To study the effect of dexmedetomidine priming on convulsion reaction induced by lidocaine.The New Zealand white rabbits were applied for the mechanism study of dexmedetomidine priming for preventing convulsion reaction induced by lidocaine. The influence of dexmedetomidine priming with different doses on the time for convulsion occurrence and the duration time of convulsion induced by lidocaine, as well as contents of excitatory amino acids  and inhibitory amino acids  in the brain tissue were investigated.With 3 and 5 μg/kg dexmedetomidine priming, the occurrence times of convulsion were prolonged from 196 seconds to 349 and 414 seconds, respectively. With dexmedetomidine priming, the contents of excitatory amino acids  were much reduced at occurrence time of convulsion comparing with that without dexmedetomidine priming, while content of inhibitory amino acids Gly was much enhanced.The application of dexmedetomidine before local anesthetics can improve intoxication dose threshold of the lidocaine, delay occurrence of the convulsion, and helped for the recovery of convulsion induced by lidocaine. The positive effect of dexmedetomidine on preventing convulsion would owe to not only the inhibition of excitatory amino acids , but also the promotion of inhibitory amino acids Gly secretion. However, excessive application of lidocaine or infiltration into blood vessels can result in local anesthetics poisoning. The central nervous system toxicity convulsion induced by local anesthetics is a common clinical complication. Early symptom usually exhibited excitation, such as lip tongue numbness, headache, blurred vision, tinnitus, aphasia, etc., and without treatment would developed into central nervous system inhibition, circulatory collapse, and even respiratory stop, coma, seriously threating to life safety.Previous studies have demonstrated that benzodiazepines or barbiturates as preanesthetic medication were benefit for the prevention and reduction of local anesthetics toxicity. Improving the safety threshold value of lidocaine and prevention the convulsion reaction induced by lidocaine are important clinical problems urgently to be resolved. The pentothal sodium and midazolam are conventional anticonvulsants. The pentothal sodium has strong vascular stimulation and significantly inhibition effect on respiration and circulation, while midazolam would result in respiratory depression and aggravating hypoxia when quickly injected. Moreover, high dose using midazolam could cause ataxia. Hence, new anticonvulsant with less side-effect should be developed.2-adrenergic receptors, dexmedetomidine is an anxiolytic, sedative, and analgesic medication, with advantages of mild influence on respiration, stress response inhibition, nonirritating, and nerve protective effect.[8]As a highly selective central agonist of αe effect.–8The current study suggests that application of dexmedetomidine before local anesthetics can reduce the occurrence of local anesthetics poisoning.In this work, the influence of dexmedetomidine priming with different doses on the convulsion time of the New Zealand white rabbits induced by lidocaine, as well as contents of excitatory amino acids  and inhibitory amino acids  in the brain tissue was investigated. And the preliminary action mechanisms were discussed.22.1A total of 40 male New Zealand white rabbits (Wanqianjiaxing Co. Whhan) with weight of 2 to 2.5 kg were housed as standard protocols and feeding in clean environment, free drinking, and eating. The temperature maintained in 22 to 24 °C, and the relative humidity 50% to 60%.Group D1: normal saline priming and lidocaine injection for convulsion.Group D2: 3 μg/kg dexmedetomidine priming and lidocaine injection for convulsion.Group D3: 5 μg/kg dexmedetomidine priming and lidocaine injection for convulsion.Group C: normal saline.The rabbits were randomly divided into 4 groups with 10 pregroup.2.2The ethics committee of the First Affiliated Hospital of Nanchang University approved this study. The white rabbits were anesthetized by intraperitoneal injection of 40 mg/kg 1% sodium pentobarbital. Auricular vein catheters were set and fixed. Then the white rabbits were fixed in the prone position in the operating table, shearing the head villus and sterilization operation area. After longitudinal incision of the skin and subcutaneous tissue (about 1.5 cm) and exposed skull, the operating point selection sited at the middle line about 0.3 cm distance near the occipital protuberance. Drill hole in the direction of the 45° and epidural catheter to the direction of the foramen magnum. Clear cerebrospinal fluid flows along the tube. Cut-off at 5 cm from the skull, and connected to the epidural catheter injection head. Suture the skin and subcutaneous tissue, and fixed the catheter and the rabbit hair. Right thigh shearing, femoral artery catheter placed with heparinized saline heparin (20 mg/L). The depth was about 2 to 3 cm. The catheter was fixed to the surrounding tissue, and then the distal end of the catheter sealed with heparin cap.After 24 hours, the white rabbit was fully conscious. Then the microsyringe pumps were connected onto auricular vein for continuous infusion of drugs. For group D1, 8 mL 0.9% normal saline were pumped in 10 minutes, then 2% lidocaine was pumped with the speed of 4 mg/kg/minute, and stopped immediately when convulsion. For Group D2, 8 ml 0.9% normal saline containing 3 μg/ kg dexmedetomidine was pumped and then pumped lidocaine in similar manner. Group D3: 8 ml 0.9% normal saline containing 5 μg/ kg dexmedetomidine was pumped and then pumped lidocaine in similar manner. For group C, 0.9% normal saline was pumped with average liquid dosages of group D1, D2, and D3. The white rabbits were all mask for oxygen during the drug infusion, and the dead ones were eliminated from the experiment.The times for convulsion occurrence (t) and the duration times of convulsion (tt) of the rabbits in groups D1, D2, and D3 were recorded. The recovery of convulsion was determined by the time that the white rabbits could self-stand up.For white rabbits in groups D1, D2, and D3, when they were recovery from convulsion, 2 mL arterial blood was pumped out from the femoral artery, stationary on the ice for 1 hour and then centrifugation for 10 minutes. The plasma was stored in −20 °C refrigerator. The lidocaine content in plasma of each group was measured by high efficiency liquid chromatography.The 0.5 mL cerebrospinal fluid was collected at the times of drill catheter (T0), convulsion occurrence (T1), and 30 minutes after convulsion (T2). The cerebrospinal fluid put on the dry ice for freezing and then centrifuged for 20 minutes . The supernatant was stored in −20 °C refrigerator. The contents of excitatory amino acids  and inhibitory amino acids  in the cerebrospinal fluid were tested by reversed phase high performance liquid chromatography fluorescence (RP-HPLC) method.2.3P value of less than 0.05 was considered statistically significant.All statistical analyses were performed using SPSS version 13.0 . Measurement data were expressed as the mean ± SD and compared with single factor analysis of variance among groups. A 3Figure + channel blocker 4-aminopyridine.2+-NO signaling pathway. The central inhibitory neurons would be inhibited by local anesthetics lidocaine, resulting in the dominant of excitatory neuron. The excitatory amino acid Glu released by neuronal was increased and the receptor channel was opened. In condition of the dexmedetomidine, presynaptic α2A adrenoceptors are participated inhibition of release. With chelating of extracellular Ca2+ ions and dexmedetomidine associating with the vesicular transporter inhibitor bafilomycin A1, the Glu release was blocked. Via Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels,2+ entered into the cell along the concentration gradient and activated calmodulin, which had effect on nitric oxide synthase (NOS). Excitatory amino acid, NMDA, Ca2+, and NO had interaction and reciprocal causation mediated convulsion. As the increasing ratio depolarization-induced intrasynaptosomal Ca2+ levels would be prevented, inhibitory effect dexmedetomidine would be abolished by blocking the Cav2.2 and Cav2.1 channels. Moreover, Glu release was also promoted by mitogen-activated/extracellular signal-regulated kinase (MEK) inhibitors via effect of dexmedetomidine.2A adrenoceptors, the inhibitory effect of dexmedetomidine on the Glu release was directly associated with the blocking of voltage-dependent Ca2+ channels, kinase activity of mitogen-activated protein, as well as NMDA-Ca2+-NO signaling pathway.To explore the mechanism of dexmedetomidine priming for the prevention of convulsion reaction induced by lidocaine, the contents variation of excitatory amino acids  and inhibitory amino acids  in the cerebrospinal fluid at the times of drill catheter (T0), convulsion occurrence (T1), and 30 minutes after convulsion T2) were tested. It can be seen in Fig.  were tesFigure In summary, application of dexmedetomidine before local anesthetics can improve intoxication dose threshold of the lidocaine, delay occurrence of the convulsion, and helped for the recovery of convulsion induced by lidocaine. The positive effect of dexmedetomidine on preventing convulsion would owe to not only the inhibition of excitatory amino acids , but also the promotion of inhibitory amino acids Gly secretion. This study provides more safe and effective therapy and theoretical basis for clinical prevention and treatment of anesthetics poisoning by lidocaine.


