Appropriate regulation of canonical BMP signaling is critical for the development and homeostasis of numerous human organ systems, as aberrations in the BMP pathway or its regulation are increasingly associated with diverse human pathologies. In this review, we provide a wide-perspective on strategies that increase or decrease BMP signaling. We briefly outline the current FDA-approved approaches, highlight emerging next-generation technologies, and postulate prospective avenues for future investigation. We also detail how activating other pathways may indirectly modulate BMP signaling, with a particular emphasis on the relationship between the BMP and Activin/TGF-β pathways. Bone morphogenetic proteins (BMPs) constitute the largest subdivision of the TGF- To date, approximately thirty distinct human proteins are named BMPs and some have additionally been assigned as Growth/Differentiation Factors (GDFs). However, important differences exist among these molecules with regard to pathway mechanics and effects on cellular behavior. This imprecise nomenclature can cause confusion when discussing BMP ligands and their role in human physiology or disease. Clarification may come, however, by focusing on the downstream pathway activated by each ligand rather than name alone. The intracellular effectors SMAD1/5/8 actuate the “bone morphogenetic protein” activity  originally described by Urist [ bona fide components of the canonical BMP signaling cascade. On this basis, it is possible to identify approximately thirteen bone fide BMP ligands in humans. Bona fide human bone morphogenetic proteins (BMPs)  are as follows: BMP2 . BMP4 . BMP5. BMP6 . BMP7 (OP-1). BMP8A. BMP8B (OP-2). BMP9 . BMP10. BMP15 . GDF5 . GDF6 . GDF7 (BMP12).It is this narrow definition of BMP signaling that we utilize in this review article.Bone morphogenetic proteins (BMPs) constitute the largest subdivision of the TGF-by Urist . Protein Nanog and Sox2 [ Bmp7 is essential for the maintenance of nephron progenitor cells and its absence promotes premature arrest of nephrogenesis [β, and Wnt signals [Bone morphogenetic proteins (BMPs) are unequivocally involved in the modulation of several stem cell populations including embryonic stem cells (ESCs), induced pluripotent stem cells, intestinal stem cells, and mesenchymal stem cells (reviewed in –6). For and Sox2 . Mouse Eand Sox2 . Geneticogenesis . Additioogenesis . These fogenesis . The sam signals . And, BM signals . Thus, d per se, though numerous reported applications are noted in the main text and supplemental tables. We briefly outline the current FDA-approved approaches, highlight emerging technologies, and postulate prospective avenues for future investigation. We also detail how activating other pathways may indirectly modulate BMP signaling, with a particular emphasis on the relationship between the BMP and Activin/TGF-β pathways.This widespread yet context-dependent role of BMP signaling in modulating stem cell behavior requires appropriate regulation of BMP signaling for the development and homeostasis of numerous human organ systems . AberratIn this section, we highlight several strategies to activate the BMP pathway. These different approaches are schematized in https://clinicaltrials.gov/).The potential for clinical application of the BMP pathway was discovered decades prior to the identification of the BMP ligands , 2. In t E. coli [ in vivo . Several of these studies accomplished cell type specific and/or regulated BMP synthesis. One very interesting idea put forth involves ingesting bacteria that express BMPs for localized production in the gastrointestinal tract [Recombinant BMPs have a high production cost for clinical use, which raises concern about their cost-effectiveness , 19. As  E. coli –40. Addial tract , which mPart of the high cost of rhBMPs is related to the fact that large amounts of protein have been required for clinical use, leading multiple groups to engineer versions that have higher activity than the naturally-occurring ligand . For ins in silico screen has identified several compounds that could bind to Noggin to disrupt its interaction with BMP ligands [ in vivo at present. The FDA-approved immunosuppressant tacrolimus (Astellas Pharma), which is also known as FK506, activates BMP signaling by inhibiting FKBP12 and is being tested in a clinical trial for the treatment of PAH (NCT01647945).BMP pathway activation is regulated by a large number of soluble antagonists . Because ligands  and lead ligands –79. We a in vivo [Enhancing the expression of BMP pathway components could serve as a means to increase signaling. Numerous stimuli have been reported to increase expression levels of BMP ligands or receptors (Table S8). Notably, several kinds of clinically relevant physical stimuli, such as pulsed electromagnetic fields, ultrasound, and mechanical loading, can positively modulate the BMP pathway at multiple levels –89. Addi in vivo –95. BMP2 in vivo , 97. Pan in vivo –104. in vitro and in animal models (Recent years have brought considerable attention to the role that microRNAs (miRNAs) play in gene expression, and several miRNAs have been implicated in negatively regulating the expression of BMP pathway components . This opl models . This teIn this section, we will highlight several strategies to inhibit the BMP pathway. These different approaches are schematized in NCT01458392, NCT01642082, NCT01720173, NCT01727336, and NCT02024087); ALK1-ECD preferentially sequesters BMP9 and BMP10 [The fact that BMP ligands are present in the extracellular environment makes them vulnerable to sequestration upstream of receptor binding on target cells, and the extracellular antagonists Noggin, Gremlin, and Chordin might be used to regulate BMP signaling in this manner . Numeround BMP10 –111. Grend BMP10  or via bnd BMP10 . in vivo efficacy of these four molecules head-to-head. Similarly, Tsugawa et al. concluded that differential type 1 receptor targeting underlies the finding that LDN-193189, DMH2, and VU5350 are effective in promoting liver regeneration in a rodent model while 1LWY is not [BMP receptors are serine/threonine kinases, which makes them attractive targets for small molecules that block the kinase pocket and inhibit their activity. Considerable attention has been focused upon type 1 BMP receptors (ALK1/2/3/6) and the first kinase inhibitor reported was Dorsomorphin . Though Y is not .It should be noted that some of these small molecules also target type 2 BMP receptors BMPR2, ACVR2A, and ACVR2B , which m in vivo has been accomplished by gene transfer in multiple scenarios and by various methods . Notably, one emerging gene therapy strategy uses allele-specific RNA interference (ASP-RNAi) to selectively silence a single protein isoform, such as a constitutively active (ca) mutant [ in vitro to knock down disease-causing caALK2 expression [ BMPR2 that are found in some heritable PAH patients and are associated with earlier onset and more severe disease than nonexpressed mutants [As mentioned in ) mutant . Two seppression , 127. Thpression . ASP-RNA mutants . in vitro and decreases ossification in an ectopic assay [https://clinicaltrials.gov/).In comparison to stimuli that positively modulate the BMP pathway, relatively few agents have been described to reduce expression and/or pathway activity (Table S9). Notably, the FDA-approved antianginal drug perhexiline reduces BMP signalingic assay . BMP inhA large body of literature describes effects on the BMP pathway when other signaling pathways are targeted. Many of these studies were designed to augment BMP signaling, especially in orthopedic and dental applications (Table S1) though other scenarios have also been evaluated (Tables S2–S7) and several ways that the cellular or tissue microenvironment can be altered to be more permissive to BMP signaling have come to light. One example of this is the synergy observed when intermittent parathyroid hormone therapy is combined with BMP2 or BMP7 in bone healing , 132. in vivo. One significant exception to this is the wide range of contexts in which the Activin/TGFβ and BMP pathways elicit distinctly opposing effects on the same cell type. Some examples of this includes early body patterning [β superfamily cytokines are increasingly associated with human diseases, including pulmonary and kidney fibrosis [β pathway inhibition, for example, on skeletal muscle mass or bone volume, could in part be due to reducing antagonism of the BMP pathway. Support for this idea comes from the fact that increasing the BMP pathway can have similar effects to inhibiting TGFβ signaling . Wh. Wh in veoblasts  and BMP eoblasts , most sthttp://pubmed.com/ by combining the following search terms: antagonism; antagonist; bmp; bone morphogenetic protein; gene therapy; inhibition; inhibitor; siRNA. Articles retrieved were indexed to MEDLINE prior to January 6, 2016. Clinical trials were identified on https://clinicaltrials.gov/ and https://www.clinicaltrialsregister.eu/ prior to January 21, 2016. Specific applications highlighted are meant to be representative rather than exhaustive of the field and no endorsement by the authors of any particular application should be inferred.Studies germane to this topic were identified in Specific reports of BMP pathway modulation and related applications are provided in the supplemental material. Specific applications highlighted are meant to be representative rather than exhaustive of the field and no endorsement by the authors of any particular application should be inferred.


