreme apex and the subapical F-actin fringe could not be clearly detected in any of the analyzed normally elongating Lifeact-YFP or YFP-mTn expressing pollen tubes. Despite the essentially identical labeling patterns obtained with Lifeact-YFP and YFP-mTn, but consistent with the slightly lower potential of Lifeact-YFP to affect pollen tube growth , F-actin imaging in normally elongating tobacco pollen tubes appeared to be somewhat more effective with this marker. Presumably, a slightly larger proportion of all transformed pollen tubes displayed non-invasive F-actin labeling when Lifeact-YFP was expressed. Furthermore, cytoplasmic background labeling and actin filament bundling in the shank appeared to be a little less pronounced in pollen tubes expressing Lifeact-YFP. By contrast, YFP-mTn expression seemed to visualize the sub-apical F-actin fringe somewhat more clearly. However, none these minor differences were sufficiently substantial to allow confirmation by quantitative analysis.Serial confocal optical sectioning showed that YFP was diffusely distributed throughout the pollen tube cytoplasm , which were elongating at reduced rates , substantially differed from the Lifeact-YFP and YFP-mTn labeling patterns in normally growing pollen tubes described above . Although the shank of YFP-FABD2 expressing pollen tubes also contained primarily longitudinally oriented actin fibers, these fibers generally appeared to form a more fuzzily labeled and less regularly arranged network, suggesting that YFP-FABD2 may induce F-actin reorganization in the pollen tube shank already at minimal detectable expression levels. Most strikingly, in none of the pollen tubes expressing YFP-FABD2 a subapical F-actin fringe or actin fibers extending from the longitudinally oriented F-actin network in the shank to the subapical cell cortex were observed, suggesting that these F-actin structures are either disrupted or not labeled by this marker. In fact, almost no F-actin labeling was detectable in a region covering the first 15–30 μm at the tip of YFP-FABD2 expressing pollen tubes.F-actin organization as observed by confocal imaging of pollen tubes expressing YFP-FABD2 at lowest detectable levels , demonstrated that under these conditions each of the two markers induced massive F-actin bundling and reorganization . In the shank of all imaged pollen tubes a dense network of brightly labeled, thick F-actin fibers was observed, which appeared to be more curved and less regularly arranged than the finer F-actin fibers in the shank of normally elongating pollen tubes expressing Lifeact-YFP or YFP-mTn at minimal levels . Furthermore, small F-actin rings with a diameter of a few micrometers were often observed in the shank of pollen tubes expressing Lifeact-YFP or YFP-mTn at high-levels . Interestingly, in most of these pollen tubes neither a subapical F-actin fringe nor actin fibers connecting the subapical cell cortex to the F-actin network in the shank were visible, whereas fine F-actin fibers were often found to extend into the apical cytoplasm all the way to the extreme tip . Like the small F-actin rings observed in the shank, these fine F-actin fibers in the in apical cytoplasm clearly need to be considered artifacts induced by high-level Lifeact-YFP or YFP-mTn expression, as neither of these two structures was ever observed in normally growing pollen tubes expressing one of the two markers at minimal levels .Confocal imaging of brightly fluorescent pollen tubes displaying morphological aberrations , which expressed Lifeact-YFP or YFP-mTn at high levels that inhibited cell expansion , and reaches substantially lower maximal expression levels than Lifeact-YFP or YFP-mTn in these cells . Comparing F-actin organization in pollen tubes expressing YFP-FABD2 at minimal detectable  or high  levels indicates that YFP-FABD2, like Lifeact-YFP and YFP-mTn, in a dose-dependent manner can stimulate F-actin bundling and the formation of thick actin fibers in the pollen tube shank. However, in addition to labeling these fibers highly expressed YFP-FABD2 generally also massively accumulated in diffusely labeled cytoplasmic regions, indicating local F-actin disruption . At maximal expression levels, YFP-FABD2 labeled F-actin structures frequently collapsed into one large aggregate near the pollen tube tip . Interestingly, irrespectively of expression level YFP-FABD2 failed to label any of the F-actin structures that can be visualized at the tip of pollen tubes expressing Lifeact-YFP or YFP-mTn either at low  or high  levels.As discussed above, YFP-FABD2 interferes with the growth of pollen tubes already at minimal