Phaeocystis. In the spring 2015 abundant gypsum crystals embedded in Phaeocystis aggregates were collected throughout the water column and on the sea floor at a depth below 2 km. Model predictions supported by isotopic signatures indicate that 2.7 g m−2 gypsum crystals were formed in sea ice at temperatures below −6.5 °C and released into the water column during sea ice melting. Our finding indicates that sea ice derived (cryogenic) gypsum is stable enough to survive export to the deep ocean and serves as an effective ballast mineral. Our findings also suggest a potentially important and previously unknown role of Phaeocystis in deep carbon export due to cryogenic gypsum ballasting. The rapidly changing Arctic sea ice regime might favour this gypsum gravity chute with potential consequences for carbon export and food partitioning between pelagic and benthic ecosystems.Mineral ballasting enhances carbon export from the surface to the deep ocean; however, little is known about the role of this process in the ice-covered Arctic Ocean. Here, we propose gypsum ballasting as a new mechanism that likely facilitated enhanced vertical carbon export from an under-ice phytoplankton bloom dominated by the haptophyte  However, excess density by incorporation of ballast minerals  can significantly increase the sinking speed of fresh organic matter and magnitude of carbon export5. Thus, mineral ballasting plays an important role in strengthening the biological carbon pump that transfers particulate organic carbon (POC) from the surface to the deep ocean6. Ballasting by lithogenic material plays a minor role in the deep Central Arctic Ocean8 and the scarce sediment-trap data available indicate that mineral ballasting of organic carbon in the ice-covered Arctic Ocean is three orders of magnitude lower than the global average6. The resulting POC flux to depths >1000 m in the ice-covered Arctic Ocean is usually 0.17–1 gC m−2yr−1 (refs10)  has recently been reported in Arctic sea ice22, but this mineral has never been implicated as a potential ballasting material for POC. Here, we report cryogenic gypsum ballasting of an under-ice phytoplankton bloom dominated by the haptophyte Phaeocystis north of Svalbard: this is the first report of cryogenic gypsum ballasting.The precipitation of gypsum  expedition of RV Polarstern  systematically investigated the sea-ice ecosystem north of Spitsbergen  showed remains of ume Fig. . No biomume Fig. . HoweverPhaeocystis aggregates on the sediment surface had settled through the water column, plankton (>55-µm) and aggregates from the water column (in the upper 2000 m) were collected during two Multi Plankton Sampler (MultiNet) casts (see method). Microscopic analyses of the Rose Bengal–ethanol treated, washed, and dried residues confirmed that remains of Phaeocystis aggregates with embedded crystals occurred in samples from all water depths. However, quantification of the relative aggregate/crystal number and volume was hampered by the sampling technique itself. The use of MultiNets could have allowed re-aggregation of gypsum crystals and aggregates, as well as loss of an unknown quantity of thin needle-like crystals through the nets. Nevertheless, the finding of needle-like crystals entangled in algal aggregates caught with the MultiNets supports the observation of an export event occurring during the video recording and very recent sedimentation of the algal aggregates retrieved from the seafloor.To confirm that the 34S = +21.3‰) was consistent with the enrichment expected for abiotic gypsum precipitation from a marine sulphate source 25. This observation indicates that the gypsum crystals were precipitated from marine sulphate within sea ice rather than from oxidation of reduced sulphur in biogenic pyrite or organic matter within the algal aggregates, which would have yielded lower δ34S values (−50‰ to +15‰)29.The crystals retrieved from the aggregates were identified as gypsum by means of confocal Raman microscopy   and POC (9.4 g m−2) standing stocks at Station PS92/47  with 50% gypsum would increase the density from ~1 to 1.65 g cm−3 with gypsum ballasting. This density increase could enhance the amount and speed of surface-derived organic carbon export to the abyssal ocean32.To estimate the amount of gypsum in the local sea ice, we reconstructed its thermal history and applied FREZCHEM, a chemical–thermodynamic model that was explicitly developed to quantify aqueous electrolyte properties at sub-zero temperatures; it calculates the precipitation of solids by solving the equations of thermodynamic equilibrium using the Pitzer approach30 model does not account for the time scales of gypsum dissolution outside the stability range. The direct observation of gypsum crystals at a depth of more than 2 km presented here indicates that the relatively large gypsum crystals observed were stable enough to survive the descent through the water column. Even so, our current knowledge of the kinetics of gypsum dissolution under the conditions at the sampling site is insufficient to exclude the possibility that some smaller crystals dissolved during the descent. Gypsum crystals might have been expelled from the sea ice during warming, even before melting, when brine pockets interconnect at T > −5 °C (ref.33), which is near the warm gypsum precipitation window. The release of crystals to the water column coincided with a large Phaeocystis bloom 13.The genus Phaeocystis has been found in surface sediments in the Southern Ocean37, it has been proposed that this alga is remineralized within the upper 50–100 m (refs39). However Phaeocystis export events have recently been observed below 300 m at the ice edge in the Arctic Ocean40, but they were not related to a release of gypsum crystals and thus did not demonstrate the ballasting export mechanism presented here. Phaeocystis can form large blooms in its colonial stage36. In Phaeocystis colonies (which reach sizes of up to 2 mm), the cells are embedded in a matrix of polysaccharides – they are known to excrete large amounts of mucopolysaccharides and heteropolysaccharides36. This combination of large size and the sticky polysaccharide gel matrix41 make Phaeocystis colonies in the water column an ideal trap for gypsum crystals released from sea ice. Our video recording of the MUC deployment and aggregate accumulation on the sea floor clearly indicates downward flux of the gypsum-ballasted Phaeocystis , abundant particles in the water column, and gypsum-ballasted algal aggregates at the sediment surface at a depth of 2146 m, as observed at station PS92/47  for the upper 50 m at station PS92/47. The total NPP was 0.24 ± 0.02 g C m−2 d−1, with a Phaeocystis contribution of 56%  corresponding to a daily production share of 0.13 ± 0.02 g C m−2 d−1  to the seafloor during the short-term gypsum ballasting export event would rival the annual carbon export in various deep-sea regions of the Arctic Ocean Unless stated otherwise, all treatments, measurements and analyses were carried out at the Alfred-Wegener-Institut Helmholtz-Zentrum für Polar- und Meeresforschung in Bremerhaven, Germany.−1) mounted to the MUC frame routinely recorded the descent and ascent of the MUC (Supplementary Video A multicorer (MUC) with eight tubes of 10-cm inner diameter was used for retrieving undisturbed sea-floor surface sediment samples. Simultaneously, a Sanyo HD400P video camera , with five nets, each with an opening of 50 × 50 cm, a length of 250 cm and a 55-µm mesh was used for plankton sampling. At station PS92/47 the water column was sampled above 100, 200, 600, 1000 and 2000 m water depths at a speed of 1 m sIn the home laboratory, all samples were washed with tap water over a 63-µm mesh sieve, dried at room temperature (MUC samples) or 50 °C (MultiNet samples), and examined under a stereo microscope .et al.44. The taxonomic structure of the phytoplankton groups was calculated from marker pigment ratios using the CHEMTAX program45. Pigment ratios were constrained as suggested by Higgins et al.46 based on microscopic examination of representative samples during the cruise, and the input matrix published by Fragoso et al.47 was applied. The resulting Phaeocystis contribution represents a percentage of the total chlorophyll a (Chl a) biomass and is expressed as µg Chl a L−1. The standing stocks of Phaeocystis, in terms of carbon biomass, were calculated by multiplying the chlorophyll concentrations with a conversion factor of 31.4, which was estimated for this bloom by Assmy et al.13.For pigment analysis with high pressure liquid chromatography (HPLC), seawater samples (1–2 L) were taken with Niskin bottles from seven depths in the upper 100 m attached to a rosette water sampler with a package of electronic instruments (SBE911plus) that continuously measures conductivity, temperature, and depth (CTD). The sample handling and pigment measurement processing were carried out as described in Kilias 48 using phase contrast and at three different magnifications. Phytoplankton cells were identified to the genus level, and the phytoplankton carbon content was obtained by multiplying the counts by the carbon values for individual cells. The phytoplankton carbon content was calculated