Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.The endo/lysosomal system in cells provides membranous platforms to assemble specific signaling complexes and to terminate signal transduction, thus, is essential for physiological signaling. Endocytic organelles can significantly extend signaling of activated cell surface receptors, and may additionally provide distinct locations for the generation of specific signaling outputs. Failures of regulation at different levels of endocytosis, recycling, degradation as well as aberrations in specific endo/lysosomal signaling pathways, such as mTORC1, might lead to different diseases including cancer. Therefore, a better understanding of spatio‐temporal compartmentalization of sub‐cellular signaling might provide an opportunity to interfere with aberrant signal transduction in pathological processes by novel combinatorial therapeutic approaches. J. Cell. Biochem. 117: 836–843, 2016. © 2015 The Authors.  ARL8BADP‐ribosylation factor‐Like 8BBMPRbone morphogenetic protein receptorEGFRepidermal growth factor receptorGEFguanine nucleotide exchange factorGLP‐1Rglucagon‐like peptide‐1 receptorGPCRsG protein—coupled receptorsHGFhepatocyte growth factorIQGAP1IQ motif containing GTPase activating protein 1LAMTORlate endosomal/lysosomal adaptor and MAPK and mTOR activatorMethepatocyte growth factor receptorMVBmultivesicular bodiesmTORmechanistic target of rapamycin kinasemTORC1mTOR kinase complex 1PDGFRplatelet‐derived growth factor receptorPAC1Rpituitary adenylate cyclase 1 receptorPTHRparathyroid hormone receptor typeRTKsreceptor tyrosine kinasesSTAT3signal transducer and activator of transcription 3TGFtransforming growth factorTSCtuberous sclerosis complexTSHRthyroid‐stimulating hormone receptorV2Rvasopressin type 2 receptoren route to lysosomes. By a third mechanism, signaling components are exclusively localized to endosomes/lysosomes but not directly connected to receptor endocytosis.Cells respond to different environmental stimuli by initiation of signal transduction at the plasma membrane. The binding of ligands to specific receptors at the cell surface, such as Receptor Tyrosine Kinases (RTKs) or G protein—coupled receptors (GPCRs), triggers the activation of many downstream signaling pathways, which are important for normal tissue homeostasis. For integration and regulation of complex signaling networks the endo/lysosomal system is essential [Miaczynska et al., The general principle of spatio‐temporal regulation of receptor‐mediated signal transduction is that the cellular response to different types of ligands is maximal and transient at the plasma membrane and sustained in endosomes. In this review we try to look at cellular signaling from an organelle‐centric point of view and present selected examples of deregulation of endosomal signaling in disease progression such as cancer. Hence, we discuss also perspectives in development of combinatorial therapies based on our current knowledge on compartmentalized signal transduction.Endocytosis of cell surface receptors is one of the control mechanisms of signal transduction initiated by extracellular stimuli. For a long time it has been assumed that receptors signal from the plasma membrane until they are internalized, endocytosed and sent to lysosomes for degradation. Stimulation of cells in vitro with appropriate ligands as, for instance, EGF reaches maximal EGFR activation levels within the first minutes of stimulation [Stasyk et al., Notably, different ligands can have diverse effects on recycling of the same receptor. Again the EGFR is a well‐established example for this. The receptor is directed for lysosomal degradation if induced by EGF but is recycled upon transforming growth factor (TGF)‐α stimulation. TGF‐α leads to sustained EGFR signaling and, therefore, is more mitogenic than EGF [Waterman et al., Signaling from endosomes has been demonstrated for a number of cell surface receptors from different receptor families such as the RTKs , serine/threonine kinase receptors (transforming growth factor‐β (TGF‐β), the bone morphogenetic protein (BMP) and the activin receptors), GPCRs, toll‐like receptors, as well as interferon, Wnt and Notch receptors. Endosomal signaling of these receptors is well characterized and was extensively reviewed elsewhere [Hupalowska and Miaczynska, There are several important characteristics of endosomal signaling that is spatially and temporally separated from signaling at the plasma membrane: 1) signaling complexes on organelles are different from those at the plasma membrane; 2) receptor endocytosis and active signaling from organelles are required for the full activation of their downstream effectors; and 3) there could be specific targets or distinct pathways stimulated by the same receptor depending on which endosome it is localized. These properties of endosomal signaling were very recently shown for Hepatocyte growth factor (HGF) receptor (Met) signaling from two different populations of endosomes in human breast cancer models [Menard et al., Scaffolds and adaptor proteins often organize specific endosomal signaling complexes. For example, such complexes containing activated endocytosed EGFR are organized on early endosomes by APPL, which recruits AKT and its substrate GSK3 [Miaczynska et al., Another well‐established example is activated transforming growth factor‐β receptor (TGFβR), which upon internalization interacts with Smad anchor for receptor activation (SARA) on early endosomes. In case of bone morphogenetic protein receptor (BMPR) another specific scaffold, endofin, organizes signaling from endosomes. These and many other scaffolds that make possible activation of their downstream targets at specific subcellular locations, were previously comprehensively summarized in [Palfy et al., An interesting mechanism of early endosomal EGFR signaling, sustained by the non‐receptor tyrosine kinase PTK2B/PYK2, was reported recently [Verma et al., Recent data suggest that GPCRs mediate the production of cyclic AMP not only from the plasma membrane but also from endosomal membranes. Originally, two studies have shown that two GPCRs, thyroid‐stimulating hormone receptor (TSHR) [Calebiro et al., All examples discussed above, of specific endosomal signaling complexes, contain internalized and endocytosed receptors of different families and scaffolds specific for different receptors and (sub‐)populations of endosomes. In addition to this well established and commonly accepted type of endosomal signaling there is an emerging field of organelle signaling independent from endocytosis, if the latter one is defined as active transportation of cell surface components into the cell. In most eukaryotic cells the plasma membrane makes up only for a minor part of all cellular membranes. The endo/lysosomal system, as a component of cellular endomembranes, provides membranous platforms to assemble specific signaling complexes at specific subcellular locations. Here we will briefly discuss endolysosomal signaling of the mechanistic  target of rapamycin (mTOR) kinase.Endolysosomes are the fusion product between late endosomes and lysosomes and the place where most of the hydrolysis of endocytosed cargo takes place [Huotari and Helenius, It has become evident already some time ago that endosomes play a role in cell migration [Sadowski et al., Another interesting example of endosomes as vehicles of signals over long distance is transportation of activated STAT3 from the plasma membrane into the nucleus upon activation with EGF and PDGF [Bild et al., Above‐mentioned examples from different fields compose quite a significant body of evidence for spatial and temporal consequences of endosomal signaling. It is particularly exciting task for the future to demonstrate different physiological responses produced by activated receptors at the cell membrane and at endosomes. Better understanding of how to specifically interfere with the two waves of signaling might be beneficial for therapeutic purposes to treat different diseases, including cancer.Deregulations of signal transduction at different subcellular levels, caused by defective trafficking of growth factor receptors, can drive tumorigenesis Table . MutatioOne well‐established oncogenic deregulation of cell surface receptors is their escape from ligand‐stimulated ubiquitination by the Cbl family of adaptor proteins and ligases that function as negative regulators of many signaling pathways. Such mechanism was described for EGFRvIII, which is the most common mutation of EGFR gene. This tumor‐specific deletion in the extracellular domain of EGFR contributes to the formation of many epithelial malignancies. In some brain tumors this mutation of EGFR occurs at extremely high frequency. Interestingly, although EGFRvIII possesses only about 10% of the intrinsic activity of wild‐type EGFR its net signaling rate is enhanced by the delay of its endocytosis. The low level of EGFRvIII signaling causes hypo‐phosphorylation of the receptor that prevents engaging of Cbl and, therefore reduced polyubiquitination and degradation of the receptor, and, therefore, increased recycling. This may also play a critical role in the development of resistance to tyrosine kinase inhibitor treatments as observed in the clinics [Han et al., Aberrant activity of GPCRs is frequently associated with tumorigenesis. Recent deep sequencing studies show that GPCRs are mutated in nearly 20% of human cancers [O'Hayre et al., Upregulation of lysosomal mTORC1 signaling is one of the most common hallmarks of human cancers [Zoncu et al., Since patients often do not respond to conventional anti‐cancer drugs or develop resistance, alternative therapies are required. According to the current sequential protocol, the patient is administered one by one to different drugs, for example the tyrosine kinase inhibitors, until the optimal response is reached. Unfortunately the risk of multidrug resistance in this approach is increased. An alternative therapeutic approach by administering combinations of different drugs at the same time could provide a cure for patients that did otherwise not respond to conventional therapies. Appropriate combinations should potentially enhance response and decrease development of drug resistance. Combinations of well‐established drugs, which inhibit receptors on the cell surface with specific inhibitors of endosomal signaling as well as more general inhibitors of endocytosis, which act on different levels of the process such as internalization, recycling or degradation see Fig.  might opMet signaling from late endosomes was recently shown to be crucial for sustained Rac1 activation in breast cancer cells, responsible for cell spreading and metastasis formation [Menard et al., Another combinatorial approach could be the simultaneous inhibition of several receptors or the targeting of common signaling pathways, in the context of crosstalk between different receptors. As discussed above, EGFinduced early endosomal PYK2 signaling, which leads to PYK2‐STAT3‐Met positive feedback [Verma et al., All those inhibitors of endocytosis are still pretty far from preclinical development since they might target too common cellular mechanisms with potentially high toxicity at the organism level. However, the mode of action with targeting endocytosis would suggest that chemical libraries could be developed in high‐contents screening setups that act like molecular chaperons by selectively only disturbing endocytosis and recycling of certain mutated oncogenic receptors.H. pylori eradication in the clinics and have also been used recently in tumor models. Proton pump inhibitor pretreatment inhibited V‐type H+‐ATPase activity and increased both extracellular pH and pH of lysosomal organelles. Hence, in human/mouse xenograft models oral pretreatment with proton pump inhibitors is able to sensitize human solid tumors to anticancer drugs [De Milito and Fais, Margalef et al. have shown recently that inhibition of the endosomal V‐ATPase is a potential therapeutic strategy for the treatment of colorectal cancers with mutant BRAF, which is predictive of poor prognosis and therapeutic resistance [Margalef et al., An approach combining the mTORC1 inhibitor Everolimus with an aromatase inhibitor improved progression‐free survival in patients with hormone‐receptor‐positive advanced breast cancer [Baselga et al., We anticipate that selective targeting of the endosomal signaling, especially in combination with conventional anti‐cancer therapy and targeted small molecules, may offer more effective treatments. The here suggested link between endosomal signaling and cancer increases expectations from this type of combinatorial therapeutics in future.