detectable expression levels . In addition, under the same conditions actin fibers connecting these two structures, which extend from the subapical cell cortex into the shank, are often visualized by each of the two markers . Together, these structures appear to constitute the tobacco pollen tube actin cytoskeleton as it can be non-invasively visualized using in vivo markers tested in this study at normal growth rates at which F-actin organization is unlikely to be substantially affected. The same structures were already described in the original reports establishing YFP-mTn as an in vivo F-actin marker  a network of primarily longitudinally oriented fine actin fibers in the shank, which are helically arranged in the cell cortex, and (b) a subapical F-actin fringe , in which no subapical F-actin fringe, but brightly labeled, thick F-actin fibers in the shank as well as fine F-actin fibers extending into the apical cytoplasm are visible. Information concerning the growth rates of imaged pollen tubes expressing “enhanced” GFP-mTn, or the level of expression of this marker in these cells, has not been reported  we employed for the work presented here are not expected to perform substantially differently , which shows a somewhat reduced self-association tendency  or were labeled with phalloidin-derived fluorescent probes . Like GFin vivo marker of the tobacco pollen tube actin cytoskeleton. This fusion protein substantially inhibits tobacco pollen tube growth already at the lowest detectable expression level and therefore clearly does not allow non-invasive F-actin observation in these cells. Furthermore, as previously reported  At low expression levels, Lifeact-YFP and YFP-mTn non-invasively label the same key components of the actin cytoskeleton in normally growing tobacco pollen tubes: (a) a network of primarily longitudinally oriented actin fibers in the shank, (b) a subapical F-actin fringe and, less effectively, (c) actin fibers connecting these two structures.(2) At high expression levels, Lifeact-YFP, YFP-mTn and YFP-FABD2 inhibit tobacco pollen tube growth, stimulate actin filament bundling and induce F-actin reorganization. High-level Lifeact-YFP or YFP-mTn expression disrupts F-actin structures observed in normally growing pollen tubes  and induces the formation of structural artifacts including actin fibers in the apical cytoplasm and small F-actin rings.(3) Lifeact-YFP is the marker of choice for non-invasive F-actin visualization in tobacco pollen tubes, as it has a somewhat lower potential than YFP-mTn to generate diffuse cytoplasmic background, to alter F-actin organization and to interfere with tip growth. YFP-FABD2 is not suitable as an F-actin marker in tobacco pollen tubes, because it affects F-actin organization and tip growth already at the lowest detectable expression levels, and fails to label parts of the actin cytoskeleton.in vivo F-actin markers, are expected to affect F-actin organization and actin-dependent cellular processes in all cell types. However, at low expression levels at least Lifeact-YFP and YFP-mTn are likely to allow non-invasive F-actin visualization not only in normally growing pollen tubes, whose actin cytoskeleton is essential for cell expansion and extremely sensitive to F-actin disrupting drugs, but also in most other cell types. As in other cell types possible effects of markers on F-actin structure and functions are generally more difficult to assess than in pollen tubes, whose growth rate highly sensitively reacts to F-actin perturbation, markers should always be expressed at the lowest detectable level for F-actin imaging. Different GFP-based markers may label distinct F-actin structures in specific cell types, possibly because they compete for separate F-actin binding sites with differentially expressed endogenous actin binding proteins. To optimize F-actin visualization in a specific cell type, testing the performance of different markers may therefore be helpful. When comparing different markers, it is essential to ensure each marker is expressed at an optimal level.(4) With caution, key results of our analysis of the performance of Lifeact-YFP, YFP-mTn and YFP-FABD2 in tobacco pollen tubes can probably be extrapolated to other cell types. When expressed at high levels, these three markers, as well as presumably all other available GFP-based AM-R acquired all data and contributed to the design of the work, to data analysis as well to the writing of the MS. BK conceived the work, was responsible for data analysis and interpretation, and wrote the final version of the MS.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.


Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and the most common cause of female infertility. However, its etiology and underlying mechanisms remain unclear. Here we report that a transgenic obese mouse (Mito-Ob) developed by overexpressing prohibitin in adipocytes develops polycystic ovaries. Initially, the female Mito-Ob mice were equally fertile to their wild-type littermates. The Mito-Ob mice began to gain weight after puberty, became significantly obese between 3-6 months of age, and ∼25% of them had become infertile by 9 months of age. Despite obesity, female Mito-Ob mice maintained glucose homeostasis and insulin sensitivity similar to their wild-type littermates. Mito-Ob mice showed morphologically distinct polycystic ovaries and elevated estradiol, but normal testosterone and insulin levels. Histological analysis of the ovaries showed signs of impaired follicular dynamics, such as preantral follicular arrest and reduced number, or absence, of corpus luteum. The ovaries of the infertile Mito-Ob mice were closely surrounded by periovarian adipose tissue, suggesting a potential role in anovulation. Collectively, these data suggest that elevated estradiol and obesity per se might lead to anovulation and polycystic ovaries independent of hyperinsulinemia and hyperandrogenism. As obesity often coexists with other abnormalities known to be involved in the development of PCOS such as insulin resistance, compensatory hyperinsulinemia and hyperandrogenism, the precise role of these factors in PCOS remains unclear. Mito-Ob mice provide an opportunity to study the effects of obesity on anovulation and ovarian cyst formation independent of the major drivers of obesity-linked PCOS. Summary: Obesity-related overgrowth of periovarian adipose tissue and elevated estradiol levels cause anovulation and ovarian cyst formation in mice, independent of hyperinsulinemia and hyperandrogenism. The incidence rate of infertility is on the rise worldwide. Ovulatory defects and unexplained causes account for more than 50% of infertile etiologies . It is paP2) gene promoter  developed by overexpressing prohibitin in adipocytes . At 6 moaP2 gene promoter is active in the female reproductive tract and ovary in Mito-Ob mice, which might have contributed to ovarian dysregulation, we determined prohibitin levels using western immunoblotting with anti-prohibitin antibody , which aromatizes testosterone to estradiol . In rodeHere we report a novel and spontaneous transgenic mouse model of obesity-associated anovulation and polycystic ovary independent of insulin resistance. The ovarian phenotype in Mito-Ob mice provides evidence that obesity and elevated estradiol levels are sufficient to cause anovulation and ovarian cyst formation. Furthermore, polycystic ovary might arise due to primary defect in the ovary and overgrowth of periovarian adipose tissue, independent of factors normally implicated in its development such as increased androgens and insulin levels. Altered follicular dynamics, as observed in the ovaries of Mito-Ob mice, is consistent with the notion that ovulation is a crucial event in the normal functioning of the follicular cycle, and that anovulation negatively affects follicular dynamics and leads to preantral follicular arrest and cyst formation . In thisHyperandrogenism is the most consistent feature of women with PCOS . HoweverIn addition to Mito-Ob mice, we have recently reported a mutant Mito-Ob mouse expressing a phospho mutant (Y114F) form of prohibitin in a similar manner . The mutIt is anticipated that the spontaneous models of obesity-related anovulation and polycystic ovary described here will be valuable to explore the underlying mechanisms involved in obesity-related anovulation and ovarian disorders. Thus, Mito-Ob mice will be helpful to discern the relative contribution of different factors generally co-existing in obesity-related ovarian disorders.ad libitum.The generation of transgenic Mito-Ob mice overexpressing prohibitin in adipocytes has been described elsewhere . All aniThe uterus and ovary lysates from Mito-Ob and wild-type mice containing equal amounts of proteins (15 mg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently analyzed by immunoblotting using anti-prohibitin antibody .Ovaries from 3- to 9-month-old Mito-Ob and wild-type mice were fixed overnight in 10% buffered formaldehyde and subsequently dehydrated in a graded series of ethanol, before being embedded in paraffin. Five-micrometer ovarian sections were cut and processed for Hematoxylin and Eosin (H&E) staining .Serum insulin and adipokine levels were measured using mouse Bio-Plex Pro™ Diabetes Assay Panels and Bio-Plex 200™ multiplex suspension array systems (Bio-Rad) as per the manufacturer's protocols , 2016. LGlucose and insulin tolerance tests in 9-month-old Mito-Ob mice and their corresponding wild-type littermates were performed as per the standard procedures described in our previous publications .t-tests were performed to compare Mito-Ob and wild-type littermates, unless indicated otherwise. P<0.05 was considered significantly different.All statistical analyses were performed using GraphPad Prism 6 . Experimental results are shown as mean±s.e.m. Two-tailed Student's unpaired