as described in Edler49.To verify the HPLC pigment measurements, the phytoplankton taxonomic composition was analysed by light microscopy in samples from the chlorophyll maximum  just below the sea ice was derived from the incoming global radiation measured every 10 minutes over a period of 24 hours by the Polarstern weather station. Hourly PAR values were then estimated by integrating the measurements taken during each hour (n = 6). For each hour, the PAR at different depths  was estimated using a 4% transmittance through snow and sea ice, as determined from radiometric measurements taken by a remotely operated vehicle (ROV) and the attenuation coefficient (kdPAR) of 0.15 m−1, which was estimated from vertical light profiles measured during the time at ice station PS92/47.To calculate photosynthetic parameters, seawater samples from the Niskin bottles were incubated at different light levels in the presence of et al.51 in order to account for photoinhibition. The non-linear fitting was done using the Levenberg–Marquardt algorithm from the minpack.lm R package52.The hourly primary production at each depth was then estimated using fitted photosynthetic parameters that were derived from photosynthesis versus irradiance (P/I) curves by fitting the model of Platt At each depth, primary production was estimated using photosynthetic-parameter and PAR estimates. Finally, daily primary production (PP) was calculated by integrating production over time and depth. For error estimation of PP, a Monte Carlo procedure was used to propagate the uncertainty of the fitted photosynthetic parameters on the estimated daily primary production. At each depth, a total of 10,000 simulations  were performed by randomly sampling parameter values based on a multivariate normal distribution of the fitted parameters. Using the generated curves, the depth and daily integrated primary production rates were calculated as described above. The standard deviation of the 10,000 integrated daily primary production rates was then used as a measure of the uncertainty around the estimated value of primary production.Seawater samples for suspended POC were collected from eight discrete depths between 0 and 200 m, with Niskin bottles mounted on a rosette water sampler with a CTD system (SBE911plus). Triplicate samples (300–500 mL depending on concentration) were filtered onto glass microfiber filters (GF/F) filters (Whatman), precombusted at 500 °C, and immediately frozen at −20 °C for laboratory analysis at UiT, the Arctic University of Norway, Tromsø, which occurred within 6 months of sample collection. For analysis, the frozen filters were dried at 60 °C for 24 h and subsequently placed in an acid fume bath (concentrated hydrochloric acid) for 24 h to remove all inorganic carbon. The filters were then placed into a 60 °C desiccator for an additional 24 h and finally put into nickel capsules for analysis. The samples were analysed for POC using an Exeter Analytical CE440 CHN elemental analyser.54. As seen from the Raman spectrum  using an excitation wavelength of 488 nm (pinhole size: 50 µm) and a 20×  lens. This type of instrument is ideal for unambiguous determination of the mineral phase of small (few µm) crystals43S) of the gypsum extracted from the MUC samples was measured in the Godwin Laboratory at the University of Cambridge (United Kingdom). Gypsum microcrystals (~300 mg) were handpicked and combusted in a tin capsule in the presence of V2O5 at 1030 °C by a Flash Elemental Analyser . The sulphur dioxide produced was measured by continuous-flow gas source isotope ratio mass spectrometry . The sample run was bracketed by three standards . A blank (no sample) was analysed before and after the gypsum sample and the block of standards, to avoid a memory effect. No drift was detected during the run. The reproducibility of the method was better than 0.1‰ (1 sd) based on the analysis of the six NBS-127 standards. All δ43S values are relative to V-CDT (Vienna-Canyon Diablo Troilite). Consistent with Thode et al.55 and Pierre56, the sulphur isotope value (δ43S) of +21.3‰ determined for the gypsum is regarded as indicating a marine source of sulphur (+20.3‰), with an offset of approximately 1‰ due to fractionation during precipitation.The sulphur isotopic composition .The weight of the gypsum crystals was calculated from a 3-mL volume of algal aggregates pipetted from the MUC sediment surface that was stored in the Rose Bengal–ethanol solution. In the laboratory, the 3-mL sample of algal aggregates was sieved through a 63-µm mesh screen, and the residue was dried at room temperature. All gypsum crystals were picked from this residue, and the weight was determined with a high-precision Sartorius SE2 ultra-microbalance. Using a density value of 2.32 g cmPhaeocystis aggregates increased the mass density due to ballasting, i.e., 50% (by volume) Phaeocystis (density φ ~ 1) plus 50% gypsum (density φ = 2.3) is equivalent to φ~1.65 g cm−3.The incorporation of gypsum in the 42. This process provided a trajectory of the surveyed ice parcel from its initial formation to the location where it was sampled. This trajectory was used to extract the appropriate temperature and six-hourly meteorological forcing data from the European Centre for Medium-Range Weather Forecasts (ECMWF) ERA-Interim reanalysis product57. Together with the assumption of a constant ocean heat flux of 5 W m−2, these data were used to force the SNOWPACK thermodynamic model60 in a newly developed variant SNOWPACK SeaIce Branch model (http://models.slf.ch/p/snowpack/) to derive the ice growth and the internal temperature field. The total ice thickness of 95 cm (sea ice plus superimposed ice/snow ice) calculated by the model is in good agreement with the modal ice thickness of 90–100 cm measured by electromagnetic induction sounding  that was provided in the expedition report23. The fact that the modelled and observed ice thicknesses correspond well strongly suggests that this approach is suitable for reconstruction of the thermodynamic evolution of the ice parcel, despite all the uncertainties involved in backtracking and reanalysis data42.To evaluate the potential for gypsum precipitation in sea ice and quantify the amount of precipitated crystals, we combined ice-floe backtracking, reanalysis forcing data, a thermodynamic sea-ice model and a geochemical freezing chemistry model. Although each of the individual models has shortcomings and uncertainties, we are confident that we achieved the best possible, conservative estimate of precipitated gypsum mass. Solid precipitation in sea ice is mostly driven by temperature evolution. To reconstruct gypsum growth, we reconstructed ice temperatures of the surveyed area over the winter season before sampling. As no ice-mass-balance buoy was within a reasonable distance during period before sampling, ice temperatures were reconstructed using a thermodynamic model forced by reanalysis data. To assess the correct location of the surveyed ice floe throughout the winter, we used a Lagrangian backtracking algorithm employing a variety of sea-ice drift products30. This model is frequently used for calculations of seawater freezing, and is thus the best tool for an absolute estimate of total precipitated gypsum mass. Seawater freezing was calculated at 0.1 °C intervals for standard seawater, starting at 0 °C. As the precipitation kinetics of gypsum are largely unknown, especially at low temperatures, we had to base our calculations on two assumptions: first, the fact that we found sea-ice-precipitated gypsum in algal aggregates on the deep-sea floor proves that, once they have formed, gypsum crystals remain stable during the melting phase of sea ice. Hence, once gypsum is produced, it will not be dissolved by further thermal changes in the ice cover. Second, we assume that gypsum precipitation occurs immediately when the temperature threshold is reached. As almost nothing is known about the kinetics of gypsum formation, and the FREZCHEM model does not offer a temporal dimension, this is the only suitable assumption. Earlier observations22 that gypsum crystals form within a limited time frame support the validity of this assumption.To calculate the potential for gypsum precipitation, we used the FREZCHEM freezing chemistry model−2) were divided by the ratio of the seawater salinity of approximately 35 and the observed sea-ice bulk salinity of 5. This results in an estimate of potential gypsum precipitation to 2.7 g gypsum m−2.By adding the maximal gypsum formation potentials of all modelled horizontal layers, we estimated the potential gypsum precipitation on our surveyed ice parcel. To account for brine rejection during sea-ice formation, the final precipitated gypsum masses . The plot for Phaeocystis (a) is based on the samples from discrete depths in the upper 50 m  and is gridded with the weighted average method in ODV. The temperature  and salinity (b) contours are based on CTD sensor data23 with 1-m vertical resolution and are gridded with the DIVA method in ODV.The data shown in Supplementary Fig. Supplementary video 1Dataset 1