The HERV-K(HML-2) family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the general biological properties of this ancient virus family. The expression of consensus sequences and sequences of elements with reverted post-insertional mutations has therefore been very instrumental in overcoming this limitation. We investigated the particle morphology of a recently reconstituted HERV-K113 element termed oriHERV-K113 using thin-section electron microscopy (EM) and could demonstrate that strong overexpression by substitution of the 5'LTR for a CMV promoter and partial codon optimization altered the virus assembly type and morphology. This included a conversion from the regular C-type to an A-type morphology with a mass of cytoplasmic immature cores tethered to the cell membrane and the membranes of vesicles. Overexpression permitted the release and maturation of virions but reduced the envelope content. A weaker boost of virus expression by Staufen-1 was not sufficient to induce these morphological alterations. HERVs comprise over 8% of the human genome and there are more than 500,000 individual elements scattered throughout our chromosomes. These elements are derived from former exogenous retroviruses that infected germ cells or their progenitor cells and have since been vertically transferred from generation to generation . HERVs ai.e., the most recently integrated) retroviral elements belong to the HERV-K(HML-2) family. As they use a lysine (K) tRNA to prime their reverse transcription and are related to the mouse mammary tumor virus (MMTV), these proviruses, together with nine other families, are termed HERV-K(HML). This relationship is reflected in the acronym HML, which stands for “human MMTV-like”. Elements of the HERV-K(HML-2) family are found exclusively in Old World primates including humans. Indeed, several members are specific to humans, suggesting integration after the separation of the human and chimpanzee lineages about five million years ago.The youngest  described previously [®, Agilent Technologies, Inc.). pcDNA-oricoHERV-K113 (“oricoHERV-K113”) consists of the oriHERV-K113 genome with regions codon-optimized for enhanced protein expression in mammalian cells. The complete proviral oricoHERV-K113 genome sequence was synthesized  and cloned behind the CMV promoter in pcDNA3.1 . Construction of the human Staufen-1 expression vector has been described previously [The generation of pBSK-oriHERV-K113 (“oriHERV-K113”) has been described elsewhere  and cloneviously .l-glutamine (2 mM), penicillin (50 U/mL) and streptomycin (50 μg/mL).HEK 293T and HeLa cells were cultured in complete Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 6 or 5 × 105 HEK 293T or HeLa cells were seeded into 100 mm dishes or 6-well plates , respectively and subsequently transfected with the plasmids using calcium phosphate or the Qiagen Polyfect Kit  according to the manufacturer’s instructions. At two to four days post transfection, samples of culture media were harvested, clarified at 3345 × g for 8 min and filtered (0.45 μm) to remove cell debris. For samples derived from 100 mm dishes, supernatants were then centrifuged at 175,000 × g for 3 h at 4 °C through a 20% sucrose cushion in a Beckman SW32Ti rotor. Virus pellets were resuspended in 60 μL of 0.05 M Hepes buffer, pH 7.2 for Western blot analysis. To prepare cell lysates, transfected cells were resuspended in cell lysis buffer  containing complete protease inhibitor cocktail .For electron microscopy analysis and Western blots, 2.4 × 104 , block-stained with uranyl acetate , dehydrated stepwise in graded alcohol, immersed in propylenoxide and embedded in Epon  with polymerisation at 60 °C for 48 h. Ultrathin sections (60–80 nm) were cut using an ultramicrotome  and stained with 2% uranyl acetate and lead citrate. For post-embedding immunostaining, cells were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde. They were dehydrated by graded alcohol and embedded in Lowicryl (HM20) according to the progressive lowering temperature technique. Thin sections applied to uncoated 300hexa Mesh nickel grids were incubated on droplets of blocking buffer (5% milk powder in PBS) for 30 min, and subsequently in blocking buffer with rat anti-CA, rat anti-p15  or goat anti-GFP  and a 5 nm gold-conjugated anti-rat/goat antibody  for 1 h. After fixation with 0.1% glutaraldehyde in 0.05% Hepes buffer for 15min, samples were counterstained with 2% uranyl acetate and 0.2% lead citrate. Transmission electron microscopy was performed with an EM 902 (Zeiss) operating at 80 kV and the images were digitized using a slow-scan charge-coupled-device camera .The procedure for preparing samples for electron microscopy has been published elsewhere . In shorViral lysates or pelleted virus particles were mixed with 5× sample buffer and heated for 10 min at 98 °C before being subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred onto a PVDF membrane  by semi-dry electrophoresis . After blocking in PBS with 5% skim milk powder and 0.1% Tween, the membranes were probed with specific rat anti-CA sera as described previously  and a seWe previously described 25 non-synonymous post-insertional mutations in the HERV-K113 provirus originally characterised by Turner and co-workers ,15,16. RcoHERV-K113”  encoded proteins Rec and Gag with the human Staufen-1 protein and noticed a 20-fold increase in oriHERV-K113 production in cells overexpressing Staufen-1 .i.e., with a 4:1 ratio of oriHERV-K113 to Staufen-1 DNA.In order to boost oriHERV-K113 expression by Staufen-1 as strongly as possible and to evaluate a potential switch in morphology, we first optimized the transfection ratio of oriHERV-K113 and Staufen-1 expression plasmids. As shown in The morphology of viral particles produced by transfected HEK 293T cells under optimized Staufen-1 mediated enhancement conditions were also analyzed by thin-section EM. Despite Staufen-1 mediated enhancement resulting in significantly more viral particles, there was no evidence for a C- to A-type switch B. As expCodon-optimization of retroviral sequences is commonly used to enhance protein expression in eukaryotic cells ,21. For Upon transfection into eukaryotic cells, a dramatic increase in the levels of cytoplasmic Gag was detected compared to those in cells transfected with oriHERV-K113. Interestingly, in oricoHERV-K113 transfectants, thin-section electron microscopy revealed the presence of a multitude of cytoplasmic particles with apparently completely assembled Gag shells. Such intracellular particles with immature cores had never been seen in cells expressing oriHERV-K113, where only budding stages or released immature and mature virions were seen. This is consistent with earlier observations in teratocarcinoma cell lines ,22 and wIn contrast to the situation with vesicles, the release of oricoHERV-K113 virions into the supernatant is very efficient. Thin-section EM of virus pellets from supernatants of oricoHERV-K113 expressing cells reveals high numbers of particles. It is, however, currently unclear to which extent type A-particles are able to leave the cell or whether the pelleted particles were released by C-type budding occurring in parallel to the formation of A-type particles. Whatever the dominant budding phenotype, the particles released have mature cores. Efficient egress and maturation of oricoHERV-K113 has been confirmed by Western blotting and the measurement of RT activity in the supernatants (data not shown). These findings indicate that the partial codon-optimization of oriHERV-K113 does not prevent the switching of reading frames, the production of Gag-Pro and Gag-Pro-Pol precursor proteins or the release of active protease and reverse transcriptase. These results are consistent with those we obtained earlier by expressing the Gag-Pro-Pol region of oricoHERV-K113 alone . The useDespite a significant boost, it was not possible to drive oriHERV-K113 into the production of type A-particles by overexpression of Staufen-1, a binding partner of the Rec protein . Howeverin vivo and whether they have a relevant biological role is also a matter of speculation. It is conceivable in this context that such cores might be potentially transferred from cell-to-cell or might play a role in retrotransposition events contributing to HERV-K(HML-2) proliferation following endogenization. Whatever the biological role and the underlying molecular mechanism of the morphological switch reported in this paper is, it is certain that EM will continue to play a pivotal role in future studies aimed at shedding light on this issues.Whether this type of particle is formed


HTLV-1 expression is maintained at low levels in vivo through unknown mechanisms. Interferon-α (IFN-α) has been used in combination with zidovudin (AZT) to treat adult T-cell leukemia/lymphoma (ATL) patients with favorable effects, although the mechanism is also unclear. We previously reported that HTLV-1 gene expression could be markedly suppressed by stromal cells through a type-I IFN response in IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients. Here, we found that treatment with IFN-α alone produced suppressive effects on HTLV-1 gene expression in ILTs and also on the spontaneous HTLV-1 induction in short-term cultured primary ATL cells. Following IFN-α treatment, the levels of intracellular Tax protein decreased earlier than those of HTLV-1 mRNA in ILTs. An RNA-dependent protein kinase (PKR) inhibitor reversed IFN-α-mediated suppression of Tax in ILTs, suggesting the involvement of a post-transcriptional mechanism mediated by PKR in the suppression. IFN-α also induced cell cycle arrest at the G0/G1 phase in ILTs. We further found that AZT combined with IFN-α induced cell apoptosis in ILTs, associated with phosphorylation of p53 and enhanced expression of genes responsive to p53, whereas AZT alone did not affect cell viability or viral expression. Our results suggest that ILTs and ATL cells retain susceptibility to type-I IFNs, which suppress HTLV-1 gene expression primarily at a post-transcriptional level and potentially allow for signaling through the p53 pathway in combination with AZT. These findings would partly explain how HTLV-1 gene expression is regulated in vivo, and how AZT/IFN-α produces therapeutic effects in ATL.