Here we investigated whether the different monocyte subsets are potential HTLV-1reserviors that contribute to viral infection and persistence. PBMCs from 17 HTLV-1 infected patients (IP) and 11 normal donors (ND) were phenotypically analyzed by flow cytometry. Classical monocyte frequency was lower in HTLV-1-IPs compared to NDs (p=0.048). Moreover, we found a positive correlation between PVL and intermediate monocyte frequency . Focusing on the presence of HTLV-1 provirus DNA in the different monocyte subsets, cell populations were isolated from PBMCs of 16 HTLV-1-IP. When we analyzed by nested PCR genomic DNA isolated from sorted CD4+, CD8+ CD14++CD16–, CD14+CD16++, and CD14++CD16+, we found that HTLV-1 patients with high PVL, all monocyte subsets as well as CD4+ and CD8+ cells were positive for HTLV-1. In contrast, the intermediate monocytes were negative or very weakly positive for HTLV-1 in patients with low PVL. To test whether natural STLV-1 infection recapitulates what we find in HTLV-1-IPs, we analyzed the monocyte subsets distribution in 8 STLV-1 infected Rhesus macaques and 16 naïve animals. Consistent with human infection, the frequency of intermediate monocytes was higher in infected macaques compared to naïve animals (p=0.0001) with a positive correlation between PVL and intermediate monocytes frequency . In conclusion, our results suggest that monocytes play an important role in viral dissemination and persistence and are potential viral reservoirs.Peripheral blood monocytes can be classified into three main subsets: CD14


The detection limits for UA and Xa were 3.00 × 10−8 M and 5.00 × 10−8 M, respectively. Finally, the proposed method was applied to simultaneously determine UA and Xa in human urine with good selectivity and high sensitivity.Poly(methylene blue) and electrochemically reduced graphene oxide composite film modified electrode (PMB-ERGO/GCE) was successfully fabricated by electropolymerization and was used for simultaneous determination of uric acid (UA) and xanthine (Xa). Based on the excellent electrocatalytic activity of PMB-ERGO/GCE, the electrochemical behaviors of UA and Xa were studied by cyclic voltammetry (CV) and square wave voltammetry (SWV). Two anodic sensitive peaks at 0.630 V (versus Ag/AgCl) for UA and 1.006 V (versus Ag/AgCl) for Xa were given by CV in pH 3.0 phosphate buffer. The calibration curves for UA and Xa were obtained in the range of 8.00 × 10 Graphene oxide (GO) and graphene are promising materials with many interesting properties for applications ; for exaπ-π noncovalent interaction, some electroactive water-soluble aromatic dyes could absorb on the surface of carbon material, such as the combination of Toluidine blue O and multiwall carbon nanotube  and 1.0 M KCl, followed by sweeping with cyclic voltammetry. The effective area of electrode can be calculated from the Randles-Sevcik plot: Ip = 2.69 × 105n3/2AD1/2v1/2C, wherein ip is current in amps (A), n is number of electrons transferred of K3[Fe(CN)6] in the redox event , A is electrode effective area, D is diffusion coefficient (7.6 × 10−6 cm2·s−1), C is concentration (5 mM), and ν is scan rate (0.05 V·s−1). The effective areas for GCE, PMB/GCE, ERGO/GCE, and PMB-ERGO/GCE were 0.0941 cm2, 0.1906 cm2, 0.2088 cm2, and 0.2291 cm2, respectively. PMB-ERGO/GCE contains the largest effective area, which presumably accounts for its excellent electrocatalytic activity.GCE, PMB/GCE, ERGO/GCE, and PMB-ERGO/GCE were immerged into the solution containing 5.0 × 10−5 M UA and 1.00 × 10−4 M Xa were recorded from pH 2.0 to 8.0. As shown in In most cases, the electrolyte pH is an important condition to the electrochemical reaction. The cyclic voltammograms of 5.00 × 10−5 M) on PMB-ERGO/GCE, cyclic voltammograms were performed by various potential scan rates. To investigate the effect of scan rate on the electrochemical behavior of UA and Xa  RT/. The real reaction electron numbers of UA and Xa were calculated to be 1.964 and 1.913, respectively. Their theoretical reaction electron numbers should be 2. According to literatures [The slope values approximate to 0.5 in equations oftionship ; herein,eratures , 37, 38,Figures The results in −5 M UA and 1.00 × 10−4 M Xa were SWV measured for 20 parallel experiments. The relative standard deviations (RSD) of the peak currents for UA and Xa were 3.1% and 2.7%, respectively, implying remarkable reproducibility. After the modified electrode was stored in humid environment at room temperature for 15 days, it retained 94.5% of its original response and held the similar shape of the original curves, suggesting an acceptable stability of PMB-ERGO/GCE.The mixed solution of 5.00 × 10−5 M UA and 5.00 × 10−5 M Xa. The tolerance limit was set as the maximum concentration of the foreign substances that caused an approximately ±5% relative error in the determination. The results showed that K+, Na+, Ca2+, Fe2+, Mg2+, Ba2+, Zn2+, Al3+, Cl−, NO3−, SO42−, C2O42−, starch, L-arginine, L-threonine, L-serine, L-histidine, dopamine (≥1.0 mg) and L-cysteine (0.6 mg), Cu2+ (0.6 mg), Ag+ (0.05 mg), I− (0.03 mg), and ascorbic acid (0.5 mg) had no interference with the determination of UA and Xa. Therefore, it is possible to simultaneously determine UA and Xa in a sample on PMB-ERGO/GCE.The influences of various foreign species were investigated in a mixture solution containing 2.00 × 10 M PBS (pH 3.0) before measurement. The obtained results are summarized in Human urine sample was selected as the real sample to examine the reliability of the proposed SWV method. 1 mL urine sample was diluted 10 times with 0.1PMB-ERGO/GCE was prepared by electropolymerization. The modified electrode exhibited good conductivity and excellent electrocatalytic activity toward UA and Xa. The introduction of ERGO enhanced the effective surface areas on modified electrode compared with PMB film. The real reaction electron number of UA and Xa were calculated to be 1.964 and 1.913, respectively, in pH 3.0 phosphate buffer solution. The oxidation of UA and Xa were both controlled by diffusion. PMB-ERGO/GCE displayed desirable properties including excellent stability, reproducibility, selectivity, and sensitivity. The results demonstrated that the proposed method is a rapid, sensitive, and reproducible method for determination of UA and Xa in human urine sample. Therefore, PMB-ERGO/GCE would act as a promising sensor for a wide range of electrochemical sensing and biosensing applications.


Human T cell Lymphotropic Virus type 1 (HTLV-1) is the etiological agent of a severe form of neoplasia designated Adult T cell Leukemia/Lymphoma (ATLL) It is widely accepted that the viral transactivator Tax-1 is the major viral product involved in the onset but not in the maintenance of neoplastic phenotype as only 30-40% of ATLL cells express Tax-1. It has been recently demonstrated that HBZ (HTLV-1 bZIP factor), a protein encoded by the minus strand of HTLV-1 genome, constantly expressed in infected cells and in ATLL tumor cells, is also involved in the pathogenesis of leukemia. The full role played by HBZ in oncogenesis is still to be explored in detail mainly owing to the unavailability of tools to assess quantitative expression, subcellular location and interaction of HBZ with host factors in ATLL. By the use of the first reported monoclonal antibody against HBZ, 4D4-F3, generated in our laboratory it has been possible to carefully assess for the first time the above parameters in HTLV-1 chronically infected cells and, most importantly, in leukemic cells from patients. Endogenous HBZ is expressed in speckle-like structures localized in the nucleus. The calculated number of endogenous HBZ molecules varies between18.0 to 36.0 molecules per cell, 22-44 fold less than the amount expressed in HBZ transfected cells used by most investigators to assess the expression, function and subcellular localization of the viral protein. HBZ interacts in vivo with p3 and JunD and co-localizes only partially, and depending on the amount of expressed HBZ, not only with p3 and JunD but also with CBP and CREB2. The possibility to study endogenous HBZ in detail may significantly contribute to a better delineation of the role and presence of HBZ during HTLV-1 infection and cellular transformation.


