Methylation-mediated silencing of inhibitors of Wnt signaling provided evidence for epigenetic activation of the pathway in placental tissues and choriocarcinoma cells. Similarly, abundant nuclear expression of β -catenin in invasive trophoblasts of complete hydatidiform moles suggested a role for hyper-activated Wnt signaling. In contrast, upregulation of Wnt inhibitors was noticed in placentae of women with preeclampsia, a disease characterized by shallow trophoblast invasion and incomplete spiral artery remodeling. Moreover, changes in Wnt signaling have been observed upon cytomegalovirus infection and in recurrent abortions. In summary, the current literature suggests a critical role of Wnt signaling in physiological and abnormal trophoblast function.Wingless ligands, a family of secreted proteins, are critically involved in organ development and tissue homeostasis by ensuring balanced rates of stem cell proliferation, cell death and differentiation. Wnt signaling components also play crucial roles in murine placental development controlling trophoblast lineage determination, chorioallantoic fusion and placental branching morphogenesis. However, the role of the pathway in human placentation, trophoblast development and differentiation is only partly understood. Here, we summarize our present knowledge about Wnt signaling in the human placenta and discuss its potential role in physiological and aberrant trophoblast invasion, gestational diseases and choriocarcinoma formation. Differentiation of proliferative first trimester cytotrophoblasts into invasive extravillous trophoblasts is associated with nuclear recruitment of β -catenin and induction of Wnt-dependent T-cell factor 4 suggesting that canonical Wnt signaling could be important for the formation and function of extravillous trophoblasts. Indeed, activation of the pathway was shown to promote trophoblast invasion in different  Figure 1A). After formation of the lacuna system, the ancestor of the intervillous space, cytotrophoblasts (CTBs) emanating from the trophectodermal layer generate primary villi by proliferation and invasion through the primitive syncytium . Throughout pregnancy, these primary villi transform into secondary and tertiary villi characterized by invasion of extraembryonic mesenchymal cells, villous branching, and vascularization. During the first trimester of pregnancy two types of mature villi can be discriminated, which are floating and anchoring villi . Floating villi connected to the intervillous space represent the transport units of human placenta. After establishment of blood flow nutrients and oxygen pass the epithelial layers of these villi ensuring appropriate fetal development and growth. Multinucleated syncytiotrophoblasts covering the surface of floating villi are continuously generated by asymmetrical cell division, differentiation and fusion of villous cytotrophoblasts (vCTBs) with the developing syncytium  cell types are generated. At early stages of pregnancy, invasive endovascular cytotrophoblasts (eCTBs) plug the maternal arterioles to prevent premature onset of blood flow into the intervillous space . Failurein vitro can only be studied upon access to first trimester placental samples, which in general is limited due to ethical considerations. Growth of the trophoblast cell column harboring progenitor cells for the invasive differentiation pathway may involve paracrine factors released from the underlying placental mesenchyme such as IGF molecules which also promote proliferation of vCTBs in vitro , urokinase plasminogen activator (uPA) and cathepsins, which are thought to degrade decidual extracellular matrix proteins and thereby facilitate cell invasiveness. The respective inhibitors, tissue inhibitors of metalloproteinases (TIMPs) and plasminogen activator inhibitors (PAIs) are produced by EVTs as well as decidual cells to limit the extent of trophoblast invasion. Numerous soluble factors expressed at the fetal-maternal interface including chemokines, cytokines and angiogenic factors were shown to promote trophoblast motility in an autocrine or paracrine manner . As a coll lines . Moreovell lines .Furthermore, published literature suggests key signaling pathways that are involved in trophoblast motility. Abundant growth factors such as hCG, EGF, HGF, or IGF2 activate MAPK kinase (MEK)/extracellular regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling, whereas prostaglandins were shown to act through the Rho-Rock pathway . BesidesTo identify novel genes and pathways controlling trophoblast motility and differentiation we and others recently performed comparative gene expression studies of CTBs and EVTs isolated from first trimester placental tissues. Chip-based profiling of EGFR-positive CTBs, isolated by flow cytometry, and EVTs, generated by seeding of the CTBs on fibronectin for 12 h, resulted in the identification of 3433 mRNAs which are at least two-fold differentially expressed between the two cell populations . Using iDrosophila to human, the particular signal transduction cascade controls early axis formation, limb patterning and organogenesis  pathway  or secreted frizzled-related proteins (sFRPs) binding to Wnts and LRP, respectively, have been identified  and Rho-associated kinase (ROCK), respectively. Wnt/PCP signaling is critically involved in the formation of embryonic tissues and organs and aberrant activation of the pathway was shown to promote metastasis of different cancer types , which blocks Ca2+ release in unstimulated cells and activates phospholipase C (PLC) and elevation of inositol 1,4,5-trisphosphate (IP3) thereby releasing Ca2+ from the endoplasmic reticulum , calcium/calmodulin-dependent kinase II (CamKII) and calcineurin which provoke nuclear recruitment of nuclear factor κ B (NFκ B) and of nuclear factor of activated T cells (NF-AT) (2+ accumulation upon Wnt5a stimulation can induce TGF-β -activated kinase (TAK1) and Nemo-like kinase (NEMO), which block TCF through phosphorylation and antagonize canonical Wnt signaling  . In addiignaling . Hence,  and LRP . Moreove and LRP . In concAs outlined above and discussed elsewhere, early placental development is associated with rapid generation of several trophoblast subtypes forming distinct functional villi in mice and men . LikewisThis review focuses on function of Wnt signaling in trophoblast and placental development and differentiation, whereas a number of different papers summarize the role of canonical and non-canonical Wnt signaling in female reproductive tract development and differentiation, uterine function and decidualization .2+ pathway could be involved . Hence, expression of canonical  as well as non-canonical Wnts  suggests that different Wnt pathways may operate during human trophoblast development. Wnt1, Wnt7b, Wnt10a, and Wnt10b were downregulated from first trimester to term suggesting roles in trophoblasts of early pregnancy . Considering that Wnt7b is required for mouse placental development . Moreover, stimulation with a Wnt ligand increased invasion of primary CTBs and trophoblastic SGHPL-5 cells in transwell assays and promoted migration in villous explant cultures seeded on collagen I, which was inhibited upon treatment with recombinant Dkk1 , transcript levels did not change upon Wnt stimulation suggesting that the pathway induces MMP-2 secretion or affects its stability .Although canonical Wnt signaling is strongly elevated upon EVT formation, CTBs also respond to Wnt signals. Wnt-dependent activation of TCF/β -catenin provoked increased proliferation and expression of the cell cycle regulator cyclin D1 as well as induction of Wnt signaling components . AlthougFurthermore, epigenetic analyses provided evidence for a general activation of Wnt signaling in placental tissues and isolated trophoblasts. Genes encoding negative regulators of the pathway, i.e., APC, sFRP2 and engrailed-1 were shown to be hypermethylated in trophoblasts, whereas these changes were not observed in placental fibroblast or leukocytes . This suIn summary, the present literature implicates canonical Wnt signaling in human trophoblast proliferation and invasion. However, the question of whether TCF molecules are indeed regulators of EVT formation, in other words control the switch from proliferation to cell cycle arrest and differentiation is still unknown and currently under investigation in our laboratory. So far, canonical Wnt signaling has been identified as a regulator of trophoblast cell fusion. GCM1, the most critical factor in syncytialisation controlling expression of the fusogenic proteins syncytin-1 and -2, harbors TCF binding sites in one of its introns and silencing of TCF-4 or β -catenin impaired cAMP-induced cell fusion of BeWo choriocarcinoma cells . Moreove2. ITGA6 and EGFR are highly expressed in the five different CTB cell pools but weakly present in the six EVT preparations confirming the published literature . In contrast, HLA-G1, and the pro-migratory genes integrin α1 (ITGA1), integrin α5 (ITGA5) and