In this study, we found that HTLV-1 bZIP factor (HBZ) is responsible for the suppressed c-fos transcription in ATL cells. The results of reporter assay implied the roles of LXXLL motif of HBZ on the suppression of c-fos. HBZ has been reported to suppress AP-1 and NFAT signaling pathways through the direct interaction with c-Jun and NFATc2, respectively. We found c-Fos overexpression impairs the suppressive effects of HBZ on AP-1 and NFAT, suggesting that HBZ overcomes the inhibitory effects of c-Fos by suppressing its transcription. HBZ is known to bind to c-Jun instead of c-Fos. Suppressed transcription of c-fos facilitates HBZ to interact with c-Jun, and enhances suppressive effect of HBZ on AP-1 pathway. AP-1 and NFAT signaling pathways are activated by T-cell receptor (TCR) stimulation, leading to T cell activation. We found that TCR stimulation induces the c-fos up-regulation and the expression of the activation marker CD69 on CD4+ T cells of wild type mice, but not of HBZ transgenic mice, indicating that the activation of the signaling pathways initiated from TCR are suppressed by HBZ. We hypothesize that c-fos suppression by HBZ may contribute to impaired activation of T cells and pathogenesis of HTLV-1 infection.c-Fos forms AP-1 heterodimer with JUN family proteins such as c-Jun and functions as a transcription factor to regulate diverse biological processes including T cell activation. Although an HTLV-1-encoded oncoprotein Tax induces the expression of c-


The second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) transduce many neuromodulatory signals from hormones and neurotransmitters into specific functional outputs. Their production, degradation and signaling are spatiotemporally regulated to achieve high specificity in signal transduction. The development of genetically encodable fluorescent biosensors has provided researchers with useful tools to study these versatile second messengers and their downstream effectors with unparalleled spatial and temporal resolution in cultured cells and living animals. In this review, we introduce the general design of these fluorescent biosensors and describe several of them in more detail. Then we discuss a few examples of using cyclic nucleotide fluorescent biosensors to study regulation of neuronal function and finish with a discussion of advances in the field. Although there has been significant progress made in understanding how the specific signaling of cyclic nucleotide second messengers is achieved, the mechanistic details in complex cell types like neurons are only just beginning to surface. Current and future fluorescent protein reporters will be essential to elucidate the role of cyclic nucleotide signaling dynamics in the functions of individual neurons and their networks. The cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are ubiquitous second messengers present in most cell types. Within the brain, cyclic nucleotides transduce neuromodulatory signals into functional outputs for individual neurons leading to changes in neural networks themselves or their function. The importance of cyclic nucleotide signaling pathways is well appreciated in the field of clinical neuroscience and psychiatry, with many drugs targeting the G-protein coupled receptors to modulate neuronal activity Lim, . The effThe idea of compartmentalized signaling is an essential component to the cyclic nucleotide signaling model. To elicit their diverse functional effects in a highly specific manner, cAMP and cGMP signaling is thought to be spatially compartmentalized . The reporting unit can be made up of two fluorescent proteins flanking the sensing unit as is the typical arrangement for fluorescence resonance energy transfer (FRET)-based biosensors. In FRET, energy is transferred non-radiatively from an excited donor molecule to an acceptor molecule. For a fixed FRET donor-acceptor pair, the efficiency of FRET is dependent on the distance and orientation of the two fluorophores. In a FRET-based biosensor, cyclic nucleotide binding induces conformational changes of the CNBD sensing unit, which acts as a molecular switch to change the physical separation or orientation between the fluorescent protein pair resulting in a change in FRET Figure . In inteFluorescein-labeled PKA Catalytic subunit and Rhodamine-labeled Regulatory subunit) was based on chemically labeled regulatory and catalytic subunits of the PKA holoenzyme . A newer version was generated using CFP fused to the RII regulatory domain and YFP fused to the catalytic domain  was flanked by an N-terminal CFP and a C-terminal YFP  instead. Both biosensors generated a decrease in the yellow to cyan emission ratio upon binding cAMP, indicating a decrease in FRET. In an attempt to generate a more sensitive cAMP probe called Epac2-camps300, Norris et al. introduced a K405E mutation into Epac2-camps that decreased the EC50 from ~0.9 μM to ~0.3 μM  have been developed in parallel. First in the series, ICUE1 contained full-length Epac1 sandwiched between ECFP and the YFP variant Citrine   and used mTurquoise2 as the FRET donor and a single circularly-permuted Citrine as the FRET acceptor -YFP using Epac150 ~6 μM) and provides another tool to monitor cAMP in cells with larger basal or fluctuating concentrations.In some cases, the concentration of cAMP in a cell can saturate a highly sensitive reporter. To accommodate large fluctuations in signaling, Nikolaev et al. used the hyperpolarization-sensitive cyclic nucleotide gated channel 2 (HCN2) to generate HCN2-camps and image large changes in cAMP concentration in cardiomyocytes  can act as an acceptor for both CFP and YFP. ICUE2 was redesigned as a YFP-RFP FRET sensor (YR-ICUE) and co-imaged with a CFP-RFP-AKAR (CR-AKAR) to analyze the temporal relationship between cAMP and PKA signaling following receptor activation in HEK-293 cells , the first 77 amino acids of PKG1α (PKG1αΔ1–77) were truncated. PKG1αΔ1–77 was flanked by a reporting unit comprised of ECFP and EYFP at the N- and C- termini, respectively. Cygnet-2, the catalytically inactive version of cygnet-1, was generated by introducing mutation T516A to PKG1αΔ1–77. The pH-insensitive EYFP variant Citrine was used to replace YFP in cygnet-2 to generate cygnet-2.1. cGMP binding to the cygnet reporters induces a decrease in FRET. Cygnet-1 and cygnet-2 have an affinity of 1.5 μM and 1.9 μM for cGMP, respectively, although endogenous PKG1α has a cGMP affinity of ~100 nM. It is conceivable that fusion of the fluorescent proteins or the Δ1–77 N-terminal truncation and catalytic site mutations affected their cGMP binding affinity. Around the same time, Sato et al. generated a similar PKG1α based probe called CGY-Del1 which had an N-terminal truncation of the first 47 amino acids  makes it potentially suitable for detecting low cGMP concentrations but may also be confounded by variations in experimental conditions when determining the EC50. The probe was used to develop a method for simultaneous measurements of signaling activities by using a single excitation light that excites both T-sapphire and CFP, four channel detection and linear unmixing.Nikolaev et al. developed smaller probes utilizing a single CNBD from PKG1α79–336-YFP, they elongated the N- and C- termini of PKGIα79–336 and screened for constructs based on cGMP affinity and the FRET response amplitude. Using this approach, they generated a series of cGMP biosensors cGi-500 (EC50 = 500 nM), cGi-3000 (EC50 = 3.0 μM), and cGi-6000 (EC50 = 6.0 μM).Russwurm and colleagues generated a series of FRET based cGMP sensors in order to achieve faster kinetics and provide an array of probes with a range of affinities  and a dark fluorescent protein acceptor , a secreted molecule that guides axon/dendrites growth and neuronal migration  mediated gene transcription Kandel, . Modulatin vivo brain imaging will include studies of cyclic nucleotide signaling dynamics in live animals.From shedding light on the kinetics of neuromodulatory signaling events to uncovering new aspects of signal transduction, fluorescent biosensors have been an invaluable tool for neuroscience research. Over the past decade, FRET-based biosensors have been used to dissect the intricacies of cyclic nucleotide signaling in the nervous system with unprecedented detail. Second messenger signaling is now being studied in more physiologically relevant samples like brain slices and transgenic animals with FRET-based biosensors  by fusing a bacteriophytochrome BphG1 to a bacterial adenylate cyclase CyaB1. IlaC was expressed in Caenorhabditis elegans cholinergic neurons and was shown to induce locomotion with exposure to red light (650 nm) as measured by the number of body bends per minute . This valuable addition to the toolbox allows for active user-controlled manipulation of cAMP and cGMP levels in specific locations within the cell. These blue-light (450 nm wavelength) sensitive nucleotidyl cyclases respond to light via conformational changes in the light-oxygen-voltage (LOV) or sensors of blue light using FAD (BLUF) photoreceptor protein domains. EuPAC from 2+. How do multiple signaling pathways interact to form a coherent output instead of chaos? Despite the advances in monitoring and manipulating cyclic nucleotides, we still do not have a clear understanding and additional tools such as mathematic modeling are needed to understand the intricacies of second messenger crosstalk and compartmentation (Saucerman et al., A fundamental question in cyclic nucleotide research is the maintenance of specific signaling in the midst of multiple inputs and outputs. For example, within neurons there is substantial cross-regulation of cAMP, cGMP and CaWith the multitude of neuromodulatory inputs affecting cyclic nucleotide levels, signaling information needs to be encoded specifically to produce specific cellular responses. Encoding can occur on spatial, temporal, and digital levels using molecular distribution, enzyme kinetics, and signal thresholding to elicit various physiological outputs from cAMP and cGMP signal transduction (Rich et al., 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.


We treated wild-type mice with NO to activate soluble guanylate cyclase (sGC), a key enzyme of cGMP signaling. Compared to untreated mice, NO-treated mice had higher red blood cell counts and total hemoglobin but reduced leukocyte counts, demonstrating that when activated, NO-cGMP signaling exerts hematopoietic effects on multiple types of blood cells in vivo. We next generated mice which overexpressed rat sGC in erythroid and myeloid cells. The forced expression of sGCs activated cGMP signaling in both lineage cells. Compared with non-transgenic littermates, sGC mice exhibited hematologic changes similar to those of NO-treated mice. Consistently, a membrane-permeable cGMP enhanced the differentiation of hematopoietic progenitors toward erythroid-lineage cells but inhibited them toward myeloid-lineage cells by controlling multiple lineage-specific transcription factors. Human γ-globin gene expression was induced at low but appreciable levels in sGC mice carrying the human β-globin locus. Together, these results demonstrate that NO-cGMP signaling is capable of stimulating erythropoiesis in both in vitro and vivo settings by controlling the expression of multiple lineage-specific transcription factors, suggesting that cGMP signaling upregulates erythropoiesis at the level of gene transcription. The NO-cGMP signaling axis may constitute a novel target to stimulate erythropoiesis in vivo.Much attention has been directed to the physiological effects of nitric oxide (NO)-cGMP signaling, but virtually nothing is known about its hematologic effects. We reported for the first time that cGMP signaling induces human γ-globin gene expression. Aiming at developing novel therapeutics for anemia, we examined here the hematologic effects of NO-cGMP signaling  Nitric oxide (NO) plays a critical role in the regulation of vascular tone ,2 and haThis study was undertaken to test our hypothesis that if HU exerts hematological effects on blood cells at least in part through NO-cGMP signaling, then mice in whose blood cells NO-cGMP signaling was activated should demonstrate hematological changes similar to those of SCD patients treated with HU. We activated cGMP signaling in mouse blood cells by (1) treating mouse blood cells with low-dose NO, an sGC activator , and (2)c•T diff analyzer, Beckman Coulter, Fullerton, CA, USA). All animal studies were approved by the Institutional Animal Care and Use Committees of the Georgia Regents University and the University of California San Francisco.Wild-type mice  were housed in a viral-free environment in standard approved chambers (5 mice/cage). Mice were allowed to breathe 8 parts per million (ppm) NO gas for 8 weeks as described . To deteCell surface antigens of mouse BM mononuclear cells (BMMNC) obtained from mice treated or not treated with 8 ppm NO were analyzed by flow cytometry  as described . Fluores+ cells were provided by NHLBI PEGT Hematopoietic Cell Processing Core . Human erythroid and myeloid cells were isolated from semi-solid cultures in which CD34+ cells (4 × 105 cells) were plated in the presence of 8-bromo-cGMP as described , li, li38], A recent study reported that oral administration of sodium nitrate suppresses erythropoiesis in rats , which aOur results have clinically relevant implications for anemic disorders such as SCD. First, hematologic changes such as increased total hemoglobin and HbF expression as well as reduced leukocyte count occur among SCD patients treated with HU . BecauseOur finding that NO-cGMP signaling plays a role in modulating erythropoiesis as well as γ-globin gene expression may account for some puzzling clinical and experimental observations. First, the negative correlation found between HbF and zinc protoporphyrin levels in SCD  may be eSeveral lines of clinical evidence suggest that the mechanisms regulating leukocyte count may be involved in HU-mediated HbF induction in the context of SCD. First, robust HbF induction by HU is associated with a pronounced reduction in leukocyte and reticulocyte counts . We receThe HU-mediated reduction in leukocyte count may occur via one or more pathways : the genIn conclusion, this study shows for the first time that activation of NO-cGMP signaling in blood cells induces erythropoiesis, which is associated with increases in RBC counts and total hemoglobin levels, but suppresses myelopoiesis by reducing leukocyte counts. Altered expression of lineage-specific transcription factors caused by cGMP signaling may contribute to these hematopoietic changes in NO-treated mice and sGC transgenic mice. Our finding that peripheral blood leukocyte counts can be reduced by activating cGMP signaling suggests that leukocyte count could serve as a good surrogate marker for evaluating the sGC-cGMP pathway in SCD patients who receive a phosphodiesterase 5 inhibitor or other sGC-cGMP pathway activator . AdminisS1 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