In vivo, subcutaneous human tumor xenografts were evaluated. Primary cultures of mucin- and mixed-IHCCA were characterized by a different pattern of expression of cancer stem cell markers, and by a different drug sensitivity. Gemcitabine and the Gemcitabine-Cisplatin combination were more active in inhibiting cell proliferation in mixed-IHCCA while Cisplatin or Abraxane were more effective against mucin-IHCCA, where Abraxane also enhances apoptosis. 5-Fluoracil showed a slight inhibitory effect on cell proliferation that was more significant in mixed- than mucin-IHCCA primary cultures and, induced apoptosis only in mucin-IHCCA. Among Hg inhibitors, LY2940680 and Vismodegib showed slight effects on proliferation of both IHCCA subtypes. The tyrosine kinase inhibitors, Imatinib Mesylate and Sorafenib showed significant inhibitory effects on proliferation of both mucin- and mixed-IHCCA. The MEK 1/2 inhibitor, Selumetinib, inhibited proliferation of only mucin-IHCCA while the aminopeptidase-N inhibitor, Bestatin was more active against mixed-IHCCA. The c-erbB2 blocking antibody was more active against mixed-IHCCA while, the Wnt inhibitor, LGK974, similarly inhibited proliferation of mucin- and mixed-IHCCA. Either mucin- or mixed-IHCCA showed high sensitivity to nanomolar concentrations of the dual PI3-kinase/mTOR inhibitor, NVP-BEZ235. In vivo, in subcutaneous xenografts, either NVP-BEZ235 or Abraxane, blocked tumor growth. In conclusion, mucin- and mixed-IHCCA are characterized by a different drug sensitivity. Cisplatin, Abraxane and the MEK 1/2 inhibitor, Selumetinib were more active against mucin-IHCCA while, Gemcitabine, Gemcitabine-Cisplatin combination, the c-erbB2 blocking antibody and bestatin worked better against mixed-IHCCA. Remarkably, we identified a dual PI3-kinase/mTOR inhibitor that both in vitro and in vivo, exerts dramatic antiproliferative effects against both mucin- and mixed-IHCCA.We investigated the sensitivity of intrahepatic cholangiocarcinoma (IHCCA) subtypes to chemotherapeutics and molecular targeted agents. Primary cultures of mucin- and mixed-IHCCA were prepared from surgical specimens (N. 18 IHCCA patients) and evaluated for cell proliferation (MTS assay) and apoptosis (Caspase 3) after incubation (72 hours) with increasing concentrations of different drugs.  CCA proGenistein, Cyclopamine and 5-Fluorouracil (5-Fu) were purchased from Sigma-Aldrich Co . Cisplatin, Gemcitabine, Vismodegib, LY2940680, Imatinib mesylate, Bestatin, NVP-BEZ235, AZD6244 (Selumetinib), MK2206 and LGK974 were purchased from Selleck Chemicals . Cetuximab was purchased from Merck Serono . The c-ErbB2 blocking antibody was obtained from Spring Bioscience Corporation . Abraxane (Nab-Paclitaxel) was obtained from Abraxis BioScience .The use of human materials has been approved by our local Institutional Review Board. Specimens of human IHCCA were obtained from patients submitted to surgical resection and specifically: 18 patients with IH-CCA presenting as a single mass lesion within the liver. Patient characteristics were detailed in −4 mol/L adenine, 5 μg/ml insulin, 5 μg/ml transferrin, 2 x 10−9 mol/L triiodothyronine, 1.7 x 10−6 mol/L hydrocortisone, 1.0 x 10−6 mol/L human epidermal growth factor, 5.5 x 10−6 mol/L epinephrine , 10% fetal bovine serum , 100 U/ml of penicillin, and 100 μg/ml of streptomycin, at 37°C in a humidified atmosphere of 5% CO2 in air. Primary cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. The different drugs were tested after 20–30 passages.Human IHCCA samples were classified as mixed- or mucin-IHCCA by morphologic criteria and Pas staining, according to Komuta M. et al . Patient5 cells were analyzed using a FACS Diva software (BD).Primary IHCCA cell cultures were investigated by FC, at passages 20–30, for the expression of CSC surface markers by using the following antibodies: PE-mouse anti-human CD13 , CD90-FITC human, CD133-APC human, CD45-PE human, EpCAM-FITC human , anti-LGR5 mouse mAb PE coniugate . The fluorescence threshold between negative and positive cells was set on the basis of the reactivity of appropriate non-specific fluorochrome-conjugated isotypical controls. At, least 5 X 10TM  and 1-bromo-3-chloropropane in substitution of chloroform. The isolated RNA was dissolved in 55 μl of RNase-free water. RNA quality and quantity was controlled by the Experion Automated Electrophoresis System equipped with the RNA StSens Analysis Chip . The reverse transcription primed by the random hexamer  was conducted in a 20 μL volume with an amount of 2.5 μg of total RNA and the M-MLV reverse transcriptase (Invitrogen s.r.l.) according the manufacturer's directions. Gene expression was determined by Real-Time PCR with a MX3000P instrument  using the averaged cycle threshold (Ct) automatically computed by the built-in software from three replicas of each sample. PCR amplifications were conducted into a volume of 25 μl, with 1.0 μl of cDNA template, 12.5 μl of 2x SYBR Green Brilliant QPCR Master Mix (Stratagene), 3 pmoles each of upstream and downstream primer for the gene analyzed, and 0.3 μl of diluted reference dye . All real-time PCR amplifications were conducted with the cycling program: 10 min at 95°C followed by 40 cycles . The fluorescence detection was performed during the extension step of each cycle. The following genes of interest (GOI) were measured: Vimentin, CD13, CD90, CD133, EpCAM, and LGR5. All expression levels were normalized to the expression of GAPDH housekeeping gene. For IHC/IF, semi-confluent cultures were generated on four-chamber slides . The medium was removed, and cells were fixed in 10% buffered formalin for 10 min at room temperature. Cells were rinsed twice with PBS buffer for 2 min, blocked and incubated 1 hour with the following antibodies: Vimentin mouse monoclonal IgG1 , α-SMA mouse monoclonal IgG1, E-Cadherin mouse monoclonal IgG1 , CD326/EpCAM mouse monoclonal IgG1 , CD133/PROM1 mouse monoclonal IgG1 , LGR5 rabbit polyclonal IgG , cytokeratin-19 (CK-19) sc-6278 CK-19 mouse monoclonal IgG2a (Santa Cruz Biotechnology), Interleukin 6 (IL6) mouse monoclonal IgG2a  at room temperature. After rinsing twice with PBS for 2 min, cells were incubated for 40 min at room temperature with secondary biotinylated antibody  rinsed twice with PBS and then incubated with Vectastain ABC reagent  for 20 min. Diaminobenzidine  was used as substrate, and sections were counterstained with hematoxylin. Slides were examined by DM 2000 Light and/or Fluorescence Microscopy  equipped with a DFC450 C Videocam . For RT-PCR, cell cultures were extracted for total RNA by using the TRI REAGENT3 cells were seeded into 96-well plates in 100 μL of culture medium. After 24 hours the medium was replaced with fresh culture medium containing increasing concentrations of the tested drug and then, after 72 hours, the MTS assay was performed. Results were expressed as % changes with respect to controls considered equal to 100. Apoptosis was evaluated by Caspase-3 Kit  by following instructions of the vendor. A total of 5x105 cells were plated into flasks in 20 mL of culture medium. After 24 hours the medium was replaced with fresh culture medium containing a determined concentration of the different drugs; we tested the concentration that determined a significant inhibition of cell proliferation at the MTS assay. Apoptosis was detected after 72 hours and expressed as ratio between casapse-3 activity measured in drug-treated and control cells.Sensitivity to chemotherapeutics and molecular targeted agents was tested by evaluating cell proliferation or apoptosis in primary cell cultures exposed to increasing concentrations of different drugs. Drugs were prepared as a stock solution in DMSO and then diluted  in the culture medium at the desired final concentration; the same amount of DMSO was added in controls. Proliferation was evaluated by MTS assay . A total of 5x103, mice were treated by gavage with NVP-BEZ235  and Abraxane  for two weeks. Control mice received PBS only. The health of all mice was monitored daily throughout the study. Main criteria used to assess mice health were the evaluation of body weight and consumption of food and water, other than the essentials for assessing mouse health as described by Burkholder et al. [Male NOD/SCID mice, 4–6 weeks old, purchased from Charles River  were maintained under standard conditions and cared according to our institutional guidelines for animal care. As previously described , CD13+ ar et al.  