There are some spaces between abutment and implant body which can be a reservoir of toxic substance, and they can penetrate into subgingival space from microgap at the implant–abutment interface. This penetration may cause periimplantitis which is known to be one of the most important factors associated with late failure. In the present study, three kinds of abutment connection system, external parallel connection (EP), internal parallel connection (IP), and internal conical connection (CC), were studied from the viewpoint of microleakage from the gap between the implant and the abutment and in connection with the loosening of abutment screw.We observed dye leakage from abutment screw hole to outside through microgap under the excessive compressive and tensile load and evaluated the anti-leakage characteristics of these connection systems.During the experiment, one abutment screw for EP and two screws for IP, out of seven samples in each group, were fractured. After the 2000 cycles of compressive tensile loadings, removal torque value (RTV) of abutment screw represented no statistical differences among three groups. Standard deviation was largest in the RTV of EP and smallest in that of CC. The results of microleakage of toluidine blue from implant–abutment connection indicated that microleakage generally increased as loading procedure progressed.The amount of microleakage was almost plateau at 2000 cycles in CC, but still increasing in other two groups. The value of microleakage greatly scattered in EP, but the deviation of that in CC is significantly smaller. At 500 cycles of loading, there were no significant differences in the amount of microleakage among the groups, but at 1000, 1500, and 2000 cycles of loading, the amount of microleakage in CC was significantly smaller than that in IP. Throughout the experiment, the amount of microleakage in EP was largest, but no statistical difference was indicated due to the high standard deviation.Within the limitation of the present study, CC was stable even after the loading in the RTV of abutment screw and it prevented microleakage from the microgap between the implant body and the abutment, among the three tested connections. Although promising outcome of implant therapy has been reported, periimplantitis which is known to be one of the most important factors associated with late failure  is a serImplant–abutment connection can be divided into three types, external parallel connection (EP), internal parallel connection (IP), and conical connection which has the friction between the implant and the abutment (CC). Nobel Biocare has implant systems with these three kinds of implant–abutment connections, and these connection systems are popular even in other manufacturers and they also employ one or some of these connection systems. These systems have a lot of pros and cons and should be selected depending on the dentists’ demand.In the present study, we would like to consider these three kinds of connection system from the point of view of microleakage from the gap between the implant and the abutment and in connection with the loosening of abutment screw. We herewith performed the investigation of dye leakage from abutment screw hole to outside through microgap under the excessive cyclic load applied to cantilever superstructure. Cantilever model could simulate non-axial offset loading applied to implant–abutment complex, and toluidine blue solution was used to measure the extravasation from the gap at the implant–abutment interface.n = 7 each) . Implant–abutment connections are external, internal parallel, and internal and conical connection, respectively. Implant was embedded into steel mold and fixed with epoxy resin . A superstructure with cantilever  was fabricated using Au–Pt alloy  and fixed with abutment screw with fastening torque recommended by the manufacturer (35 Ncm), using torque wrench (Nobel Biocare). Five hundred microliter of water was poured into steel mold, the surface of water located, to small extent, superior to microgap. Then, 50 μl of 0.05% toluidine blue solution was poured at the access hole , Nobel Replace WP 5.0 × 15 mm (IP), and Nobel Parallel CC RP 5.0 × 15 mm (CC) , and load was applied to the cantilever, at 17.5 mm distance from the center of access hole.t test with Bonferroni correction for multiple comparisons. After the completion of 2000-cycle loading, removal torque value (RTV) of abutment screw was measured.As one cycle, one compressive and tensile loads (10 N each) was applied per 1 s and 2000 cycles of loading was done. Before starting load application and every 100 cycles, 100 μl solutions were collected from pool and absorbance at 627 nm was measured using a spectrophotometer . Every 500 cycles, the amount of microleakage was statistically compared using Student During the experiment, one (EP) and two (IP) abutment screws out of seven samples were fractured. In this case, abutment screw was changed to new one and experimental procedure was re-run.After the 2000 cycles of compressive tensile loadings, RTV of abutment screw was measured. There were no statistical differences in the RTV among three groups. Standard deviation was largest in the RTV of EP and smallest in that of CC  was sufficient to obtain the trends of microleakage, but it may be better to investigate using large number of samples to analyze the nature of microleakage at the implant–abutment interface in detail. Another limitation was that the fastening torque applied to screw was not necessarily accurate. To mimic clinical situation, we used dedicated beam-type toque wrench delivered from manufacturer. According to the previous studies, the wrench of Nobel Biocare only reportedly demonstrated the target torque value falling within the 95% confidence interval for the true population mean among four kinds of wrenches . In addiWithin the limitation of the present study, after the offset cyclic tensile and compressive loading, the amount of microleakage from implant–abutment interface was smaller in conical connection than in internal parallel connection.


Inhibition of VEGF signaling by the stress-induced matricellular protein TSP1 plays a role in modulating the angiogenic response to VEGF in both health and disease. TSP1 binding to CD47 inhibits VEGFR2 activation. The full implications of this inhibitory interaction are unknown. We developed a detailed rule-based computational model to inquire if TSP1-CD47 signaling through VEGF had downstream effects upon ERK1/2 and calcium. Our Simulations suggest that enhanced degradation of VEGFR2 initiated by the binding of TSP1 to CD47 is sufficient to explain the inhibition of VEGFR2 phosphorylation, calcium elevation, and ERK1/2 activation downstream of VEGF. A complementary mechanism involving the recruitment of phosphatases to the VEGFR2 complex with consequent increase in the rate of receptor dephosphorylation may augment the inhibition of the VEGF signal. The model was then utilized to simulate the effect of inhibiting external TSP1 or the depletion of CD47 as potential therapeutic strategies in restoring VEGF signaling. Results suggest that depleting CD47 is a more efficient strategy in inhibiting the effects of TSP1/CD47 on VEGF signaling. Our results highlight the utility of  VEGF is crucial in normal angiogenesis during embryonic vascular development  by TSP1  given in the Supplementary Material Section. We also included the SBML file associated with the model.Table Binding of PLCγ to pVEGFR2 and subsequent phosphorylation and dissociation of PLCγ from the receptor is described by a Michaelis-Menten type reaction as follows:pVEGFR2 denotes all the species containing phosphorylated VEGFR2 which is determined by BioNetGen. Using this approach lowers the number of reactions generated by the rules and prevents combinatorial explosion in the model.Ras activation by S1P is modeled as a Michaelis-Menten type reaction as follows:S1PRas and Km, S1PRas determining the strength of Ras activation by S1P.with parameters kCurrent through the CRAC channels is modeled according to the following equation. This model is a simplified version of the CRAC component of the mathematical model developed by Schmeitz et al.  in T-celThe steady-state current (Equation 4) as a function of ER calcium concentration is described by an empirically determined Hill function . The set of ordinary differential equations (ODEs) describing the reaction network was numerically integrated using SUNDIAL numerical solver suite  algorithm described in Marino et al. . The parTo accurately capture receptor dynamics and signaling to downstream targets, we opted to use a rule-based modeling approach where molecular details of the species and the corresponding rules for the interactions are implemented in a programing environment such as BioNetGen   via the IP3 sensitive receptors and calcium regulation involving plasma membrane and SERCA pumps. CRAC channels are also a prominent hallmark of the calcium cycling module Figure  see met. The com−1 vs. 1.24 × 10−3 s−1). This reduction in recycling rate is sufficient to explain the rapid decay of total cellular receptor levels in the absence of NRP1. In fact, it is the combined effects of internalization, recycling, and degradation that determine the rapid decay of VEGFR2 cellular level . Samples traces for four