Drosophila neuromuscular junction (NMJ), synaptic vesicles are mobile; however, the mechanisms that regulate vesicle traffic at the nerve terminal are not fully understood. Myosin VI has been shown to be important for proper synaptic physiology and morphology at the NMJ, likely by functioning as a vesicle tether. Here we investigate vesicle dynamics in Myosin VI mutants of Drosophila.At the Drosophila, Myosin VI is encoded by the gene, jaguar (jar). To visualize active vesicle cycling we used FM dye loading and compared loss of function alleles of jar with controls. These studies revealed a differential distribution of vesicles at the jar mutant nerve terminal, with the newly endocytosed vesicles observed throughout the mutant boutons in contrast to the peripheral localization visualized at control NMJs. This finding is consistent with a role for Myosin VI in restraining vesicle mobility at the synapse to ensure proper localization. To further investigate regulation of vesicle dynamics by Myosin VI, FRAP analysis was used to analyze movement of GFP-labeled synaptic vesicles within individual boutons. FRAP revealed that synaptic vesicles are moving more freely in the jar mutant boutons, indicated by changes in initial bleach depth and rapid recovery of fluorescence following photobleaching.In This data provides insights into the role for Myosin VI in mediating synaptic vesicle dynamics at the nerve terminal. We observed mislocalization of actively cycling vesicles and an apparent increase in vesicle mobility when Myosin VI levels are reduced. These observations support the notion that a major function of Myosin VI in the nerve terminal is tethering synaptic vesicles to proper sub-cellular location within the bouton. Drosophila NMJs exhibited rapid recovery times following photobleaching Although new synaptic vesicles are known to be transported to the nerve terminal from the cell body along microtubule tracks, less is known about the regulation of vesicle traffic within the nerve terminal itself. It was previously thought that upon delivery to the nerve terminal, synaptic vesicles remained relatively static until they were mobilized for neurotransmitter release Drosophila melanogaster, shares the well-conserved basic structural conformation of other Myosin proteins Drosophila NMJ Myosin VI plays an important role in maintaining proper peripheral vesicle localization within the nerve terminal Drosophila also exhibit impaired neurotransmission, consistent with a function of Myosin VI in tethering vesicles to the bouton periphery Myosins are a superfamily of actin-based motor proteins that use energy derived from ATP hydrolysis to move along actin filaments in vivo imaging methods were used to investigate intra-bouton synaptic vesicle localization and mobility at the Drosophila third instar larval NMJ: FM dye labeling and fluorescence recovery after photobleaching (FRAP). FM dye labeling revealed vesicle mislocalization of actively cycling vesicles following stimulation in the nerve terminals of Myosin VI mutants, consistent with previous Synaptotagmin labeling of fixed specimens. We also show, by way of FRAP analysis, that a reduction in Myosin VI expression corresponds to an increase in synaptic vesicle mobility. These data lend strong support to the idea that Myosin VI acts as an anchor to restrict vesicles in Drosophila boutons and ensure proper vesicle localization and trafficking.The present study was undertaken to further investigate the role of Myosin VI in synaptic vesicle localization and mobility. Two http://flystocks.bio.indiana.edu/bloom-food.htm) supplemented with yeast paste at room temperature. The Myosin VI loss of function alleles used in this study were 322jar and Df(3R)crb87-5 . Oregon R (OreR) flies served as wild-type controls. During handling, flies were temporarily anesthetized using CO2. To visualize synaptotagmin dynamics in vivo, control C155Gal4; UAS-synaptotagmin-GFPelav flies were used and were crossed to jar mutant genotypes. C155Gal4; UAS-synaptotagmin-GFPelav stocks (from herein abbreviated to C155SytGFP) contain the nervous system Gal4 driver, C155Gal4elav, and a UAS-synaptotagmin-GFP transgene in the same fly All fly strains and crosses were maintained on Bloomington standard medium  was used to make recordings. All muscles used for analysis had a resting potential of at least −50 mV. To ensure release of the reserve pool of vesicles, high frequency stimulation was performed at 10 Hz for 10 minutes. During the first stimulation protocol, larval fillets were bathed in HL3 solution with 1.5 mM CaExperiments were performed on third instar larval fillets glued to Slygard-coated glass slides in HL3 saline. All images were acquired within 2 hours from the time of dissection. Larvae were glued to plates using a topical tissue adhesive . Recordings of vesicle dynamics were taken from type Ib boutons on NMJ 6/7, with a maximum of 6 boutons from the same larvae used. For the first FRAP protocol, a total of 60 images were taken over a period of 2 minutes, with a 1 second delay between image acquisition. Images were captured as 512×512-pixel frames with the 488-nm line of the argon laser on an LSM510 (Carl Zeiss) confocal laser microscope. These images consisted of four baseline images taken prior to bleaching at 5% of full laser power, followed by the fifth scan consisting of 9 rapid iterations of bleaching at 97% of full laser power in the selected region of interest (ROI). The remaining 56 scans were then completed at 5% of full laser power. In a second series of experiments the protocol was adjusted to capture additional data points for fluorescence recovery immediately following bleaching. In this protocol, a total of 45 images were taken over a period of 45 seconds with no delay between image acquisition. Images were captured as 256×256-pixel frames with the 488-nm line of the argon laser on an LSM510 (Carl Zeiss) confocal laser microscope. Again, these images consisted of four baseline images taken prior to bleaching at 5% of full laser power, followed by the fifth scan consisting of 12 rapid iterations of bleaching at 97% of full laser power in the selected ROI. The remaining 41 scans were then completed at 5% of full laser power. Constant imaging parameters were maintained between samples and the size of the ROI was kept constant at 24×30 pixels in both FRAP protocols.Fluorescence intensity was measured in Image J for the bleached region, the background and the total bouton area using the Time Series Analyzer plug-in. To account for photobleaching during the experiment and the movement of bleached molecules, recovery curves were generated following double normalization of the raw fluorescence intensity Differences between recovery curves were assessed by fitting the data with a double exponential curve. A double exponential curve was used to account for the two phases in fluorescence recovery observed with respect to vesicle mobility, the initial more rapid recovery immediately post bleaching and the later plateau All statistical analyses were performed in GraphPad Prism Software 5. A significance level of p<0.05 was used for all experiments.Drosophila, via immunofluorescent detection of Synaptotagmin . Here we seek to investigate vesicle localization and dynamics in living specimens.We previously reported on mislocalization of synaptic vesicles in Myosin VI mutants of in vivo following 10 Hz nerve stimulation for 10 minutes. Boutons were labeled with two concentrations of FM 1–43 dye, 4 µM and 10 µM, in 1.5 mM and 2 mM Ca2+ HL3 saline respectively. Differences in FM dye uptake were quantified by measuring the relative difference in fluorescence intensity across a bouton's plot profile and calculating a profile index [(max intensity-min intensity)/max intensity]. For the first labeling protocol using 4 µM dye and 1.5 mM calcium saline, the characteristic plot profile for OreR boutons has two peaks indicating a peripheral distribution of FM labeled vesicles (322/Df(3R)crb87-5jar mutant larvae indicating a diffuse distribution of FM labeled vesicles (/Df(3R)crb87-5 mutant boutons compared to OreR control boutons . There was no significant difference in mean fluorescence across the entire bouton between OreR controls and 322/Df(3R)crb87-5jar mutant larvae .FM dye labeling was used to examine synaptic vesicle distribution vesicles . In contvesicles . We foun2+ solution with the same 10 Hz, 10 minutes stimulation protocol. Similarly, this protocol revealed that the 322/Df(3R)crb87-5jar mutant larvae exhibited a greater proportion of boutons with diffuse FM dye labeling throughout the bouton area in contrast to OreR control boutons. Calculating the plot profile index of fluorescence intensity under these conditions also revealed a significantly lower index for 322/Df(3R)crb87-5jar mutant boutons (n = 34) compared to OreR control boutons . Again, this is consistent with the majority of 322/Df(3R)crb87-5jar mutant boutons exhibiting the diffuse synaptic vesicle distribution phenotype observed as a single maxima when a plot profile is made from the bouton area , we compared FRAP recovery curves and found a significant increase in synaptic vesicle mobility in the jar mutant boutons.To examine vesicle dynamics at jar mutants to controls. Interestingly, we found that heterozygous jar mutants, C155Gal4; UAS-Synaptotagmin-GFP/<; jar322/+elav (n = 18 boutons) and C155Gal4; UAS-Synaptotagmin-GFP/<; Df(3R)crb87-5/+elav (n = 18 boutons), exhibited a greater extent of fluorescence recovery compared to the control, C155Gal4; UAS-Synaptotagmin-GFP/<elav  and C155Gal4; UAS-Synaptotagmin-GFP/<; Df(3R)crb87-5/+elav (n = 15 boutons), compared to the control C155Gal4; UAS-Synaptotagmin-GFP/<elav  and C155Gal4; UAS-Synaptotagmin-GFP/<; Df(3R)crb87-5/+elav .In such image sequences, we noticed that there was less bleaching in the mutant genotypes compared to controls. This observation is also consistent with more freely moving vesicles in the mutants. We therefore adjusted our acquisition protocol to account for this difference by employing a greater number of bleach iterations and more rapid image acquisition, to capture additional information about fluorescence recovery in the first part of the recovery curve. With this protocol we confirmed that the diffuse boutons of the jar heterozygotes with normal and diffuse vesicle distribution (C155Gal4; UAS-synaptotagmin-GFP/<elav boutons compared to diffuse boutons of jar loss of function heterozygotes (Bleach depth was found to be significantly greater for control boutons immediately following 9 bleach iterations compared to the boutons of Drosophila type Ib boutons and that the center of the bouton is relatively free of vesicles Drosophila larval synaptic terminal has confirmed that vesicles in different functional pools are spatially intermixed at the bouton periphery Ultrastructural studies have reliably shown that synaptic vesicles have a peripheral distribution within jar alleles of Drosophila, FM1-43 dye uptake was induced through a high frequency nerve stimulation protocol using two different dye concentrations. Imaging of vesicle distribution immediately following electrical stimulation and washing of dye revealed that vesicles were localized throughout the bouton area in jar loss of function mutant boutons while they remained restricted to the bouton periphery in wild-type controls. Given that FM dye labeling was visualized immediately following high frequency loading and that total bouton fluorescence did not differ between mutants and controls, the central localization of vesicles in jar mutants cannot be attributed to an increase in vesicle abundance. Rather, it is suggestive of an active role of Myosin VI in peripheral vesicle clustering at the nerve terminal.In jar mutants and suggest that Myosin VI plays an active role to maintain peripheral vesicle distribution during neural activity.This result is consistent with our previously published Synaptotagmin staining of fixed tissue, which indicated that Myosin VI is important for proper vesicle localization within the bouton. The present FM labeling reveals that actively cycling vesicles also have altered distribution in Drosophila NMJ Synaptic vesicles are mobile at the jar alleles. FRAP analysis revealed a significant enhancement of vesicle mobility in jar mutant boutons that showed both normal and diffuse vesicle distribution phenotypes. However, the enhancement in vesicle mobility compared to the controls was even greater for diffuse boutons than the jar boutons with proper torus-shaped localization of vesicles. In addition, bleach depth was significantly less for mutant boutons compared to controls, which is indicative of fluorescence recovery occurring so rapidly it is taking place during bleaching itself Drosophila nerve terminal Drosophila embryonic nervous system jar mutants have a lower level of Myosin VI expression and therefore, a reduced ability to anchor vesicles resulting in their enhanced mobility at the synapse. The FRAP data is important because it provides the mechanism of Myosin VI function at the synapse. Myosin VI may maintain proper synaptic physiology by tethering vesicles to ensure their proper localization for efficient release.If Myosin VI functions in synaptic vesicle localization to the bouton periphery by acting as a tether, it should normally restrain vesicle movement at the synapse. To test that idea, we undertook FRAP analysis of vesicle movement in the living nerve terminal of the jar synapses? We previously reported that there was only a detectable change in low-frequency synaptic strength, observed as a ∼⅓ reduction in EJP amplitude and a small reduction in mEJP frequency, in the most severe combination of jar alleles. Whereas in this report we show that there are measurable changes in FRAP recovery, even in the