A recent survey of 79 countries showed that fertility knowledge was lower in Japan than in any other developed country. Given the fertility decline in Japan and the importance of fertility knowledge, we conducted an online survey to examine fertility knowledge and the related factors for effective public education.We studied people aged 18-59 years old, n = 4,328 , and also people who had been trying to conceive for at least six months, 18-50 years old, n = 618 (the “Triers” group). Fertility knowledge was assessed using the Japanese version of the 13-item Cardiff Fertility Knowledge Scale (CFKS-J). All participants provided socio-demographic and fertility information. Participants also completed a 14-item health literacy scale and an 11-item health numeracy scale. We asked participants who were aware of age-related decline in fertility when and where they first acquired that knowledge.The average percentages of CFKS-J items answered correctly were 53.1% in the Triers group and 44.4% in the General group (p < 0.001). Multivariate linear regression models showed in the Triers group greater fertility knowledge was associated with greater health literacy and prior medical consultation regarding their fertility. In the General group greater fertility knowledge was associated with being female, younger, university educated, currently trying to conceive, non-smoking, having higher household income, having higher health literacy and having higher health numeracy. Of those who were aware of the age-related decline in fertility, around 3% first learned the fact “at school”, and around 65% first learned it “through mass media” or “via the Internet”. More than 30% of the respondents first learned it “less than 5 years before” the survey.Although fertility knowledge had improved since a previous study, possibly due to recent media coverage of age-related infertility, it was still low. Educational interventions, both in schools and in the community, may be needed to increase fertility knowledge in the general population because most people obtain fertility knowledge from mass media, which has been shown to often present distorted and inaccurate fertility information. The trend to delay childbearing and the decline in fertility are serious concerns regarding reproductive-health in developed countries , 2. In aIn Japan, the total fertility rate has been decreasing 1.41 in 2012) and the parental age at first birth has been increasing  . These t12 and thThe aims of the present study were to examine fertility knowledge in the general population and to investigate the related factors, which will provide important background for effective public education. We investigated knowledge in two groups: a representative sample of the general population  and a sample consisting of people who were currently trying to conceive (‘Triers’ group). The Triers group was studied to replicate the IFDMS research in Japan. The General group was recruited to represent people who would be the target group in future public-education campaigns. We included future, current, and post-reproductive generations in the target population because older people generally transmit information, whether it is correct or incorrect, to younger generations. In line with the previous studies examining factors associated with fertility knowledge , 22–24, Participants were recruited via online social research panels (SRPs). Inclusion criteria for the General group were that respondents were men and women aged between 18 and 59 years old . The inclusion criteria for the Triers group were that respondents were men and women aged between 18 and 50, currently married or living with their partner, currently trying to conceive (Triers) for at least 6 months, and not pregnant (n = 618). The criteria for the Triers group were the same as those used in the IFDMS .An online market research company , which has a nationwide research panel (SRP) of more than 1 million registrants, sent recruitment emails to people who were randomly selected from its registrants   (CFKS-J)The CFKS consists of 13 items that measure knowledge about fertility facts, risks, and myths. All items were rated on a three point scale of ‘true’, ‘false’, or ‘do not know’. A correct answer was assigned one point and an incorrect or ‘do not know’ answer was assigned zero points. Scores are reported as percents of the highest possible score. Internal consistency coefficient alpha of the original CFKS was 0.79, and an exploratory factor analysis showed that all items loaded >0.30 on one general factor that accounted for 30% of between-item variance .The first item of the CFKS-J asks whether the statement “A woman is less fertile after the age of 36 years” is true or false. We asked participants who answered that item correctly when and where  they first acquired that knowledge. We asked them when and where they first learned about prevention of pregnancy and STIs as well.Gender, age in years, and categorized annual household income of the participants were provided by the online market research company. Annual household income was categorized into 4 groups: low, < 4 million Japanese Yen (JPY); moderate, 4 – 7 million JPY; high, ≥ 8 million JPY; and “unknown”. At the time of the study 1 Japanese Yen = 0.0076 Euro. Participants also indicated their academic background  and marital status .Participants reported whether they had given birth or fathered a child. Participants in the General group indicated whether they were currently trying to conceive (yes/no) and whether they hoped to have children in the future (yes/no). Participants in the Triers group stated the number of months they had been trying to conceive, and whether they had sought a medical consultation or treatment regarding fertility (yes/no).We used the 14-item health literacy scale (HLS-14) for measuring health literacy . The HLSHealth numeracy was measured using the Japanese version of the Lipkus scale (the Lipkus-J) , 26. TheAdditionally, we asked participants about tobacco smoking, as an indicator of health behaviour (smoke more than a few times a week: yes/no).Internal consistency coefficient alpha, bi-serial item correlations, and tetrachoric correlations among items were used to evaluate the CFKS-J. A factor analysis was conducted using the matrix of tetrachoric correlations. Factors with loadings ≥ 0.3 were retained.t-test for independent samples was used to compare results from the present study with results from the IFDMS [Descriptive statistics were used to describe performance on CFKS-J and the named sources of information. T-tests and analysis of variance (ANOVA) were used to compare the total scores on the CFKS-J between socio-demographic categories and between fertility-status categories. Pearson’s correlation was used to examine the association between age in years and CFKS-J score. Spearman’s correlation was used to explore the associations among scores on the HLS-14, the Lipkus-J, and the CFKS-J. The he IFDMS .Multivariate linear regression using the robust estimator of variance was used to assess the relation between the total score on the CFKS-J and the variables described above. All the analyses were performed using STATA12-SE .Ethical review and approval was carried out at the institutional ethics committee of the National Center for Child Health and Development (Heisei 25 nendo-10), Daito Bunka University , and the University of Tokyo (10346). Collection of online data complied with the “Code of Conduct of Marketing Research” and “Requirements for Compliance Program on Personal Information Protection (JIS Q 15001)” of the Japan Marketing Research Association.Table rtetrachoric = 0.89), because the eigenvalues of the factors changed to 3.71, 0.88, 0.35 when item 3 was dropped from the analysis. The biserial item correlations with the underlying factor ranged from 0.36 to 0.55 and the factor loadings for the first factor ranged from 0.30 to 0.67. The results in the Triers group were similar , 0.72 (Triers group). A single to 3-factor solution was computed to examine the dimensionality of the 13 items. In the General group, the eigenvalues of the factors were 4.14, 1.33, and 0.56, which accounted for 72%, 23%, and 10% of the variance, respectively. The high eigenvalue of the second factor was attributed to a high tetrachoric correlation between item 3  and item 4  (The average percent correct scores on the CFKS-J were 53.1 SD = 23.4) in the Triers group and 44.4 (SD = 23.1) in the General group . Figure 3.4 in th2 for the model = 0.095)  Table .Table 32 for the model = 0.13) (Table As shown in Table 3) Table .A total of 3,334 participants  answered the question about the age-related decline in fertility correctly  . To dateOur findings are consistent with those of previous studies that investigated the relationship between fertility knowledge and background factors. The IFDMS reported that greater fertility knowledge was associated with female gender, university education, paid employment, very high Human Development Index, and prior medical consultation for infertility . Other pInterest in childbearing was strongly associated with fertility knowledge: In the General group, people who were currently trying to conceive had scores that were 6.5 percentage points higher than those who were not currently trying to conceive, and in the Triers group those with a history of medical consultation or treatment for fertility had scores that were 11.6 percentage points higher than the others. However, to prevent infertility due to a lack of fertility knowledge, people without current interest in childbearing need to know the facts while they have time to protect their own health and to make choices about childbearing. We should also be aware of the fertility knowledge of older people. If they have misperceptions or biases regarding fertility, those could be transmitted to younger people. The older people in this study were relatively young when they had first become parents, but their levels of fertility knowledge were low.Although referring to female age and fertility was once taboo in Japan, the issue has recently been taken up by the media since the national television channel NHK aired a documentary program discussing age-related infertility in 2012 . SubsequSome countries are conducting educational interventions to spread accurate information , 9, 12. There are several limitations to be noted. First, this was a cross-sectional survey and thus we cannot infer causality. Second, the use of SRPs could have caused selection bias. Acquiring health-related information via the Internet is associated with higher education levels and higher household incomes , 39 and Although fertility knowledge among people in Japan had improved, possibly due to the recent governmental movements and the media coverage of age-related infertility, it was still lower than the average in developed countries. Given the fact that fertility decisions should be based on correct and complete information, we note that multiple factors are associated with fertility knowledge, especially education and the personal relevance of childbearing. Importantly, many people obtained knowledge through mass media or via the Internet. To prevent infertility and broaden people’s choices about childbearing, educational interventions may be needed both in schools and in the community.