fibronectin 1 (FN1) were upregulated in EVTs. Interestingly, various mRNAs such as BAMBI, a marker of metastasis in colon cancer . Indeed, these genes have already been implicated in the control of trophoblast motility  which have stopped proliferation allowing for differentiation to take place. Although invasive trophoblasts do not induce mesenchymal vimentin, at least in vitro, and maintain expression of the epithelial marker cytokeratin 7, they upregulate typical EMT-associated mRNAs such as heat shock protein 47 (HSP47), Snail, ITGA5, ITGAB1, fibroblast-specific protein 1 (FSP1), MMP-2, and secreted protein acidic and rich in cysteine (SPARC) and downregulate genes associated with cell-cell adhesion and epithelial polarity such as the tight junction proteins occludin (OCLN) and ZO1. Also, transient loss of adherens junction proteins, i.e., membrane-bound β -catenin and E-cadherin, has been detected in the proximal invasion zone of anchoring villi using immunofluorescence in first trimester placental tissues , in which epithelial cells lose their polarity and gain fibroblast-like properties promoting invasion and migration . Interes tissues . Elevate tissues . Hence,  tissues .Changes in gene expression as well as in epigenetic modifications of Wnt signaling components were shown to be associated with different gestational diseases. Compared to normal tissues, higher numbers of β -catenin-positive EVT nuclei were detected in placentae of complete hydatidiform mole (CHM) suggesting that aberrant Wnt signaling could contribute to abnormal invasion in this pregnancy disorder . Genes eFigure 4). Differentiation of CTBs into growth-arrested invasive EVTs is associated with increased expression of TCF-4, nuclear recruitment of β -catenin and elevated canonical Wnt activity controlling trophoblast motility potentially through Fzd6. Furthermore, TCF-4/β -catenin could activate a set of genes promoting invasion as well as the EMT-like features of migratory trophoblasts. Hence, Wnt signaling likely contributes to the strong, intrinsic differentiation program of EVTs and their inherently invasive properties. Hyperactivation of autocrine Wnt signaling could play a role in trophoblast disorders with elevated proliferation and invasion such as CHM and choriocarcinomas whereas downregulation of the pathway could be a cause of impaired placentation and trophoblast invasion observed in preeclampsia. Epigenetic changes such as methylation of negative regulators of Wnt signaling could contribute to the induction of Wnt signaling likely promoting normal placentation as well as trophoblast tumor progression. Besides autocrine control decidual Wnt ligands could modulate trophoblast function via canonical and/or non-canonical pathways. The fact that Dkk1 is expressed in the decidua and increased upon progesterone treatment could suggest a mechanism to restrain the extent of trophoblast invasion. Further studies are needed to delineate specific Wnt-Fzd interactions in human trophoblasts and to define the role of non-canonical Wnt signaling in normal and aberrant trophoblast proliferation, invasion and differentiation.In conclusion, the present literature suggests that Wnt signaling is critical for physiological processes of human trophoblasts. The pathway could play a role in blastocyst adhesion, implantation and early trophoblast lineage decisions. Moreover, Wnt signaling could regulate trophoblast cell fusion as well as development of the anchoring villus controlling proliferation, invasion and differentiation. In first trimester placenta numerous canonical and non-canonical Wnt ligands are expressed in CTBs and likely regulate proliferation or other functions of these cells through the most abundant receptors, i.e., Fzd5 and Fzd10 (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A considerable high proportion of HTLV-I/II seroindeterminate blood donors have been documented in many countries including Argentina. In 5-10% of indeterminate Western Blot (WB) cases proviral sequences are detected. The aim of the study was to evaluate HTLV-I proviral load (pVL) in WB indeterminate PCR positive cases.A total of 87 indeterminate WB samples (HTLV blot 2.4 assay- Genelabs Diagnostics-) were studied over a 10 year period referred from the Blood Bank at Garrahan Hospital (n=83) and from the Virology Unit at Muñiz Hospital (n=4). HTLV-I and –II tax and pol proviral sequences were amplified by in-house nested PCR assays. HTLV-I pVL was estimated by a quantitative real-time PCR assay targeting the HTLV-I pol gene and the albumin gene as normalizer. The limit of detection of the assay was 2.6 log10 copies of HTLV-I/106 PBMCs (0.04% copies/100 PBMCs).In 8/87 samples HTLV proviral sequences were amplified, 7 HTLV-I and one HTLV-II. Proviral load was detectable in 4 of the 7 HTLV-I positive samples ranging from 2.72 log10 copies/106 PBMCs (0.05%) to 4.65 log10 copies/106 PBMCs (4.5%), but was undetectable in the remaining 3 (<0.04%). One of the subjects followed for 5 years remained with low pVL  and without changes in the WB profile.Detectable HTLV-I proviral load in half of the cases with indeterminate WB profiles with positive PCR underlies the importance to conduct follow-up studies to evaluate the evolution of the infection.


Human T-cell lymphotropic virus type 1 is endemic to central Australia among Indigenous Australians. However, virologic and clinical aspects of infection remain poorly understood. No attempt has been made to control transmission to indigenous children. We report 3 fatal cases of adult T-cell leukemia/lymphoma caused by human T-cell lymphotropic virus type 1 Australo-Melanesian subtype c. The human T-cell lymphotropic virus type 1 (HTLV-1) currently infects at least 5–10 million persons worldwide; however, this oncogenic retrovirus is not ubiquitous, and areas of high endemicity are typically separated by areas where infection is uncommon  with atypical lymphoid cells. The patient was seropositive for HTLV-1 by Western blot and positive for hepatitis B surface antigen. Flow cytometry of cells obtained by bone marrow and lymph node biopsies revealed a population of CD2+, CD3+, CD4+, CD5–, CD7−, CD8−, CD25+ cells (wwwnc.cdc.gov/EID/article/19/10/13-0105-F1.htm).The first case-patient, a 68-year-old indigenous Australian man, reported a several-month history of limb pain, abdominal pain, and diarrhea. Peripheral blood examination revealed lymphocytosis  caused by CD2+, CD3−, CD4+, CD5+, CD7−, and CD8− atypical lymphoid cells  and 28 primate T-lymphotropic virus type 1 sequences. These sequences cluster with the Australo-Melanesian c-subtype (Molecular characterization of the viral strain was possible for patients 1 and 2. The entire HTLV-1 -subtype  and are -subtype .Elsewhere, HTLV-1–associated ATLL directly causes death in as many as 1 in 20 HTLV-1 carriers (Strongyloides stercoralis roundworms, which shortens the latent period for malignant transformation (S. stercoralis parasites contributed to the development of ATLL in the patients reported here is uncertain. However, S. stercoralis parasites are endemic to central Australia (S. stercoralis parasite–endemic areas (The paucity of clinical information regarding HTLV-1–associated diseases among indigenous Australasians might reflect these persons’ limited access to medical care rather than intrinsic differences in the oncogenic potential of the HTLV-1c Australo-Melanesian subtype. Indeed, social deprivation can increase the risk for adverse outcomes among HTLV-1 carriers (The occurrence of 3 cases of ATLL in indigenous central Australian adults, together with previous studies that have demonstrated high HTLV-1 seropositivity rates (


Mycobacterium tuberculosis infection represents pathological attributes of the host immunity to infection and is required for the containment of infection. Granuloma formation is a complex process involving initiation and development of organized multicellular structures comprised of components of extracellular matrix [Granuloma formation during r matrix . ActivatTo investigate the molecular mechanisms underlying Phosphatidyl-myo-inositol dimannosides triggered MMP-9 expression in macrophages.We show possible implications of Notch signaling on immunological parameters associated with interaction of macrophages with novel cell wall antigen of Mycobacteria. We present evidences that PIM2 triggered expression of MMP-9 involves the activation of PI3K and Notch1 signaling in TLR2- MyD88 dependent manner.PIM2 triggers the activation of PI3K and Notch1 signaling leading to MMP-9 expression. Notch1 signaling perturbations demonstrate the involvement of a cross-talk with members of PI3K and MAPK pathway. PIM2 triggered significant p65 NF-κB nuclear translocation that was dependent on activation of PI3K or Notch1 signaling. MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NF-κB to respective promoters.PI3K and Notch1 signaling are obligatory early proximal signaling events during PIM2 induced MMP-9 expression in macrophages.