Chrysodeixis (formerly Pseudoplusia) includens nucleopolyhedrovirus (ChinNPV), is a new Alphabaculovirus pathogenic to Chrysodeixis includens. Here, we report the complete genome sequences of six ChinNPV isolates. The availability of these genome sequences will provide information on ChinNPV molecular genetics, promoting understanding of its pathogenicity, diversity, and evolution.The baculovirus,  Chrysodeixis includens nucleopolyhedrovirus isolates, obtained from Chrysodeixis includens larvae collected from cotton and soybean crops from Brazil and Guatemala exhibited genetic and phenotypic diversity in previous studies (Pseudoplusia includens single nucleopolyhedrovirus (PsinSNPV). However, the nomenclature of this new baculovirus species was reviewed and its name revised to Chrysodeixis includens nucleopolyhedrovirus , MIRA v4.0.2 , and opeThe six isolates exhibited genome sizes ranging from 138,869 bp (ChinNPV-IB) to 140,859 bp (ChinNPV-IF), with an average G+C content of 39.2%. The isolates showed high nucleotide sequence identity with both ChinNPV-IC and ChinNPV-ID sharing 96.4% identity and ChinNPV-IF and ChinNPV-IG sharing 99.4% identity with the PsinSNPV-IE genome  (GenBankbro genes (bro-a and bro-b), similar to the PsinNPV-IE genome. However, an insert of approximately 1,700 bp was mapped between the pif-3 and sod29 genes which resulted in the identification of a third bro gene (bro-c) in the ChinNPV-IA, ChinNPV-IC, and ChinNPV-ID genomes.Structural rearrangements such as large-scale inversions and translocations were absent. Genetic heterogeneity was observed among these seven virus isolates with the presence of indels (deletions or insertions), single nucleotide polymorphisms (SNPs), and small fragment insertions throughout the genome. The genomes of all the ChinNPV isolates exhibited two Single nucleotide polymorphisms observed among the ChinNPV genomes may also significantly contribute to phenotypic diversity and different degrees of pathogenicity previously reported among the seven isolates herein analyzed .The complete genome sequences presented here will be invaluable for further molecular genetic studies that could provide new insights into the virulence, insect-host interactions, and phenotypic diversity observed among ChinNPV isolates. In addition, this genomic data will contribute to advances in the understanding of genetic diversity and to functional studies of baculovirus genes.KU669289 (for ChinNPV-IA), KU669290 (for ChinNPV-IB), KU669291 (for ChinNPV-IC), KU669292 (for ChinNPV-ID), KU669293 (for ChinNPV-IF), and KU669294 (for ChinNPV-IG).ChinNPV isolate genome sequences have been deposited in GenBank under the following accession numbers:


Gankyrin deficiency in myeloid cells, but not in epithelial cells, reduced the activity of mitogen activated protein kinase and the expression of stem cell markers, leading to attenuated tumorigenic potential. These findings provide important insights into the pathogenesis of CAC and suggest that gankyrin is a promising target for developing therapeutic and preventive strategies against CAC.Although long-standing colonic inflammation due to refractory inflammatory bowel disease (IBD) promotes the development of colitis-associated cancer (CAC), the molecular mechanisms accounting for the development of CAC remains largely unknown. In this study, we investigated the role of gankyrin in the development of CAC since gankyrin is overexpressed in sporadic colorectal cancers. We analyzed gene expression of colon tissues obtained from 344 patients with IBD and CAC and found that expression of gankyrin was much higher in colonic mucosa of patients with refractory IBD than in those with IBD in remission. Expression of gankyrin was upregulated in inflammatory cells as well as tumor cells in colonic mucosa of patients with CAC. Over-expressing studies utilizing tagged ganlyrin-cDNA identified physical interaction between ganlyrin and Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1). Importantly, the interaction between ganlyrin and SHP-1 leads to