In social Hymenoptera , various chemical compounds present on the cuticle have been shown to act as fertility signals. In addition, specific queen-characteristic hydrocarbons have been implicated as sterility-inducing queen signals in ants, wasps and bumblebees. In Corbiculate bees, however, the chemical nature of queen-characteristic and fertility-linked compounds appears to be more diverse than in ants and wasps. Moreover, it remains unknown how queen signals evolved across this group and how they might have been co-opted from fertility signals in solitary ancestors.Centris analis, and the highly eusocial stingless bee Scaptotrigona depilis. Our results demonstrate the presence of fertility-linked compounds belonging to 12 different chemical classes. In addition, we find that some classes of compounds  were already present as fertility-linked signals in the solitary ancestors of Corbiculate bees, while others appear to be specific to certain species.Here, we perform a phylogenetic analysis of fertility-linked compounds across 16 species of solitary and eusocial bee species, comprising both literature data as well as new primary data from a key solitary outgroup species, the oil-collecting bee Overall, our results suggest that queen signals in Corbiculate bees are likely derived from ancestral fertility-linked compounds present in solitary bees that lacked reproductive castes. These original fertility-linked cues or signals could have been produced either as a by-product of ovarian activation or could have served other communicative purposes, such as in mate recognition or the regulation of egg-laying.The online version of this article (doi:10.1186/s12862-015-0509-8) contains supplementary material, which is available to authorized users. In order to be able to effectively organize themselves, insect societies require reliable information and communication systems . In sociFriesella schrottkyi, it has recently been shown that specific linear and methyl branched alkanes were characteristic for the queen and that non-polar cuticular queen extracts inhibited worker reproduction [The important role of cuticular hydrocarbons as queen pheromones in ants, wasps and the bumblebee contrasts with what is known from the honeybee, where most existing studies have indicated that mandibular gland compounds, including the keto acid 9-oxo-decenoic acid (9-ODA), act as the principal sterility-inducing queen signals . Neverthoduction .Lasius ants [In order to gain more insight into the origin and evolution of social insect queen pheromones, several studies have started to apply formal phylogenetic methods, and these show that there is remarkably high evolutionary conservation of queen signals , 16, 33.ius ants . These rius ants , 24. Furius ants . Yet desius ants , how queius ants  did not Centris analis, which is the sister taxon of all Corbiculate bees [Scaptotrigona depilis. Including Centris as an outgroup in our analysis allowed us to study character states present just before the transition from a solitary to a social lifestyle. In this way, we are able to obtain important insight with respect to what ancestral signals queen pheromones could have evolved from, and provide important clues as to what could be bioactive queen pheromones across different groups of bees.In the present study, we test the hypothesis that queen signals evolved from fertility-linked cues present in solitary ancestors  and perfate bees , 36, as Centris analis, which is the closest extant relative of the Corbiculate bees [Scaptotrigona depilis. The oil collecting bee Centris analis was sampled using trap-nests for solitary bees [n = 5) were sampled when they visited their nest cavities whereas virgin females (n = 8) were collected on the day they emerged from trap-nests that were placed in the laboratory. Egg-laying queens (n = 5) and workers (n = 15) of the stingless bee Scaptotrigona depilis were sampled from the experimental meliponary from the University of São Paulo. Cuticular chemical data for the two bee species were obtained through gas chromatography and mass spectrometry analysis. Detailed sampling methods and details on the chemical analyses are provided in supplementary material.Chemical data on the identity of fertility signals across different species of solitary and social bees were collected from the literature through a systematic review of published studies, in which we compiled chemical data from fourteen different species . According to the country’s law, research with bees is exempted from ethics committee approval when performed in research institutions.Centris and the Corbiculate bees -9-octadecenoic acid, hexadecanoic and octadecenoic acid methyl ester or decyl decanoate [In general, our phylogenetic analysis adds credence to the idea that also in bees, compounds present on the cuticle, including both saturated and unsaturated hydrocarbons as well as other classes of compounds, such as ester and fatty acids, may act as conserved and ancient queen pheromones , 24. In elopment , 21 but elopment , even thelopment . Similar as well . Finally as well –56 in pr as well , 29 and  as well , 31, als as well , such asecanoate , 58, areApis honeybees, the major queen pheromone component 9-ODA [Lasioglossum malachurum, a synthetic blend of the fertility-linked saturated lactones applied onto dummy bees was able to induce male inspection and pouncing behaviour  [Colletes cunicularius, several fertility-linked long-chain alkenes have also been shown to act as contact sex pheromones [Megachile rotundata, alkene fraction extracts from virgin females, containing also the compounds linked to fertility, were shown to attract males in bioassays [Lasioglossum zephyrum, L. malachurum and Osmia rufa [If it is correct that some groups of social insect queen pheromones were co-opted from fertility signals that were already present in solitary ancestors, the question arises what function these fertility-linked compounds had in the solitary species. Previously, three main possibilities have been suggested, namely that they originally served as sex pheromones, that they were a by-product of ovarian activation, or that they were involved in regulating egg-laying, reviewed in . Althougnt 9-ODA , 59, 60 nt 9-ODA –63, and nt 9-ODA  as well nt 9-ODA . Moreovey acids) . In addieromones , 68 and ioassays . In all ioassays , 71, altioassays  or transioassays , or the mia rufa , 74. Eitmia rufa . Possiblmia rufa , 76.In the future, several of the hypotheses supported by our current dataset may be further validated by looking at the degree of evolutionary conservation in the expression levels of the actual underlying enzymes involved in the biosynthesis of different fertility-linked compounds , 40, 43,In a recent study, it was suggested that queen pheromones in different groups of social insects had been co-opted from ancient fertility signals present in common solitary ancestors . Based o


This study evaluated the prevalence of skin diseases among HTLV-1 infected and the relation between HTLV-1 proviral load, and CD4+ and CD8+ T cells count among, regardless of clinical status, with or without associated skin disorders.193 HTLV-1-infected subjects were studied. Patients were submitted to a complete dermatological examination, lymphocyte proliferation assay (LPA), assay for HTLV-1 proviral load, CD4+ and CD8+ T cells count. A total of 147 patients had an abnormal skin condition; 116 (79%) of these patients also had skin disorder associated with HTLV-1 infection (SD-HTLV-1) , followed by seborrheic dermatitis (27%). Three of them had the association of adult onset IDH and HAM/TSP. The patients with SD-HTLV-1 were older (51 vs. 47 years), had a higher prevalence of myelopathy/tropical spastic paraparesis (HAM/TSP) (p=0.015), higher HTLV-1 proviral load (p=0.009) and had an increased 3-day basal LPA compared with patients without SD-HTLV-1 (p=0.008). T CD4+ and CD8+ cells counts show no significance. When HAM/TSP patients were excluded from the analysis, the HTLV-1 proviral load showed a significant difference (p=0.021), while LPA showed no difference. There was a high prevalence of skin disorders (76%) among HTLV-1-infected individuals, regardless of clinical status Initial HTLV-1 proviral load and age was higher in SD-HTLV-1 individuals, but the LPA showed an increase only in SD-HTLV-1 subjects with HAM/TSP.


HTLV-I and HTLV-II are endogenous and have shown high prevalence rates among blood donors (1.4%), pregnant women (1.1%), Guarani amerindians (5.8%), HIV-HTLV-I/HTLV-II coinfected patients (16.3%) and spastic paraparetic individuals (21.6%) among indivividuals living in the city of Porto Alegre, southern Brazil. Tropical spastic paraparesis/HTLV-I associated myelopathy is a immune-mediated inflammatory disease in mid-thoracic spinal cord determining a myelopathy, with spastic-ataxic gait, intestinal and micturitional disorders. No medication was proved to be a disease modifying drug, just physical therapy and symptomatic management. No antiretroviral drug could benefit the patients. Fampridine, a selective neuronal potassium channel blocker, was licenced in USA and other countries, since April 2010, for the treament of gait disorders in Multiple Sclerosis. Some clinical trials have shown some benefit in patients with other kind of spinal cord diseases. Since mid-2012 seven patients monoinfected by HTLV-I and HAM/TSP are regularly using. Six of them improved in ataxia, gait and strengh in the lower limbs. Micturictional dysfunction improved in two. This off-label observational experience offer a preliminary data for a broad discussion.


In order to clarify the HTLV introduction and dispersion in the south of Bahia, we analyzed 29 samples from HTLV-1 seropositive women. Before the blood collection, all of them answered a standardized questionnaire. The DNA was extracted by QIAgen Kit. It was performed a nested-PCR to the LTR region of the HTLV. The sequences were submitted to the LASP HTLV-1 Automated Subtyping Tool. The phylogenetic analyses were generated using the neighbor-joining e máxima-verossimilhança methods. The evolutionary model of Tamura-Nei with gamma distribution was selected as the best adaptive model by software Modeltest 3.7. The likelihood ratio was used to calculate the statistical support for the branches in trees. Bayesian tree were constructed to verify the posterior probability statistical parameter. To evaluate genetic ancestry of the population, it was analyzed mtDNA ancestry markers and β-globina haplotypes. From the total, 21 samples were successfully amplified and sequenced and they were classified as HTLV-1 aA (bootstrap support of 100%). The phylogeny analysis showed multiple introductions of the virus in Brazil. In addition, for the first time, Mozambique sequences were grouped with Brazilian and South Africa sequences, supporting the hypothesis that Africans infected with the virus have been brought from the southern regions of Africa. In relation to the genetic ancestry, the African ethnicity was predominantly found by mtDNA markers. In addition, the type benin was detected by β-globin analyses. These data corroborate to clarify the introduction and dispersion of this virus in Brazil, especially in the Bahia.