milder jar alleles. Together these data suggest that the process of synaptic transmission is relatively insulated from the intermediate increase in vesicle mobility we observe here with the mild jar alleles, but that the greatest increase in mobility correlates with a modest decrease in synaptic strength. This result implies that there may be a small reduction in the immediately releasable pool of vesicles in the jar mutant. These data also show that FRAP measurements are more sensitive for observing phenotypic changes in vesicle physiology, than EJP measurements alone.Correct vesicle organization is important for subsequent steps in neurotransmitter release following neural stimulation. What is the relationship of these vesicle mobility results to the known physiological phenotypes of jar alleles showed greater increases in synaptic transmission in 1 mM Ca++ and a greater extent of synaptic depression in 10 mM Ca++ than either control or less severe jar combinations, which behaved similarly. This observation indicates that although vesicles are moving rapidly throughout the bouton they can transition from the reserve pool to the readily releasable for participation in synaptic transmission and indeed appear to be able to sustain high rates of transmitter release. Although the mechanisms that regulate the translocation of vesicles between different functional pools during high frequency stimulation have not been fully resolved, Myosin motors have been implicated in this function in other preparations jar alleles lead to concomitant increased vesicle mobility, reduced synaptic output at low frequencies of stimulation and enhanced plasticity at higher frequencies of stimulation.In addition, we previously demonstrated that high-frequency stimulation paradigms revealed enhanced frequency-dependent increases in synaptic strength. At 10 Hz facilitation, severe In summary, the present work shows that Myosin VI plays a critical role in restraining synaptic vesicles to the bouton periphery. In the absence of Myosin VI function, peripheral vesicle localization is disrupted and vesicle mobility is enhanced at the synapse. The changes in vesicle mobility that we report here may, in part, account for the physiological changes to synaptic transmission we previously described in Myosin VI loss of function mutants. Future studies aimed at understanding dynamic regulation of Myosin VI function will yield information about the control of synaptic vesicle pools during synaptic transmission.


In vivo single unit recordings from anaesthetized NRG1-pretreated mice revealed increased spike bursting of nigral dopamine neurons. In slice preparations from NRG1-pretreated mice, spontaneous firing was elevated relative to controls. The relative increase in firing rates was abolished by a GABAA receptor antagonist. Whole-cell recording showed that perinatal NRG1 pretreatment diminished inhibitory miniature synaptic currents as well as GABAA receptor sensitivity. These results collectively suggest that perinatal exposure to neuregulin-1 results in the disinhibition of nigral dopamine neurons to influence their firing properties at the adult stage when the behavioral deficits are evident.Aberrant neuregulin-1 (NRG1) signals are suggested to associate with the neuropathophysiology of schizophrenia. Employing a mouse schizophrenia model established by neonatal neuregulin-1 challenge, we analysed postpubertal consequence of the NRG1 pretreatment for the electrophysiological property of nigral dopamine neurons.  Neuregulin-1 (NRG1) is one of candidate genes for schizophrenia susceptibility3In the central nervous system, a NRG1 receptor, ErbB4, is highly expressed in neocortical GABA neurons and midbrain dopaminergic neurons, whereas another NRG1 receptor, ErbB3, is mainly expressed in glial cells such as oligodendrocytes6781011121313According to the neurodevelopmental hypothesis for schizophrenia, we established a mouse schizophrenia model by treating neonatal mice with NRG1. Following peripheral administration, a mature form of NRG1 or an epidermal growth factor (EGF)-like core domain of NRG1 (eNRG1) penetrates the blood–brain barrier of the neonates and activates ErbB4 in the midbrain and neocortex14151614in vivo extracellular single unit recordings (in the anaesthetized state) and by in vitro single unit recordings and whole-cell recordings in midbrain slices. To distinguish the intrinsic effects from the extrinsic effects of synaptic inputs, we measured miniature synaptic currents in this cell population. These electrophysiological results implicate NRG1-mediated alterations in dopaminergic activities in the schizophrenia-associated behaviours of this model. We also discuss provisional similarities of the present dopaminergic pathophysiology to the mechanism of opioid addiction.In the present study, we characterized the long-term effects of perinatal eNRG1 pretreatment on the electrophysiological properties of adult nigral dopamine neurons by in vivo single unit recordings from anaesthetized adult controls and eNRG1-pretreated mice . No spike bursting was observed in slice conditions, consistent with previous studiesA receptor antagonist, picrotoxin , marginally reduced mean spike frequency in eNRG1 group  but not in control group . Accordingly, PTX perfusion abolished the frequency difference between eNRG1 and control groups (The shift in firing mode (an increase in bursting activity) may result from changes in intrinsic firing properties and/or changes in synaptic inputs onto dopaminergic neurons. To distinguish these possibilities, we performed single unit recordings in midbrain slice preparations. Dopamine neurons in slices from eNRG1-pretreated mice exhibited a significantly higher mean spike frequency than dopamine neurons in control slices  . The ampA receptors.To explore the mechanism(s) underlying these decreases in inhibitory synaptic events, we measured GABA-induced inward currents in dopaminergic neurons of midbrain slices . SuperfuIh (IAHP) , membrane resistance (Rm) or membrane capacitance (Cm) between control and eNRG1-pretreated mice . We alsoIh , and an h (IAHP) , both ofin vivoTo assess the contribution of excitatory neurotransmission to the irregularity of dopaminergic firing in vivo; (2) higher spike frequency in vitro which was abolished by pharmacological blockade of the GABAA receptor; (3) less remarkable impact of in vivo microinfusion of a GABAA receptor agonist; (4) no changes in membrane characteristics ; (5) decreases in mIPSC amplitudes and frequencies, (6) a substantial decline of GABAA receptor agonist-evoked currents, and (7) no significant differences in NMDA effects in slice. These results suggest that perinatal NRG1 treatment results in the reduction in synaptic GABA neurotransmission, which presumably involves the decreased channel activity of GABAA receptors in these neurons and may contribute to the elevated burst firing activity of dopamine neurons in vivo. The in vivo and in vitro effects of eNRG1 on firing properties of dopaminergic neurons were both stimulatory for their function but qualitatively differed. Thus, this controversy remains to be characterized further.Our previous reports demonstrated that NRG1 penetrates the immature blood–brain barrier following subcutaneous administration to mouse pups, directly acts on ErbB4 receptors in the midbrain and promotes terminal arborization of midbrain dopaminergic neurons78in vivo effects on dopaminergic bursting may include extrinsic influences as well (see below). As we failed to find any significant influence of eNRG1 on Ih or IAHP , as well as on NMDA-receptor sensitivity, the intrinsic influences of eNRG1 appear to be limited to GABAA receptor function. These pharmacological results from the GABAA receptor agonist and antagonist agree with the decreases in mIPSC amplitudes, although the decreases in mIPSC frequency may implicate the additional extrinsic influence of inhibitory afferents.Given the enrichment of the ErbB4 receptor in midbrain dopaminergic neurons, we postulate that the observed intrinsic effects on GABA receptor sensitivity mainly represent the direct actions of NRG1 on nigral dopamine neurons, whereas the et al.A receptorsA receptor mRNAs. This down-regulation appeared to require prolonged treatment with NRG123Burst firing was observed many weeks after eNRG1 exposure, while acute eNRG1 application had no significant effects on the spiking properties of dopaminergic neurons . Why didin vivo and in vitro dopaminergic neurons are regulated by intrinsic channel properties as well as by extrinsic synaptic inputsIh and GABAergic synaptic inputs but also by NMDA receptors and several potassium channels1921262728293031in vitro recording. The denervation of major excitatory inputs to nigral dopamine neurons may illustrate the observed controversy that the eNRG1 effects in vitro appeared in firing rates but not in burst rates24in vivo data. In this context, the impact of the local disinhibition on nigral dopamine neurons remains to be compared with that of GABAergic afferents distantly projecting to these cells.Firing patterns and excitability of A similar phenomenon was reported in response to local infusion of another neurotrophic factor, glial derived neurotrophic factor (GDNF), for which specific receptors are also enriched in midbrain dopaminergic neurons. The GDNF-induced enhancement of dopaminergic firing results from the alteration of GABAergic afferents as well as from postsynaptic GABA sensitivity28333537Disinhibition of midbrain dopaminergic neurons is also strongly implicated in the pathophysiology of opioid addiction424445141547Pregnant C57Bl/6NCr mice (at gestation day 15 or 16) were purchased from SLC . Experimental pups were administered daily subcutaneous (s.c.) injections of the human recombinant EGF-like core domain of NRGβ1  from postnatal (P) days 2–10et al.Male mice  were anaesthetized with an intraperitoneal (i.p.) injection of trichloroacetaldehyde monohydrate  and placed in a stereotaxic apparatus  with an auxiliary ear bar for mice . Body temperature was maintained at 36–37 °C using a heating pad. The skull overlying the midbrain was removed [Anterior–Posterior (AP) −2.4 to −3.8 mm from bregma; Medial–Lateral (ML) 0.2 to 1.6 mm from bregma). Extracellular action potentials were recorded with glass microelectrodes filled with 2% Direct Blue 1  in 0.5 M NaCl. Electrode tips were broken to adjust the resistance to 10–18 MΩ. Extracellular potentials originating from putative dopaminergic neurons were identified by well-established criteria49Each dopaminergic neuron was recorded for 2 min after a 2-min stabilization period. After recording from each track, Pontamine Sky Blue dye was iontophoretically injected (−20 μA for 15 min). After cardiac perfusion of 10% formalin (Wako) in 0.1 M phosphate buffer, the brain was removed and fixed with the same solution. To determine the recording position, midbrain slices (100-μm thickness) were prepared and bright field images of dye deposits were photographed using a digital microscope with reconstruction mode . Stereotaxically determined ML levels were corrected by measuring the distance from the dye-injection site to the midline. We analysed cells located at ML 0.8–1.0 mm right, identified as the medial SNc and anterior parabrachial pigmented nucleus see 51.Spike units originated from dopaminergic neurons were identified by well established criteria, i.e., their rather longer duration; ≥1.1 msec from an onset to a negative peak and occasional bursting activities with lower firing ratesA double-barrelled theta glass microelectrode  was employed for iontophoretic application to a recording site. One of the barrel for drug application was filled with 10 mM muscimol Adult mice  were anaesthetized with halothane and decapitated. Horizontal midbrain slices (thickness: 400 μm) at the level of the interpeduncular fossa were prepared as described53Single unit recordings were obtained from the anterior region of the medial terminal nucleus of the accessory optic tract (MT), which contains dopaminergic cell bodies of the substantia nigra pars compactaIh activated by hyperpolarizing pulses  from a holding potential of −55 mV as well as by post hoc immunohistochemical analysis of biocytin labelled cells (see below).One or two midbrain horizontal slices (300-μm thick) were prepared from each mouse (10–15 weeks olds). Whole-cell patch-clamp recordings were made at room temperature from putative dopaminergic neurons located in the medial or anterior region of MTet al.mIPSCs were recorded at a holding potential of −60 mV using patch pipettes filled with a high chloride internal solution modified from Mansvelder B receptor antagonist CGP 52432 . After recording, slices were fixed and processed for immunostaining with anti-tyrosine hydroxylase (TH) monoclonal antibody to confirm somatic positions and the TH-immunoreactivity (dopaminergic phenotype) of biocytin-labelled cells.GABA currents evoked by superfusion of 1 mM GABA (Wako) were recorded at a holding potential of −55 mV in the presence of the GABAIAHP, unclamped action potentials were evoked by depolarizing pulses  from the holding potential to +40 mVTo elicit and measure Results are presented as mean ± S.E.M. Electrophysiological data from eNRG1-pretreated and vehicle-pretreated mice were compared by Mann–Whitney U tests and/or Kolmogorov–Smirnov tests. P < 0.05 was considered statistically significant. Group effects of coefficient variation values were estimated with an analysis of variance (ANOVA). Statistical analysis was performed using SPSS software .How to cite this article: Namba, H. et al. Perinatal Exposure to Neuregulin-1 Results in Disinhibition of Adult Midbrain Dopaminergic Neurons: Implication in Schizophrenia Modeling. Sci. Rep.6, 22606; doi: 10.1038/srep22606 (2016).