inhibition of STAT3 activation and to enhancement of TNF-α and IL-17 in inflammatory cells. To further address the role of gankyrin in the development of CAC, we created mice with intestinal epithelial cell-specific gankyrin ablation ( Colorectal cancer is one of the most leading causes of cancer-related death worldwide , 2. InflGankyrin  is an oncoprotein overexpressed in many malignancies including hepatocellular carcinoma, cholangiocellular carcinoma, colorectal cancer and lung cancer . Given tf/fVil-Cre;Gankyrin) mice and deletion of gankyrin in both myeloid and epithelial cells  mice, were employed. We found that expression of gankyrin in myeloid cells rather than in epithelial cells is required for efficient tumorigenesis. Mechanistically, binding of gankyrin to SH2-containing protein tyrosine phosphatase-1  enhances the pro-inflammatory cytokine responses in myeloid cells through activation of signal transducer and activator of transcription-3 (STAT3) [Here we examined whether gankyrin plays a role in inflammation-associated tumorigenesis in the colon using a murine CAC model in which two kinds of tissue-specific gankyrin-deficient mice, intestinal epithelial cell-specific gankyrin ablation ( (STAT3) , 18 and In our previous study, we showed that longstanding intestinal inflammation increases the expression of stress response proteins including HSPA4 in the colonic mucosa of IBD patients. Interestingly, such increased expression of HSPA4 was accompanied by that of gankyrin, which suggests possible involvement of ganlyrin in the development of long-standing colonic inflammation . Based oWe then performed immunohistochemical studies to identify the cells expressing gankyrin in the human colon tissues. Gankyrin expression was enhanced in inflammatory cells as well as epithelial cells in chronically inflamed mucosa Figure . In addiGankyrin and IL-17A or IL-23p19 mRNA expression in UC patients  is a prototypical marker of the self-renewing multipotent adult stem cell populations residing in intestinal crypts that mediate regeneration of the intestinal epithelium . Lgr5 exf/fGankyrin mice .Chronic inflammation increases intestinal cancer risk in IBD . To invee Figure  and thenf/fMx1-Cre;Gankyrin mice compared with f/fGankyrin controls  into f/fMx1-Cre;Gankyrin mice and deleted gankyrin after the initiation of DSS treatment. Inflammation was slightly attenuated by gankyrin deletion after the DSS treatment . Non-transplanted irradiated mice survived less than 2 weeks after irradiation, indicating successful deletion of BM cells by gamma irradiation. Irradiated animals that received BMT were allowed to recover for 2 months prior to placing them on the DSS-induced colitis protocol. Inflammatory cell infiltration scores of irradiated gankyrin-intact mice harbouring gankyrin-deficient BM cells was lower than those harbouring gankyrin-intact BM cells , which ireatment . Consistpression .f/fMx1-Cre;Gankyrin mice, gankyrin was deleted in the liver as well. To confirm the role of gankyrin expression in the colon, we created f/fAlbumin-Cre;gankyrin mice where gankyrin is deficient only in the liver  and compared f/fAlbumin-Cre;gankyrin mice and f/fgankyrin mice in colitis model. No difference in DSS-induced colonic inflammation was seen between f/fAlbumin-Cre;gankyrin mice and f/fgankyrin mice  and their littermate controls (f/fGankyrin mice) were challenged with AOM and DSS to confirm a role of myeloid cells expressing gankyrin. Deletion of gankyrin in enterocytes did not show gross abnormalities. No significant differences were seen in tumor numbers or maximum sizes between f/fVil-Cre;Gankyrin mice and control f/fGankyrin mice in the murine CAC model  is linked to the development and progression of several cancers including colorectal cancer [f/fMx1-Cre;Gankyrin mice, but not from f/fVil-Cre;Gankyrin mice, exhibited marked down-regulation of Pim1 mRNA relative to those isolated from f/fGankyrin mice , which signals directly within transformed colonic epithelial cells to promote early tumor development by activating MAP kinase, especially ERK signaling [Previous studies report that upregulation of gankyrin was detected in many tumor cell lines and primary tumors. Such enhanced expression of gankyrin has been considered to promote the prolifearion of cancer cells by inhibiting apoptosis and cell cycle in cell lines –14. Howeignaling . ConsistAnother pro-inflammatory cytokine associated with the development of CAC through activation of gankyrin is TNF-α. Expression of TNF-α was positively correlated with that of gankyrin in human IBD samples. In addition, TNF-α expression was significantly lower in