Animal wEthics Committee of Hospital Policlinico Umberto I of Rome/Sapienza University of Rome , and was conducted according to the principles expressed in the Declaration of Helsinki. Subjects have been properly instructed and have indicated that they consent to participate by signing the appropriate informed consent paperwork.The research protocol was reviewed and approved by the Ethics Committee of Hospital Policlinico Umberto I of Rome/Sapienza University of Rome . Animal welfare was carefully ensured constantly by experienced operators every day. Mice were then killed by cervical dislocation. All efforts were made to minimize suffering of the animals along all the duration of their life and during the sacrifice.The experiment on animals was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the European Commission. The protocol was reviewed and approved by the The processing was compliant with Good Manufacturing Practice.t-test as appropriate or the analysis of the variance (ANOVA) when multiple comparisons were performed. A p value < 0.05 was considered statistically significant. The half maximal inhibitory concentration (IC50) values were calculated by nonlinear regression analysis.Data are presented as arithmetic mean ± S.D. Statistical analysis was conducted using the paired or unpaired Student’s Primary cultures prepared from human mixed- and mucin-IHCCA specimens were characterized for mesenchymal, epithelial and CSC surface markers by IH, FC and RT-PCR. As shown in 50 = 0.37 ± 0.04 μM) than mucin-IHCCA primary cultures . Cisplatin, in contrast, was more active (50 = 4.7 ± 1.3 μM) than mixed-CCA . With respect to the two drugs alone, the Gemcitabine-Cisplatin combination induced a higher inhibition of cell proliferation (50 = 0.7 ± 0.2 μM) but not in mixed-IHCCA  and mucin-IHCCA (IC50 = 0.1 ± 0.02 μM) primary cultures, although the effect on mucin-IHCCA was predominant (p< 0.05). Abraxane (0.05 μM) induced a significant increase of apoptosis only in mucin- but not mixed-IHCCA  , showed ed-IHCCA .50 = 9.8 ± 0.3 μM) and mucin-IHCCA (IC50 = 34.9 ± 5.7 μM) primary cultures, although the effect on mixed-IHCCA was predominant (p< 0.05). 5-FU (20 μM) induced apoptosis but only in mucin-IHCCA  and mixed-CCA .Cyclopamine, a natural occurring alkaloid , displayApoptosis was unaffected by Vismodegib (50 μM) or LY2940680 (20 μM) in primary cultures of mucin- and mixed-IHCCA 50: mucin-IHCCA = 10.5 ± 3.6 μM; mixed-IHCCA = 13.0 ± 1.3 μM, 50: mucin-IHCCA = 7.8 ± 0.9 μM; mixed-IHCCA = 6.3 ± 0.4 μM; We tested the following tyrosine kinase inhibitors: Genistein that is a broad spectrum tyrosine kinase inhibitor ; ImatiniApoptosis was significantly enhanced only by Imatinib Mesylate (10 μM) and only in mixed-IHCCA .50 = 6.1 ± 0.7 ng/ml) and mixed-IHCCA (IC50 = 3.4 ± 0.3 ng/ml), but with a slight predominant effect on the latter , had no effect neither on mucin- nor mixed-IHCCA . In contp< 0.05, . Cetuximp< 0.05,  showed n50 = 1.1 ± 0.14 nM) and mixed-IHCCA (IC50 1.26 ± 0.88 nM) primary cultures (50 = 4.5 ± 2.3 μM) and mixed-CCA (IC50 5.6 ± 0.53 μM) but at a significant lower extent in comparison with NVP-BEZ235.NVP-BEZ235, a dual PI3K and mTOR inhibitor , showed cultures . Also thcultures  in primaAs far as apoptosis is concerned, only the AKT inhibitor, MK2206, but not NVP-BEZ235, induces significant apoptotic effects, at 10 μM, without differences between mucin-IHCCA and mixed-IHCCA .50 = 18.7 ± 6.1 μM) with respect to mixed-IHCCA   inhibits50 of 103 ± 2.7 μM for mucin-IHCCA and of 150± 17 μM for mixed-IHCCA , and from 590 ± 55 mm3 to 1450 ± 76 . Similar findings were obtained when CD13+ spheroids were implanted, since the tumor volume remained almost stable following the treatment with NVP-BEZ235 . At the histo-pathological evaluation  among chemotherapeutics, Gemcitabine and the Gemcitabine-Cisplatin combination were more active in inhibiting cell proliferation in mixed-CCA while Cisplatin or Abraxane were more effective against mucin-CCA, where abraxane was also able to induce apoptosis. 5-FU exerted a slight inhibitory effect on cell proliferation, predominating in mixed-IHCCA, and also induced apoptosis but only in mucin-IHCCA; ii) among Hg inhibitors, Ciclopamine was ineffective while LY2940680 and Vismodegib showed slight effects on proliferation but not on apoptosis; iii) the tyrosine kinase inhibitor, Genistein was completely ineffective while either Imatinib Mesylate or Sorafenib showed significant inhibitory effects on proliferation; iv) the MEK 1/2 inhibitor, Selumetinib, was active in inhibiting proliferation only against mucin-CCA while the aminopeptidase-N inhibitor, Bestatin was more active against mixed-CCA; v) the c-erbB2 blocking antibody was more active against mixed- than mucin-IHCCA while, Cetuximab failed to show significant effects; vi) either mucin- or mixed-IHCCA showed high sensitivity to PI3-kinase inhibitors and particularly to NVP-BEZ235 that markedly inhibited cell proliferation at nanomolar concentrations and, also in situ differences in terms of CSC composition and EMT markers between the two CCA subtypes. Also primary cultures showed large differences since CD90+ cells predominated in mucin-CCA while CD13+ cells in the mixed–CCA subtype. In spite of these clinical and patho-biologic differences, no report investigated the drug-sensitivity of the two CCA subtypes and, clinically, the need of a different therapy has been so far completely neglected. We used primary cultures of mixed- and mucin-CCA to test drug sensitivity and to calculate IC50. These cultures are characterized by the predominance of cells expressing mesenchymal markers and EMT trait. We firstly demonstrated the different sensitivity of the two CCA subtypes to chemotherapeutics since Gemcitabine and the Gemcitabine-Cisplatin combination were more active against mixed-CCA while Cisplatin or Abraxane were more effective in inhibiting cell proliferation in mucin-CCA primary cultures. Abraxane is an albumin bound formulation of paclitaxel where albumin favors drug internalization and indeed, according to a recent report, this formulation showed promising results in advanced pancreatic cancer [in vivo, in subcutaneous xenografts, Abraxane showed a similar inhibitory effect on the growth of both mucin- and mixed-IHCCA.The current standard of care for unresectable IHCCA is the Gemcitabine/Cisplatin regimen , 19 thatc cancer . This isc cancer . HoweverA critical point, in the era of targeted therapies, is the scarcity of basic or preclinical studies evaluating molecular targeted therapies in relation with biologic or genetic CCA signatures. Among the molecular target agents, we firstly tested the Hg signaling inhibitors since different studies suggested how the Hg signalling pathways are of relevance for CCA biology , 24. TheEGFR and/or ErbB2 are overexpressed in human and rodent CCA cells and EGFR has been implicated with CCA pathogenesis , 32. EGFin vivo, in subcutaneous xenografts, NVP-BEZ235, blocked the progression of mucin- and mixed-IHCCA. Also the pan-AKT inhibitor, MK2206, exerted antiproliferative affects and, this compound also enhanced apoptosis. Thus our findings support future clinical trials testing PI3k/mTOR inhibitors.Signaling mediated by PI3K and mTOR play a critical role in CCA cell proliferation, as well as in modulating authophagy and drug resistance –38. IncrSelumetinib is an allosteric inhibitor of MEK1 and MEK2 phosphorylation of ERK and of interleukin-6 secretion, the latter playng a major role in modulating CCA cell proliferation , 12% of We also tested Bestatin, a CD13+ CSC antagonist, that showed antitumoral activity in different cancers . We founFinally, we tested a Wnt signalling inhibitor, since recent evidence highlighting the role of Wnt signaling in sustaining CCA growth and progression . StartinIn conclusion, our findings indicate that other than biologically different, mucin and mixed-CCA subtypes are also different as far as the sensitivity to chemotherapeutics and targeted-agents is concerned. Future therapeutic strategies triggering IHCCA should take in consideration these differences and, therefore, discrimination between the two subtypes is fundamental also for therapeutic decisions.


Among human T-lymphotropic virus type 1 (HTLV-1)-infected individuals, the lifetime risk of developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) differs between ethnic groups, also, there is an association between HTLV-1 tax gene subgroups  and the risk of HAM/TSP. In an attempt to understand the molecular mechanism responsible for this association, we studied the full-length proviral genome sequences of various HTLV-1-infected cell lines and patient samples, the functional difference in the viral transcriptional regulator Tax and HBZ between each subgroup, and the relationship between tax subgroups and the clinical and laboratory characteristics of HAM/TSP patients. The results indicated that (1) the distinct nucleotide substitutions corresponding to each tax subgroup are also associated with the nucleotide substitutions of viral structural, regulatory, and accessory genes; (2) mRNA expression of HBZ in HTLV-1-infected cells was significantly higher in HAM/TSP patients with tax-B than those with tax-A; (3) there was a positive correlation between HBZ and the expression of its target FoxP3 in HAM/TSP patients with tax-B but not in patients with tax-A; (4) transcriptional changes in inducible cells that express each subgroup of Tax or HBZ protein under the control of an inducible promoter showed different target gene profiles; however, (5) there were no clear differences in clinical and laboratory characteristics between HAM/TSP patients with tax-A and tax-B; and (6) there were no functional differences in Tax and HBZ between each subgroup evaluated by reporter gene assays. Our results indicate that although different HTLV-1 tax subgroups cause different patterns of viral and host gene expression in HAM/TSP patients via independent mechanisms of direct transcriptional regulation, these differences do not significantly affect the clinical and laboratory characteristics of HAM/TSP patients. Further studies on other viral genes, as well as clinical samples of asymptomatic carriers and adult T-cell leukemia (ATL) patients, are in progress to clarify the role of HTLV-1 tax subgroups.