Juvenile Systemic Lupus Erythematous (JSLE) is characterised by auto-antibody production directed against nuclear auto-antigens. Toll-like receptors (TLRs) are pattern recognition receptors of the innate immune system responsible for initiating an immune response against invading pathogens. TLR 3, 7-9 have been studied in SLE due to their unique ability to detect nuclear antigen. Their expression is increased in JSLE and significantly associated with disease activity and anti-dsDNA titres. Upon recognition of an extracellular pathogen, neutrophils may release neutrophil extracellular traps (NETs) containing antimicrobial peptides to capture and neutralise pathogens. Nuclear material including DNA and histones comprise the major structural components of NETs and may act as a source of nuclear auto-antigen in SLE. The mechanism by which NETs may induce an auto-inflammatory response has not been elucidated. Our hypothesis is that neutrophil NETs are a source of nuclear auto-antigen in JSLE being detected through the TLR pathway leading to an auto-inflammatory response.To investigate whether NETs are able to activate the TLR pathway, using pIRAK1, a signalling protein specific to the TLR pathwayNeutrophils were isolated from children with JSLE and paediatric & adult non-inflammatory controls and were either left unstimulated or incubated with 100ng IFN-alpha for 2 hours. Induction of NETs was visualised using confocal microscopy. Extracellular DNA was measured using the Quant-iT Picogreen assay .PBMCs were isolated from healthy adult controls and either left unstimulated or induced with LPS, TLR7/TLR9 agonist, as a positive control, or NETs derived from JSLE, paediatric or adult control neutrophils with +/- IFN-α for 30 minutes; cell protein was then extracted. pIRAK1 protein expression was determined by Western blot and normalised to β-actin expression.Cells incubated with LPS (×1.4 fold), TLR 7 (×1.7) & 9 (×1.7) agonists and NETs showed increased pIRAK protein expression as compared to unstimulated PBMCs. PBMCs incubated with NETs containing higher concentrations of dsDNA showed a greater fold increase in pIRAK1 protein expression. Increased expression did not seem to be influenced by origin of NET.Neutrophil NETosis has been proposed as a potential mechanism for auto-antigen exposure in SLE and has been shown to be dysregulated in lupus and correlate with lupus nephritis. Here we have shown that NETs are capable of activating the TLR 7/9 pathway and suggest that this is one mechanism by which an autoimmune response is driven. We have shown this to occur in a dose dependent manner to dsDNA. This data adds to the growing body of evidence supporting the role of TLRs in JSLE and the potential benefit of using TLR inhibition therapy in JSLE.None declared.


Cimex lectularius L. has re-emerged as a serious and growing problem in many parts of the world. Presence of resistant bed bugs and the difficulty to eliminate them has renewed interest in alternative control tactics. Similar to other haematophagous arthropods, bed bugs rely on their olfactory system to detect semiochemicals in the environment. Previous studies have morphologically characterized olfactory organs of bed bugs’ antenna and have physiologically evaluated the responses of olfactory receptor neurons (ORNs) to host-derived chemicals. To date, odorant binding proteins (OBPs) and odorant receptors (ORs) associated with these olfaction processes have not been studied in bed bugs. Chemoreception in insects requires formation of heteromeric complexes of ORs and a universal OR coreceptor (Orco). Orco is the constant chain of every odorant receptor in insects and is critical for insect olfaction but does not directly bind to odorants. Orco agonists and antagonists have been suggested as high-value targets for the development of novel insect repellents. In this study, we have performed RNAseq of bed bug sensory organs and identified several odorant receptors as well as Orco. We characterized Orco expression and investigated the effect of chemicals targeting Orco on bed bug behavior and reproduction. We have identified partial cDNAs of six C. lectularius OBPs and 16 ORs. Full length bed bug Orco was cloned and sequenced. Orco is widely expressed in different parts of the bed bug including OR neurons and spermatozoa. Treatment of bed bugs with the agonist VUAA1 changed bed bug pheromone-induced aggregation behavior and inactivated spermatozoa. We have described and characterized for the first time OBPs, ORs and Orco in bed bugs. Given the importance of these molecules in chemoreception of this insect they are interesting targets for the development of novel insect behavior modifiers.Recently, the bed bug,  Cimex lectularius L. (Hemiptera: Cimicidae), are obligate hematophagous insects that have become a serious and growing global problem in the last decade Bed bugs, Control of bed bugs is primarily based on intensive application of a limited number of insecticides, mainly pyrethroids Aedes aegypti mosquitoes have roughly 2000 olfactory sensilla on each antenna flagellum and olfactory clues play a critical role in mosquito host-finding behavior Bed bugs have nocturnal habits and during day-time, they remain hidden and aggregated in cracks or crevices.This state of immobility is induced by aggregation pheromones present in frass and body secretions Odorants are thought to interact with at least two different classes of insect proteins when entering an olfactory sensillum: odorant-binding proteins (OBPs) and odorant receptors (ORs) Anopheles gambiae and the dengue mosquito, Ae. aegypti were activated in the presence of an Orco agonist, VUAA1, while this effect was inhibited by addition of the Orco antagonist VU0183254 , which suggested that Orco expression  in sperm is conserved across different insect orders In addition to olfactory organs, Orco is expressed in spermatozoa of mosquitoes and other insects. A recent study by Pitts et al. VUANT in . These aBed bugs were maintained at 25°C, 65±5% RH, and a photoperiod of 14∶10 (L:D) h. The bed bug colony was collected from a human dwelling in New Jersey City, NJ. Bed bugs were collected by a pest control company and the resident or owner of the property gave permission to collect bed bugs from the site. Insects were fed with a parafilm-membrane feeder containing defibrinated citrated rabbit blood  that was heated to 39°C with a circulating water bath Labia, antenna, maxillae and mandibles were dissected from 50 males and 50 females, placed in Trizol and stored at –80°C until ready for use. Total RNA was then extracted from these samples according to the W.M. Keck Foundation protocol. A Nanodrop 1000  was used to quantify total RNA concentration. Starting RNA quality was assessed visually by an RNA gel. RNA was pooled to approximately 500 nanograms of total RNA and was then used to prepare a library using the Illumina TruSeq RNA Sample Preparation Kit v2 according to the manufacturer protocol for low-throughput sample preparation, with a few exceptions. The protocol was followed beginning with Purify and Fragment mRNA through Normalize and Pool libraries. Libraries were indexed separately for multiplexing. Differences in the protocol and our preparation procedure were: using PCR strip tubes instead of PCR plates. The addition of step 26 in ligate adapters, transfer 20 µl of supernatant from each well to a new 0.3 ml PCR plate labeled with PCR barcode. Elute, Prime, Fragment mix was thawed and mixed into each well of the RBP plate on ice. Ligation mix was thawed on ice and mixed into each well of the ALP plate on ice.The resulting libraries were quantified using a Thermo Scientific Nanodrop 1000 and Agilent Bioanalyzer 2100 and sent for sequencing to the National Center for Genome Resources . The sequencing lab further analyzed the libraries and pooled them for sequencing on a HiSeq2000, 1×100 bp reads. Reads were deposited in the NCBI sequence read archive under accession number SRP018037.Rhodnius odorant receptors and odorant binding proteins against using tblastn.Reads were assembled in two ways; the first via assembly with Velvet with a kmer setting of 31 followed by minia, and the second by Trinity alone Rhodnius prolixus (RPRC000476-PA) was retrieved from Vectorbase and used to search the read database. Reads aligning to the Rhodnius Orco sequence were then assembled with CAP3 Reads obtained from sequencing were used to construct a blast database. The Orco protein sequence from D. melanogaster, R. prolixus and A. aegypti (retrieved from Genbank) with Clustal Omega and highlighted using the sequence manipulation suite. Transmembrane domain regions were manually annotated using the regions described for DrosophilaTo create Ixodes scapularis, retrieved from Genbank Schistocerca gregaria gi|371444780, Locusta migratoria gi|371444778, Lygus hesperus gi|421991706, Rhodnius prolixus RPRC000476-PA (vectorbase), Drosophila melanogaster gi|14285640, Aedes aegypti gi|157111190, Anopheles darlingi ADAR011157 (vectorbase), Culex quinquefasciatus gi|167869857, Glossina moristans GMOY005610 (vectorbase), Pediculus humanus corporis gi|212509555, Stomoxys calcitrans gi|193795139, Chrysomya rufifacies gi|384503150, Musca domestica gi|384503152, Lucilia sericata gi|440631705, Haematobia irritans irritans gi|193795141, Bactrocera cucurbitae gi|302171921, Tribolium castaneum gi|226334904, Ixodes scapularis gi|215504237.To create C. lectularius, downloaded 3/19/2013 A blast database was created using scaffolds from the i5k sequencing project for TCA TGT CCC TGA GCC ATG CAA ACT; qCLRPL18R: GCT TCG TGT GCG AGC GAG GGG; qCLOrcoF: TCA TGT CCC TGA GCC ATG CAA ACT; qCLOrcoR: TGG CAC TAC AGA ATC CAA AGC TGC A.Total RNA was obtained from adult male and female bed bugs using TRIzol reagent . RNA from different tissues was isolated from 30 unfed males and 30 unfed females. Relative transcript levels were quantified on an Eppendorf Mastercycler ep realplex  using PerfeCTa SYBR Green FastMix . Primers were as follows: qCLRPL18F: The Orco agonist VUAA1 2 and all antennal segments  were removed with fine-tip forceps 24 hours before the bioassay. For the analysis, we focused our attention on two potential behavioral responses; the proportion of individuals wandering the arena and the proportion of individuals that preferred the pheromone tented area if they chose a tented area. These two responses act at different scales for the bed bugs. The first shows an ability to find the areas of interest, while the second investigates how well the pheromone signal is perceived by the treated individuals. The data were non-normal and were rank transformed and assayed with a MANOVA followed by separated ANOVAs for interpretation. A Tukey's Studentized Range (HSD) Test was conducted on the separated