The increase might be due to the production of siderophore-producing capacity of the tested actinobacteria, which was confirmed in our previous studies by q-RT PCR on siderophore genes expressing up to 1.4- to 25-fold increased relative transcription levels. The chickpea seeds were subjected to processing to increase the mineral availability during consumption. The processed seeds were found to meet the recommended daily intake of FDA by 24–28 % for Fe, 25–28 % for Zn, 28–35 % for Cu, 12–14 % for Ca, 160–167 % for Mn and 34–37 % for Mg. It is suggested that the microbial inoculum can serve as a complementary sustainable tool for the existing biofortification strategies and substantially reduce the chemical fertilizer inputs.The present study was evaluated to test the potential of plant growth-promoting actinobacteria in increasing seed mineral density of chickpea under field conditions. Among the 19 isolates of actinobacteria tested, significant ( Among the 19 tested actinobacteria, ten strains were deposited in the National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau, Uttar Pradesh, India, and the accession numbers  were obtained.All the tested PGP actinobacteria were previously identified by 16S rDNA sequencing as genus −1 soil of available nitrogen, 8.6 mg kg−1 soil of available phosphorous and 298 mg kg−1 soil of available potassium. The field was kept fallow except for this post-rainy season crop. The fields were prepared into broad beds and furrows with beds 1.2-m wide flanked by 0.3-m furrows in both seasons. Surface application and incorporation of 18 kg N ha−1 and 20 kg P ha−1 as di-ammonium phosphate were performed. The experiment was laid out in a randomized complete block design (RCBD) with three replicates and plot sizes of 4 m × 3 ridges (rows).The field trial conditions used were as follows. This experiment was carried out during 2013–2014 post-rainy cropping season at ICRISAT, Patancheru , in peninsular India. Soils at the experimental site are classified as Vertisols  having 52 % clay, 21 % silt and 26 % sand, with an alkaline pH of 7.7–8.5 and an organic carbon content of 0.4–0.6 %. The soil depth of the field used was ≥1.2 m, and this soil retained approximately 200 mm of plant-available water in a 120-cm (maximum rooting depth by chickpea) soil profile. The mineral content of the top 15 cm of rhizosphere soil includes 24.7 mg kg−1) were treated with PGP actinobacteria (containing 108 CFU mL−1) for 50 min and sown by hand during November 2013 in rows 30-cm apart at a depth of 4–5 cm to achieve an estimated plant stand density of at least 26 plants m−2. The PGP actinobacteria  was applied once every 15 days on the soil close to the plant until the flowering stage. Control plots were maintained without the application of PGP actinobacteria. The plots were irrigated on the 21st and 49th day after sowing. The crop was kept weed free by manual weeding. No serious insect pest or phytopathogen attacks were observed during the cropping period. The crop was harvested manually during February 2014.The 19 test strains of PGP actinobacteria were cultured individually on a starch casein broth at 28 °C for 5 days. Seeds of chickpea variety ICCV 2  and on par with the uninoculated control. The highest increase of 38 and 36 % was noticed in the CAI-21 and MMA-32 treatments with Fe values of 6.8 and 6.7 mg/100 g seeds, respectively. In case of Zn, all the isolates (except CAI-13 and CAI-26) treated chickpea were found to increase its content significantly (p < 0.05) than the uninoculated control plots. The increases were found to be 13–30 % with the highest by CAI-21 treatment. Similarly the Ca content was also increased (14–26 %) by all the PGP treatments except for KAI-27, KAI-32 and KAI-90 treatments. The highest Ca content of 158.3 mg/100 g seed was noticed on CAI-13 treatment. The other minerals such as Cu, Mn and Mg were not influenced by most of the PGP treatments as observed on Fe, Zn and Ca contents. Still, significant increases were documented on 50 % PGP actinomycete treatments, i.e., about nine isolates on Cu and ten isolates on Mn and Mg. The highest increases were shown by CAI-93 for Cu (54 %), CAI-68 for Mn (35 %) and CAI-17 for Mg (24 %). The best strain was observed to be CAI-21, as it showed the highest increase of both Fe and Zn than other isolates and this was followed by CAI-68 and MMA-32.The estimated mineral values for Fe, Zn, Ca, Cu, Mn and Mg of PGP actinobacteria-treated and field-harvested chickpea seeds are shown in Table p < 0.05) increased mineral density for all the tested minerals on par with the control treatment, but showed different expression profile of siderophore genes, i.e., CAI-68 and CAI-93 showed 10.8- and 1.4-fold increased transcription levels, whereas CAI-78 showed insignificant expression. The genus Streptomyces is well known for its siderophores, including its own characteristic types such as hydroxamate siderophores: desferrioxamine and coelichelin  plant biometric parameters. Further studies of Rana et al. (Providencia sp., having P, Zn, Fe solubilization capacity increased the Fe content by 105 %.The present study is an initial observation showing the potential of previously reported plant growth-promoting actinobacteria on increasing seed mineral density of chickpea. The increases observed might be due to their mineral-mobilizing capacity through the production of siderophores, which we previously observed in our in vitro biochemical studies and also at gene-level studies. Among the 19 isolates tested by q-RT PCR analysis on the expression profile of siderophore genes, 11 showed 1.4- to 25-fold increase of relative transcription levels (Gopalakrishnan et al. a et al.  on wheatThe other possible reason for increased mineral contents could be modification of the root system observed in our previous studies. The isolates CAI-13, CAI-85, CAI-93, CAI-140, CAI-155 and KAI-180 were found to increase root length, weight and volume on rice (Gopalakrishnan et al. In the context of processing effects on seed mineral content, the processed chickpea seeds showed reduced mineral content irrespective of the PGP actinobacteria treatment and also in control plot seeds, which lies in the range of 5–30 % for Fe, 1–11 % for Zn, 2–20 % for Ca, 4–46 % for Cu, 9–18 % for Mn and 2–17 % for Mg Table . The recThis study suggests the use of PGP inoculums which could lead to the development of a complementary sustainable tool for the influence of existing biofortification strategies. Improved soil health and crop growth induction are the other benefits. Use of these in-kind microbial inoculums can reduce the fertilizer inputs and also decrease the dependence on few selected crop varieties. Further studies on determining the actual microbial mechanisms behind the mineral transfer from soil to seed are required.


Ursolic acid (UA), a natural pentacyclic triterpenoid, exerts anti-tumor effects in various cancer types including hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying this remain largely unknown.Secrete-Pair™ Dual Luminescence Assay Kit. In vivo nude mice xenograft model and bioluminescent imaging system were used to confirm the findings in vitro.Cell viability and cell cycle were examined by MTT and Flow cytometry assays. Western blot analysis was performed to measure the phosphorylation and protein expression of p38 MAPK, insulin-like growth factor (IGF) binding protein 1 (IGFBP1) and forkhead box O3A (FOXO3a). Quantitative real-time PCR (qRT-PCR) was used to examine the mRNA levels of IGFBP1 gene. Small interfering RNAs (siRNAs) method was used to knockdown IGFBP1 gene. Exogenous expressions of IGFBP1 and FOXO3a were carried out by transient transfection assays. IGFBP1 promoter activity was measured by We showed that UA stimulated phosphorylation of p38 MAPK. In addition, UA increased the protein, mRNA levels, and promoter activity of IGFBP1, which was abrogated by the specific inhibitor of p38 MAPK (SB203580). Intriguingly, we showed that UA increased the expression of FOXO3a and that overexpressed FOXO3a enhanced phosphorylation of p38 MAPK, all of which were not observed in cells silencing of endogenous IGFBP1 gene. Moreover, exogenous expressed IGFBP1 strengthened UA-induced phosphorylation of p38 MAPK and FOXO3a protein expression, and more importantly, restored the effect of UA-inhibited growth in cells silencing of endogenous IGFBP1 gene. Consistent with these, UA suppressed tumor growth and increased phosphorylation of p38 MAPK, protein expressions of IGFBP1 and FOXO3a in vivo.Collectively, our results show that UA inhibits growth of HCC cells through p38 MAPK-mediated induction of IGFBP1 and FOXO3a expression. The interactions between IGFBP1 and FOXO3a, and feedback regulatory loop of p38 MAPK by IGFBP1 and FOXO3a resulting in reciprocal pathways, contribute to the overall effects of UA. This in vitro and in vivo study corroborates a potential novel mechanism by which UA controls HCC growth and implies that the rational targeting IGFBP1 and FOXO3a can be potential for the therapeutic strategy against HCC. Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths globally characterized by high malignancy, aggressive progression, clinical difficulty and limited therapeutic options, resulting in poor prognosis and remaining a significant clinical challenge –4. UsualUrsolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid obtained from TCM herbs and edible plants, exhibits potential anticancer effects through multiple mechanisms in various human cancers including HCC –14. Our Insulin-like growth factor (IGF) binding protein 1 (IGFBP1), a pivotal protein of the IGF system, has been shown to be implicated in many cellular functions including proliferation, development, apoptosis, DNA damage repair, and tumor growth through IGF-dependent and -independent mechanisms –18. EarlHuman forkhead box class O (FOXO) transcription factors implicated in a wide variety of cellular activities, such as differentiation, cell cycle, metabolism, stress resistance, mitogenic signaling, and tumor suppression . Among fIn this study, we further explore the potential mechanism by which UA controls growth of HCC cells. Our results in vitro and in vivo indicated that UA inhibited growth of HCC cells through p38 mitogen-activated protein kinase (MAPK)-mediated induction of IGFBP1 and FOXO3a expressions.2. The culture medium consisted of RPMI1640 medium  supplemented with 10 % (v/v) heat-inactivated fetal bovine serum , 100 μg/ml streptomycin and 100 U/mL penicillin. When cells reached 70 % confluence, they were digested with 0.25 % trypsin for the following experiments.Monoclonal antibodies specific for total p38 MAPK and the phosphor-form (Thr180/Tyr182) were purchased from Cell Signaling Technology Inc. . The IGFBP1 and FOXO3a antibodies were obtained from Epitomics . SB203580 was purchased from Merck Millipore . MTT powder was purchased from Sigma Aldrich . Ursolic acid was purchased from Chengdu Must Bio-technology Company . Lipofectamine 3000 reagent was purchased from Invitrogen . The drugs were freshly diluted to the final concentration in culture medium before experiment. Human HCC cell lines HepG2, Bel-7402, QGY-7703, HMCC97L and HMCC97H were obtained from the Cell Line Bank at the Laboratory Animal Center of Sun Yat-sen University  and the Chinese Academy of Sciences Cell Bank of Type Culture Collection . The cells were cultured at 37 °C in a humidified atmosphere containing 5 % CO3 cells/well) were treated with increasing concentrations of UA for up to 72 h. After incubation, 10 μL MTT solution (5 g/L) was added to each well and HCC cells were incubated at 37 °C for an additional 4 h. Supernatant was removed, then 100 μL solvent dimethyl sulfoxide (DMSO) was added to each well and oscillated for 5 min. Absorbance at 570 nm was determined by ELISA reader . Cell viability (% of control) was calculated as (absorbance of test sample/absorbance of control) × 100 %.Cell viability was measured using the 3--2, 5-diphenyltetrazolium bromide (MTT) assay and described previously , 30. BriThis procedure was reported previously . In brieThe detailed procedure was reported previously . For the5 cells/well in 6-well dishes and grown to 60 % confluence. For each well, 2 μg of the desired N1-GFP or FOXO3a-GFP plasmid DNA, kindly provided by Frank M. J. Jacobs  and was reported previously [pCMV6) purchased from OriGene Technologies, Inc.  at a final concentration of 2 μg/mL were transfected into the cells using the lipofectamine 3000 reagent according to the manufacturer’s instructions for up to 24 h, followed by treating with UA for an additional 24 h. In separated experiment, cell were transfected with pEZX-PG04-IGFBP1 promoter construct linked Gaussia luciferase (GLuc) gene and secreted alkaline phosphatase (SEAP) internal control obtained from GeneCopoeia, Inc. . The preparation of cell extracts and measurement of luciferase activities were determined using the Secrete-Pair Dual Luminescence Assay Kit . Gaussia luciferase activity was normalized with SEAP within each sample.The detailed procedure was reported previously . In brieeviously  and the A quantitative real-time PCR (qRT-PCR) assay was used to detect IGFBP1. The primers used in this study were designed as follows: IGFBP1 forward 5′- TCACAGCAGACAGTGTGAGAC −3′; reverse 5′- CCCAGGGATCCTCTTCCCAT −3′; GAPDH forward 5′- AAGCCTGCCGGTGACTAAC −3′; reverse 5′- GCGCCCAATACGACCAAATC −3′. In brief, qRT-PCR was performed in a 20 μL mixture containing 2 μL of the cDNA preparation, 10 μL 2X SYBR Green Premix ExTaq (Takara), and 10 μM primer on an ABI 7500 Real-Time PCR System . The PCR conditions were as follows: 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. Each sample was tested in triplicate. Threshold values were determined for each sample/primer pair, the average and standard errors were calculated.The detailed procedure was reported previously , 30. Bri6 cells) in 100 μL PBS were injected subcutaneously in nude mice. Xenografts were allowed to grow for over one week when the initial measurement was made with calipers and with bioluminescence imaging (BLI) using the IVIS-200 Imaging System . The mice were randomly divided into control, low (25 g/kg), and high doses (50 g/kg) of UA treatment groups, which based on other studies [n = 12/group).In order to explore the effects and mechanisms of UA on tumor growth in vivo, a xenografted nude mouse model of HCC cells was established. Animal experiments were approved by Institutional Animal Care and Use Committee Animal Care of Guangdong Provincial Hospital of Chinese Medicine . A total of 36 female nude mice (eight-week-old) obtained from Guangdong Provincial Research Center for Laboratory Animal Medicine , were obtained and maintained at the Animal Center of Guangdong Provincial Hospital of Chinese Medicine in a specific pathogen-free environment with food and water provided. HepG2 cells carrying luciferase report gene  (1x10 studies –36. The 2 × length). The body weights of the mice were measured once a week. All mice were sacrificed on 30 days after each treatment using CO2 for euthanasia. The corresponding xenografted tumors were processed for detecting the phosphorylation of p38 MAPK, IGFBP1 and FOXO3a proteins by Western blot.For bioluminescence imaging (BLI) procedure, the mice were anesthetized by inhalation of 2 % isoflurane at the end of experiment. Each set of mice were injected subcutaneously  with 150 mg/kg D-luciferin  in approximately 200 μL. Imaging and quantification of signals (photons/sec) were controlled by the acquisition and analysis software living image . Tumor volume measurements were calculated using the formula for an oblong sphere: volume = (widthp) value of <0.05 was considered to be significant.The data are reported as means ± SD of at least three repeated experiments in triplicate measures. Differences between groups were assessed by one-way ANOVA and significance of difference between particular treatment groups was analyzed using Dunnett’s multiple comparison tests . The results were presented relative to the control. Asterisks showed in the figures indicate significant differences of experimental groups in comparison with the corresponding control condition. A probability  showed a significant growth-inhibitory effect as assessed by the Xenogen IVIS200 System Fig. . In addiChinese herbal medicines and its components have drawn a great attention for their potential impact in the treatment of many cancer types. Increasing numbers of studies demonstrated that ursolic acid, a pentacyclic triterpenoid found in medicinal herbs and fruits, inhibited the proliferation and induced the apoptosis in several types of cancers including HCC cells. We previously showed that UA inhibited growth and induced apoptosis of HepG2 HCC cells through AMPKα-mediated inhibition of Sp1; this in turn results in inhibition of DNA methyltransferase 1 . In thisUA was found to inhibit growth in multiple HCC cell lines in the current study confirming the tumor suppressing properties of this agent in HCC cells. We did not observe the time-dependent effects of cell growth inhibition by UA and the reasons remained unclear. Whether longer time treatment (>72 h) showed significant changes needs to be determined. On the other hand, the possible desensitization of prolonged exposure of UA to the cells may also be responsible for this occurrence, which required to be confirmed. In our study, compared to the untreated control cells, there was actually 19 % more G0/G1 phase cell arrest by UA treatment. We believed that this may explain the significant inhibition of cell growth by UA, while the possible UA-induced apoptosis of HCC cells may also occurred, which required to be tested further. It was possible that the inhibition of proliferation could be in part a consequence of increased cell apoptosis. In addition, we demonstrated that UA inhibited growth of HCC cells through not only AMPKα but also p38 MAPK indicating that activation of these two signaling pathways may be required for UA induced HCC growth inhibition . These pMoreover, our data supported crucial function of IGFBP1 in the current study. Increasing evidence suggested an important role of IGFBP in the development and progression of several types of cancers –18, 47. We also demonstrated the involvement of FOXO3a that mediated the anti-HCC cell growth by UA. As a potential tumor suppressor, FOXO3a has been associated with many physiological and pathological processes, including proliferation, differentiation, cell cycle arrest, apoptosis and tumorigenesis , 48, 49.Furthermore, our data also implicated in the correlation between FOXO3a and IGFBP1, implying that IGFBP1 may be upstream of FOXO3a and mediated the positive feedback regulatory loop of p38 MAPK by FOXO3a. This contributed to the UA-inhibited HCC cell growth. The association between FOXO family and IGFBP1 has been shown in other studies , 50, 51.More importantly, our in vivo data were consistent with the findings from that in vitro, confirming the effect of UA on liver cancer growth inhibition and regulation of IGFBP1, FOXO3a expression, and p38 MAPK phosphorylation. The doses used for UA in the current study were similar to other reports demonstrating the significant effects in inhibiting growth of several cancer types including HCC –36. NeveOverall, our results show that UA inhibits HCC cell proliferation through p38 MAPK-mediated induction of IGFBP1 gene expression and upregulation of FOXO3a. The inter-correlation between IGFBP1 and FOXO3a, and positive feedback regulatory loop of p38 MAPK by IGFBP1 and FOXO3a resulting in reciprocal pathways, contribute to the overall effects of UA Fig. . This in


Drosophila eyes and wings, which depends on dFoxO, a transcription factor known to be involved in cell death. In addition, dFoxO is required for ectopic and endogenous Wg signaling to regulate wing patterning. Moreover, dFoxO is necessary for activated Wg signaling-induced target genes expression. Furthermore, Arm is reciprocally required for dFoxO-induced cell death. Finally, dFoxO physically interacts with Arm both in vitro and in vivo. Thus, we have characterized a previously unknown role of dFoxO in promoting Wg signaling, and that a dFoxO-Arm complex is likely involved in their mutual functions, e.g. cell death.The Wnt/β-catenin signaling is an evolutionarily conserved pathway that regulates a wide range of physiological functions, including embryogenesis, organ maintenance, cell proliferation and cell fate decision. Dysregulation of Wnt/β-catenin signaling has been implicated in various cancers, but its role in cell death has not yet been fully elucidated. Here we show that activation of Wg signaling induces cell death in  Drosophila retina and ommatidia development910The Wnt/β-catenin signaling represents one of the most intensively studied pathways, which is tightly associated with cancers, especially colorectal cancer134367C. elegans to mammalsFoxO gene in invertebrates, four members have been identified in mammals including FoxO1, FoxO3a, FoxO4 and FoxO613141512171813The Forkhead box O (FoxO) transcription factors belong to the large forkhead family proteins, which are characterized by a winged helix DNA binding domain called ‘Forkhead box’12C. elegans2224β-catenin has been reported to interact with FoxO and enhance its transcriptional activity in mammalian cells and Drosophila melanogaster, which has reduced genome redundancy and many available genetic tools, we found that activation of Wingless  signaling induces intensive cell death in Drosophila eyes and wings, which depends on dFoxO (Drosophila FoxO homolog). In addition, dFoxo is required for Wg signaling to activate target genes expression and execute its endogenous functions in wing patterning. Furthermore, loss of armadillo  or pangolin  could also suppress dFoxO-triggered cell death, suggesting a reciprocal effect. Finally, dFoxO physically interacts with Arm, providing a molecular mechanism for the role of dFoxO in promoting Wg signaling. Thus, contradict to the previous studies that FoxO proteins inhibit Wnt signaling, our data point to a positive role of dFoxO in modulating the canonical Wg signaling in Drosophila, suggesting FoxO differentially regulates Wnt signaling in a cell context-dependent manner.Here using Drosophila eye, and found that ectopic expression of Wg, Dsh or Arm resulted in massive cell death in 3rd instar eye discs as revealed by acridine orange (AO) staining  phenotype  and head involution defective (hid) are important pro-apoptotic genes regulating cell deathrpr and hid, we ectopically expressed Arm in the wing pouch driven by sd-Gal4. Compared with the controls , a known Wg pathway target gene that mimic wg expression pattern, using a wf-LacZ reporter , which is required for bristle formationdfoxo suppressed sd > Arm induced wf and sens expression in wing discs , UAS-ArmS2 (4783), UAS-pan-IR#2 (26743) and bsk2 (5283) lines were obtained from the Bloomington stock center.The following lines have been described previously: rd instar larva stage and fixed in 4% formaldehyde for 15 minutes, rinse the tissue once in PBST buffer containing 3.3 mM K3Fe(CN)6 and 3.3 mM K4Fe(CN)6, then incubate in the above PBST buffer with 0.2% X-gal at room temperature for 24 hours, photographs were taken under light microscope.X-Gal staining was done as describedrd instar larva stage, and stained in 1 × 10−5M AO solution for 5 minutes, photographs were taken under fluorescence microscope.AO staining was done as describedrd instar larva stage, and stained for TUNEL using the Fluorescein Cell Death Kit (Boster), photographs were taken under Leica confocal microscope.TUNEL staining was done as describedrd instar larva stage in PBS, fixed in 4% formaldehyde for 20 minutes and washed in 0.3% PBST for 3 times, then label with primary antibody overnight, and secondary antibody for 2 hours. Primary antibodies used were 1:100 mouse-anti-BrdU (Sigma) and 1:400 rabbit-anti-PH3 , secondary antibodies used were 1:1000 goat anti-mouse CY3 (CST) and 1:1000 goat anti- rabbit CY3 (CST). Photographs were taken under fluorescence microscope.Discs were dissected at 3rd instar larva stage in Schneider’s medium (Sigma), incubated for 40 minutes in Schneider’s medium containing 0.2 mg/ml of BrdU, and fixed in 4% formaldehyde, then washed in PBST and hydrolyzed in 2 N HCl. After that the discs were blocked in 10% horse serum and labeled with mouse anti-BrdU and anti-mouse CY3 antibodies, photographs were taken under fluorescence microscope.BrdU incorporation was done as describedS2 cells were transfected with indicated plasmids for 48 h, immunoprecipitation and western analyses were performed as previously describedrd instar larva, for each genotype, more than 100 discs were collected and total RNA was isolated using TRIzol (Invitrogen). qRT-PCR was performed as previously described and rp49 was used as internal controlWing discs were dissected at 3wf fwd: AAGTCGAGCAATGGGCAATGAT,rev: TGGAGGAGCGTGTCTTCTG;sens fwd: CCGAAAAGGAGCATGAACTC,rev: CGCTGTTGCTGTGGTGTACTrp49 fwd: CATCCGCCCAGCATACAG,rev: CCATTTGTGCGACAGCTTAGP value of less than 0.05 was considered significant.For pupal retina AO staining, wing size measurement and PH3 staining, unpaired t-text analysis was used. For qRT-PCR, one-way analysis of variance test followed by the post Dunnett test was used. A How to cite this article: Zhang, S. et al. dFoxO promotes Wingless signaling in Drosophila. Sci. Rep.6, 22348; doi: 10.1038/srep22348 (2016).