gankyrin-deficient mice treated with AOM-DSS than in gankyrin-intact mice. Furthermore, ganlyrin-knowdown leads to a diminished TNF-α production by human monocytic cells through inhibition of STAT3 and MAP kinase activation, which suggests involvement of gankyrn-mediated signaling pathways for optimal production of TNF-α. Given the fact that TNF-α produced by myeloid cells is a critical player for the longstanding chronic inflammation and CAC , the resf/fMx1-Cre;Gankyrin mice and f/fVil-Cre;Gankyrin mice. Deletion of gankyrin was confirmed in hemapoietic and non-hematopoietic cells in f/fMx1-Cre;Gankyrin mice whereas deletion was confirmed in epithelial cells in f/fVil-Cre;Gankyrin mice. The development of CAC induced by AOM-DSS treatment was significantly attenuated in the colon of f/fMx1-Cre;Gankyrin mice as compared with control f/fGankyrin mice. In contrast, the development of CAC induced by AOM-DSS treatment was comparable between f/fVil-Cre;Gankyrin mice and control f/fGankyrin mice. Similarly, f/fMx1-Cre;Gankyrin mice exhibited attenuated inflammatory scores upon acute DSS colitis model. In line with this, the development of acute DSS colitis was attenuated in irradiated-gankyrin-intact mice transplanted gankyrin-deficient BM cells as compared with thoses transplanted with gankyrin-intact BM cells. These studies utilizing two kinds of conditional gankyrin-deficient mice together with BMT experiments strongly suggest a pivotal role played by hematopoietic cells expressing gankyrin in the development of chronic colitis and colitis-associated cancer. Compatible to this idea, gankyrin expression was detected in immune cells of the colon tissue of human CAC and refractory IBD samples. Moreover, pro-inflammatory cytokine responses such as TNF-α and IL-17 were significantly reduced in the colon tissues of AOM-DSS-treated f/fMx1-Cre;Gankyrin mice, which effects were again accompanied by a reduction in expression of cancer stem cell markers and in activation of STAT3 and MAP kinase activation. Thus, it is very likely that myeloid cells expressing gankyrin play an indispensable role in the development of chronic colitis and CAC through the enhancement of pro-inflammatory cytokine responses, activation of STAT3 and MAP kinases, and finally expression of cancer stem cell markers. Therefore, present findings show for the first time that as a tumor promoter gankyrin can enhance tumor development through the interaction between tumor cells and immune cells. It should be noted, however, that we cannot exclude a role played by gankyrin-expressing epithelial cells. Gankyrin in epithelial cells would at least partly contribute to tumorigenesis in another type of cancer such as sporadic colorectal cancer rather than CAC.In this study, we created two kinds of conditional gankyrin-deficient mice; One question arising from the present study is whether gankyrn expression is up-regulated not only in IBD but also in non-chronic inflammatory diseases such as infectious or ischemic colitis in human samples. In this regards, we do not have enough specimens of non-chronic inflammatory diseases and thus cannot provide the data of gankyrin expression in non-chronic inflammatory diseases. However, our preliminary assay utilizing biopsy samples from Amoebatic dyscentery revealed that the gankyrin expression was found to be lower than that in IBD patients.The most feared long-term complication of IBD is CAC as patients with IBD have an increased risk of colorectal cancer . Most caf/fMx1-Cre;Gankyrin mice and f/fVil-Cre;Gankyrin mice. As mentioned above, f/fMx1-Cre;Gankyrin mice lacking gankyrin in hematopoietic cells and epithelial cells, but not f/fVil-Cre;Gankyrin mice lacking gankyrin in epithelial cells alone, were resistant to the development of acute colitis and inflammation-associated colon tumorigenesis. Importantly, such resistance to inflammation-associated colon tumorigenesis in f/fMx1-Cre;Gankyrin mice was associated with a significant reduction of pro-inflammatory responses (IL-17 and TNF-α) and expression of cancer stem cell markers (Bmi1 and Sox9). A final step in the chain of evidence consisted of yeast-two hybrid, over-expression, and gene-silenecing studies showing that gankyrin interacts with SHP-1 to induce activation of STAT3 and MAP kinases and then to induce TNF-α production. In accordance with this, a physical interaction between gankyrin and SHP-1 was detected in immune cells of the colon tissue of gankyrin-intact mice treated with AOM and DSS. Collectively, these data strongly suggest that gankyrin-expressing myeloid cells produce pro-inflammatory cytokines through its interaction with SHP-1 