Adult T-cell leukemia/lymphoma (ATL) is a rare and often fatal outcome of infection with human T-lymphotropic virus type I (HTLV-I). Altered host immunity in HTLV-I carriers has been postulated as a risk factor for ATL, but is not well understood.We prospectively examined well-validated serologic markers of HTLV-I pathogenesis and host immunity in 53 incident ATL cases and 150 carefully matched asymptomatic HTLV-I carriers from eight population-based studies in Japan, Jamaica, the United States and Brazil. We used multivariable conditional logistic regression, conditioned on the matching factors , to evaluate the biomarkers’ associations with ATL in all subjects and by years  from blood draw to ATL diagnosis.In the pooled population, above-median soluble interleukin-2-receptor-alpha levels , 95% confidence interval (CI)=4.08, 1.47-11.29) and anti-Tax seropositivity , which indicate T cell activation and HTLV-I replication, respectively, were independently associated with an increased ATL risk. Above-median total immunoglobulin E levels , which indicate type 2 (B cell) activation, predicted a lower ATL risk. The sIL2R and anti-Tax associations with ATL were stronger in samples collected ≤5 years pre-diagnosis.The biomarker profile predictive of ATL risk suggests a role for heightened T cell activation and HTLV-I replication and diminished type 2 immunity in the etiology of ATL in HTLV-I carriers. Translation of these findings to clinical risk prediction or early ATL detection requires further investigation.


Plants are frequently attacked by herbivores and pathogens and therefore have acquired constitutive and induced defenses during the course of their evolution. Here we review recent progress in the study of the early signal transduction pathways in host plants in response to herbivory. The sophisticated signaling network for plant defense responses is elicited and driven by both herbivore-induced factors  and plant signaling  in response to arthropod factors. We describe significant findings, illuminating the scenario by providing broad insights into plant signaling involved in several arthropod-host interactions. Coordination of defensive actions against attacking pests results from interactions between the plant and herbivore-derived elicitors and effectors which are followed by rapid activation of sophisticated plant signaling cascades. However, the molecular mechanisms in the hosts that regulate the balance between activation and suppression of resistance are not fully understood. Despite the high number of known plant responses to herbivory, there are only a few known classes of animal-derived defense elicitors .N-(17-hydroxylinolenoyl)-Lglutamine], a hydroxy fatty acid-amino acid conjugate (FAC), which was identified in beet armyworm (Spodoptera exigua) oral secretions [2+ channels to transmit the signal [Pieris brassicae) [P. brassicae [Schistocerca americana) [Spodoptera frugiperda) [+ICDINGVCVDA−] and the related peptides [+(GE)ICDINGVCVDA−] are derived from chloroplastic ATP synthase gamma-subunit regulatory regions. These peptides elicit rapid and sequential production of phytohormones, and consequently volatile emissions   ), severae [MeJA] ,104, andl acetate,105–108.l acetate, but it 2+ influx, protein phosphorylation/dephosphorylation and the action of ROS [Some of those volatiles can activate defense genes and this is likely mediated via well-known signaling processes such as Can of ROS ,109. It n of ROS . Corn sen of ROS . Changesn of ROS ,112–114 n of ROS . It is tn of ROS . Howevern of ROS .Exposure to structurally similar compounds often results in different defensive responses in plants ,117, sugA network of both plant cellular signaling (via phytohormone-dependent/independent pathways) and extra-cellular signaling (via plant VOCs) is induced and modulated in plants in response to herbivory. This highly coordinated and sophisticated network has probably been acquired in order for host plants to respond effectively when damaged by a wide range of feeding attackers. Thus, such complexity appears to exist and act differentially in programs controlling defense genes for acquiring certain kinds of immunity.


Oreta rosea Walker 1855 (Lepidoptera: Drepanidae), that differs morphologically from previously studied species. Caterpillars of this species produce three distinct types of vibrational signals during territorial encounters with conspecifics — mandible drumming, mandible scraping and lateral tremulation. Signals were recorded using a laser-doppler vibrometer and characterized based on temporal and spectral components. Behavioural encounters between a leaf resident and a conspecific intruder were staged to test the hypothesis that signaling functions as a territorial display. Drumming and scraping signals both involve the use of the mandibles, being hit vertically on, or scraped laterally across, the leaf surface. Lateral tremulation involves quick, short, successive lateral movements of the anterior body region that vibrates the entire leaf. Encounters result in residents signaling, with the highest rates observed when intruders make contact with the resident. Residents signal significantly more than intruders and most conflicts are resolved within 10 minutes, with residents winning 91% of trials. The results support the hypothesis that vibrational signals function to advertise leaf occupancy. Signaling is compared between species, and evolutionary origins of vibrational communication in caterpillars are discussed.Abstract Vibrational communication in hook-tip moth caterpillars is thought to be widely used and highly variable across species, but this phenomenon has been experimentally examined in only two species to date. The purpose of this study is to characterize and describe the function of vibrational signaling in a species,  Acoustic communication in adult Lepidoptera has been broadly studied and serves a variety of social and defensive functions . HoweverSparganothis pilleriana,Drepana arcuata,Caloptilia serotinella,Drepanabilineata,There is increasing evidence that larval Lepidoptera employ an acoustic sense for communication, primarily in the form of vibration. Although anecdotal reports e.g.  suggest D. arcuata and D. bilineata), there is abundant suggestive evidence  residents should maintain exclusive use of their territory, (ii) residents should defend their territories against conspecifics, and (iii) intruders should only rarely displace residents. The aim of this study is to test for territorial behaviour and vibrational signaling, and if present, compare it with previously studied species. Life-history traits relevant to territoriality and spacing will also be compared to provide insight into some of the factors underlying the evolution of signaling in the Drepaninae.The goal of this study is to examine one of these species, nal oars . To the tars see , we hypoOreta rosea Walker 1855 (Lepidoptera: Drepanidae) moths were collected from the wild at ultraviolet collecting lights between May and August 2007 in Dunrobin, Ontario, Canada. Females oviposited on cuttings of viburnum (Viburnum lentago) and larvae were reared indoors on V. lentago or V. opulus under a LD 18:6 photoperiod at 21–26°C. Early- (first and second) and late- (third to fifth) instar larvae were used for life-history and behavioural observations. Late-instars were further used for morphological analysis of sound-producing structures, laser vibrometry recordings and behavioural trials.www.olympus.com) equipped with a Zeiss camera , or with a Nikon Digital SLR camera .Behavioural observations relevant to communication and spacing were recorded daily. These included the position on the leaf, presence of silk on the leaf, mode of feeding, and interactions between individuals. Photographs of eggs, larvae and adults were obtained with an Olympus dissection microscope  were digitized and recorded onto a Marantz Professional portable solid state recorder . Vibrations perpendicular to the leaf surface were measured at the location of a circular piece of reflective tape (2.0 mm in diameter) positioned 1 – 2.5 cm from the resident caterpillar. All recordings were made in an acoustic chamber . These recordings were used to determine the types of signals produced and to measure temporal and spectral characteristics of signaling. Temporal characteristics, including mean signaling bout duration, mean interval duration between signaling bouts and number of signals per bout were measured using Raven Bioacoustics Research Program . Bouts were defined as any combination of signals that was preceded and followed by feeding, walking or at least 1 s of inactivity. Durations of each signal type were calculated from 20 individuals. Power spectra were made using a 512-point Fourier transform  in Raven Bioacoustics Research Program. Signals were not filtered and a power spectrum of background noise was included for comparison.Vibrational signals were monitored and characterized using two recording methods - a microphone and laser-doppler vibrometer (LDV). Both methods involved recording late-instar larvae