different degradation rates are also shown in Figure We simulate the direct effect of TSP1 by assuming that CD47 binding to TSP1 leads to accelerated VEGFR2 degradation. It is assumed that TSP1/CD47/VEGFR2 undergo internalization together and either recycle back to the membrane or degrade with a higher degradation rate . In cells devoid of C47, VEGFR2 signaling is not affected by TSP1. This implies that TSP1/CD47 interaction is necessary for VEGFR2 degradation. For the simulations here, cells are exposed to 2 nM TSP1 for 10 min, followed by 40 min exposure to 50 ng/ml VEGF. This is similar to the experimental protocol in Kaur et al. . Accordi2 (7000 receptors per cell) to 2.2 receptors/μm2 (3100 receptors per cell). Figure 2 does not affect TSP1 inhibition of VEGFR2 signaling.The effect of VEGFR degradation and CD47 surface levels on ERK1/2 activation is investigated in Figure Overall, the results here demonstrate that the enhanced degradation of VEGFR2 by TSP1/CD47 interaction is a viable mechanism to explain the global shutdown of VEGFR2 signaling. The dependence on TSP1 concentration is also consistent with the experimental data indicating the viability of the enhanced degradation mechanism.We next consider the hypothesis that TSP1 might insert some of its inhibitory effects on VEGFR2 by recruiting phosphatases to the receptor complex. For the simulations here the cells are exposed to 2 nM TSP1 for 10 min, followed by the addition of VEGF for 40 min. It is assumed that the increase in dephosphorylation rate is the sole mechanism. As shown in Figure Next, we consider a different layer of TSP1 effects involving the experimentally suggested alteration of calcium signaling by TSP1  is modeled in a simple manner by lowering the concentration of TSP1 and monitoring the recovery of calcium and pERK1/2 signals. We consider a mixed mechanism involving enhanced VEGFR2 degradation (10-fold) and increased VEGFR2 dephosphorylation (51-fold). These values are consistent with the two-parameter scan in Figures The effect of depleting CD47 as a therapeutic strategy is summarized in Figures The simulations demonstrate that CD47 depletion may be a more effective strategy than targeting TSP1 in restoring VEGF signaling to calcium and ERK1/2. CD47 depletion is particularly effective in restoring ERK1/2 activation downstream of VEGFR2.in silico approach to computationally investigate potential mechanisms of inhibition. The model incorporated detailed molecular mechanisms using a rule-based model for receptor-receptor interaction and a coarse-grained model for signaling to calcium and ERK1/2 from the activated receptors. The inclusion of detailed calcium cycling module in the model allowed us to study the role of TSP1 in inhibiting calcium signaling and the potential crosstalk with ERK1/2 activation. Fluorescene resonance energy transfer based (FRET-based) experiments in endothelial cells had shown the preassociation of VEGFR2 with CD47 in endothelial and T cells . Indeed, only 18% depletion of CD47 was needed to restore ERK1/2 activation signal compared with 72% TSP1 inhibition. The results highlight the important role of efficient and sustained therapeutic agent delivery to affected tissues to ensure above-threshold inhibition of TSP1 and CD47. They further suggest that CD47 depletion should be considered as an initial approach in pro-angiogenic therapeutic intervention. It is plausible that different endothelial cell types may exhibit differential inhibition thresholds to TSP1 and CD47 which would further necessitate the need for sustained delivery of anti-TSP1 and anti-CD47 agents.Simulations here also proposed the possibility of inhibiting TSP1 or CD47 as potential interventions for restoring VEGF-mediated angiogenic response. The model showed that sequestering TSP1 effectively normalized calcium signaling and ERK1/2 activation, albeit requiring at least 80% inhibition. This high threshold for inhibition might preclude the Further complicating anti-TSP1 therapy, platelets contain significant pre-formed TSP1 that are a depot source of protein. Thus, targeting CD47 is an alternative strategy expected to be more efficient at preserving pro-angiogenic signals. Additionally, VEGF can also signal via NO while CD47 can interact with cell membrane SIRPα  and has equity interest in the same and in Tioma Therapeutics . The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


These findings together suggest that oleamide has protective effects against excitotoxicity-induced neuronal death and behavioral seizure, partly via its direct calpain inhibitory activity.Oleamide was first known as a sleep-inducing fatty acid amide, and later shown to have wide range of neuropharmacological effects upon different neurochemical systems. However, the effects of oleamide on brain damage have scarcely been studied, and the molecular mechanisms and sites of its action remain elusive. Kainic acid (KA) has been used to produce an epileptic animal model that mimics human temporal lobe epilepsy and to induce calpain-activated excitotoxicity, which occurs in numerous neurodegenerative disorders. In this study, we examined whether oleamide protects against the KA-induced excitotoxic brain damage accompanied by behavioral seizure activity and neuronal cell death. Moreover, whether these effects of oleamide were mediated by calpain activity-related cellular mechanisms was investigated. KA-induced epileptic rats were produced by an intrastriatal injection of KA (5 nmole). Oral administration of oleamide  30 min prior to the KA injection showed dose-dependent inhibition of the KA-induced behavioral seizure activities that were monitored starting from 60 to 180 min post-surgery. Further repetitive oral administration of oleamide (once per day) for the next 4 consecutive days post-KA injection produced significant neuroprotection against the disrupted neuronal integrity that resulted from KA-induced excitotoxic damage that was also demonstrated by staining of striatal tissue sections with cresyl violet, hematoxylin/eosin, and fluoro-Jade B. In addition, oleamide blocked the KA-induced cleavage of cyclin-dependent kinase-5 coactivator (Cdk5-p35) and collapsin response mediator protein-2, which are believed to be mediated by calpain activation in striatal tissues dissected from KA-induced epileptic rats. Oleamide also reversed the KA-induced reduction in expression of an endogenous calpain inhibitory protein, calpastatin, and a marker of synaptic activity, synapsin-II. The hypothesis that oleamide could induce direct calpain inhibition was further investigated using  Cis-9,10-octadecenoamide) is a centrally acting fatty acid amide that belongs to the family of endogenous lipid signaling molecules that includes endocannabinoids, anandamide, N-palmitoylethanolamine, and N-oleoylethanolamine  receptor (1 receptor (A) receptors  has been used to produce an epileptic animal model that mimics human temporal lobe epilepsy  and induchanisms . An incrll death  that incll death , and alsll death  or autopll death . Therefoll death , and KA-ll death .in vivo epileptic rat models and in vitro neuronal systems.The present study investigates the antiepileptic and neuroprotective effects of oleamide through calpain inhibition as a potential intracellular mechanism. The ability of oleamide to inhibit calpain activity was demonstrated in both brain tissues of KA-induced trans-(ethoxycarbonyloxirane-2-carbonyl)-L-leucine-(3-methyl butyl) amide] was obtained from Enzo Life Sciences, Inc. . The μ-calpain was purchased from Calbiochem . Oleamide was suspended in 0.2% methyl cellulose.Oleamide, KA, carbamazepine, calpeptin, cresyl violet, hematoxylin and eosin were purchased from Sigma–Aldrich . Fluoro-jade B was purchased from Histo-Chem Inc. . E64d [2S,3S-Sprague Dawley rats (230–240 g body weight) were purchased from Orient Bio Department . The animals were housed individually in a temperature- (20 ± 1°C) and relative humidity-controlled environment and maintained on a 12-h light/12-h dark cycle. All animal experiments were conducted according to ethical procedures and approved by the Institutional Animal Care and Use Committee of Ewha Womans University .For surgery, rats were anesthetized with zoletil (20 mg/kg) and xylazine (9.5 mg/kg) and placed in a stereotaxic apparatus. A Hamilton syringe was used with a mini-pump  to inject the rats with KA  or vehicle  intrastriataly at coordinates of 1.2 mm posterior, ± 2.5 mm lateral, and ± 5.5 mm ventral, relative to the bregma. Rats were sacrificed 5 days after surgery. Oleamide  or vehicle (0.2% methyl cellulose) was orally administered 30 min before the surgery, and administered daily for 4 days after the surgery.Rats were continuously observed throughout the 3-h period by observers blinded to the treatment. Behavioral seizures were monitored and scored starting from 60 min through 180 min post-surgery. The seizure counts and score were recorded every 10 min to produce representative counts of seizure expression during that period. The convulsive behavior scale consisted of the following seven stages: stage 0, normal behavior; stage 1, wet dog shakes and mouth or facial movements; stage 2, head nodding; stage 3, forelimb