ANOVAs to compare between treatments Male bed bugs were placed in individual cells of 6–well cell culture plates . The bottoms of the cells were covered with tightly fitting filter papers. Insects were temporarily immobilized by placing plates on ice for ∼5 min. 100 male bed bugs were used for each experimental group. Topical applications (2 µl) of Orco agonist  or antagonist  were made onto the dorsal surface of the abdomen. We chose delivery to the abdomen because we found that dipping bed bugs head first in acetone solution interfered with their sense of olfaction for several hours. Similar delivery methods have been successfully used to deliver other lipophilic molecules into insect hemolymph, for example juvenile hormone Each group of 25 virgin male bed bugs was temporarily immobilized by placing plates on ice for ∼5 min and topical applications (2 µl) of 35 mM VUAA1 or acetone were made onto the dorsal surface of the abdomen. Each individual male was placed with a recently fed female for mating. After mating was observed (at least one mating occurred within one hour) the female was transferred to individual cells of 24-well cell culture plates whose bottom was covered with tightly fitting black filter paper to facilitate egg count. After six days, number of eggs was recorded and the females were given another blood meal. Females remained at 25°C, 65±5% RH for two weeks and egg number, hatch rate and nymph viability was recorded at the end of this period. For the analysis, we removed replicates where individuals either did not blood feed at a time point or never produced eggs. This resulted in 18 replicates for the 35 mM VUAA1 treatment and 22 replicates for the acetone treatment. The data for eggs produced at day 6 and day 14 as well as the number of hatchlings at day 14 was normal and assayed with a MANOVA followed by separate ANOVAs for interpretation 2, 1.3 mM CaCl2, 5 mM D-glucose, 10 mM HEPES pH 7.4 containing 10% [v/v] DMSO) with or without indicated compound was immediately put on the specimen. A coverslip was placed on the specimen and gently pressed to expose the sperm to the assay buffer. Sperm movement was observed under phase-contrast compound microscope  equipped with digital camera , image was video-fed by INFINITY ANALYZE software (Lunenera Corp.), and screen video was recorded by QuickTime Player . To assess drug (VUAA1) delivery to the seminal vesicles via topical application, male bed bugs were applied on abdominal tergites with 2 µL of 35 mM VUAA1 in acetone or acetone alone as control. The bugs recovered at room temperature for 5–10 min, and seminal vesicles were dissected for the assay. Experiments were repeated six to ten times. Sperm activity was assessed and scored a qualitative “motility index” (MI) similar to “activation index” described in Pitts et al. Since most bed bug sperm can be found in the seminal vesicles, we used sperm in seminal vesicles for our assay. Seminal vesicles were dissected and kept in 1× PBS on ice on the day of the experiment. Since a seminal vesicle is sufficiently large, one seminal vesicle was used for multiple assays which gave us an advantage that sperm from the same seminal vesicle could be used for different treatments . A part of seminal vesicle was transferred to a microscope slide and 20 µL of assay buffer  at 1∶500 dilution in PBTG at room temperature for 3 h. After washing the sections in PBT three times at room temperature for 5 min, sections were mounted with ProLong Diamond Antifade Mountant with DAPI (Life Technologies), coverslips were put on the slides, sealed with nail polish and cured at room temperature in dark condition overnight. Sections were imaged by Leica TCS SP5 II confocal microscope (Leica). Control and treatment images were acquired with the same settings.Male head and reproductive system were dissected and fixed with 4% paraformaldehyde (PFA) in PBT (0.1% Triton X-100 in PBS) overnight at 4°C. The fixed samples were washed in PBS three times for 5 min at room temperature and cryoprotected with 30% sucrose in PBT overnight at 4°C. Heads and reproductive organs were frozen in Tissue-Tek OCT compound , and 10 µm thickness sections were made with Leica CM1850 cryostat microtome  and mounted on Superfrost Plus microscope slides . The slides were kept at –80°C until used. Sections were dried at room temperature for 30 min and fixed in 4% PFA in PBT for 30 min. The slides were washed in PBT three times for 5 min. After blocking in 5% goat serum in PBT (PBTG) for 1 h at room temperature, the sections were incubated in anti-Our six libraries produced a total of 190.6 million, 100 cycle reads that passed quality control. Our assemblies generated 10,309 contigs with an N50 of 1,133 nucleotides using the velvet-minia assembly program and 219,383 contigs with an N50 of 817 using Trinity.Tribolium sequences. Analysis of our Trinity contigs using blast yielded 12 contigs which matched against 6 of 13 R. prolixus OBPs that are annotated in Vectorbase  annotated in Vectorbase, somewhat fewer than the number of ORs in Drosophila (62) and An. gambiae (79), while significantly less than A. aegypti (131) Based on these results we hypothesize that we have identified partial cDNAs of at least six Rhodnius Orco protein sequence. Using this in silico sequence, we assembled a putative cDNA. Primer covering the full length open reading frame were developed and used to amplify full-length Orco. The PCR product was cloned and sequenced and we published the nucleotide sequence in Genbank (GenBank accession number KM275232). Cimex lectularius Orco is a 451 amino acid protein, the TMHMM software An. gambiae Orco, AgOR7 An. gambiae and D. melanogaster Orco which have eight and six exons, respectively, and the three C-terminal exons with identical size An. gambiae or D. melanogaster Orco.Using BLAST, we mapped 22 reads against the After mapping our sequenced full length Orco cDNA to the i5k genome sequence, we identified a premature stop codon located in exon three of the i5k Orco sequence. This result in a predicted protein with only 209 amino acids in the i5k genome data compared to a 451aa sequence from our cloning. We hypothesize that this might be due to a sequencing or assembly mistake.Cimex Orco is most closely related to that of Rhodnius (89.8% identity) and other members of Hemiptera (true bugs), vs. other orders of insects. Amino acid sequence identity with D. melanogaster Orco was 59.1%. The phylogenetic tree shown in The amino acid sequence of Orco is highly conserved in insects with the highest degree of identity in the C-terminus of the protein . Of the Drosophila Orco antibody to detect Orco protein expression in antennae and sperm by immunofluorescence. Orco is expressed in the antennae, and localized in a cluster of cells that most probably represent olfactory receptor neurons (ORN) (C. lectularius sperm cells An. gambiae (We used an anti-ns (ORN) . Orco exns (ORN) . Hemipte gambiae . For mor gambiae  and 3D m gambiae  are provDrosophila legs An. gambiae, another holometabolous insect, G-Protein Receptor (GPR) odorant receptors were found to be expressed in olfactory tissues and legs Locusta migratoria. Mouthparts, tarsi and wings also show expression of Orco, demonstrating a ubiquitous expression in varying tissues throughout L. migratoriaQuantitative-RT-PCR was used to determine Orco expression in unfed adult male and female bed bugs. Orco is highly expressed in the antennae and legs of the female bed bugs with slightly reduced expression in reproductive organs, head, and the gut . There wDrosophila melanogaster and mosquito species (An. gambiae and Ae. aegypti) and interact with these insect odorant receptors The chemicals VUAA1 and VU0183524 have been demonstrated to be agonist and antagonist, respectively, for Orco in 10,212 = 16, P<0.001) with shifts in both the proportion of individuals making a choice  and the proportion of individuals preferring the pheromone area having made a choice . At the lowest doses of VUAA1 (25 mM), there was not a significant difference present as compared to the control in either of the two responses . The number of eggs produced  and the number of hatched nymphs  was always similar between the two treatments (The application of (2 µl) of 35 mM VUAA1 or acetone did not collectively alter mating efficiency  T. infestans that has a broader host spectrum ranging from rattlesnakes to birds to humans In this study we present a series of experiments designed to identify odorant binding proteins, odorant receptors and odorant receptor co-receptor (Orco) from sensory organs of a medically important urban pest, We have described and characterized the first time odorant receptor co-receptor (Orco) in a hemimetabolous insect. The striking conservation of Orco observed across insect taxa is an important feature when proposing a role for these genes in olfactory signaling in insects. We also observed expression of Orco in various bed bug organs, which indicate that Orco is functionally required for a broad range of chemosensory driven behavior. We evaluated the potential role of Orco in sperm motility with an Orco agonist and an Orco antagonist. Assays with the agonist VUAA1 showed that, contrary to what occurs in mosquitoes, sperm motility of bed bug males is dramatically reduced, however no effect on fecundity was observed. The mechanism for reduction of sperm motility is unclear and further work with other agonists and antagonists is needed to fully elucidate the role of Orco in bed bug sperm function and explore new ways to interfere with bed bug reproduction. We also explored the potential of an agonist an antagonist for disrupting aggregation behavior in bed bugs. Our results showed that the Orco agonist VUAA1 changed bed bug pheromone-induced aggregation behavior. The results of this study suggest that bedbug Orco is a high-value target for the development of novel behavior modifiers for bedbug control.Figure S1These panels are larger version of. A, B, C correspond to, respectively. Red arrowheads in E indicate a portion of a flagellum. Scale bar, 10 µm.(TIF)Click here for additional data file.Table S1Contigs of OBP genes potentially involved in olfaction.(XLSX)Click here for additional data file.Table S2Contigs of OR genes potentially involved in olfaction.(XLSX)Click here for additional data file.File S1Synthesis of VUAA1 and VU0183524.(DOCX)Click here for additional data file.Video S13D projection of multiple optical sections of seminal vesicle shown in . Continuous Orco labeling (green) along flagella is visible.(MOV)Click here for additional data file.Video S2Control bed bug sperms in assay buffer.(MOV)Click here for additional data file.Video S3Bed bug sperms treated with 10 mM VUAA1 in assay buffer.(MOV)Click here for additional data file.