with a videocamera and a microphone or LDV during encounters with conspecific intruders (see below). Vibrations measured using a Polytec LDV .Structures associated with signal production and the last abdominal segments (A8–A10) were examined in early- and late-instars preserved in 80% ethanol. For scanning electron micrographs, mandibles and head capsules were dissected, mounted on aluminum stubs and air-dried. Specimens were sputter-coated with gold-palladium and examined using a JEOL scanning electron microscope  and a remote Sony audio microphone (ECM-MS907) placed 1–2 cm behind the leaf or with the LDV.Once it was established that n et al. . Brieflyhttp://rsb.info.nih.gov/ij/).Videotapes from 22 trials were analyzed to measure the durations and outcomes of contests, and to monitor changes in signaling rates in both residents and intruders throughout each trial. Durations of trials in which the intruder signaled were compared to those in which only the resident signaled using a Wilcoxon rank sum test. To compare average signaling rates of residents and intruders during encounters, signals from 21 encounters  were counted at 5-s intervals during the 80-s period prior to and the 80-s period following the time at closest distance between the resident and intruder. The distances between the head of the intruder and closest point of the resident were measured at each interval using ImageJ software , mid (20-s period following the mid-way point between the far and close distances) and close . Time intervals did not overlap in any of the trials. Signal escalation was analyzed by calculating the mean number of signals at each distance category for each type of signal and each individual. The data were square-root transformed and the means were compared using an analysis of variance (ANOVA). O. rosea, signaling rates for D. arcuata and D. bilineata were obtained from staged encounters from previous studies using similar methods . Signaling was initiated when a resident of a leaf is approached by a conspecific. Signaling was not ever observed in response to agitating the leaf or disturbances caused by a paintbrush. Overall, signaling typically occurred in bouts . Bouts typically comprised more than one signal, averaging 4.0 ± 2.0 signals per bout . Time intervals between bouts were highly variable, ranging from 1.7 – 15.4 s . Spectral analysis revealed that all signals are broadband with most energy ranging from 0.5 – 2.0 kHz .drumming  is produdrumming  to produMandible scraping.Mandible scraping .scraping  is produLateral tremulation.Lateral tremulation , and although highly variable, is much longer than a single mandible scrape or drum. One lateral tremulation event typically occurred at the beginning of a bout, followed by any combination of mandible drums and scrapes. Bouts rarely contained more than one lateral tremulation event.mulation  was onlyn = 44), but were similar between contestants in a given trial . Residents won 91.0% of trials, intruders won 4.5% and 4.5% were ties. Contests lasted 457.4 ± 330.7 s in trials where a winner was decided (n = 21). The only contest won by an intruder was of average duration (510.0 s).A total of 22 encounters were staged between a resident and an intruder of equal weight. Weights of the contestants ranged from 7.5 – 244 mg  from the beginning of the trial and at a mean distance of 6.97 ± 9.91 mm (n = 15) from the intruder's head to the closest point on the resident's body. Residents remained in the same approximate position on the leaf during trials. Signaling did not occur at all in three trials. Intruders signaled in 47.4% of trials where signaling occurred, but were the only contestants to signal in 15.8% of trials. Overall, residents signaled at significantly higher rates than intruders .Residents remained silent until they detected an intruder , 6. ResiF = 22.6, P = 0.001; MS: F = 6.1, P = 0.43; V: F = 22.6, P < 0.001). Lateral tremulation did not vary significantly with distance, perhaps due to the fact that it was rarely observed in comparison to the other signals .The rate of signaling in residents escalated as the intruder approached . Very li% of trials . Biting was never observed.A fourth type of behaviour that lacks a vibrational signal was observed in 31.8 f trials . LateralDrepana arcuata, D. bilineata and O. rosea are all solitary in their late-instars and defend territories against conspecifics. D. arcuata is the only species that makes a silken leaf shelter, while the others produce minimal silk by laying mats on the leaf surface . When compared to D. bilineata, O. rosea mandible drums significantly less . Lateral tail contact was also compared between species, and it was found that O. rosea contacts conspecifics with its caudal projection at similar rates to D. bilineata  whose caudal projection is about 10 times smaller  differ significantly between species, D. arcuata signaling significantly more than D. bilineata and O. rosea, and D. bilineata signaling significantly more than O. rosea .In terms of signaling rates,  smaller . Unlike The purpose of this study was to examine a variation on a theme — vibrational signaling in hook-tip moth caterpillars. The Drepaninae subfamily shows interesting diversity in vibrational signaling and morphology of the terminal abdominal segment. While all species lack anal prolegs , only soO. rosea produces three types of substrate-borne signals upon encountering a conspecific — mandible drumming, mandible scraping and lateral tremulation. The only morphological structure employed by O. rosea to produce vibrational signals are the mandibles, which do not appear to be specifically differentiated for sound production. There is mounting evidence demonstrating that the use of mandibles for acoustic signaling may be common in caterpillars . Furthermore, O. rosea larvae were not observed to produce vibrational signals during encounters with predators (unpublished data). The latter alternative hypotheses can also be discounted in O. rosea larvae as they do not produce secretions, are not associated with ants and are not gregarious at any stage of their life cycles. In the future, playback experiments may provide further insight into the function of vibrational signaling in these caterpillars.Results from staged encounters support the hypothesis that vibrational signaling in  animals . Signali animals , mutuali animals  and cons animals . The apoO. rosea and other species of Drepaninae is thought to have evolved to avoid the costs associated with physical fighting, as territorial encounters in other larval species often end in serious injury or death to one of the contestants (D. arcuata, the only species that produces a leaf shelter, invests significantly more in leaf defense via vibrational signaling than O. rosea and D. bilineata. Of the three species, D. arcuata is also the only species that lives gregariously in the early-instar stage. Therefore, the chances of encountering a wandering caterpillar from the same brood is expected to be higher in D. arcuata than in O. rosea and D. bilineata because the latter disperse earlier in development. Therefore, higher rates of ritualized signal production in D. arcuata may have evolved to avoid incurring physical injury to relatives.Ritualized vibrational signaling in testants . The invtestants . This isOreta rosea and D. bilineata share similar life-histories in that they live solitarily at all instars, do not build leaf shelters, and produce comparable amounts of silk. Signaling rates would thus be expected to be similar between these two species if signaling was linked to nest investment. However, this is not the case as D. bilineata signals at a significantly higher rate than O. rosea. To determine the cause for the difference in signaling rates between these two species, future comparative studies examining caterpillar behaviour in natural conditions are required to assess other lifehistory traits that may be linked to signaling. Comparison of territorial behaviour in O. rosea and D. bilineata also suggests that the elongated caudal projection found in O. rosea did not evolve for a defensive function against conspecifics because no significant difference was found in rates of lateral tail contact between species, despite the distinct difference in caudal projection size. This does not discount its use as a defense against heterospecifics, such as parasitoids, and further studies examining its specific function are needed.The present study contributes to the understanding of vibrational signaling in the Drepaninae, describing signaling in a novel morphological form. It also provides evidence that signaling in the Drepanoidea may be widespread and highly variable. Each species possesses unique characteristics that can contribute to their vibrational signaling repertoire. Further behavioural and morphological observations in a number of Drepanoidea species mapped onto a molecular phylogeny are now underway and will provide additional insights into the ultimate and proximate mechanisms underlying the evolution of ritualized signaling in these caterpillars.