During melanoma cell extravasation through the vascular endothelium, melanoma cells interact with endothelial cells through secretion of cytokines and by adhesion between proteins displayed on opposing cell surfaces. How these tumor cell associated signals together regulate the dynamics of intracellular signaling pathways within endothelial cells leading to endothelial cell-cell junction disruption is not well understood. Here, we used a combination of experimental and computational approaches to examine the individual and combined effects of activation of the vascular cell adhesion molecule (VCAM)-1, interleukin (IL)-8, and IL-1β signaling pathways on the integrity of vascular junctions. Our simulations predict a multifaceted interplay of signaling resulting from individual activation of VCAM-1, IL-8 and IL-1β pathways that is neither synergistic nor additive compared to all inputs turned on simultaneously. Furthermore, we show that the levels of phosphorylated proteins associated with actinomyosin contractility and junction disassembly peak prior to those related to actin remodeling. The results of this work provide insight into the dynamics of tumor-mediated endothelial junction disassembly and suggest that targeting proteins downstream of several interaction pathways may be the most effective therapeutic approach to reduce melanoma extravasation. The spread of cancer cells from a primary tumor site to distant organs, metastasis, is one of the most devastating aspects of cancer accounting for 90% of cancer-related deaths. A key event during tumor metastasis is the extravasation of a cancer cell through the blood vessel wall The maintenance and stability of endothelial cell-cell junctions is thought to be regulated by the balance between cell-cell adhesion and cellular contractility The cadherin-catenin complex dynamically links adherens junctions with the actin cytoskeleton and this interaction is mediated by association with α-catenin and actinin. Treatment of endothelial monolayers with hyperpermeability inducing agents leads to actin reorganization into linear, parallel bundles known as stress fibers across the cell interior 4β1) which binds vascular cellular adhesion molecule (VCAM)-1, an integrin receptor displayed on the surface of endothelial cells Melanoma cells express the ligand very late antigen (VLA)-4  were obtained from American Type Culture Collection (ATCC) and cultured in F-12K medium supplemented with 10% fetal bovine serum , 100 units/ml penicillin-streptomycin , 30 µg/ml endothelial cell growth supplement , and 50 µg/ml heparin . A2058 melanoma cells were obtained from ATCC and cultured in DMEM/F-12 (Life Technologies) supplemented with 10% FBS and 100 units/ml pen-strep. All cells were grown at 37°C and 5% CO5 cells to 25-mm diameter glass coverslips coated with 1 µg/ml human fibronectin (BD Biosciences) for all experiments. When 95–99% confluent, monolayers were incubated with F-12K media supplemented with 2% FBS for 12 hours prior to co-culture. For co-culture, A2058 melanoma cells were lifted from tissue culture dishes using a thin coating of 0.5% trypsin (Life Technologies). The A2058 cells were then suspended in fresh medium and rocked for 1 hour in a 37°C incubator. A2058 melanoma cells were then plated at 1.8×105 cells/cm2 to the endothelial monolayers. Endothelial cell-cell junction integrity was monitored following 45 minutes of co-culture.HUVECs were seeded at 3×10HUVEC monolayers were incubated with the following inhibitors or carrier solution for 30 minutes prior to co-culture with A2058 melanoma cells: blebbistatin , cytochalasin D , PP1 , ML-7 , or dimethyl sulfoxide . Following incubation with inhibitors, HUVEC monolayers were rinsed once with F-12K/2% FBS culture medium and were then co-cultured with A2058 melanoma cells.Following co-culture, HUVEC monolayers were gently rinsed once with 1× phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Cells were then washed with PBS, permeabilized with 0.3% Triton X-100 and blocked with 5% goat serum (Sigma). Samples were then incubated with VE-cadherin primary antibody  overnight at 4°C. The next day samples were thoroughly rinsed with PBS to remove unbound primary antibody, incubated with AlexaFluor secondary antibodies (Life Technologies), and then counterstained with Hoechst 33342 (Life Technologies) to visualize cell nuclei. Coverslips were mounted to microscope slides using Fluoromount G (Southern Biotechnology) prior to imaging.2 charge coupled device camera. Images were taken from 9 randomly chosen locations within the monolayer. Gaps between endothelial cells were quantitatively measured from fluorescence images of VE-cadherin using ImageJ software (NIH) as the ratio of pixels within all monolayer gaps to the total number of pixels in one image.Samples were imaged with a 40× air or 100× oil objective (N.A. = 1.3) using a Nikon Eclipse Ti-E inverted fluorescence microscope equipped with a Photometrics CoolSNAP HQStatistical analysis was performed with KaleidaGraph 4.1 software (Synergy) using analysis of variance (ANOVA) followed by a Tukey Honestly Significant Difference (HSD) post-hoc test. Differences were considered significant for p<0.05. Values are reported as mean ± standard error of the mean. Three independent replicates were performed for all experiments.An intracellular signaling network was created to connect the VCAM-1, IL-8, and IL-1β inputs and their respective signaling cascades to factors that are responsible for regulating endothelial junction breakdown. The interactions were altered to represent enzyme kinetics and a system of ordinary differential equations was developed using these kinetics to construct a model using a MATLAB systems biology toolbox developed by Schmidt and coworkers −1 s−1, respectively Kinetic parameters and initial concentrations were obtained from literature and experimental data. If the specific value of the concentration or parameter was not available, then they were set to 1 µM and 0.1 µMFor time course simulations, the time at which the peak protein concentration occurred was assumed to be the optimal activation of that protein.Due to the complexity of inhibitory kinetics and the lack of knowledge of the type of inhibitions on the proteins in the model, inhibitory reactions were neglected.Melanoma cell signals including soluble cytokines and cell surface adhesive molecules can impact the barrier properties of the vascular endothelium. To examine how melanoma cues affect the integrity of endothelial cell-cell junctions, A2058 melanoma cells were co-cultured with HUVEC monolayers for 45 minutes. Cell-cell junctions were monitored by examining VE-cadherin localization and it was observed that co-culture of melanoma cells with HUVECs resulted in dissolution of VE-cadherin junctions local to the tumor cells . QuantifTo examine the roles of endothelial cellular contractility, junction disassembly, and actin remodeling in melanoma-mediated gap formation, we experimentally targeted proteins within endothelial cells linked to these outputs. Cellular contractility is controlled by the actomyosin cytoskeleton, which is regulated by signaling through the Rho GTPases and MLCK. Decreasing cellular contractility through treatment with the non-muscle myosin ATPase inhibitor blebbistatin significantly reduced A2058-induced gap formation in HUVEC monolayers . CytochaTo examine the dynamic response of the endothelium to multiple melanoma cell signals simultaneously, we developed a model of the signaling networks involved in melanoma-induced gap formation. The model included melanoma cell signals  as well as downstream p38 MAPK, Src, PKC, and MLC signaling pathways within the endothelium . The sigTo investigate the relative timing of activation of signaling cascades leading to actin remodeling, cellular contraction, and junction disassembly we monitored the levels of phosphorylated heat shock protein (HSP)-27, MLC, and VE-cadherin, respectively, within the simulation. Turning on all three inputs, VCAM-1, IL-8, and IL-1β, represented the melanoma cell stimulus. Previous studies have linked disruption of VE-cadherin junctions to phosphorylation of VE-cadherin and reduced junction strength The time courses of each output were viewed over 45 minutes (2700 seconds) since melanoma-induced endothelial gap formation is observed to occur over 45 minutes experimentally . With alTo determine whether VCAM-1, IL-8, and IL-1β have an additive, synergistic, or antagonistic effect on endothelial gap formation we also performed simulations with VCAM-1, IL-8, and IL-1β signaling pathways individually activated and compared these results to simultaneous activation of all three inputs. Each simulation was performed for 2700 seconds and the concentrations of pMLC, pHSP-27, and pVE-cadherin were monitored as a function of time. At early time points, pMLC dynamics associated with all inputs on was recapitulated by IL-8 alone suggesting that the IL-8 signaling cascade is likely responsible for the initial increase in pMLC levels within endothelial cells as well as for the induction of increased contractility. At intermediate and late time points, the case with all three inputs turned on did not produce the highest concentration of pMLC therefore suggesting antagonistic effects between the different individual signaling inputs . For pHSWe next investigated the effects of p38 MAPK, MLCK, and PKC signaling on gap formation since these molecules are influential regulators of actin remodeling, actinomyosin contraction, and junction disassembly, respectively Given the important role of MLCK in controlling cellular contractility, we next examined the role of MLCK in regulating the dynamics of protein phosphorylation within endothelial cells in response to melanoma cues. Knockout of MLCK within the simulations resulted in a substantial decrease in the concentration of phosphorylated MLC across all time points although MLC phosphorylation was not completely abrogated . FurtherSignaling through PKC has been linked to endothelial barrier dysfunction and transendothelial migration In this study, we examined the roles of the melanoma cell inputs, IL-8, IL-1β, and VCAM-1 activation, and the endothelial cell outputs, actinomyosin contraction, junction disassembly, and actin remodeling, in melanoma-induced endothelial cell-cell junction breakdown. Actinomyosin contraction, junction disassembly, and actin remodeling have all been correlated with increased gap formation, but the importance that each output possesses in gap formation and whether there is synergy between the three to optimize gap formation is not clear. Experimentally, we demonstrated the importance of key signaling proteins in melanoma-induced gap formation. We also created an intracellular signaling network to connect the three inputs and their respective signaling cascades to factors that are responsible for regulating endothelial junction breakdown. Within the simulations, we quantitatively monitored the roles of the inputs and outputs involved in gap formation. Moreover, through the simulations we investigated the signaling dynamics that coordinate endothelial junction breakdown. Overall, our data suggest that cellular contractility, junction disassembly, and actin remodeling are all significant contributors to melanoma-induced endothelial gap formation.2β1 integrin displayed on the surface of breast cancer cells mediates phosphorylation of VE-cadherin in endothelial cells via Ras/MLC signaling Since it is difficult to monitor the dynamics of multiple signaling cascades simultaneously in direct co-culture experiments, we developed a model of the interactions between melanoma cells and endothelial cells to enable examination of signaling dynamics. Using this model, we investigated the phosphorylation dynamics of MLC, HSP-27, and VE-cadherin as read-outs of cellular contractility, actin remodeling, and junction disruption, respectively. We found that over the 45 minute (2700 second) window for gap formation, that the concentration of pMLC is maximized at early time points (from 300 to 1300 seconds) followed by pVE-cadherin at approximately 1000 seconds with the concentration of pHSP-27 peaking at 2700 seconds. Interestingly, pMLC reaches its peak concentration around the same time that pHSP-27 and pVE-cadherin concentrations start their initial increase. This suggests that crosstalk between the signaling pathways may be important to gap formation with actinomyosin contraction or pMLC possibly impacting or promoting phosphorylation of HSP-27 and VE-cadherin, actin remodeling, and junction disassembly. Indeed, recent studies show that during transendothelial migration monocytes activate the HRas/Raf/MEK/ERK signaling cascade which induces MLC phosphorylation followed by Src-mediated phosphorylation of VE-cadherin Investigation of the role of the individual activation of VCAM-1, IL-8, and IL-1β signaling cascades revealed that the effects of these cues are neither additive nor synergistic on the phosphorylation dynamics of downstream signaling molecules, as activation of all inputs never resulted in additive effects or large increases in the levels of pMLC, pHSP-27, or pVE-cadherin over levels from individual activation during the time course of gap formation. The phosphorylation dynamics of HSP-27 and VE-cadherin resulting from VCAM-1 signaling alone most closely matched that produced by activation of all three pathways simultaneously suggesting that intracellular signaling cascades initiated by VCAM-1 likely have the most influence on actin remodeling and junction disassembly. Previous studies report that crosslinking VCAM-1 with antibodies is sufficient to induce stress fiber formation and gaps in HUVEC monolayers and blocking VCAM-1 activation on endothelial cells by monocytes through the use of specific antibodies attenuates phosphorylation of VE-cadherin Previous studies have shown that PKC, p38 MAPK, and MLCK are influential regulators of endothelial cell junction disassembly, actin remodeling, and actinomyosin contraction, respectively This study focused on melanoma-induced endothelial cell-cell junction disruption, however, the results described may not be unique to the interaction between melanoma and the endothelium. Indeed, a variety of cancer cell types including melanoma In summary, we present integrated experimental and computational analyses that show actinomyosin contraction, junction disassembly, and actin remodeling are all important factors in mediating melanoma-induced endothelial junction breakdown and we demonstrate that MLCK and p38 MAPK have significant roles in gap formation. Simulations showed that the peak levels of phosphorylated proteins associated with actinomyosin contraction and junction disassembly occur towards the beginning of gap formation while the peak level of phosphorylated proteins linked to actin remodeling occurs at later stages of gap formation. In addition, activating VCAM-1, IL-8, and IL-1β signaling cascades simultaneously has neither an additive nor synergistic effect on the concentration of phosphorylated proteins associated with contractility, junction disassembly, and actin remodeling individually in comparison to activation of each signaling pathway alone. Thus, melanoma-mediated endothelial junction disruption arises from a complex interplay of multiple tumor cell-associated signals as well as multiple intracellular signaling cascades within endothelial cells. Analysis of the interplay between these signaling pathways will enable a clearer understanding of how these cues act in concert to influence endothelial cell-cell junction breakdown and may suggest novel approaches for blocking cancer metastasis.Table S1Initial concentrations (greater than zero) for signaling network model.(DOCX)Click here for additional data file.Table S2Kinetic parameters used within the signaling network model.(DOCX)Click here for additional data file.