P < 0.05). Univariate analysis showed a significant association between the decreased expression of PTPN12 and adverse overall survival and disease-free survival (P < 0.05). More importantly, multivariate analysis identified the PTPN12 expression in NPC as an independent prognostic factor. The decrease expression of PTPN12 might be important in conferring a more aggressive behavior in NPC. Thus, PTPN12 expression may be used as a novel independent prognostic biomarker for patients with NPC.Tyrosine-protein phosphatase nonreceptor type 12 (PTPN12) has been proposed to predict prognosis of various human cancers. However, the clinicopathologic and prognostic significance of PTPN12 expression in NPC has not yet been elucidated. The objective of this study was to investigate the clinicopathological and prognostic implication of PTPN12 in nasopharyngeal carcinoma (NPC) patients. Protein expression levels of PTPN12 were explored by semiquantitative immunohistochemical staining on archival formalin-fixed, paraffin-embedded pathological specimens consisting of 203 NPCs, and 40 normal nasopharyngeal mucosa tissues. Receiver operating characteristic (ROC) curve analysis was employed to determine the cutoff score of PTPN12 expression in NPCs. The PTPN12 immunohistochemical staining results were then correlated with various clinicopathological features and patients’ prognosis using various statistical models. Our results showed that decreased expression of PTPN12 was more frequently observed in NPC tissues compared with the normal nasopharyngeal mucosa. Further correlation analyses indicated that the decreased expression of PTPN12 was significantly associated with tumor T classification, N classification, distant metastasis, and clinical stage in NPCs ( Nasopharyngeal carcinoma (NPC) is the most common cancer originating from the nasopharynx, which is prevalent in south-east Asia, especially in southern China, where it constitutes a significant health burden . Most ofTyrosine-protein phosphatase nonreceptor type 12 (PTPN12) is one of ubiquitously expressed cytosolic protein tyrosine phosphatases (PTPs) family with an amino (N)-terminal PTP domain and a carboxyl (C)-terminal region involved in protein–protein interactions . PTPs plThe study was approved by the Institute Research Medical Ethics Committee of Sun Yat-sen University. No informed consent  was obtained for use of retrospective tissue samples from the patients within this study, most of whom were deceased, since this was not deemed necessary by the ethics committee, who waived the need for consent. All samples were anonymized.In this study, 203 specimens of NPC were collected in Sun Yat-sen University Cancer Center and in Guangdong Provincial People’s Hospital, Guangzhou, China, between January 1991 and August 2000. The selection of cases was based on the following criteria: pathologically confirmed as nonkeratinizing carcinoma of nasopharynx  with available biopsy specimens for tissue microarray (TMA) construction; no previous malignant disease or a second primary tumor; without radiotherapy, chemotherapy history before biopsy; Karnofsky ≥70; received radiotherapy (RT), induction chemotherapy/radiotherapy (IC/RT), or induction chemotherapy/chemoradiotherapy (IC/CRT) regimen; and follow-up regularly. Patients with unavailable biopsy tissues for constructing TMA were excluded from our study to provide adequate samples for pathological diagnosis. We chose 203 primary NPCs from the Department of Pathology of our institutes, and 40 samples of normal nasopharyngeal mucosa were used for controls. The routine staging workup was composed of a detailed physical examination, including fiber optic nasopharyngoscopy, magnetic resonance imaging (MRI) of the entire neck, chest X-ray, abdominal ultrasonography, a complete blood count, and a biochemical profile. The clinical stage was defined on the basis of the International Union Against Cancer (UICC) and the American Joint Committee on Cancer (AJCC) . The InsThe response of RT or CRT was assessed clinically for primary lesion based on fiber optic nasopharyngoscopy and MRI 1 month after treatment. In our study, the patients were followed every 3 months for the first year and then every 6 months for the next 2 years and finally annually, thereafter. The diagnostic examinations consisted of fiber optic nasopharyngoscopy, MRI, CT, chest X-ray, abdominal ultrasonography, and bone scan when necessary to detect recurrence and/or metastasis. The total follow-up period was defined as the time from diagnosis to the date of death or the last date censored if patients were still alive.TMA was constructed in accordance with a previously described method . In brieFormalin-fixed, paraffin-embedded NPC samples were cut into 4-μm thick sequential sections and processed for immunohistochemistry (IHC) according to the previously described protocol . The TMAAccording to our previous study , proteinThe plot of sensitivity versus 1-specificity across varying cutoffs generates a curve in the unit square called an receiver operating characteristic (ROC) curve, the optimal cutoff value can be determined using ROC curve analysis by the point  or  , 19, at P value of less than 0.05 was considered as statistically significant in all cases.Statistical analyses were performed using SPSS software, version 16.0 . The correlation between PTPN12 expression and clinicopathological features of NPC patients was assessed by Chi-square test. Univariate analysis of overall survival  and disease-free-survival (DFS) data were performed using the Kaplan–Meier method. The Cox proportional hazards regression model was used to identify the independent prognostic factors. A two-tailed The clinicopathological characteristics of NPC patients were detailed in Table Since the IHC scores were evaluated semiquantitatively, in our study, we utilized ROC curve analysis to avoid the use of predetermined and often arbitrarily set cutoff values. ROC curves are commonly used in clinical oncology to evaluate and compare the sensitivity and specificity of diagnostic tests. Moreover, they allow one to identify the threshold value above which a test result should be considered positive for some outcome Fig. . In immuP < 0.001).To select an optimal PTPN12 cutoff score for further analysis, the ROC curves for each clinicopathological feature  on NPC patients’ survival rates  of patients with normal PTPN12 expression was significantly higher than that of patients with decreased PTPN12 expression (55 %), suggesting the patients with normal expression of PTPN12 had better prognosis than those with decreased expression of PTPN12.NPC is characterized by poorly or undifferentiated carcinoma. It differs from nonnasopharyngeal head and neck squamous cell carcinomas in several ways, including its association with the Epstein–Barr virus (EBV) and strong sensitivity for radiotherapy and chemotherapy. High death rate is mainly due to tumor metastasis despite the new treatment that combines radiotherapy with chemotherapy . ClinicaPTPN12 is one of the PTPs families regulating the equilibrium of tyrosine phosphorylation and plays a prominent role in tumor suppression. The PTPN12 protein is thought to act as an important regulator in controlling cell adhesion, motility, and metastasis by interacting with and inhibiting multiple oncogenic tyrosine kinases . RecentlWe found that the expression of PTPN12 protein could be influenced by some factors including missense mutation of PTPN12 gene . This liIn summary, our study showed that the examination of PTPN12 expression by IHC could serve as an effective tool in identifying those NPC patients at increased risk of tumor invasiveness and metastasis, and also elaborate decreased PTPN12 expression as a novel adverse independent prognostic factor in NPC, which may help us find new therapeutic target.