While slb and tri mutants do not display alterations in canonical Wnt signaling, we found that they are hypersensitive to it. Overexpression of the canonical Wnt/β-catenin ligand Wnt8a in slb or tri mutants resulted in dorsalized embryos, with tri mutants being much more sensitive to Wnt8a than slb mutants. Furthermore, the hyperdorsalization caused by Wnt8a in tri could be rescued by Nkd1. These results suggest that Nkd1 functions as a passive antagonist of Wnt signaling, functioning only when homeostatic levels of Wnt signaling have been breached or when Wnt signaling becomes destabilized. Wnt signaling is involved in many aspects of development and in the homeostasis of stem cells. Its importance is underscored by the fact that misregulation of Wnt signaling has been implicated in numerous diseases, especially colorectal cancer. However, how Wnt signaling regulates itself is not well understood. There are several Wnt negative feedback regulators, which are active antagonists of Wnt signaling, but one feedback regulator, Nkd1, has reduced activity compared to other antagonists, yet is still a negative feedback regulator. Here we describe our efforts to understand the role of Nkd1 using Wnt signaling compromised zebrafish mutant lines. In several of these lines, Nkd1 function was not any more active than it was in wild type embryos. However, we found that Nkd1’s ability to antagonize canonical Wnt/β-catenin signaling was enhanced in the Wnt/Planar Cell Polarity mutants  The regulation of these pathways must be tightly controlled in order for normal development to proceed. Regulation can occur at several different levels, but one important mechanism is the ability of a signaling pathway to invoke a negative feedback loop. The Wnt signaling pathway is a major player in development and in homeostasis of stem cells –4 and seotochord –20. Overotochord . Howevernkd is expressed at multiple other stages of development in domains of active Wnt signaling, its loss of function does not appreciably affect these other Wnt signaling events [nkd1 and nkd2 in mouse results in subtle alterations in cranial bone morphology, but are otherwise normal and fertile [The passive effect of Nkd1 may be a universal phenomenon. In flies, absence of Nkd results in a naked cuticle phenotype (hence its name) at larval stages, but even though g events . Double  fertile  and ubiq fertile ,17,21. Tsilberblick (slb/wnt11) and trilobite (tri/vangl2) mutants, both of which are specific for the Wnt/Planar Cell Polarity pathway. These mutant lines are sensitive to canonical Wnt signaling, but Nkd1 can rescue this sensitivity.In vertebrates it has been well established that the Wnt/PCP pathway can antagonize canonical Wnt/β-catenin signaling, but at what level in the pathway remains controversial –27. Nonenkd1, either at the start ATG or at a splice site, are toxic beginning at late somitogenesis, which is obvious by the neural necrosis at 1 dpf ) mutant, which has a mutation in the tcf7l1a transcription factor gene. In the absence of β-catenin, tcf7l1a is a transcriptional repressor and only in the presence of activated Wnt signaling and stabilized β-catenin is the inhibition released and transcription initiated. However, a frameshift mutation in the hdl mutant results in a truncated protein that can still bind β-catenin but cannot bind DNA [hdl mutant mimics the Wnt8a overexpression phenotype in that both lack eyes , has a highly dorsalized phenotype [vangl2 (strabismus or tri mutants) results in embryos highly sensitive to canonical Wnt signaling [Previously we determined that Nkd1 can inhibit non-canonical Wnt/PCP signaling independent of Nkd1’s role in canonical Wnt/β-catenin signaling . Furtherhenotype  and lossignaling ,36. Imposilberblick, slb) homozygous parents did not have a dorsalizing effect on somitogenesis stage embryos . However, by measuring the width of gsc expression at 30% epiboly, Nkd1 MO resulted in a slightly expanded organizer . The surviving embryos also had varying degrees of dorsalization and slight rescue of cyclopia (not shown). Taken together, this suggests that slb mutant embryos are more sensitive to canonical Wnt signaling and that this sensitivity increases the antagonistic activity of Nkd1. Furthermore, Wnt8 may also affect the Wnt/PCP pathway, which is currently being investigated.We also tested the sensitivity of this mutant to canonical Wnt signaling. Overexpression of Wnt8a resulted in a slight increase in organizer gene expression but had other curious effects at 1 dpf. In multiple experiments, 50% of the embryos died by 1 dpf and of the 50% that remained, 37% had no eye whereas wild type embryo controls had 75% with no eye (knypek/glypican 4 (kny/gpc4). Overexpression or knockdown of Nkd1 did not have any effect on the development or morphology of somitogenesis stage or 1 dpf embryos (aside from MO toxicity)  . In additrilobite/vang-like 2 tri/vangl2)  mutant, which has a mutation in the transmembrane vangl2 gene [tri mutants is consistent with Nkd1 affecting Wnt/PCP signaling [slb mutants, Nkd1 affects the expressivity of the Wnt/PCP mutant phenotypes, but in the case of kny, Nkd1 has no effect at all.Lastly we determined the effect of Nkd1 in the gl2 gene ,38. Vanggl2 gene . Knockdogl2 gene ,43. It iignaling ,44,45. Tslb mutant, we observed a significant decrease in gsc gene expression in more than 50% of the embryos obtained from tri heterozygote crosses injected with nkd1 mRNA  to have a phenotype, the remaining 75%  to be similar to nkd1 injections into wild type embryos and have slightly expanded gsc expression [tri embryos are haploinsufficient for the tri gene with respect to its ability to regulate canonical Wnt signaling, but the genotype of the individual embryos has not been determined. The effect of Nkd1 on dorsal gene expression in tri mutants is similar to, but greater than, that observed in slb mutants from homozygous mutant parental crosses. We conclude that Nkd1 function in canonical Wnt signaling is more active in vangl2 mutants compared to slb mutants and wild type embryos, potentially as a result of reduced Wnt/PCP control over canonical Wnt/β-catenin signaling. Curiously, and as mentioned above for the slb mutants, the majority of the embryos with reduced or absent gsc expression must recover later in development, because we did not observe significant D–V patterning defects at 1 dpf . Consistent with the effect of Nkd1 on gsc expression in these embryos, this suggests that a reduction in the maternal contribution of Vangl2 is sufficient to sensitize these embryos to Wnt signaling. At the lower dose of wnt8a, approximately 15% of the embryos had a strong dorsalization phenotype which phenocopies the maternal wnt5a mutant, ppt , similar to the effect of Wnt8a in tri mutants. We also determined the effect on dorsal-ventral patterning by assaying the expression of gsc. Injection of vangl2 MO or wnt8a individually did not have a significant affect on the size of the gsc expression domain , but the endogenous domain was not significantly perturbed  was extremely sensitive to canonical signaling having significantly higher levels of luciferase in the presence of Wnt8a. In agreement with our observations, they found that by itself the vangl2 mutant does not exhibit increased Wnt signaling [gsc  according to Kimmel et al [hdl, slbtz215, knym119, trim207 or trim747, knym119 homozygous or heterozygous embryos  were collected from pairwise matings of homozygous or heterozygous adults, respectively.Adult zebrafish and embryos were raised at 28.5el et al . Materna°C in phosphate-buffered saline solution containing 4% paraformaldehyde. In situ hybridization was carried out as described by Thisse et al. (1999). The following antisense digoxigenin-labeled probes were used: chordin [goosecoid [bozozok [Staged embryos were fixed overnight at 4 chordin , goosecooosecoid ,52 and b[bozozok . Probes Width of gsc expression was measured by ImageJwnt8a [kny and slb mutant injections (not shown); nkd1 mRNA: 800 pg, except for nkd1 ATG MO 4 ng (5’-GAAGTTTACCCATTTCTCTGCATCG-3’); Vangl2 MO has been previously described [Capped mRNA was synthesized using Ambion’s mMessage mMachine kit. Following transcription, the RNA was purified over a G-50 sephadex column (Roche) and diluted in RNase free water, and its quantity and quality was analyzed as described above. The following mRNA concentrations were used: wnt8a ,53: Figuescribed . 4 ng of