Protein kinase D 1 (PKD-1) is a signaling kinase important in fundamental cell functions including migration, proliferation, and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease, and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis. PRKD) is a serine threonine kinase consisting of three isoforms PKD-1,-2, and -3 , fibroblasts, and dendritic cells. This indicates that PKD-1 is a key regulator in tissue homeostasis and cellular functions. PKD-1 mediates signaling pathways important in cardiovascular diseases, immune functions and cancer  signaling  and a pleckstrin homology (PH) domain inserted between the cysteine-rich motif and the catalytic domain. Different from protein kinase PKC, PKD contains PH domain within the regulatory region. The catalytic domain is distantly related to Ca2+-regulated kinase, and a highly hydrophobic stretch of amino acids is located in its N-terminal region  is a member of a zinc-finger-like transcription factor subfamily. VEGF activates PKD-1 via a VEGFR2-PLCγ-PKD-dependent pathway. Moreover, PKD-1 signaling interacts with HDAC5 to promote transcriptional activation of myocyte enhancer factor-2 (MEF2) and a specific subset of gene expression in response to VEGF including NR4A1, an orphan nuclear receptor involved in angiogenesis . HDACs are critical to inhibiting acetylation of nucleosome histones. Interestingly, HDAC5 and HDAC7, highly expressed in ECs , a lipid signaling mediator, regulates angiogenesis in a chicken chorioallantoic membrane assay and via mediating the formation of a nuclear complex comprised of FoxO1 and HDAC7. PKD-1 signaling activation might specifically turn off the FoxO1 switch in the CD36 gene locus for suppressing CD36 transcription, whereas arteriogenic gene reprogramming is initiated once CD36 transcription is turned off in response to LPA . VEGF interactions with VEGFR2/KDR (kinase insert domain receptor) are critical for normal blood vessel development β(1) trafficking -6, CXC chemokines IL-8, and growth-related oncogene-alpha (GRO-alpha; Hao et al., via producing signaling molecules in the tissue microenvironments. Interestingly, the isoform PKD2 also regulates proliferation and migration in HUVECs, the two important process of angiogenesis, and this may be mediated by modulation of the expression of VEGFR-2 and fibroblast growth factor receptor-1 (Hao et al., PKD-1 regulates collagen I-induced vascular morphogenesis Tumor angiogenesis is critical, not only in rapidly growing macroscopic tumors, but also in microscopic premalignant phase of neoplastic progression, and thus is considered as an integral hallmark of cancer (Hanahan and Weinberg, via LPA/PKD-1 signaling-mediated angiogenesis (Dong et al., PKDs regulate both hypoxia-induced VEGF expression/secretion by the tumor cells and VEGF- stimulated angiogenesis, which are essential for the malignant progression of tumors. Studies show that PKD-1 promotes anchorage-independent growth, invasion, and angiogenesis in human pancreatic cancer including PDAC (Guha et al., The PKD-2 also mediates production of various angiogenic factors in human pancreatic cancer cells and stimulates the angiogenic response of the host vasculature (Azoitei et al., via a nuclear HDAC7-FoxO1 complex, implicating microvascular remodeling and tumor arteriogenesis (Kohlenberg et al., via this axis. Activation of this pathway in vivo may promote vascular remodeling and the functional stability of the arterial networks (Ren et al., PKD-1 regulates the expression of a variety of angiogenic genes. Interestingly, LPA/PKD-1 signaling suppresses CD36 transcription and reprograms MVECs for arteriogenic gene expression via regulation of αvβ3 integrin recycling (Woods et al., via cross-talking with integrin αvβ3 and CD36, subsequently turning on angiogenic switch for arterial differentiation of ECs to promote arteriogenesis. ECs is essential for developmental and adult arteriogenesis (Ren et al., Furthermore, PKD-1 signaling is essential for VEGF signaling-mediated angiogenic functions and is implicated in tumor arteriogenesis. VEGF signaling is regulated by the tight control of intracellular VEGFR2 localization (Bhattacharya et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It has been sporadically reported that HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) develops after living-donor kidney transplantation from HTLV-1 positive donors (D+) to negative recipients (R-). However, these details have been unknown. Thus we evaluated the incidence and clinical characteristics of HAM/TSP after living-donor kidney transplantation from D+ to R-(D+R-transplantation). Using data obtained from the Japanese Renal Transplant Registry, we analyzed 13,299 cases of living-donor kidney transplantation between 2000 and 2013 in Japan. In addition, we have collected information about 5 patients who developed HAM/TSP after D+R-transplantation. The incidence of HAM/TSP after D+R-transplantation was calculated as the ratio of “the number of recipients who developed HAM/TSP” to “the number of cases of D+R-transplantation”. The characteristics of HAM/TSP in D+R-transplant recipients such as time from transplantation to disease onset and rate of disease progression are investigated. About 70% of all 13,299 donors took a HTLV-1 antibody test and 64 cases were positive for HTLV-1 antibody. Although the remaining 4,072 donors didn't take the antibody test, we estimated the number of HTLV-1 positive donors as 36 according to the HTLV-1 prevalence in Japan. As a result, the estimated incidence of HAM/TSP after D+R-transplantation was 5%. All the 5 cases of HAM/TSP after D+R-transplantation showed an early onset after transplantation. Four out of five cases developed rapidly and had difficulty walking in one or two years. This study demonstrated that incidence rate (5%) of HAM/TSP in recipients after D+R-transplantation is extremely higher compared to the lifetime risk (0.25%) of HAM/TSP in an HTLV-1-infected person. Further, this study suggested that HAM/TSP after D+R-transplantation is characterized by rapid onset and progression. Therefore, we need to conduct nationwide survey to assess the risk of D+R-transplantation.