Methanocella paludicola) NHEJ nuclease and polymerase enzymes, demonstrating their strict structural conservation with their bacterial NHEJ counterparts. Structural analysis, in conjunction with biochemical studies, has uncovered the molecular basis for DNA strand displacement synthesis in AP-NHEJ, revealing the mechanisms that enable Pol and PE to displace annealed bases to facilitate their respective roles in DSB repair.The non-homologous end-joining (NHEJ) pathway repairs DNA double-strand breaks (DSBs) in all domains of life. Archaea and bacteria utilize a conserved set of multifunctional proteins in a pathway termed Archaeo-Prokaryotic (AP) NHEJ that facilitates DSB repair. Archaeal NHEJ polymerases (Pol) are capable of strand displacement synthesis, whilst filling DNA gaps or partially annealed DNA ends, which can give rise to unligatable intermediates. However, an associated NHEJ phosphoesterase (PE) resects these products to ensure that efficient ligation occurs. Here, we describe the crystal structures of these archaeal ( Cellular life must overcome a vast array of undesirable genomic alterations in order to replicate and divide. One of the most catastrophic types of DNA damage are double-strand breaks (DSBs) . There aThe canonical mechanism of NHEJ in mammalian cells is initiated by the binding of the Ku70/80 heterodimer to the broken DNA ends, along with the DNA-dependent protein kinase catalytic subunit (DNA–PKcs), reviewed in references  and 4)..4). Ku70Mycobacterium tuberculosis (Mtu) AP–NHEJ complex is the most extensively characterized system  demonstrated that both species possess equivalent core AP–NHEJ components, with orthologous proteins possessing near identical substrate specificities  Pol and PE with those of the established bacterial (M. tuberculosis and P. aeruginosa) DSB repair complexes .We recently elucidated a role for PE in AP–NHEJ, demonstrating that it acts as as a regulator of the polymerase activity. We discovered that Pol has DNA strand displacement activity during RNA synthesis and that PE resects excessive incorporation to promote efficient break ligation . In thisomplexes ,16,20,21Mpa PE was purified as described in . The diffraction data were processed with SCALA (Full-length ribed in . 0.7 μl th SCALA  with addth SCALA . The staMpa Pol was purified as described in (Mpa Pol were collected in-house on a Rigaku Micromax 007-HF. The diffraction data were processed with SCALA (Full-length ribed in . 0.7 μl th SCALA  with addth SCALA . The staMpa PE was determined by molecular replacement using the program PHASER (Pseudomonas aeruginosa PE (PDB: 3N9B) was used as a molecular replacement search model  was used as a molecular replacement search model. Initial refinement was carried out against 95% of the data with REFMAC5  of the overexpression plasmids for wild-type (wt) Mtu PolDom and Mpa PE, respectively (mutagenesis primers listed in Supplementary Table S1). The DNA constructs were then sequenced to verify accurate mutation and subsequently transformed into E. coli B834(DE3)pLysS. Purification of all Mtu PolDom proteins was performed as described in  and then boiled at 95ºC for 10 min. The DNA products were separated by electrophoresis in 8 M urea-containing 15% polyacrylamide gels in 1X TBE buffer for 2 h. Fluorescently labelled DNA and DNA/RNA oligomers were detected by scanning using a Fujifilm FLA-5100 fluorescent image analyser.RNA extension of DNA primers was performed as described in . BrieflyRibonuclease and phosphatase assays were performed as described in . BrieflyM. paludicola phosphoesterase (Mpa PE), which is operonic with an NHEJ RNA/DNA primase–polymerase and ligase, is a functional component of the archaeal NHEJ repair apparatus. Mpa PE remediates DNA displacement synthesis by the NHEJ polymerase, enabling efficient ligation of broken DNA ends to restore the integrity of the genome. To understand more about the molecular mechanism of these atypical nucleases, we elucidated the crystal structure of Mpa PE at a resolution of 1.79 Å . Th. ThM. pa10 helix forming one side of the active site . Allflexible . The arcflexible . This stMpa PE was used as a template to identify other potential AP–NHEJ PEs using PSI-BLAST. We aligned some known bacterial and archaeal PEs with several putative bacterial, archaeal, fungal and plant PEs that were identified during our search  may be one of the key residues in positioning the scissile phosphate in the active site . Next, we assayed this mutant for its ability to perform phosphomono- and diesterase activities on DNA/RNA primers annealed to a DNA template. The reaction scheme is shown in Figure H82A was completely unable to cleave the DNA/RNA substrate, in contrast to the wt Mpa PE control  in the absence of this histidine  during RNA synthesis to incorporate up to 3 or 4 bases  hydrogen bonds with the 5′-phosphate (5′-P) of the downstream DNA strand (D-strand)  (K16A). We then compared the displacement activities of wt PolDom and PolDomK16A on four different DNA substrates   . This doure S3B) . We hypo) Figure , B and CF63A and PolDomF64A mutants are capable of filling in 1 nt and 2 nt gaps but both mutants showed reduced displacement synthesis compared to wt Pol, especially on the nicked substrate. However, neither of these residues appears critical to displacement synthesis, since the activity still occurs to some degree in their absence, even on DNA nick substrates. Given the combined template-splaying activity of these phenylalanine residues, it is difficult to unpick their role in DNA extension from a potential role in displacement synthesis. We conclude that F63 and F64 may encourage the separation of the downstream dsDNA but are not essential for the process since it still occurs in a limited manner after their substitution to alanine.As previously reported, loss of the prominent base stacking residues F63 and F64 significantly reduces, but does not ablate, strand synthesis Figure  and E 3. PolDomFP55A) had little impact on the activity , whilst loss of the arginine (PolDomR53A) produced the most striking results of all the mutants with all three gapped and nicked DNA substrates  but R53A did not add any additional nucleotides to ‘over-fill’ gapped DNA, and it was unable to insert any nucleotides in the nicked DNA substrate. Together, these results establish that R53 plays a pivotal role in strand displacement synthesis, likely acting to disrupt the annealed downstream DNA strand. Examination of the PolDom–DNA complex structure revealed that R53 intercalates into the annealed DNA interface, making hydrogen bond contact with the ribose of the ultimate base of the D-strand  enable these polymerases to directly promote break synapsis by MMEJ. During this break annealing process, each side of the DSB is bound by a polymerase and the surface loops promote break synapsis . In the in trans ,37. The Mtu Pol), that contacts the annealed juncture of 5′-DNA end, plays a critical role in enabling the local unwinding of the DNA helix. This forms part of a molecular wedge that allows displacement synthesis activity to occur, resulting in ribonucleotide incorporation into the previously annealed duplex. An analogous process appears to be utilized by other polymerases to promote base unpairing (Thermococcus gorgonarius family B DNA polymerase (Tgo PolB) revealed that an arginine residue contacts the primer/template junction in a similar way to R53 in Mtu PolDom and, together with a neighbouring tyrosine side chain, disrupts base stacking on the primer strand. This allows strand displacement to occur that facilitates subsequent removal of misincorporated bases by the 3′ exonuclease activity of this replicative polymerase thus promoting proofreading. In the case of the AP–NHEJ polymerases, the arginine residue physically promotes separation of the dsDNA to allow for displacement synthesis activity.Both bacterial and archaeal NHEJ polymerases readily perform strand displacement synthesis, when operating on ‘gapped’ DNA substrates possessing a downstream strand. A conserved arginine residue (R53 in npairing ,39. A stThe structures of these NHEJ polymerases bound to DNA, combined with biochemical evidence, demonstrates that strand displacement activity is not an artifact but results from the presence of a prominently conserved wedge structure that induces a significant distortion in the template strand DNA that it is an intrinsic part of its synthesis mechanism. However, the exact requirement of this conserved displacement activity in DSB repair is not immediately obvious and, in fact, is a potentially dangerous activity to possess. One possibility is that displacement synthesis increases flexibility in DSB repair by increasing the variety of reconfiguration options to suit the potentially huge array of mismatched or damaged DNA ends that may occur following a break Figure . DisplacHowever, although these possibilities are feasible and may partially account for the necessity to perform displacement synthesis, they do not adequately explain the strong tendency of these polymerases to ‘over fill’ gaps at MMEJ-synapsed termini. A crucial step in polymerase-mediated MMEJ process is the formation of stably synapsed DNA intermediates. This is not an energetically unfavourable task when DSB overhangs are long and complementary and probably require minimal intervention to promote break annealing. However, MMEJ can occur using homologies as little as 1–2 base pairs and, in such scenarios, these intermediates can readily unanneal if this ‘foothold’ is not reinforced. It is likely that strand displacement synthesis plays an important role in stabilizing such unstable MMEJ intermediates. This process can be likened to the procedure of bringing two sides of a zipper back together Figure . The firIt has recently been reported that Pol θ is required for MMEJ repair in mammalian cells . Human PMpa phosphoesterase and Mpa polymerase domains, respectively.Atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5DMP and 5DMU for the


Results obtained by the proposed method were compared with those using the pharmacopoeia reference method and significant difference was not observed. Therefore, proposed method allowed a fast, precise, and accurate determination of propranolol hydrochloride in pharmaceutical preparations. Furthermore, it is possible to carry out on-line analysis of this active principle in pharmaceutical formulations with use of fiber optic probe.A method for determination of propranolol hydrochloride in pharmaceutical preparation using near infrared spectrometry with fiber optic probe (FTNIR/PROBE) and combined with chemometric methods was developed. Calibration models were developed using two variable selection models: interval partial least squares (iPLS) and synergy interval partial least squares (siPLS). The treatments based on the mean centered data and multiplicative scatter correction (MSC) were selected for models construction. A root mean square error of prediction (RMSEP) of 8.2 mg g Therefore, the proposed method for propranolol hydrochloride allowed a suitable linearity when it is compared to the reference method.Correlation between the values using s2i20PLS model and the reference method was evaluated. Ideally the intercept “equation . For prot-test was performed between s2i20PLS model values and UV reference values. The experimental t value  was smaller than the critical t value  and the s2i20PLS model using FTNIR/PROBE data may be considered accurate.Accuracy was evaluated by comparison of results obtained using the s2i20PLS model with those obtained by UV reference method. Paired Additionally, accuracy was also evaluated using the weighted least square (WLS) to obtain the elliptical joint confidence region (EJCR). The WLS method was used with results obtained for s2i20PLS model (prediction set) to verify the accuracy and to check if the proposed methodology is free of systematic error. The WLS results include the theoretical expected value , indicating that the proposed methodology is accurate and free of systematic errors.Results obtained demonstrated that FTNIR/PROBE associated with multivariate analysis is a convenient method for propranolol hydrochloride determination in pharmaceutical preparations. Results obtained using siPLS models for determination of propranolol hydrochloride in powder pharmaceutical preparations showed suitable prediction capacity (lower RMSEP). Variable selection methods were able to produce better models in comparison to the PLS full-spectrum model. Results obtained for propranolol hydrochloride using the better siPLS (s2i20PLS) model were in agreement with results obtained by reference method. The proposed procedure using FTNIR/PROBE and PLS algorithms is faster, less expensive, accurate, and precise and minimizes solvent use when compared to pharmacopoeia UV method.