Paeonia lactiflora Pall.) and were followed in three cultivars over different developmental stages as measured by high performance liquid chromatography (HPLC). OA and UA levels in leaves and stems demonstrated an overall upward trend from May 1 to September 15 except for UA in the leaves of “Hong Feng”. The maximum values of OA and UA in leaves of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” were 852.98, 575.60, 290.48 μg/g FW and 924.94, 827.36, 432.67 μg/g FW, respectively. The maximum values of OA and UA in stems of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” were 359.28, 90.49, 43.90 μg/g FW and 326.86, 82.25, 56.63 μg/g FW, respectively. OA and UA contents in leaves of 47 different herbaceous peony cultivars ranged from 66.73–618.12 and 36.23–665.14 μg/g FW, respectively, with average values of 171.62 and 227.57 μg/g FW, respectively. The results suggested that the aboveground parts of herbaceous peony may be used as an alternative source of OA and UA for medicinal purposes in addition to its ornamental purposes.Oleanolic acid (OA) and ursolic acid (UA) have been proven to possess many biological activities, and much attention is focused on the search for plants which are rich in OA and UA. In this report, the OA and UA accumulation characteristics were investigated in 47 cultivars of Chinese herbaceous peony ( Paeonia lactiflora Pall.), belonging to the Paeoniaceae family [Herbaceous peony  and ursolic acid (UA), which are usually widespread within the plant kingdom in the form of free state or glycosides. Many reports have shown that OA and UA mainly exist in sinensis , Crataegnatifida , Diospyrros kaki ,19, Eriojaponica ,21, Eucaglobules , Ligustr lucidum , Macrocaicinalis , Zizyphus jujuba , and oths jujuba , anti-dis jujuba , anti-ins jujuba , anti-tus jujuba  and hepas jujuba . Previous jujuba ,12,14,31In this study, the OA and UA accumulation characteristics were investigated in 47 cultivars of Chinese herbaceous peony and specifically followed in three cultivars over different developmental stages based on high performance liquid chromatography (HPLC). The results may provide a theoretical basis for herbaceous peony as an alternative source of OA and UA.Herbaceous peony is usually divided into medicinal and ornamental types. Medicinal herbaceous peony is cultivated mainly for harvesting roots, while ornamental herbaceous peony is planted as cut flowers or pot plants. Therefore, investigation of OA and UA contents in herbaceous peony leaves and stems at different developmental stages, and determination of a reasonable harvest time, will be of great significance in ensuring comprehensive utilization of these resources.The tendency for OA and UA in the leaves to change at different developmental stages is shown in The changes of OA and UA contents in the leaves of herbaceous peony were generally similar among the three cultivars. The overall trend was upward from May 1 to September 15 with a small trough on July 15 for “Yangfei Chu Yu” and “Fen Zhu Pan”. For “Hong Feng”, the small trough of OA and UA in the leaves appeared earlier than the two other cultivars on July 1, with very little change in OA content, and by September 15, OA content maintained a relatively stable level, while the UA level even decreased.The OA content in leaves of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” was lowest on May 1, with 9.66, 2.76 and 0.94 μg/g FW, respectively. It reached the highest peaks on September 15, with 852.98, 575.60 and 290.48 μg/g FW, respectively, which was 88.30, 208.55 and 309.02 fold of the lowest value, respectively. The UA content in the leaves of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” was also lowest on May 1, with 109.91, 93.30 and 51.96 μg/g FW, respectively. The highest UA content in leaves of “Yangfei Chu Yu” and “Fen Zhu Pan” appeared on September 15, with 924.94 and 827.36 μg/g FW, respectively, while those of “Hong Feng” reached the highest peak on September 1 with 432.76 μg/g FW. The highest UA content in leaves of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” was 8.42, 8.87 and 8.33 fold of the lowest level, respectively.The ratio of UA to OA in the leaves obviously changed at the different developmental stages. For instance, the UA content in leaves of “Yangfei Chu Yu” was significantly higher (3.5–11.3 fold) than OA in the first three sampling times (May 1 to June 1) and then the ratio of UA to OA decreased to 1.1–2.1 fold, except for the samples on July 15 .The OA and UA contents in stems of the three cultivars significantly changed at different developmental stages as shown in The OA content in stems of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” was lowest on May 1, with 1.85, 1.89 and 0.25 μg/g FW, respectively. It reached the highest peaks on September 15, with 359.28, 90.49, and 43.90 μg/g FW, respectively, which was 194.21, 47.88 and 175.60 fold of the lowest value, respectively. The UA content in stems of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” was also lowest on May 1, with 2.14, 8.88 and 0.49 μg/g FW, respectively. The highest UA content in stems of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” appeared on September 15, with 326.86, 82.25 and 56.63μg/g FW, respectively, which was 152.74, 9.26 and 115.57 fold of the lowest level, respectively.Very little difference in both OA and UA content was observed in the stems as compared with leaves. For instance, the ratio of UA to OA in stems in “Yangfei Chu Yu” was 0.9–2.6 from May 1 to September 15 .Great differences existed between the OA and UA contents of leaves of 47 investigated herbaceous peony cultivars. The contents of OA and UA were 54.26–618.12  and 36.23–665.14  μg/g FW, respectively. The highest contents of OA and UA in leaves were 11.39 and 18.34 fold of the lowest levels, respectively. Among 47 cultivars, “Hong Xiu Qiu” and “Yangfei Chu Yu” contained the highest OA and UA contents, with 618.12 and 665.14 μg/g FW, respectively, while the lowest OA and UA contents were observed in “Fen Zi Lu Jin” and “Jiangshan Ru Hua”, with 54.26 and 36.23 μg/g FW, respectively .The UA content in leaves was generally higher than OA with some exceptions among the 47 cultivars. OA content was mainly concentrated between 100–200 μg/g FW, while the UA content was more than 200 μg/g FW in over half the cultivars. The ratio of UA to OA in leaves of the different herbaceous peony cultivars ranged from 0.52–3.72, with an average value of 1.43.Similarly, the total OA and UA content in leaves of the different cultivars also largely differed with a range of 102.60–1251.38 μg/g FW . From thet al. [Forsythia suspensa (0.5490% and 0.406%) were significantly higher than those in young (0.3805% and 0.3275%) and mature fruits (0.417% and 0.346%). Our study indicated that OA and UA contents of old leaves and stems of herbaceous peony were also higher than those of young leaves and stems. Therefore, a mode of OA and UA content change may exist in plants such that the contents of OA and UA in old organs are higher than those in young ones during development.Lu et al.  found thet al.  reportedet al. [et al. [The contents of OA and UA in the same part of different cultivars differed in varied degrees. Wang et al.  determin [et al.  demonstrThere were some reports about the relationship between flower color and the OA and UA contents in plants. Huang  found siCallicarpa nudiflora leaves [Pterocephalus hookerie underground and aboveground parts [Hedyotis diffusa whole plant [Fructus chaenomeles fruits [Ligustrum lucidum fruits [OA and UA are widely distributed in plants with great variations between different families, genera and species. The results of our experiment showed that the contents of OA and UA in herbaceous peony leaves were in the range of 54.26–618.12 μg/g (0.0054%–0.0618% FW) and 36.32–633.26 μg/g (0.0036%–0.0633%) based on fresh weight, respectively. Considering the roughly 90% water content of plant leaves, OA and UA contents in dry leaves would increase ten-fold over that in fresh samples, with about 0.054%–0.618% and 0.036%–0.633%, respectively; these levels are higher than or similar to those of nd parts , Hedyotile plant , Fructuss fruits , and Ligm fruits .2O) was obtained with a Milli-Q water purification system from Millipore . All other reagents used in the present study were of analytical grade.OA  and UA  standards were purchased from Nanjing TCM Institute of Chinese Materia Medica . HPLC grade methanol was obtained from Caledon Laboratories Co. . The deionized distilled water  was planted in the Germplasm Nursery of Herbaceous Peony at College of Horticulture and Plant Protection, Yangzhou University, Jiangsu Province, China . The leaves and stems of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” were picked every 15 days from May 1 to September 15, 2009. The leaves of 47 cultivars were collected on September 1, 2009. All the samples were frozen in liquid nitrogen immediately after taking back to laboratory, and then stored in refrigerator at −20 °C until analysis.Herbaceous peony . The sample was centrifuged at 8000 g for 10 min, and then the filtrate was collected. Precipitates were extracted again and both filtrates were pooled and evaporated to dryness at 35 °C. The residue was dissolved in 1.5 mL methanol and transferred to a 2 mL tube. The crude extract was filtered through a 0.22 μm micro-filter before HPLC analysis. Each sample was repeated in triplicate.et al. [Quantifications of OA and UA were performed on a Waters-2695 HPLC system  equipped with Waters 2695 pump and 2487 UV detector according to the method of Zhou et al.  with somAll data are means of three replicates with standard deviations. Microsoft Excel  was used to calculate standard deviations.Traditional cultivation of both medicinal and ornamental herbaceous peony often causes a serious waste of resources due to discarding the aboveground biomasses. For medicinal purpose, herbaceous peony roots are usually harvested in August or September. During the same period, the aboveground parts of ornamental herbaceous peony will complete assimilate transportation and start to wilt in August to September. The OA and UA contents in the aboveground parts, mainly including the stems and leaves, reached a higher level from August to September. Therefore, the aboveground parts of herbaceous peony may be used as an alternative medicinal source of OA and UA in addition to its ornamental purposes.