followed by STAT3 activation and then promote CAC through induction of stem cell markers. Thus, we propose gankyrin as a tumor promoter that plays a role in the interface between immune cells and epithelial cells. It should be noted, however, that confirmation of this notion awaits future studies addressing the relationship between gankyrin expression and colon cancer prognosis as well as those addressing the role of gankyrin in immune cells by using myeloid, T cell, or B cell-specific gankyrin deletion mice.In this study, we propose that gankyrin expressed in myeloid cells promotes CAC through induction of pro-inflammatory cytokine responses and expression of cancer stem cell markers. The chain of evidence supporting this conclusion consisted first of the fact that gankyrin expression is correlated with the expression of immune cell-derived pro-inflammatory IL-17-related cytokines and with that of stem cell marker (Lgr5) in the colonic mucosa of IBD patients. Moreover, immuno-histochemical analysis revealed the expression of gankyrin in immune cells migrated into the colonic mucosa of patients with refractory IBD and CAC. These results obtained from human samples were further corroborated in experimental studies utilizing Taken together, gankyrin, whose expression is upregulated by chronic inflammation, increases STAT3 activity and the production of TNF-α and IL-17 by binding to SHP-1 in inflammatory cells, leading to augmented inflammation. Such pro-inflammatory cytokine responses may enhance MAP kinase activity in the tumor and upregulate expression of stem cell markers in the colon. These factors would eventually promote CAC Figure . Thus, sUC and CD were diagnosed as previously described . In totaflox/wtGankyrin mice (Accession No. CDB0944K) [flox/wtGankyrin, Mx1-Cre, and Villin-Cre (Jackson laboratory) mice were used to create tissue-specific conditional gankyrin knockout mice, designated here as f/fMx1-Cre;Gankyrin and f/fVil-Cre;Gankyrin mice. Induction of Mx1-Cre was achieved by 3 intraperitoneal injections of 300 μg of poly(I:C)  every other day to 4–8-week-old mice.Targeted ES clones were isolated from HK3i ES cells . ChimeriDB0944K) ; Gankyrif/fMx1-Cre;Gankyrin mice and f/fGankyrin mice were treated with poly(IC) as described previously [f/fMx1-Cre;Gankyrin mice , poly(IC)-treated f/fGankyrin mice, f/fVil-Cre;Gankyrin mice and f/fGankyrin mice (8–16 weeks old) received 2.5% (w/v) dextran sodium sulfate  in drinking water for 7 days. Inflammatory cell infiltration score was assessed as described previously [eviously . Sex- anTHP-1 cells, an immortalized line of human monocyte, were maintained in RPMI-1640 medium  supplemented with 10% fetal bovine serum, containing penicillin (100 U/ml) and streptomycin (100 mg/ml) and transfection of gankyrin siRNA and SHP-1 siRNA  was carried out using X-treme GENE siRNA Transfection Reagent . Caco2 and U-2 OS cells were maintained in DMEM medium (Gibco) supplemented with 10% fetal bovine serum, containing penicillin (100 U/ml) and streptomycin (100 mg/ml) and transfected with plasmid using Fugene  and X-treme GENE HP DNA transfection reagent (Roche). Cell viability was assessed using Cell Counting Kit-8 . Jurkat cells were transfected with a plasmid containing 3HA-gankyrin cDNA.Real-time qPCR, immunoblotting, immunoprecipitation and immunohistochemistry were previously described , 44. LisDuolink fluorescence method was employed as per manufacturer's recommendations (Sigma Aldrich). Human gankyrin antibody and SHP-1 antibody (R&D systems) were used to assess gankyrin/SHP-1 interactions under confocal laser microscope.Using the full length of gankyrin as bait, a cDNA library from human fetal intestine was screened by the yeast two-hybrid method, which was done by Invitrogen . Thirty-seven clones of yeast clones transformed with a human intestinal cDNA library were identified. Each clone proliferated on media containing the histidine inhibitor 3AT and was positive for β-galactosidase staining. DNA sequencing of the rescued plasmids revealed that two clones encoded SHP-1.In Situ Apoptosis Detection Kit as described by the manufacturer  and previous report [Immunofluorescent TUNEL staining was performed to measure apoptosis in paraffin-embedded sections using the s report . Nuclei t-test. To compare variables of more than 2 conditions, analysis of variance (ANOVA) with post-hoc Tukey-Kramer honestly significant difference (HSD) multiple comparison was applied. The relationship between the expression of several genes was analyzed by Spearman's rank correlation test. P values <0.05 were considered significant.Differences were analyzed using Student's