clonus; stage 4, rearing; stage 5, rearing and falling; stage 6, tonic seizure or death .g for 10 min at 4°C. Protein concentration was determined using a BCATM protein assay kit  and assessed by Western blotting. Equal aliquots of the samples were denatured at 100°C, separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and blotted onto polyvinylidene fluoride (PVDF) membranes . Membranes were incubated in a blocking buffer containing 5% BSA in TTBS for 1-h at room temperature. Immunodetection was performed by incubating membrane blots overnight at 4°C separately with the following primary antibodies (1:1000): anti-CRMP-2 , anti-Cdk5-p35/25, anti-calpastatin, and anti-synapsin-II . For chemiluminescent detection, membrane blots were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000) for 2-h at room temperature. Data collection and processing of the integrated optical density of the bands were performed with a luminescent image analyzer (LAS-3000) and IMAGE GAUSE software .Striatal tissues were collected in cold lysis buffer containing 1% Triton X-100, 1 mM EDTA in phosphate-buffered saline (PBS), protease inhibitor cocktail, homogenized, and centrifuged at 10,000 × n = 3 per group) were anesthetized with zoletil (20 mg/kg) and xylazine (9.5 mg/kg) and transcardially perfused with PBS followed by 4% paraformaldehyde in PBS. Perfused brains were post-fixed in 4% paraformaldehyde in PBS overnight and subjected to increasing concentrations of alcohol overnight. The brain tissue blocks were embedded in paraffin and the paraffin blocks were cut into a series of 5-μm-thick slices and stained with 0.1% cresyl violet and H&E. All sections were coverslipped with Permount  and were examined with a light microscope , and photographs were taken with an AxioCam HRC digital camera .Cresyl violet and hematoxylin/eosin (H&E) staining were used to stain tissue sections for histological examination and measurement of neuronal loss. Rats (Fluoro-Jade B (FJB) staining was used to identify degenerating neurons. Briefly, the slides were immersed in 100% ethanol for 3 min, followed by 70% ethanol for 2 min and distilled water for 2 min. The slides were then transferred to 0.06% potassium permanganate for 15 min and gently agitated. After rinsing in distilled water for 2 min, the slides were incubated for 30 min in 0.001% FJB, which was freshly prepared by adding 20 ml of a 0.01% stock FJB solution to 180 ml of 0.1% acetic acid with gentle shaking in the dark. After rinsing three times for 1 min in distilled water, the slides were dried, dehydrated in xylene, and coverslipped.2+ for activation. In contrast, μ-calpain, composed of the catalytic subunit calpain II, is located in the cytosol or near the membrane and is activated by 2–80 μM concentrations of Ca2+in vitro. Therefore, μ-calpain activity can be affected by minute changes Ca2+ concentration. The direct inhibitory effect of oleamide was examined by analyzing μ-calpain activity in calpain 1 (CAPN1)-overexpressing neuronal cells  in kinetic mode at 37°C for 210 min. Fluorescence was measured at 320 nm (ex)/420 nm (em).2 for 30 min at 37°C. Independently, oleamide  was co-incubated in the same conditions to identify that the cleavage of collapsin response mediator protein-2 (CRMP-2) and cyclin-dependent kinase-5 (Cdk5)-p35 were mediated by calpain. Western blotting was performed for detection of CRMP-2 and Cdk5-p35.Normal rat striatal tissue lysates (20 μg) were incubated with μ-calpain (0.0125 U/ug) in the presence of 1 mM CaClp-value < 0.05 was considered statistically significant. All results were expressed as the mean ± SEM of at least three independent experiments.Statistical analyses were performed with the Newman–Keuls test and analyses of variance (one-way ANOVA) with GraphPad Prism version 5.0d . The Figure 1 shows the time course of behavioral seizures in each treatment group. In the vehicle group, rats showed extreme seizures behaviors. Maximum average Racine’s score reached 4.4 points  administration of KA in rodents induced epileptic seizures similar to human temporal lobe epilepsy, with spontaneous seizures, as well as seizure-induced neuronal cell death . A clini4 points . OleamidIt has been shown that KA induces internucleosomal DNA fragmentation and loss of striatal neurons . We examFigure 2A). The number of damaged neuronal cells was measured by counting cells in each of two fields randomly selected from each of three separate striatal tissue sections of three rats per group. The general criteria used to score damaged cells included the number of hyperchromatic nuclei and cytoplasmic vacuolation. Quantitative analyses  revealed a dramatic protective effect of oleamide (10 mg/kg) following KA administration in the striatum, as assessed by both cresyl violet staining (p < 0.001) and H&E staining (p < 0.001), as compared with KA alone.H&E staining showed that striatal tissues from rats exposed to KA (5 nmole) exhibited extensive cell loss and pyknotic nuclei at the neuropil. In contrast, rats pretreated with oleamide prior to KA injection showed a significant reduction in pyknotic nuclei, while pyknotic nuclei remained in cells of vehicle-treated KA-epileptic rat striatal tissues. In addition, the protective effect of oleamide was also observed in cresyl violet staining, which enables visualization of neuronal damage and cell dispersion . No FJB-positive cells were detected in the left striatum of sham-operated and oleamide-treated control rat brains. Quantification of FJB-stained cells in the region revealed a statistically significant decrease in the number of stained cells following oleamide (10 mg/kg) administration . FJB staining data provide further evidence confirming the protective effects of oleamide against KA-induced excitotoxic brain damage.The protective effects of oleamide against neuronal damage were further confirmed using staining with FJB, which specifically stains dying neurons and becomes fluorescent green. FJB is an anionic fluoresce in derivative useful for histological staining of neurons undergoing cellular degeneration . NumerouFigure 4A). It was previously shown that CRMP-2 (∼62 kDa) is specifically cleaved into a 58 kDa protein under ischemic conditions and that CRMP-2 cleavage is mediated by calpain . Synapsin-II protein is a marker of synaptic activity and plasticity . Our data suggest a protective effect of oleamide against KA-induced disruptions in synaptic integrity. In addition, oleamide protected the expression of an endogenous calpain inhibitory protein, calpastatin, the level of which was reduced by KA .It was shown that injection of KA into the striatum increased calpain mRNA . Calpain calpain . The oleasticity  and its asticity . We obseFigure 5). This result confirms the direct calpain inhibitory effect of oleamide, suggesting the possibility that it can be used as a novel calpain inhibitor.The cell-based μ-calpain assay was performed to investigate the μ-calpain inhibitory activity of oleamide in SH-SY5Y cells. μ-Calpain is endogenously expressed in SH-SY5Y cells, but its activity is not potent enough to sufficiently cleave the exogenous substrate pep1. Therefore, the large subunit of μ-calpain, CAPN1, was additionally transfected to visibly measure the compound μ-calpain inhibitory activity in SH-SY5Y cells. E64D, an irreversible calpain inhibitor, was prepared by esterification of the free carboxylic acid group of E64C to improve cell permeability. The results showed that μ-calpain activity was highest in CAPN1-transfected cells. Treatment with oleamide  reduced μ-calpain activity with better potency than did E64D (100 μM). Oleamide (30 μM) produced an inhibitory effect to a similar degree as calpeptin (30 μM), and the higher concentration of oleamide (100 μM) significantly decreased the enhanced μ-calpain activity to the level of control cells  remarkably decreased the calpain-induced cleavage of both CRMP-2 and Cdk5-p35. These results suggest the possibility that oleamide is a novel calpain inhibitor.The in vivo antiepileptic and neuroprotective effects of exogenous oleamide against KA-induced excitotoxic brain damage, and asks whether calpain inhibition is an intracellular mechanism of those effects. We demonstrated the preventive effects of oleamide against epileptic behavior in KA-induced epileptic rat model and against excitotoxicity-induced calpain activation that leads to neuronal death. The ability of oleamide to inhibit calpain activity was examined in both brain tissue of a KA-induced epileptic rat model and cultured neurons.The present study investigates the 1 receptor signaling . Previously, the anticonvulsant effect of oleamide was only shown to produce against the behavioral seizures induced by pentylenetetrazole, the most commonly used acute seizure model  to rats was shown to produce anti-seizure effect specifically on the degree (severity) of convulsions, but not decrease the duration and latency of convulsions . The striatum has been demonstrated to express a large number of binding sites for all classes of glutamate receptors , implying that oleamide could produce antiepileptic and neuroprotective effects by the mechanism related to calpain inhibition.Since many calpain inhibitors have been