Leukotrienes (LTs) are involved in inflammatory reactions. However, there are few reports regarding the role of LTs in adipocyte differentiation. Therefore, we investigated the role of leukotriene B4-BLT signaling pathway, then the adipocyte differentiation such as lipid accumulation and the increase in triglyceride was evaluated.Mouse 3T3-L1 preadipocytes were treated with lipoxygenase (LOX) inhibitors, BLT antagonist, and small interfering RNA (siRNA) for BLT1 and BLT2 to block the LTB4-BLT signaling pathway may positively regulate preadipocyte differentiation and be a rate-limiting system to control adipocyte differentiation.Blockade of BLT signaling by treatment with a LOX inhibitor or a BLT antagonist suppressed preadipocyte differentiation into mature adipocytes. In addition, knockdown of BLT1 and BLT2 by siRNAs dramatically inhibited differentiation. These results indicate the LTB4-BLT signaling pathway provides a potent regulatory signal that accelerates the differentiation of mouse 3T3-L1 preadipocytes. Further investigations are necessary to confirm the exact role of LTB4 and BLTs signaling pathways in preadipocyte differentiation.The LTB Diabetes mellitus, hyperlipidemia, hypertension, and atherosclerosis have recently been defined as typical life style-related diseases. A common background of these diseases is obesity, which is thought to cause insulin resistance resulting in the onset of disease ,2. Recen4, C4, and D4  are generated through lipoxygenase (LOX) pathways and induce inflammatory and allergic reactions such as leukocyte activation, capillary permeability, and bronchial contraction [4 binds to specific receptors, BLT1 and BLT2, to activate signaling pathways [4 controls immature neural stem cell proliferation and differentiation via the BLT signaling pathway [4 and its signaling pathway might be involved in cell proliferation and differentiation. However, there have been few reports regarding the role of LTs in adipocyte differentiation.Leukotrienes (LTs) such as leukotriene Btraction ,7. LTB4 pathways ,9. LTs hpathways ,11. In a pathway . Thus, L4 and BLTs signaling pathways in preadipocyte differentiation in a mouse fibroblastic 3T3-L1 cell line, a widely used cell line for research of preadipocyte differentiation [4 and BLT functions on preadipocyte differentiation.In this study, we investigated the role of LTBntiation . We anal4 receptor antagonist, suppressed mouse 3T3-L1 preadipocyte differentiation , dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX) and rosiglitazone (ROSI), a specific ligand for peroxisome proliferator-activated receptor gamma (PPARγ) ,14. We un Figure C. These 4 secreted from preadipocytes into the culture medium was 31.8 ± 8.4 nmol/L . These results indicate that the BLT-signaling pathway induces the differentiation of preadipocytes to mature adipocytes.To investigate whether BLTs are expressed on preadipocytes, we performed western blot analysis, which showed that both BLT1 and BLT2 were expressed in mouse 3T3-L1 preadipocytes from the start to late phases of differentiation  and BLT2-siRNA (12.5 nM) remarkably decreased lipid accumulation and TG contents in comparison to single knockdown . Among them, we initially focused on the expressions of PPARγ and CCAAT-enhancer-binding protein, alpha (C/EBPα) which is known as important key transcriptional regulators to control adipocyte differentiation. However, both molecules did not show significant changes at the microarray analysis. Namely, increase in PPARγ expression was from 0.9 to 1.3-fold, that in C/EBPα expression was from 0.7 to 1.1-fold, respectively. Therefore, it is expected that LTB4-BLT signaling pathway promoted 3T3-L1 preadipocyte differentiation via other molecules independent to PPARγ or C/EBPα. Further investigations will be required to clarify the molecules.To investigate the potential mechanisms of BLT signaling-mediated acceleration of 3T3-L1 preadipocyte differentiation, we performed DNA microarray analysis to identify the molecules regulated by LTB4-BLT signaling pathway provides a potent regulatory signal that accelerates the differentiation of mouse 3T3-L1 preadipocytes. Our results imply a potentially important and novel role for LTB4 and BLT functions on preadipocyte differentiation. Further investigations are necessary to confirm the exact role of LTB4 and BLTs signaling pathways in preadipocyte differentiation.In conclusion, the LTBINS, DEX and IBMX were purchased from Sigma Japan . ROSI was purchased from GlaxoSmithKline K. K. . LT synthetase, LOX inhibitor, NDGA, and 5-LOX specific inhibitor, AA-861, were purchased from Sigma Japan. ONO-4057, a specific BLT antagonist, was a kind gift from ONO Pharmaceutical Co. Ltd. . Anti-BLT1 and -BLT2 polyclonal antibodies were purchased from Cayman Chemicals .Mouse 3T3-L1 preadipocytes have been frequently used to study the differentiation of preadipocytes in vitro. Cell culture and induction of differentiation of preadipocytes were performed according to the previously described method  or specific BLT antagonist (ONO-4057) was prepared in dimethyl sulfoxide  and added to the differentiation medium from day 0 to day 6  for knockdown of BLT1 and BLT2 using an siRNA system . The sequences of the sense and antisense strand for BLT1 used were 5’-CAACCUACACUUCCUAUUA-3’, and 5’-UAAUAGGAAGUGUAGGUUG-3’, respectively. The sequences of the sense and antisense strand for BLT2 used were 5’-GGGACUUAACAUACUCUUA-3’, and 5’-UAAGAGUAUGUUAAGUCCG-3’, respectively.For transfection, siRNAs or negative control siRNA  were combined with Lipofectamine RNAiMAX (Invitrogen) and incubated for 20 minutes at room temperature to produce the transfection mixture. Then, the transfection mixture was added to preadipocytes at a final concentration of 25, 50 and 100 nM siRNA . The results were considered significantly different at P <0.05.Results were expressed as the mean ± SEM. Statistical comparisons were performed using the Student’s LT: Leukotriene; LOX: Lipoxygenase; siRNA: Small interfering RNA; TNFα: Tumor necrosis factor alpha; IL-6: Interleukin 6; INS: Insulin; DEX: Dexamethasone; IBMX: 3-Isobutyl-methylxanthine; ROSI: Rosiglitazone; PPARγ: Peroxisome proliferator-activated receptor gamma; NDGA: Nordihydroguaiaretic acid; TG: Triacylglycerol; PG: Prostaglandin; C/EBPα: CCAAT-enhancer-binding protein, alpha; DMEM: Dulbecco’s modified Eagle’s medium; DMSO: Dimethyl sulfoxide; ANOVA: Analysis of variance.The authors declare that they have no competing interests.KH performed all experiments and statistical analysis, discussion of results and drafted the manuscript. KW conceived the study, participated in discussion of the results, provided additional funding for the study. YM assisted in performance of some experiments. AN, YK, TY participated in discussion of the results. All authors read and approved the final manuscript.