Human T-cell leukemia virus type 1 (HTLV-1) causes chronic infection leading to development of adult T-cell leukemia (ATL) and inflammatory diseases. Non-human primates infected with simian T-cell leukemia virus type 1 (STLV-1) are considered to constitute a suitable animal model for HTLV-1 research. However, the function of the regulatory and accessory genes of STLV-1 has not been analyzed in detail. In this study, STLV-1 in naturally infected Japanese macaques was analyzed.tax and HTLV-1 bZIP factor (HBZ). STLV-1 Tax activated the NFAT, AP-1 and NF-κB signaling pathways, whereas STLV-1 bZIP factor (SBZ) suppressed them. Conversely, SBZ enhanced TGF-β signaling and induced Foxp3 expression. Furthermore, STLV-1 Tax activated the canonical Wnt pathway while SBZ suppressed it. STLV-1 Tax enhanced the viral promoter activity while SBZ suppressed its activation. Then we addressed the clonal proliferation of STLV-1+ cells by massively sequencing the provirus integration sites. Some clones proliferated distinctively in monkeys with higher STLV-1 proviral loads. Notably, one of the monkeys surveyed in this study developed T-cell lymphoma in the brain; STLV-1 provirus was integrated in the lymphoma cell genome. When anti-CCR4 antibody, mogamulizumab, was administered into STLV-1-infected monkeys, the proviral load decreased dramatically within 2 weeks. We observed that some abundant clones recovered after discontinuation of mogamulizumab administration.We identified spliced transcripts of STLV-1 corresponding to HTLV-1 STLV-1 Tax and SBZ have functions similar to those of their counterparts in HTLV-1. This study demonstrates that Japanese macaques naturally infected with STLV-1 resemble HTLV-1 carriers and are a suitable model for the investigation of persistent HTLV-1 infection and asymptomatic HTLV-1 carrier state. Using these animals, we verified that mogamulizumab, which is currently used as a drug for relapsed ATL, is also effective in reducing the proviral load in asymptomatic individuals. Approxit period . This vit period ,6 and uvt period .The reason why most HTLV-1 carriers do not develop ATL is partly explained by the immune response of cytotoxic T cells (CTLs) against HTLV-1 proteins . Immunosin vivo. Old World monkeys are frequently infected with simian T-cell leukemia virus type 1 (STLV-1), which is closely related to HTLV-1 [Animal models that are relevant to the human immune system are required for scientists to investigate how the immune response controls the proliferation of infected cells and viral replication o HTLV-1 . Like HTo HTLV-1 . Furthero HTLV-1 ,17. Basein vivo, indicating that anti-CCR4 antibody is effective for treatment of HTLV-1-associated inflammatory diseases. These results suggest that Japanese macaques naturally infected with STLV-1 show characteristics that correlate closely with those of HTLV-1 carriers and may therefore serve as a suitable animal model for the analysis of persistent HTLV-1 infection and HTLV-1 carrier state.In the present study, Japanese macaques naturally infected with STLV-1 were investigated. We first identified the STLV-1 bZIP factor (SBZ) gene as an antisense transcript of STLV-1 similar to HBZ. Molecular analyses showed that STLV-1 Tax and SBZ have activities on various transcriptional pathways similar to those of HTLV-1 Tax and HBZ. Furthermore, we observed clonal proliferation of STLV-1-infected cells. Finally, anti-CCR4 antibody, which is currently used to treat ATL patients, was administered into STLV-1-infected Japanese macaques, and we found that this reduced the proviral load To identify STLV-1-infected monkeys, we screened plasma samples for antibody against viral STLV-1 antigens by particle-agglutination test. Out of 533 Japanese macaques examined, 320 (60%) were seropositive, while only one rhesus macaque out of 163 (0.6%) was seropositive  and Japanese macaque STLV-1, and 82% in HBZ (ATK) and Japanese macaque SBZ. Phylogenetic analyses show that Japanese macaque STLV-1 env in this study is close to Melanesian subtype C [Analysis of the STLV-1 pX region suggests the presence of e Figure . The lenrm of HBZ,18, respubtype C   of the provirus while HBZ suppresses it. As presented in Figure + T cells, as is the case with HTLV-1 infection in humans  developed anorexia and had paralysis of the lower limbs. This monkey had high proviral load (53%) in PBMCs. We suspected that this monkey has developed a disease similar to HAM/TSP because paralysis of the lower limbs is one of the major symptoms of HAM/TSP patients. Magnetic resonance imaging (MRI) revealed a high intensity lesion in the brain on a T2-weighted image Figure C. Pathol+ Figure D. In con+ Figure E, left. + Figure E, right.+ Figure F. Moreovin vitro with the fluorescently labeled antibody . After the treatment, the CCR4 positivity decreased to 1.62% and 12.4% respectively. We also measured proviral load over the course of the treatment and found that it decreased dramatically within 2 weeks  . Recentls Figure B. Thus, Eight weeks after the final administration of mogamulizumab, the proviral load started to recover Figure B. To inv+ T cells by the virus [HTLV-1 is thought to originate from STLV-1. In STLV-1-infected monkeys, investigators found clonal proliferation of STLV-1-infected cells and the preferential infection of CD4he virus ,31. Morehe virus ,17,32-35he virus ,33, demohe virus . HoweverThe amino acid sequence of STLV-1 Tax is closely homologous to that of HTLV-1 Tax, and this study demonstrated that their functions on various transcriptional pathways are similar as well. This study was the first to identify SBZ as an antisense transcript of STLV-1 and a homolog of HBZ. SBZ and HBZ share only approximately 73% identity at the amino acid level. Nevertheless, for all the functions we examined, SBZ behaves similarly to HBZ. In particular, SBZ expression could induce Foxp3 expression like HBZ expression does. This might be attributed to the following reasons. First, the N-terminal region, as well as the heptad repeats of hydrophobic amino acids in the basic leucine zipper domain, are conserved between HBZ and SBZ. This may allow SBZ to interact with and suppress NF-κB, AP-1 and other transcription factors with basic leucine zipper motifs ,38. SecoHTLV-1 increases the number of infected cells by clonal proliferation of infected cells, which likely facilitates cell-to-cell transmission of this virus. Clonal proliferation of STLV-1-infected cells in Celebes macaques was demonstrated by the conventional inverse PCR method . HoweverThis study also revealed a remarkable difference in STLV-1 seroprevalence between Japanese macaques (320/533: 60%) and rhesus macaques (1/163: 0.6%). Previous studies showed that the seroprevalence in rhesus macaques was 25%, and that in Japanese macaques was quite high -42. Simiin vivo. These growth advantages may be due to the integration site of the provirus, accumulation of genetic mutations, or epigenetic changes. The population of some clones remained constant over the course of the treatment. We speculate that these clones are negative for CCR4 expression. High proviral load is associated with risk of ATL and inflammatory diseases. Therefore, suppression of proviral load by mogamulizumab is a possible treatment for HTLV-1-associated inflammatory diseases such as HAM/TSP.In this study, we also demonstrated that mogamulizumab strongly suppressed proviral load in STLV-1-infected Japanese macaques. Proviral load was suppressed for 4 weeks after the final administration of mogamulizumab, which seems reasonable when considering that the half-life of the antibody administered at 1.0 mg/kg is approximately 18 days as measured in a clinical trial . Some STIn summary, this study is the first to show that STLV-1 Tax and SBZ have activities similar to those of Tax and HBZ, activities which likely induce clonal proliferation and T-cell lymphoma in infected monkeys. STLV-1-infected Japanese macaques appear to be a good model for studying the effects of anti-viral drugs and the immunological aspects of HTLV-1 infection.Macaca fuscata) and rhesus macaques (Macaca mulatta) used in this study were reared in the Primate Research Institute, Kyoto University. Blood samples were obtained from the macaques  under ketamine anesthesia. All animal studies were conducted in accordance with the protocols of experimental procedures that were approved (2011–095) by the Animal Welfare and Animal Care Committee of the Primate Research Institute of Kyoto University, Inuyama, Japan.Japanese macaques (tax and RAG1 as previously described [Plasma samples were screened for the presence of antibodies against HTLV-1 by particle-agglutination test using SERODIA-HTLV-1 (Fujirebio). Proviral load was measured by real-time PCR quantifying the copy number of escribed . Primerstax and SBZ was detected by PCR using primers  with the same conditions. The primers used are shown in Additional file Total RNA was extracted from STLV-1-infected Japanese macaque cell line Si-2  with TriThe PathDetect pNFκB-Luc, pAP-1-Luc and pNFAT-Luc plasmids were purchased from Stratagene. The 3TP-Lux, TopFlash reporter plasmids and WT-Luc were described previously ,49. The The SBZ coding fragment was inserted into pGCDNSamI/N utilizing the NotI and SalI sites and SBZ-expressing retroviral vector was prepared as described previously .+CD25- mouse T lymphocytes were stimulated and transduced with SBZ-expressing retroviral vector as previously described [CD4escribed . Forty-eAntibodies used in this study were as follows: anti-human CD4 (OKT4), anti-Tax MI-73 , anti-moThe provirus integration sites in the Japanese macaque genome were amplified by linker-mediated PCR as previously described , with soMacaca mulatta (MMUL 1.0) using the Burrows-Wheeler Aligner (BWA) [The host genomic sequences, located between the region immediately adjacent to the viral 3’ LTR (ACACA) and the linker sequence (AGATCG), were extracted from the reads. Reads that started with GTTGGG  were removed. Remaining reads were mapped to the reference genome of er (BWA) . Reads tTwo Japanese macaques infected with STLV-1 were treated with mogamulizumab, which is an antibody against CCR4 and is approved in Japan as a drug to treat relapsed ATL. Mogamulizumab was provided by Kyowa Hakko Kirin Co Ltd. One mg/kg mogamulizumab was diluted in 40 ml saline and infused into each monkey intravenously for 20 min. Administration was performed once a week for 4 times. Before each administration, a 10 ml of blood sample was obtained. After the fourth administration, blood samples were collected every 2 weeks until week 11. Extra samples were collected on week 15 and week 18. The two monkeys were observed for any adverse effects during the experiment.Kyowa Hakko Kirin provided us the monoclonal antibody (mogamulizumab) that was used in this study.JY and M. Matsuoka conceived of this study. JT carried out antibody screening and proviral load measurement. M. Miura, KS, GM and TZ carried out the molecular experiments and the reporter assays. AK, AW, AS and HA coordinated the macaque experiments and collected the macaque specimens. PM analyzed viral protein and surface marker expression. KO carried out immunohistochemistry and pathological analyses. M. Miura carried out massive sequencing and its data analysis. M. Miura, JY and M. Matsuoka prepared the manuscript. All the authors approved the final manuscript.Phylogenetic analyses of HTLV-1 subtypes and Japanese macaque STLV-1.Click here for fileDeep sequencing data analysis.Click here for fileIn vitro staining of Japanese macaque PBMCs with mogamulizumab.Click here for filePrimers and oligonucleotides.Click here for file