shown to produce antiepileptic effects , we post2+ homeostasis, causing calpain activation and the proteolytic degradation of axonal proteins . Additional evidence implying that oleamide can improve KA-induced synaptic dysfunction was provided by measuring the changes in the level of synapsin-II, one of the synaptic marker proteins. KA dramatically reduced the level of synapsin-II, and oleamide dose-dependently protected the synapsin-II levels . These data indicate that oleamide can block the calpain-mediated cleavage of substrate proteins that play important roles in the regulation of neuronal survival/death and neuronal activity.The KA-induced significant neuronal loss can lead to the axonal degeneration that is preceded by disruption of Caproteins . CRMP-2 proteins . In addiproteins . In our proteins , which wproteins . Since tproteins . A relatproteins . In the in vivo  showing the ability of oleamide to enhance the level of endogenous calpastatin, which is reduced in KA-induced epileptic rat brain, suggest that its antiepileptic and possible neuroprotective effects are due to calpain inhibition.We further confirmed the calpain inhibitory effects of oleamide by examining changes in endogenous calpastatin levels. An endogenous calpain inhibitory protein, calpastatin, has been proposed to play pro-survival roles in adult neurons under degenerative conditions, including in models of ischemia–excitotoxicity . Recentlin vivo . Data . In addition, the CRMP-2 and Cdk5 degradation observed in rat brain tissue lysates directly incubated with μ-calpain were inhibited by exogenous treatment with oleamide . These results provide evidence for the possibility of direct calpain inhibition by oleamide. It can be speculated that oleamide may enter into the intracellular space through an endogenous fatty acid amide membrane transporter that has been shown to transport anandamide (So far, oleamide was assumed to exert its various biological actions through as-yet undefined membrane receptors. The calpain inhibitory effect of oleamide has not been reported previously. We hypothesized that oleamide might play a role as a calpain inhibitor. To investigate this, andamide , and theTaken together, our findings reveal that exogenous oleamide attenuates not only KA-induced behavioral seizures but also calpain-mediated neuronal cell death in the brain, suggesting that oleamide is a novel promising drug candidate for various neuronal diseases including epilepsy. In addition, the antiepileptic and neuroprotective effects of oleamide could be mediated through a novel mechanism by calpain inhibition, although its direct calpain inhibitory effect needs to be further pursued.HYN performed experiments, analyzed the data, and wrote the first draft of manuscript. EJN, EL, and YK analyzed data and involved in the manuscript preparation. H-JK designed and supervised the research, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Traumatic brain injury (TBI) increases the risk of Alzheimer’s disease (AD). Calpain activation and tau hyperphosphorylation have been implicated in both TBI and AD. However, the link between calpain and tau phosphorylation has not been fully identified. We recently discovered that the two major calpain isoforms in the brain, calpain-1 and calpain-2, play opposite functions in synaptic plasticity and neuronal survival/death, which may be related to their different C-terminal PDZ binding motifs. Here, we identify the tyrosine phosphatase PTPN13 as a key PDZ binding partner of calpain-2. PTPN13 is cleaved by calpain-2, which inactivates its phosphatase activity and generates stable breakdown products (P13BPs). We also found that PTPN13 dephosphorylates and inhibits c-Abl. Following TBI, calpain-2 activation cleaved PTPN13, activated c-Abl and triggered tau tyrosine phosphorylation. The activation of this pathway was responsible for the accumulation of tau oligomers after TBI, as post-TBI injection of a calpain-2 selective inhibitor inhibited c-Abl activation and tau oligomer accumulation. Thus, the calpain-2-PTPN13-c-Abl pathway provides a direct link between calpain-2 activation and abnormal tau aggregation, which may promote tangle formation and accelerate the development of AD pathology after repeated concussions or TBI. This study suggests that P13BPs could be potential biomarkers to diagnose mTBI or AD. Many protein kinases and phosphatases have been shown to participate in the regulation of the multiple phosphorylation sites on the tau protein5. Among them, several non-receptor tyrosine kinases, such as c-Abl, have been identified to regulate tau tyrosine phosphorylation and have been implicated in tau aggregation9. However, the exact mechanisms relating tau tyrosine phosphorylation to tau pathology have not been elucidated. Calpain activation has also been implicated in many of the same disorders, and calpain-mediated p35 to p25 conversion and the resulting activation of cyclin-dependent kinase 5 (cdk5) was shown to be involved in tau phosphorylation in AD14, although this hypothesis has been challenged15. Other links between calpain and tau hyperphosphorylation involve calpain-mediated truncation of glycogen synthase kinase 3 beta (GSK-3ß)17, dual specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A)18 and protein phosphatase 2A (PP2A)19. TBI has been shown to be a major risk factor for AD21, and CTE, resulting from repeated concussions, has been associated with tau hyperphosphorylation22. However, how TBI results in tau hyperphosphorylation is still not understood.Tau hyperphosphorylation is a hallmark of several neurological disorders, including Alzheimer’s disease (AD), frontotemporal dementia, traumatic brain injury (TBI) and chronic traumatic encephalopathy (CTE)23). We have shown that these two isoforms are often activated by the same initial stimulus, although with a different time-course of activation and for different durations. Furthermore, we provided evidence that calpain-1 and calpain-2 play opposite functions under both physiological and pathological conditions. Thus, calpain-1 is required for the induction of long-term potentiation (LTP) in hippocampus and for some forms of learning and memory, while calpain-2 activation limits the magnitude of LTP and learning and memory25. Calpain-1 activation is neuroprotective through the stimulation of survival cascades but calpain-2 activation is neurodegenerative29. We postulated that these opposite functions of calpain-1 and calpain-2 were the result of the existence of different PDZ domain binding motifs in the C-terminus of calpain-1 and calpain-223. PDZ domains are conserved protein-protein interaction modules within multivalent scaffold proteins30, and thus the differential association of these 2 calpain isoforms with distinct PDZ domains could alter the activity/function of these isoforms by localizing them within different clusters of signaling proteins. In particular, calpain-1 exhibits a type II PDZ binding motif, TMFA, across the majority of vertebrates, while calpain-2 exhibits a typical type I PDZ binding domain, FSVL. In this study, we identified a key PDZ binding partner of calpain-2 to be the tyrosine phosphatase, PTPN13, aka FAP-1, which was previously shown to be involved in cell apoptosis32. We found that calpain-2 cleaves PTPN13, resulting in the inactivation of its phosphatase activity, and the generation of stable breakdown products recognized by PTPN13 antibodies (P13BPs). We also found that PTPN13 regulates the phosphorylation/activation of the protein kinase c-Abl, which was previously shown to phosphorylate tau at tyrosine 3949. Following TBI, calpain-2 cleaves PTPN13, activates c-Abl, and stimulates tau tyrosine phosphorylation and tau oligomer formation. Moreover, increased levels of P13BPs were found in postmortem brains of AD patients. Thus, calpain-2-mediated truncation of PTPN13 and the resulting c-Abl activation provide a link between TBI and tau phosphorylation, which could account for the increased risk of AD following TBI.Over the last few years, our laboratory has focused on understanding the relative roles of two of the major calpain isoforms in the brain, calpain-1 and calpain-2  in both physiological and pathological conditions  of calpain-1 and calpain-2 as GST fusion proteins and overlaid them on a PDZ domain array, which has been previously describedGST Fig.  or GST-cGST Fig. . In contGST Fig.  and S1C.GST Fig. .Figure 1We next performed a series of experiments to assess whether calpain-2 and PTPN13 were associated in neurons and in brain. Brain sections were dually stained with antibodies against calpain-2 and PTPN13 or calpain-1 and PTPN13 Fig. . High maIn parallel biochemical studies, we immunoprecipitated PTPN13 from brain homogenates with an antibody against PTPN13 under non-denaturing conditions, in order to pull down proteins that might be associated with PTPN13. As shown in Fig. 35). As shown in Fig. 2-C6H3 2)28, referred to as NA101. We previously reported that NA101 has a Ki of 25 nM against purified calpain-2 versus a Ki of 1.3 µM against calpain-1, indicating that it has a 50-fold selectivity for calpain-2 over calpain-126. In contrast, spectrin was cleaved following incubation with both