Stem cells balance their self-renewal and differentiation potential by integrating environmental signals with the transcriptional regulatory network. The maintenance of cell identity and/or cell lineage commitment relies on the interplay of multiple factors including signaling pathways, transcription factors, and the epigenetic machinery. These regulatory modules are strongly interconnected and they influence the pattern of gene expression of stem cells, thus guiding their cellular fate. Embryonic stem cells (ESCs) represent an invaluable tool to study this interplay, being able to indefinitely self-renew and to differentiate towards all three embryonic germ layers in response to developmental cues. In this review, we highlight those mechanisms of signaling to chromatin, which regulate chromatin modifying enzymes, histone modifications, and nucleosome occupancy. In addition, we report the molecular mechanisms through which signaling pathways affect both the epigenetic and the transcriptional state of ESCs, thereby influencing their cell identity. We propose that the dynamic nature of oscillating signaling and the different regulatory network topologies through which those signals are encoded determine specific gene expression programs, leading to the fluctuation of ESCs among multiple pluripotent states or to the establishment of the necessary conditions to exit pluripotency. In this regard, the dynamic interplay between signaling pathways, TFs, and epigenetic machinery plays a major role in integrating multiple inputs and switching a transient signaling event into a long-lasting phenotypic change.Stem cells balance their self-renewal and differentiation potential by integrating environmental signals with the transcriptional regulatory network (TRN) –4. AdultIn this review, we will discuss regulatory mechanisms through which signaling cascade can directly regulate histone modifications, nucleosome occupancy, and chromatin modifying enzymes. We will highlight how these chromatin modifications triggered by extrinsic signaling may affect the TFs binding, the epigenetic state, and the consequent gene expression program of stem cells. Finally, we would underline the critical role of these regulatory circuits to control the cell identity and how their misregulation may initiate pathological events such as tumorigenesis.The coordinated activation of signaling pathways impacts the epigenetic landscape by targeting TFs, chromatin regulators, or nucleosome occupancy or by directly modifying nucleosomes .α, Aurora B, and PIM1 [γ, thus promoting chromatin remodeling and transcription activation [β-mediated H3T6 phosphorylation switches the LSD1 demethylation activity from H3K4 towards H3K9 methyl group [Histones are subject to a large set of posttranslational modifications (PTMs) including phosphorylation, acetylation, methylation, ubiquitination, sumoylation, and citrullination, which influence the chromatin structure , 12. Theand PIM1 , 20–23. tivation . Other hyl group . Similaryl group .Taken together, these results illustrate how a kinase-mediated short-lived signal activates a cascade of events, which determines a long-standing output by inducing chromatin modifications and impacting gene expression. cis-regulatory elements such as promoters and enhancers [Nucleosome organization and higher order chromatin structures package genomic DNA, limiting its accessibility to most of the nuclear factors. Chromatin remodelers are multisubunit complexes that utilize ATP hydrolysis to mobilize nucleosomes and their positioning on the eukaryotic DNA, thereby being essential for modulating chromatin accessibility to transcription factors and RNA Polymerases , 28. In nhancers –32. Manynhancers –35. Anotnhancers . Apart fnhancers .These data illustrate how chromatin remodelers are influenced by environmental signals, which in turn modulate nucleosome occupancy, thereby affecting transcription regulation. cis-regulatory elements. In addition, Jak2 functions as histone tyrosine kinase by phosphorylating H3Y41 and perturbing HP1α binding [Signaling pathways can also impact the chromatin state by targeting chromatin modifying proteins. The activation of the Jak2/STAT5 pathway leads to Jak-dependent phosphorylation of STAT5, which causes its dimerization, nuclear translocation, and binding to binding .α kinase phosphorylates EZH2 on Thr372 in muscle satellite cells and promotes PRC2-mediated repression of Pax7 during myogenesis. Instead, the prosurvival PI3K-AKT signaling pathway targets EZH2 by inducing Ser21 phosphorylation, which causes the reduction of PRC2 affinity for histone H3. At the same time, AKT-mediated phosphorylation of P300 increases its H3K27-specific acetyltransferase activity, thus participating in switching from a methyl (repressive) towards an acetylated (active) K27 state.Another example of linking signaling pathways with chromatin modifications is represented by the Polycomb and Trithorax group of proteins which act antagonistically in maintaining a specific gene expression state , 40. TheOn the other hand, Myeloid/Lymphoid or Mixed-Lineage Leukemia (MLL) group of proteins mediates the trimethylation of histone 3 at lysine 4 (H3K4me3) and are core components of the Trithorax complexes. Multiple MLLs are targeted in response to signaling leading to their PTMs. For example, during the commitment of myoblasts into multinucleated myotubes, p38 MAPK signaling pathway leads to phosphorylation of Mef2d and its interaction with MLL2 complex. This signaling cascade promotes MLL2 targeting to muscle-specific genes leading to their H3K4 trimethylation and transcriptional activation .Overall, the reported examples clearly show that signaling cascades not only influence the activity of transcription factors but also perturb the chromatin state by driving dynamic chromatin changes that impact on the transcriptional program.β-catenin, Notch, Nodal/Activin, Hippo pathways, and the circadian clock, is involved both in the maintenance of stem cell homeostasis and in inducing cell lineage commitment. In general, their activation triggers the stabilization and the nuclear accumulation of their downstream effectors, which finally influence the expression of their target genes. The downstream effectors, which are activated in a controlled spatiotemporal manner by the external stimuli, provide the competence for a stem cell to adopt a particular cell fate by cooperating with the cell type-specific TFs. This concept is particularly relevant in pluripotent embryonic stem cells (ESCs), in which the same signaling pathways play a key role in the maintenance of self-renewal capacity but are also involved in lineage differentiation. This divergent stem cell responsiveness depends on the fact that signaling pathways target both TRNs and chromatin landscapes. Besides that, the integration of multiple extrinsic signals determines different transcriptional program, thus influencing the cellular response.Beside the examples described so far, developmental signaling pathways are also interconnected with the TRN and influence the chromatin state of stem cells . The dev in vivo and in vitro. Perturbation of Oct4 transcript level abrogates formation of the ICM [β-catenin [β-catenin pathway and the BMP/Smad signaling cascades, which are required to promote mESCs pluripotency, once activated in primed stem cells trigger mesoendoderm lineage commitment. In hESCs, nuclear β-catenin cooperates with SMAD2/3, leading to the activation of differentiation genes, thus inducing exit from pluripotency [β stimulation induces hESCs and EpiSC to differentiate towards mesoderm [Both mouse and human ESCs (mESCs and hESCs) are isolated from the transient pluripotent cells of the inner cell mass (ICM) , 45. The the ICM  and prom the ICM . Oct4 he the ICM , 52. Alt the ICM –55. Thes the ICM . The abi the ICM , 58–60.  the ICM , 61, 62.-catenin , 64  on pluripotency genes [α to Nanog promoter, thereby inducing its transcriptional repression. These findings underline the critical role of the direct Jak2 signaling to the chromatin in sustaining self-renewal of both embryonic and hematopoietic stem cells and how its deregulation may cause tumorigenesis [In mESCs, a LIF-independent role for Jak signaling has been demonstrated and consists in the phosphorylation of histone H3 on tyrosine 41 (H3Y41). This event leads to a reduction in the binding of heterochromatin protein 1cy genes . In hemacy genes , 71. Intigenesis . In the igenesis . Finallyigenesis . Contrarigenesis .Other mechanisms of signaling to chromatin involve the modulation of the targeting of chromatin complexes. ERK pathway regulates PRC2 deposition at developmental genes, by phosphorylating the RNA polymerase II at serine 5 and establishing poised domains . The chrSingle-cell studies on mESCs showed substantial gene expression heterogeneity with subpopulations of ESCs, which express variegated levels of pluripotency-associated factors , 84–87. The TRNs are characterized by recurring regulatory circuits, named network motifs, which define a particular pattern of interconnections, leading to a certain transcriptional outcome . Among t−/− and Dicer−/− ESCs) resulted in a more homogenous expression of pluripotency factors [−/− ESCs manifest features similar to the so-called 2i ESCs, which mirror the “naïve” or “ground state” of preimplantation epiblast cells [β-catenin [Recently, it has been shown that impairment of miRNAs production in ESCs  and naïve (2i + LIF maintained) mESCs [β should stabilize the endogenous β-catenin, thus suggesting that other regulatory circuits may modulate the dynamics of Wnt/β-catenin pathway.In pluripotent stem cells (PSCs), the ERK pathway triggers opposite cellular response: in naïve ESCs, it induces cell lineage commitment while it sustains self-renewal of primed EpiSCs Figures . This stibits it . Mechaniibits it , 113. Itd) mESCs . These rMore broadly, the observed transcriptional dynamics of pluripotency-associated TFs may reflect the integration of input at the chromatin level including histone modification, chromatin accessibility, the topology of the transcriptional regulatory networks, and activity of autocrine signaling pathways. Despite their importance, the effects of these fluctuating signaling pathways at the transcriptional and chromatin level have not been investigated so far. For example, there are no data regarding the dynamic response of the downstream effectors of the fluctuating signaling pathways in stem cells, nor on the impact on histone modifications at the target genes. Although understanding how cells decode the different dynamical patterns at the molecular level is currently a challenging goal, it is mandatory to better define these regulatory mechanisms in order to clarify their contribution to the maintenance of stem cell identity and pluripotency.The importance of the integration between signaling dynamics, TRN, and the epigenetic state is well exemplified during cell lineage choice of pluripotent