HTLV-1 is endemic in the Northwest among Kolla natives while Buenos Aires city (BA) is considered as non-endemic. In the last decades, migrations to BA have changed from Europeans to Latin-Americans from countries with endemic focus for HTLV-1 infection  and also from different African countries.To determine the phylogeographic origin of HTLV-1 strains and the ethnic background of HTLV-1 positive residents of Buenos Aires.63 HTLV-1 positive BA residents were retrospectively studied. Phylogeny of LTR region was performed by PAUP 4.0 and tree topology was visualized by TreeView. The ethnic origin of individuals was inferred by the study of mitochondrial DNA haplogroups (complete control region).All LTR sequences were classified as Cosmopolitan subtype/Transcontinental subgroup. Twenty-five of them, belonging to individuals of Amerindian and 3 of African ancestry, were included in the Big Latin American cluster with HTLV-1 references from Amerindians. On the other hand, 9 LTR sequences belonging to individuals of Amerindian ancestry, grouped with references from people of black ethnic component of Africa, French Guiana and Suriname .This study confirms the presence HTLV-1 cosmopolitan subtype/ Transcontinental subgroup in BA residents and shows HTLV-1 strains from individuals of Amerindian ancestry closely related to references from Africa and Latin-American countries with an important afro-amerindian ethnic component. Finally, these results highlight the importance of ethnic origin of HTLV-1infected people and the retroviral phylogeny to understand the influence of migration waves in the dynamics of the infection in a specific geographic area.


The overall seroprevalence of HTLV infection among pregnant women in Spain is below 0.02% and therefore universal screening is not recommended. However, given that a large proportion of immigrant pregnant women come from HTLV-1 endemic regions, this population might warrant specific screening considerations.From January 2009 to December 2010, a cross-sectional study was carried out among immigrant pregnant women attended at 14 different Spanish hospitals. An enzyme immunoassay (EIA) was used for testing serum HTLV-1/2 antibodies, being reactive samples further confirmed by Western blot.A total of 3,337 pregnant women were examined. The origin was as follows: Latin America 1579 (47%), East Europe 606 (18%), North Africa 507 (16%), Sub-Saharan Africa 316 (9%), Western Europe 116 (5%), and Asia 163 (5%). A total of 7 samples were confirmed as HTLV positive, 6 HTLV-1 and 1 HTLV-2. HTLV-1 was found in 5 women coming from Latin America and 1 from Morocco. The only single HTLV-2 woman came from Ghana.The overall HTLV seroprevalence in immigrant pregnant women was 0.2%, being 0.3% among Latin Americans and 0.2% among Africans. It was absent among women coming from other regions.The seroprevalence of HTLV infection among immigrant pregnant women in Spain is 0.2%, being all cases found among women from Latin America and Africa. Given the benefit of preventing vertical transmission of HTLV, antenatal HTLV screening could be advisable in pregnant women coming from these regions.


Understanding how signals propagate through signaling pathways and networks is a central goal in systems biology. Quantitative dynamic models help to achieve this understanding, but are difficult to construct and validate because of the scarcity of known mechanistic details and kinetic parameters. Structural and qualitative analysis is emerging as a feasible and useful alternative for interpreting signal transduction.In this work, we present an integrative computational method for evaluating the essentiality of components in signaling networks. This approach expands an existing signaling network to a richer representation that incorporates the positive or negative nature of interactions and the synergistic behaviors among multiple components. Our method simulates both knockout and constitutive activation of components as node disruptions, and takes into account the possible cascading effects of a node's disruption. We introduce the concept of elementary signaling mode (ESM), as the minimal set of nodes that can perform signal transduction independently. Our method ranks the importance of signaling components by the effects of their perturbation on the ESMs of the network. Validation on several signaling networks describing the immune response of mammals to bacteria, guard cell abscisic acid signaling in plants, and T cell receptor signaling shows that this method can effectively uncover the essentiality of components mediating a signal transduction process and results in strong agreement with the results of Boolean  dynamic models and experimental observations.http://www.phys.psu.edu/~ralbert/ESM.This integrative method is an efficient procedure for exploratory analysis of large signaling and regulatory networks where dynamic modeling or experimental tests are impractical. Its results serve as testable predictions, provide insights into signal transduction and regulatory mechanisms and can guide targeted computational or experimental follow-up studies. The source codes for the algorithms developed in this study can be found at  The normal functioning of biological organisms relies on the coordinated action of a multitude of components. The interactions between genes, proteins, metabolites and small molecules form networks that govern gene regulation, determine metabolic rates, and transduce signals ,2. InterGiven the topology (i.e. the nodes and edges) of a network, it is natural to wonder just how important  each node is to the network's connectivity and functionality. Not surprisingly the issue of node centrality and its correlation with node influence has attracted the attention of many researchers. A large number of graph measures have been developed for evaluating node centrality in complex networks, from degree centrality , closenep, of which one, the edge from A to Cp, is negative . Moreover, combinatorial regulation is ubiquitous in biological networks; this means that multiple interactions that regulate a component may act in a synergistic  fashion , with 1 indicating a node whose loss causes the disruption of all ESMs between the input and output node(s). As a comparison we also define the analogous measure using simple paths instead of ESMs (Methods).Our method ranks the importance of signaling components by the effects of their perturbation on the ESMs of the network. We characterize each node EESM) and the simple path (SP) centrality measure (denoted by ESP). We compare these measures to a traditional centrality measure, node betweenness centrality (denoted by BC) , with 1 indicating a node whose loss disrupts all ESMs between the input and output node(s).where We also consider a more traditional graph measure for the essentiality of signaling components based on the number of all simple paths (SPs) from inputs to outputs:NSP(G) and NSP(ΔvG) denote the total number of simple paths from the input(s) to the output(s) in the original expanded network G and the damaged network Δv Gafter deleting v, respectively. This SP measure also takes values in the interval , with 1 indicating a node whose loss causes the disruption of all paths between the input and output node(s). There are polynomial algorithms for computing the paths between any pair of nodes in a graph [where  a graph . Here weterval 0,, with 1 bintprog in the Matlab Optimization Toolbox which uses a branch-and-bound algorithm to solve binary integer linear programming problems. Programs are available at http://www.phys.psu.edu/~ralbert/ESM.All the algorithms in this study were coded and implemented in Matlab 7.6 . The depth-first algorithms are the Matlab implementation of the pseudo code given in the Additional file RSW and RA conceived and designed the study. RSW performed the study. RSW and RA analyzed the results. RSW and RA wrote, read, and approved the final manuscript.Algorithms developed and used in this study. This file contains the algorithms developed and used in this study, including the depth-first-search algorithm for enumerating all simple paths from the input node(s) to the output node(s) of a signaling network, the iterative integer linear programming algorithm for enumerating elementary signaling modes, the depth-first-search algorithm for estimating the number of elementary signaling modes, as well as the dynamic programming algorithm for finding shortest elementary signaling modes.Click here for fileEssentiality of the components from dynamic models of the three signaling networks. This file describes the essentiality of the signaling components obtained by dynamic simulation of Boolean models for the host immune response network and the guard cell ABA signaling network, and logical steady state analysis of the T cell receptor signaling network (Tables S1-S4).Click here for fileEssentiality of the guard cell ABA signaling components from our method. This file contains the importance values of the guard cell ABA signaling components obtained by single-node deletions  and two-node deletions , and literature support of the uncovered essential components.Click here for fileThe expanded T cell receptor signaling network. This file contains the expanded T cell receptor signaling network  and the importance values of the T cell receptor signaling components found by our method with AP as the input node .Click here for file