A133 HF mice promotes endocardial thrombosis in the acute decompensated phase of the disease, and whether alterations in von Willebrand factor (vWF) secretion from HF endocardium reduces thrombus formation as HF stabilizes.An experimental model of endocardial thrombosis has not been developed and endocardial endothelial dysfunction in heart failure (HF) is understudied. We sought to determine whether disruption of the endothelial anti-coagulant activated protein C (APC) pathway in CREBA133 mice. Endocardial thrombi incidence was confirmed with immunohistochemistry and histology. In early and acute decompensated phases of HF, CREBA133 mice had the highest incidence of endocardial thrombi and these mice also had a shorter tail-bleeding index consistent with a pro-thrombotic milieu. Both APC generation, and expression of receptors that promote APC function , were suppressed in the endocardium of acute decompensated HF mice. However, in stable compensated HF mice, an attenuation occurred for vWF protein content and secretion from endocardial endothelial cells, vWF-dependent platelet agglutination (by ristocetin), and thrombin generation on the endocardial surface.Echocardiography was used to follow HF development and to detect endocardial thrombi in CREBA133 mice develop HF and endocardial endothelial dysfunction. Attenuation of the anti-coagulant APC pathway promotes endocardial thrombosis in early and acute decompensated phases of HF. However, in stable compensated HF mice, disruptions in endothelial vWF expression and extrusion may actually reduce the incidence of endocardial thrombosis.CREB Some 60% of heart failure (HF) patients suffer thromboembolic complications that are a leading cause of mortality and morbidity . ThromboA133 mice that have a dominant negative mutation (Ser-Ala133) in the cyclic AMP response element binding protein (CREB) within cardiomyocytes. As a result of this mutation, CREB-dependent transcriptional events within cardiomyocytes are severely disrupted, mitochondrial dysfunction occurs [A133 model is also unique in that direct chemical, infective or ischemic damage is not required for endocardial thrombi formation. Further, this model has significant advantages over surgical ligation models that have inherent variability in infarct size as well as the confounding effects of inflammatory cells and cytokines. We have used these CREBA133 mice to systematically investigate the link between progressive endocardial dysfunction and thrombus formation.We focus in this study on CREBn occurs , and then occurs , 18. As n occurs . This mon occurs , 20. TheEndocardial endothelial cells (EECs) that line the cardiac chambers share many properties with vascular endothelium but they differ in that they have distinct developmental origins, have an abundance of gap junctions (sensory function), and are critical for cardiomyocyte peak force development through secretion of cardioactive factors (e.g. endothelin-1 and nitric oxide) , 22. AltThe endothelium is normally kept thrombus free through generation of activated protein C (APC) on its luminal surface , 24. APCControlling APC generation is one of the key mechanisms used by endothelial cells to suppress the coagulation cascade; these cells can also promote coagulation through their synthesis and release of von Willebrand factor (vWF). When large multimeric vWF chains are released from intracellular Weibel-Palade bodies, platelets more easily adhere to the endothelial surface . DysfuncThis study introduces an animal model in which early, acute decompensated, and stable compensated stages of HF can be investigated. Using the model we tested two hypotheses: 1) A prothrombotic endocardial surface characterizes the early and acute decompensated phases of HF as APC generation is attenuated and vWF synthesis and secretion are augmented. 2) The incidence of endocardial thrombus formation is reduced in stable compensated HF mice.A133 mice that have a dominant negative mutation (Ser-Ala133) in the cyclic AMP response element binding protein (CREB) within cardiomyocytes . CREB-dependent transcriptional events within cardiomyocytes are severely disrupted in these mice resulting in idiopathic dilated cardiomyopathy and eventual HF [A133 mice developed acute decompensated symptoms of congestive HF between 10–20 weeks (acute decompensated HF phenotype) with visible thoracic and abdominal anasarca, labored breathing, decreased activity, and dilated ventricular chambers and nutmeg liver on necropsy. HF mice with the acute phenotype were euthanized 24–48 hr after onset of significant anasarca as otherwise they were prone to sudden cardiac death. From 10–14 weeks, a mild form of the disease existed (early HF phenotype). CREBA133 mice that did not develop the acute HF phenotype were sacrificed between 20–24 weeks (chronic compensated HF phenotype). CREBA133 mice were on a CD-1 background, and wildtype CD-1 breeder females were obtained from Charles River Laboratories . Both male and female mice were used in this study. Since evidence exists that HF phenotype may be more extreme in female CREBA133 mice [A133 mice served as controls. No significant differences were seen between wildtypes from different ages and their data is presented as a combined pool. All mice were housed in a temperature controlled room (22±2°C) set to a 12-hr dark/light cycle and provided chow and water ad libitum. All experimental protocols were conducted in agreement with the NIH’s Guide for the Health and Use of Laboratory Animals, and approved by the Oregon Health & Science University Institutional Animal Care and Use Committee (protocol: IS00001994).The heart failure (HF) mice used in this study were transgenic CREBntual HF , 18. Abo133 mice , we wered) and end-systole (LVIDs) at the minor axis were measured at the mid ventricular level. Left ventricular ejection fraction (LVEF) was calculated from cavity and wall dimensions as previously described [Transthoracic echocardiography  was performed in the parasternal long axis and mid-ventricular short axis planes using a high-frequency (40MHz) phased-array transducer (n = 7–9 per group). Mice were anesthetized under isoflurane and, in order to measure dimensions precisely at end-systole and end-diastole, two-dimensional acquisitions were performed at a high frame  by ECG gating of sequential M-mode sweeps. Diastolic dimension of the septal and posterior walls, and the LV internal diameter at end-diastole (LVIDescribed . Stroke Hearts (n = 6 per group) were fixed in 4% paraformaldehyde, cryoprotected then cryosectioned in 3-stepped series of 8 slides per set  through the ventricles at the basal, medial and apical aspects giving a representative cross-section through the ventricles. One set of slides (n = 6 per group) were double immunostained with antibodies directed against vWF  and fibrin β-chain . A second set was doublestained for CD31  and vWF/fibrin/urokinase plasminogen activator . Images were visualized with a confocal microscope (Nikon A1R) with orthogonal projection of z-stack images carried out with NIS Elements software . Quantitation of vWF immunostaining was carried out with ImageJ (NIH); threshold discrimination for staining area expressed relative to total sectional area .For histology, hearts (n = 4 per group) were fixed in formalin then paraffin processed and sectioned (6 μm) followed by hematoxylin and eosin or Masson’s trichrome (Sigma-Aldrich) staining with standard histological protocols.2HPO4 0.75 mM; CaCl2 5mM) for 20 min at 37°C (75 μl per well). After incubation, 50 μl was removed from each ventricle piece and placed in a 384-well read plate, and thrombin activity inhibited in one well with 5 μl Hirudin  for 5 min at 37°C. Addition of 5 μl of 1 mM S-2238 chromogenic substrate  to each well was followed by photometer  measurements of the rate of substrate cleavage at 405 nm by thrombin. A standard curve with known concentrations of α-thrombin (0.039–25 nM) was run at the same time as experimental samples. Thrombin generation was expressed as a delta change from wells where thrombin activity was inhibited by Hirudin versus wells where thrombin generation was not inhibited. Data for APC and thrombin generation assays was normalized to LV weight as both LV and heart weights were higher in acute and chronic stable HF mice (data not shown) compared to wildtype and early HF mice.Mice (n = 4–7 per group) were anesthetized with a ketamine (75 mg/kg)/ acepromazine (2 mg/kg)/ xylazine (15 mg/kg) cocktail and the hearts immediately removed after thoracotomy. The atria were dissected free of the ventricles and the LV cut medially along its longitudinal axis to form two halves. Each LV half was placed over a well on a modified 384-well plate, endocardial surface exposed, and incubated with factors : prothrombin (50 nM), Va (50 nM), and Xa (5 nM) in HBS buffer  containing 10 nM human α-thrombin (HaemTech) and 1 μM human protein C (HaemTech) for 30 mins at 37°C. 50 μl was removed from each ventricle piece, placed in a 384-well read plate, and thrombin activity inhibited (as it can cleave the substrate) with 5 μl Hirudin  for 5 min at 37°C. Addition of 5 μl of 2 mM S-2366 chromogenic substrate (Diapharma) to each well was followed by photometer measurements of the rate of substrate cleavage at 405 nm, which is proportional to APC activity. A standard curve with known concentrations of APC  was run at the same time as experimental samples for concentration determinations. APC generation was expressed as a delta change from control wells containing all factors but not incubated with ventricular tissue. Data from technical repeat wells were averaged to obtain a mean value for APC generation from each animal’s left ventricle.Each half of the LV  was placed over a well on a modified 384-well plate  and incubated with 75 μl of reaction buffer  were placed over a modified 384-well plate (as above) and incubated with Trizol reagent  followed by a standard chloroform-ethanol extraction and purification  . 15 min The cDNA was diluted 1:4 and a quantitative real-time PCR reaction carried out with Taqman Gene Expression Master Mix and mouse Taqman forward and reverse primers with a Viia7 qRT-PCR system (Life Technologies). Assays were carried out in triplicate  and with negative controls. Manufacturer certified Taqman probe/primer sets were to TM (Mm00437014), EPCR (Mm00440992), vWF (Mm00550376), protein S (Mm0134326), and to two housekeeping genes; CD31 (Mm01242584) and Polr2a (Mm00839493). Data are expressed as a delta change in Ct expression from that of the housekeeping gene Polr2a [Protein was isolated from the endocardial surface of the LV (n = 4–9 per group) following incubation in Lysis-M buffer  in a modified 384-well plate  where only the endocardial surface was exposed to lysis buffer. 30 min lysis buffer incubation of the LV endocardial surface was chosen; this time frame gave the highest yield of protein that was positive for CD31 as an endothelial marker and negative for the cardiomyocyte marker cardiac beta-myosin heavy chain. A standard immunoblot protocol was followed as previously  with 10 Immunoblots for vWF were carried out as above except we used 3–8% tris-acetate gels (Life Technologies) under non-reducing conditions.3 and an aggregometer  used to measure ADP-induced platelet aggregation , or agglutination measured after addition of Ristocetin  [Following anesthesia (as above), blood was gently removed from the LV (n = 4–6 hearts per group) after cardiac puncture and centrifuged in a sodium citrate vial  to obtain a platelet rich plasma fraction (PRP). A platelet poor plasma (PPP) fraction was obtained after a second centrifugation step . PRP was diluted to 250, 000 platelets mmBioData) ; PPP samFor the tail clot assay (modified from ), anesthLV halves  were either stimulated with mouse thrombin  to promote maximal vWF secretion, or left unstimulated (vehicle control). Two technical replicates of 50 μl each were removed from each well and a sandwich ELISA carried out as per manufacturer's instructions  to quantitate vWF extruded from the LV endocardial surface. A standard curve with known vWF concentrations (2.5–80 mU) was run in parallel. Data from the technical replicate wells were averaged to obtain a mean value per mouse left ventricle. To measure endocardial vWF protein levels (n = 4 per group), the LV was placed endocardial surface up onto a modified 384-well plate and Lysis M buffer applied ; lysate was centrifuged then 200 μg protein per sample was processed for vWF ELISA as above.Data was analyzed  using one or two-way ANOVA (p<0.05) for multiple comparisons and a Newman-Keuls post-hoc test. All data were normally distributed and data graphically expressed as standard error of mean (+/- SEM).A133 mice  and 20–24 weeks (chronic compensated HF) did not show overt symptoms of HF such as anasarca. However, both groups of mice did have underlying HF with dilated cardiomyopathy as evidenced by smaller LVEF and LVIDs compared to wildtype mice  was as follows: 6/9 in early HF, 4/10 in acute HF, 1/8 in chronic HF. Large thrombi  generation in acute HF compared to both wildtype (65.5%) and early HF (40%) mice . In contEndocardial thrombomodulin (TM) mRNA and protein expression was decreased substantially  in all HF mice groups compared