Neural Progenitor Cells (NPCs) in developing nervous system. In the developing telencephalon, the neuroepithelial cells, which represent the earliest NPCs, proceed towards the formation of Radial Glial (RG) cells by the oscillating Notch signaling –116. AsyThe lineage-commitment factors Ascl1, Hes1, and Olig2 play opposite function in sustaining proliferation and cell differentiation of NPCs –123. ThiOver the recent past years, the massive utilization of systems biology techniques and functional genomics increased dramatically our knowledge on the regulatory networks, which control both the maintenance of cell identity and the lineage commitment. Nonetheless, a better understanding of how cells integrate multiple environmental signals and transduce them onto chromatin, in order to modulate gene expression, is still needed. in vitro multiple pluripotent states, hanging on different regulatory networks. Oscillating, spatiotemporal restricted signaling pathways represent a first causative layer for this heterogeneity (In this review, we provide multiple evidences, demonstrating how different pluripotent stem cells rely on specific extrinsic cues, which converge on transcriptional and epigenetic networks, thereby determining their cell fate Figures  and 2. Pogeneity . Althougogeneity . Importaogeneity , which togeneity . Since sMajor interest arises from the observations that the same mechanisms may take place during the onset of cell transformation. Cancer stem cells share unique biological features with embryonic and adult stem cells, such as the ability to self-renew, to indefinitely proliferate, and to give rise to aberrant multiple cell progenies. Therefore, the signaling pathways that are important to define stem cell identity have also been demonstrated to play an important role in tumor formation and maintenance. In addition, ESCs specific signatures related to epigenetic features and TFs activity have been found to be common to multiple tumors –130, sug


The Human T cell lymphotropic virus type 1 (HTLV-1)-associated infective dermatitis (IDH), is a chronic relapsing dermatitis which usually presents in children older than 2 years. A total of 300 cases have been reported worldwide . Neither IDH, nor its complications have been reported from the rest of Africa. We aimed to examine the clinical and aetiological characteristics of IDH in a cohort of South African children.Attendees at the dermatology clinic at King Edward VIII Hospital, Durban underwent clinical examination. After obtaining consent those suspected of IDH had specimens taken for blood counts, immunoglobulins, serum protein electrophoresis, viral studies (including genotyping), skin swabs and stool examinations.3. Viral genotyping of all tested samples were positive for the Cosmopolitan, Subtype A (HTLV-1a).Nineteen of 60 suspected cases recruited over 3 years met the diagnostic criteria for IDH. The male-to-female ratio was 1:2; mean age 8 years (range 0.7 to 15). Dermatitis mostly affected the scalp (78.9%) and axilla (73.7%); fewer children had nasal crusting (47.4%). Mean Ig A, IgG and IgM were raised, at 3.52 g/l, 22.6 g/l and 1.38 g/l, respectively. The median CD4 cell count was 1958 cells/mmIDH is a distinct entity which also affects South Africans. Our patients were older at presentation and the majority did not present with nasal crusting as has been described in other countries. The human T-cell lymphotropic virus type 1 (HTLV-1)-associated infective dermatitis (IDH) was first reported in Carribean children; the incidence and pathogenesis are unknown. Somewhat reminescent of seborhoeic dermatitis, the clinical presentation of IDH is that of severe exudative dermatitis with crusting of the scalp, neck, axillae, groin, external ear, and retro-auricular areas; watery nasal discharge, and/or crusting of the anterior nares, from about 2 years of age. We identified fewer than 30 publications which report less than 300 cases of IDH worldwide; reviewed in -7. We aiFollowing ethical approval by the Biomedical Research Ethics Committee, University of KwaZulu Natal, we enrolled sixty individuals with suspected IDH, screened using the established criteria, Table pol respectively. To sub-type HTLV-1 a fragment of LTR was amplified by nested PCR using primers 12P1/SK111 and 12P5/1P1/2P3 and sequenced (n = 6) as previously described [Blood counts, measurement of immunoglobulin levels, serum protein electrophoresis, viral studies, skin swabs for bacterial culture and examination of stool samples for parasites were done. All enrolled participants (n = 60) had HTLV-1 and HIV-1 serological testing done. HIV-1 serology was performed using the Vironostika HIV-1 IMPVD Microelisa system  with all positives samples confirmed by a second HIV ELISA test. Serology for HTLV-1 was performed on plasma using an enzyme-immune assay (EIA) that detects anti-HTLV-1 and anti-HTLV-2 antibodies . HTLV-1 Western blots were not available so HTLV-1 infection was confirmed by HTLV PCR. DNA was extracted from peripheral blood mononuclear cells (PBMC) of seropositive patients using the QIAamp Blood kit . Detection of a 318-bp product or a 161-bp product confirmed the presence of HTLV-1 or HTLV-2 escribed . HTLV-1 Staphylococcus aureus in 55.6% and for Streptococcal sp in 33.3%. The streptococcal species were a combination of: ß-haemolytic streptococcus (BHS) groups A, B, C and G, together with Streptococcus pyogenes. The most common of these was BHS group G. The blood count data revealed anaemia in 20% of the participants. The lymphocyte and platelet counts were within normal ranges, but the ESR was elevated at 40 mm/hr  in all participants. The immunoglobulin levels that could be measured in our laboratory were raised. The median CD4 count was elevated, with the CD4 count percentage of 56%  while the CD8 count fell within normal limits, and therefore CD4:CD8 ratio was elevated. The median HTLV-1 proviral load was 10.5% (range 1.8-29.8%). All HTLV-1 isolates genotyped were of the Cosmopolitan, Subtype A (HTLV-1a). Of the nine family members tested, five were mothers of the enrolled IDH patients. Four of the 5 mothers had clinical lesions of IDH and were HTLV-1 seropositive. One mother had no features for IDH but HTLV-1 serology was not performed. Initially patients received short courses of antibiotics but this was associated with relapses on stopping treatment. Control was maintained when penicillin-containing oral antibiotics were given simultaneously with mild-to-moderate topical steroids continuously for three to six months.Over a 3-year period, in an outpatient setting where over 3000 patients with varying skin conditions are seen, 60 participants, were enrolled. Thirty-three patients were HTLV-1 seropositive, nine of these were co-infected with HIV-1. In addition to the positive clinical findings, 19 patients underwent HTLV DNA PCR and all were confirmed HTLV-1 and therefore fulfilled the study criteria for IDH. Only HTLV-1 infected IDH confirmed subjects are included in the analysis (n = 19) and all HIV-1/HTLV-1 co-infected patients have been excluded from this analysis. The summary of the results is captured in Table HTLV-1 is endemic in KZN with a reported seroprevalence of 2.6% in the Ngwelezana  and of 3Cosmopolitan subtype A (HTLV-1 a), consistent with published data from KZN [Our cases had less severe IDH presentation than in the those from Senegal where lesions were more infective and crusted whereas the morphology of lesions in our study was more inflammatory than infective, Figures  and 2. Cfrom KZN . We had from KZN ,17. The Except for the absence of anemia in our study population in contrast to the previous reports, other laboratory parameters were in keeping with what has been documented in the previous descriptions.The data show that cases of IDH had a significantly high HTLV-1 proviral load (mean levels of 10.5%). It has been suggested that IDH act as a cofactor for the development of HTLV-1- associated diseases, such as HAM/TSP and ATLL but the mechanisms are still unclear. HTLV-1 proviral load levels is regarded as important predictor of ATLL and HAM/TSP. It would be interesting to follow all the identified cases with IDH over time and monitor the clinical evolution of the disease to determine if they develop any of the HTLV-1 associated conditions .IDH is a distinct entity that affects the African population of KwaZulu Natal, South Africa. It is predominantly a disease of childhood. The clinical features were in keeping with other series worldwide. Some differences in presentation are noted, particularly later age of onset and less nasal discharge. Interplay between genetic factors and environmental (including socioeconomy and climate) factors, may determine the age of presentation, severity of lesions and therefore explain the variation of IDH presentations between countries. The currect IDH criteria, needs revision. Crusting of the anterior nares should not be mandatory. The revised criteria should take into consideration the heterogeity this disease might have between different geographical locations.The authors declare that they have no competing interests.CH and AM conceived, designed, realized the study and analysed data. GPT and NG perfomed some of the experiments included in the results of this study. AIB perfomed nuerological examinations. CH, AM and NPK wrote the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-5945/13/11/prepub


Type I interferons represent the first line of defense against pathogens. This family of cytokines activates the expression of antiviral proteins, such as the protein kinase R (PKR), an inhibitor of viral mRNA translation, and the double-stranded RNA adenosine deaminase ADAR1. ADAR1 has the ability to convert adenosine (A) into guanosine (G), thereby introducing mutations in the viral genome during its replication. A to G editing was previously reported in cells expressing HTLV-2 or STLV-3 viruses but not investigating in HTLV-1 expressing cells . Consequently we investigated whether ADAR1 expression was associated or not with an antiviral effect in the course of HTLV-1 and HTLV-2 infections. We first show that ADAR1 expression is increased in ATL patient peripheral blood mononuclear cells, in HTLV-1 and HTLV-2 transformed cell lines as well as in activated primary peripheral blood lymphocytes. Strikingly, in cells transfected with HTLV-1 and HTLV-2 molecular clones, ADAR1 over-expression enhances viral replication and viral egress through PKR functional inhibition, as demonstrated by western-blot analyses, luciferase assays, ELISA and infection experiments. We also demonstrate that this effect is independent of ADAR catalytic activity. In addition, ADAR1 expression enhances the susceptibility of a non-infected T cell line to HTLV-1 and HTLV-2 infection. Altogether, our results demonstrate that an interferon-induced protein exerts a proviral role in the context of HTLV infection by enhancing cells susceptibility to infection and increasing viral replication.