to wildtype mice . A similThe bleeding index, reflecting time to clot and total blood loss, showed a significant decrease in clotting time in early and acute HF mice (77%) compared to wildtype mice , consistPlasma vWF showed a prominent 225 kDa vWF band that corresponded to the mature vWF protein. Mature vWF protein in plasma was increased in all HF mice groups by 378–500% over wildtype mice consistent with a pro-thrombotic phenotype . QuantitA133 transgenic mouse. Although models of atrial endocardial thrombosis exist for studies of atrial fibrillation [A133 mice, cardiomyocyte disruption is the primary initiator of HF. We have shown increased incidence of organized LV thrombi in early and acute decompensated stages of HF in these mice. Although the size of ventricular thrombi was variable, this finding is similar to clinical investigations that have also observed a broad range in LV thrombus dimensions [We set out to establish a robust model of ventricular endocardial thrombosis using the CREBillation , 36, to illation . Howeverillation , the rolmensions . A caveaEndothelial dysfunction has been implicated in the increased risk for thrombus formation within the failing heart. Given the critical endogenous anticoagulant role of APC , 26, we Having established reduced APC formation, and conversely increased thrombin generation, on HF mice endocardium, we asked whether reduced expression of the critical receptors involved in genesis of APC also occurred. Indeed, both TM and EPCR mRNA transcripts and protein were attenuated within the endocardium in both early and acute decompensated HF mice. Lower TM and EPCR expression has been shown in a number of pathological states to correlate with decreased APC generation and increased risk for thrombosis , 40. TheOur APC pathway results are congruous with endothelial dysfunction in early and acute decompensated HF mice promoting a hypercoagulable endocardial phenotype. However, in chronic compensated HF mice, endocardial thrombin generation was markedly attenuated and they did not demonstrate an increased incidence of endocardial thrombi. Despite these indices of a hypocoagulable state, the APC pathway was severely disrupted in these chronic HF mice. We postulated that another endothelial-derived factor was responsible for an overall hypocoagulable phenotype in chronic HF mice. To this end, we examined vWF expression in, and thrombin-mediated vWF secretion from, the endocardial surface. As expected, vWF expression was increased in acute decompensated HF mice. However, thrombin-mediated vWF extrusion was markedly depressed in both acute decompensated and chronic compensated HF endocardium. At a functional level, such an attenuation of vWF extrusion would likely interfere with platelet aggregation and hence blood coagulation. Indeed, platelet agglutination with ristocetin, a vWF-dependent process, was also attenuated in both acute and chronic HF mice. That platelet function was normal in these mice was confirmed with normal aggregation responses to ADP. Finally, we examined tail bleeding to look at systemic effects on coagulation. Although early and acute HF mice showed hypercoagulation compared to wildtype mice, chronic HF mice actually had bleeding times similar to wildtype mice. We suspect that endothelial dysfunction in HF initially manifests itself as a prothrombotic state with defects in the APC pathway as a primary contributor. As HF progresses, however, the once increased vWF extrusion in early HF is gradually attenuated in chronic compensated HF mice, which may actually reduce the risk of endocardial thrombi in the chronic stable condition compared to the early phase.It is not known whether there is a direct clinical relevance of HF stage-specific hypercoagulability followed by attenuated vWF secretion as shown in this mouse model. However, these studies suggest that a further understanding of the regulation of vWF, as a consequence of endothelial dysfunction, is needed in the clinical HF setting. Of interest in this context was the demonstration by Fukuchi et al  of a corAlthough chronic compensated HF mice displayed the strongest evidence for attenuated vWF secretion, the degree of attenuation likely depends on pathophysiologic factors that change as HF develops. While increased circulating vWF levels are regarded as adverse event predictors in the development of HF , 47, in There are some limitations to our study. Since we were harvesting tissue from animals we were unable to examine the same animal for thrombus incidence at the early stage of HF versus acute or chronic stages of HF. Hence, we cannot state with certainty that animals in the chronic stages, that showed low incidence of endocardial thrombi, may have had endocardial thrombi in the early HF stage. Secondly, this study focuses on endothelial dysfunction in HF and its sequelae without examining the primary drivers of this endothelial dysfunction. There may be several mechanisms for endothelial dysfunction in these HF mice; these may include as a secondary consequence of cardiomyocyte death, or due to rheological changes within the failing cardiac chambers. However, these were not the primary research questions addressed by this study.In conclusion, we were able to establish a model of endocardial dysfunction with consequent endocardial thrombosis. We found considerable evidence in favor of our first hypothesis that a prothrombotic endocardial surface characterizes the early and acute decompensated phases of HF, as APC generation is attenuated and vWF synthesis and secretion are augmented. We also generated data supporting our second hypothesis that in stable compensated HF, incidence of endocardial thrombus formation is reduced. However, the mechanism for this thrombus incidence reduction appears to be due to progressive endothelial disruption in chronic stages of the disease; this may correlate to chronic compensated HF patients that are thrombus free despite prolonged HF.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLS)Click here for additional data file.S5 Data(XLS)Click here for additional data file.S6 Data(XLS)Click here for additional data file.S7 Data(XLSX)Click here for additional data file.S8 Data(XLSX)Click here for additional data file.S9 Data(XLSX)Click here for additional data file.S10 Data(XLSX)Click here for additional data file.S11 Data(XLSX)Click here for additional data file.S12 Data(XLSX)Click here for additional data file.S13 Data(XLSX)Click here for additional data file.


The ubiquitin conjugating enzyme E2L3 (UBE2L3) gene is associated with susceptibility to many autoimmune diseases. The aim of this study was to investigate the association between UBE2L3 gene and autoimmune thyroid diseases (AITDs) and their clinical phenotypes.We genotyped five single-nucleotide polymorphisms (SNPs) rs131654, rs5754217, rs2298428, rs140489 and rs5998672 of UBE2L3 gene in case groups including 1028 patients with AITDs [676 cases of Graves’ disease (GD) and 352 cases of Hashimoto’s thyroiditis (HT)] and control group including 897 healthy individuals.The genotyping was performed with the method of polymerase chain reaction-ligase detection reaction (PCR-LDR).P = 0.031, OR = 1.441).The frequencies of allele and genotype of five SNPs in gene UBE2L3 showed no statistically significant difference between case groups and control group, respectively. Moreover, no significant differences in frequencies of allele and genotype of five SNPs of the gene were found between clinical subphenotypes of AITDs and control group. Such subphenotypes included GD, HT, and thyroid associated ophthalmopathy (TAO). The negative results were also found in the frequency of other haplotypes of the gene except the haplotype of TCGGC, which was significantly higher in HT group than in control group (The present findings indicate that TCGGC haplotype is associated with an increased risk of HT and UBE2L3 gene is likely to be a susceptibility factor to HT in a Chinese Han population.The online version of this article (doi:10.1186/s12902-016-0098-6) contains supplementary material, which is available to authorized users. Autoimmune thyroid diseases (AITDs) are a set of organ specific endocrine autoimmune diseases mediated by T lymphocyte. They mainly consist of Graves’ disease (GD), Hashimoto’s thyroiditis (HT), and thyroid associated ophthalmopathy (TAO). GD and HT are the most common phenotypes of AITDs. Now the pathogenesis of AITDs is thought to be immune dysfunction due to the interaction between genetic susceptibility and environmental factors. The recent studies have identified more than ten susceptibility loci for AITDs, which include cytotoxic T lymphocyte associated protein 4 (CTLA4) gene , thyroidUbiquintin proteasome pathway (UPP) is an important signal transduction system which involves many physiological processes. In this pathway, UBE2L3 is an ubiquitin conjugating enzyme (E2) participating in ubiquitination of substrate through sequential steps catalyzed together with activating (E1) and ligase (E3) enzymes, and exerts its degradation effects on targeting molecules . These tGenetic variation in the UBE2L3 has been demonstrated in many autoimmune diseases such as systemic lupus erythematosus (SLE) –15, rheuWe studied samples from Department of Endocrinology, the First Affiliated Hospital of Xi’an Jiaotong University Health Science Center, and Department of Endocrinology, Jinshan Hospital of Fudan University. The case groups including 1028 patients with AITDs cohort consisted of 676 in GD subgroup  and 352 in HT subgroup . The subjects in our study were not related to each other. Diagnostic basis for GD was typical high metabolic syndrome, different degrees of diffuse goiter, laboratory examination indicating hyperthyroidism and positive antibody against TSH receptor (TRAb). HT was confirmed by goiter, thyroid antibody against Tg (TgAb) or antibody against thyroid peroxidase (TPOAb) or even thyroid puncture pathological examination. The control group included 897 healthy controls , who were all screened for absence of thyroid goiter and personal or family history of thyroid diseases and any autoimmune diseases. The thyroid function was in the normal range. All subjects were ethnic Chinese Han and signed informed consent at each site. The study was approved by the Ethics Committee of Jinshan Hospital of Fudan University.We selected five SNPs rs131654, rs5754217, rs2298428, rs5998672 and rs140489 of UBE2L3 region because of their strong association with the other autoimmune diseases such as SLE, RA, Crohn’s disease, and Sjögren’s syndrome .The genomic DNA was extracted from 2 ml of peripheral blood from each subject using RelaxGene Blood DNA System  according to the manufacture’ s protocol.UBE2L3 SNPs were genotyped using polymerase chain reaction-ligase detection reaction (PCR-LDR) and analyzed with polymerase chain reaction (PCR) machine . Five pairs of primers and the target gene LDR-probe sequences for SNPs of UBE2L3 region are shown as Additional file We analyzed the case groups’ clinical characteristics and correlation between frequencies of allele, genotype and haplotype and subphenotypes on the basis of  the onse2.Allele and genotype frequencies in case and control groups were implemented using SPSS17.0 . Comparison of genotype and allele frequencies between case and control groups was made by Chi-square test. P value of < 0.05 was considered significant. Differences between groups were assessed by the odd ratio (OR) and 95 % confidence interval (95%CI). Haplotype frenquencies and linkage disequilibrium (LD) test were analyzed using Haploview 4.2 . LD testThere were no differences in allele or genotype frequency distribution in any SNP between patients (GD and HT) and control group  using Hapmap CHB data (corresponding to samples of China ancestry from Beijing) and Haploview software. Strong linkage disequilibrium was shown for five SNPs , which suggested that the haplotype TCGGC increased the risk of HT  and B cell receptor (BCR) signaling transduction. There have been reports on the correlation of five SNPs in UBE2L3 with many T cell or B cell mediated autoimmune diseases, such as SLE , 15, 26,Although various subtypes of AITDs share common genetic factors and biomarkers, they indeed have their own specific manifestations and probably specific pathophysiolgic mechanisms. We speculated that different clinical types of AITDs may have different prominent alleles and genotypes. But we did not get the predicted result after stratifying the AITDs patients into GD, HT, and TAO ones, cases with positive family history or not, and HT in euthyroid or hypothyroid state.Herein, our study provide an important clue that SNPs in UBE2L3 are involving in the pathogenesis of HT. Nevertheless, we have to admit this result may be influenced by sample size, different ethnic groups or even different research methods. Further research on UBE2L3 gene in AITDs of other races or even on UBE2L3 gene expression in AITDs is expected in the future.Thus, we may conclude that UBE2L3 gene variant may be implicated in HT in a Chinese Han population. Further investigations with other ethnic cohorts await to confirm the implication.The dataset supporting the conclusions of this article is included within the article and its additional file.