low and high calcium concentrations  or both calpain-1 and calpain-2 . After incubation, samples were immunoblotted with an antibody against PTPN13, as well as an antibody against spectrin . Moreover, PTPN13 was cleaved, generating several breakdown products, and c-Abl was phosphorylated at tyrosine 245. These effects were significantly blocked when slices were incubated in the presence of a calpain inhibitor . These results reveal that calpain-2 activation results in PTPN13 cleavage and inactivation, leading to increased c-Abl phosphorylation at tyrosine 245, a phosphorylation event that is required for c-Abl activity36.While the above results indicated that calpain-2 could cleave PTPN13 and that PTPN13 could dephosphorylate c-Abl at tyrosine 245, they did not determine whether calpain-2-mediated cleavage of PTPN13 resulted in its inactivation and increased tyrosine phosphorylation of c-Abl at tyrosine 245. To address this question, we incubated acute hippocampal slices prepared from neonatal calpain-1 knock-out mice with NMDA and determined the effects of calpain-2 activation on spectrin degradation, PTPN13 levels, and c-Abl tyrosine phosphorylation at tyrosine 245 Fig. . We prev3. We recently reported that in the controlled cortical impact model of TBI in wild-type mice calpain-2 activation was delayed and prolonged and responsible for producing cell death37. We first determined the levels of full-length PTPN13, P13BPs, c-Abl, phospho-Tyr245 c-Abl, tau and phospho-tyrosine tau in WT mice after TBI using Western Blot (WB)  Fig. . P13BPs WB) Fig. , suggestWe then used immunohistochemistry (IHC) to verify whether phosphorylation of c-Abl at tyrosine 245 was increased under these conditions. Levels of phospho-Tyr245 were significantly increased in the area surrounding the impact in the ipsilateral cortex of WT mice 6 h after TBI, as compared to 0 h Fig. . TreatmeTo verify that PTPN13 cleavage and tau tyrosine phosphorylation following TBI was mediated by calpain-2 but not calpain-1, we tested the levels of full-length PTPN13, P13BPs, tau and phospho-tyrosine tau in calpain-1 KO mice after TBI using Western Blot Fig. . PTPN13 38. Tau oligomers play a critical role in the initiation and spreading of tau pathology42. To test whether calpain-2 and c-Abl were involved in this process, we injected NA101 or a brain-penetrating c-Abl inhibitor, nilotinib, to WT mice 1 h after TBI, and examined the formation of tau oligomers 24 h after TBI using IHC, ELISA and WB with an antibody that specifically recognizes oligomeric Tau (T22). With IHC, levels of tau oligomers were dramatically increased in the ipsilateral cortex and hippocampus 24 h after TBI  domain that binds to plasma membrane and cytoskeletal elements  following calpain-2-mediated PTPN13 cleavage, and this was confirmed in hippocampal slices. C-Abl has recently been associated with both AD and Parkinson’s disease (PD). In the case of AD, c-Abl has been shown to phosphorylate tau at tyrosine 394, and this site is phosphorylated in human neurofibrillary tangles9. In the case of PD, c-Abl phosphorylates and activates parkin, leading to mitochondrial abnormalities and increased oxidative stress44.PTPN13 belongs to the family of protein tyrosine phosphatases (PTP) and has been mostly studied for its role in cancernts Fig. . This pr37. Results presented here demonstrate that PTPN13 is cleaved by calpain-2 following TBI, resulting in c-Abl and tau phosphorylation at tyrosine residues. Systemic injection of a selective calpain-2 inhibitor, NA101, 1 h after TBI at a dose of 0.3 mg/kg, which we previously showed to significantly prevent calpain-2 activation in the brain28, almost completely eliminated c-Abl activation and increased tyrosine tau phosphorylation. Moreover, injection of NA101 or of a c-Abl inhibitor, nilotinib, significantly reduced the formation of tau oligomers 1 day after TBI, suggesting that the calpain-2 → PTPN13 → c-Abl → tau phosphorylation pathway is involved in this process. Interestingly, NA101 injection significantly reduced the level of pS202/T205 after TBI. Levels of pT231 were partially but not significantly reduced by NA101 injection. In contrast, levels of pS416 were not affected by NA101. GSK-3ß and cdk5 have been shown to directly phosphorylate tau at S202, T205 and T231, while casein kinase 1 (CK1) and protein kinase A (PKA) directly phosphorylate tau at S4164. Calpain has been shown to regulate GSK-3ß45 and cdk513, and it is therefore possible that calpain-2 activation leads to increased phosphorylation of tau at multiple sites through the regulation of different protein kinases and phosphatases. Recently, accumulating evidence has shown that the propagation of tau oligomers plays a critical role in the development of tau pathology. Injection of tau oligomers derived from AD brain disrupts memory and propagates abnormal aggregation of endogenous tau in WT mice42. Further studies need to be done to examine the effect of long-term treatment a with calpain-2 inhibitor on neurofibrillary tangle (NFT) formation after TBI. Finally, our results indicate that calpain-2-mediated PTPN13 truncation is enhanced in AD brain, as compared to normal age-matched brain.We recently found that calpain-2 was activated following TBI, and that this activation plays an important role in cell death in the days following TBI26. We proposed that calpain-2 activation elicited by extrasynaptic NMDAR stimulation is the result of the association of NR2B subunit of NMDARs with RasGRF1, which provides a link between extrasynaptic NMDARs and ERK activation46. Indeed, we have previously shown that ERK activation directly phosphorylates and activates calpain-247. It has also been repeatedly shown that NR2B subunits are enriched in extrasynaptic NMDARs48, and that their activation is critical for excitotoxicity49. Thus, this pathway is likely responsible for the prolonged activation of calpain-2 upon stimulation of extrasynaptic NMDA receptors following TBI.It is worth discussing how calpain-2 is activated following TBI. Calpain-2 is located at extrasynaptic sites and activated only when extrasynaptic glutamate level increases, which occurs predominantly under pathological conditions50. While there is currently no evidence that tau is phosphorylated at tyrosine 396 in CTE brain, it is possible that repeated concussions activate the calpain-2 → PTPN13 → c-Abl → tau phosphorylation pathway, leading to NFT formation. Furthermore, as PTPN13 has previously been shown to participate in apoptosis, it is possible that calpain-2 mediated truncation/inactivation of PTPN13 is also involved in neuronal apoptosis under various pathological conditions.All these results are summarized in a schematic diagram shown in Fig. 51, and it is therefore likely that the pathway described here is also activated by mTBI. Additionally, it is possible that the P13BP(s) identified here could be further characterized and could potentially serve as novel blood biomarkers for the diagnosis of mTBI or AD. Finally, our results suggest that a selective calpain-2 inhibitor might be beneficial not only for the treatment of TBI, but also for preventing the development of CTE following repeated concussions, as well as for more chronic neurodegenerative disorders such as AD and PD.It was previously shown that blood levels of a marker of calpain activation, an N-terminal fragment of spectrin generated by calpain, were correlated with the pathological outcomes of mild TBI (mTBI)Animal use in all experiments followed NIH guidelines and all protocols were approved by the Institution Animal Care and Use Committee of Western University of Health Sciences. Calpain-1 KO mice on a C57Bl/6 background were obtained from a breeding colony established from breeding pairs generously provided by Dr. Chishti (Tufts University). C57Bl/6 mice were purchased from Jackson Labs and were the corresponding wild-type (WT).Flag-PTPN13 were generously provided by Dr. Carl-Henrik Heldin . Flag-PTPN13 D2359A was constructed based on Flag-PTPN13 using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies). EGFP-Tau  and contains exons 1, 4 and 5, 7, and 9–13, intron 13, and exon 14)(plasmid #46904), c-Fyn (human) (plasmid #16032), Flag-SYK (human)(plasmid #20646) and Flag-His-c-Abl (human) (plasmid #52684) were purchased from Addgene. GST-calpain-1 C-terminus and GST-calpain-2 C-terminus expression plasmids were constructed on pGEX-3x vector.The primary antibodies used for WB were PTPN13 , calpain-1 , calpain-2 , tau-5 , tau-1 , anti-Tau oligomer T22 , phospho-tau Ser202/Thr205 AT8 , phospho-Tau Thr231 AT180 , phospho-tau Ser416 , c-Abl , spectrin , phosphotyrosine , Phospho-c-Abl Tyr245 , Phospho-c-Abl Tyr412  and actin . The secondary antibodies used for WB were IRDye 680RD goat anti-rabbit  and IRDye 800CW goat anti-mouse .The calpain-2 C-terminal peptide was synthesized by ABI Scientific. The sequence is TIQLNLASWLSFSVL .34. Briefly, 1 μg of hexahistidine-tagged PDZ domain fusion proteins were spotted onto nitrocellulose, dried overnight, and then overlaid with GST-alone (control), GST-calpain-1-CT, or GST-calpain-2-CT (800 nM). Membranes were washed three times and incubated with an HRP-coupled anti-GST monoclonal antibody (Amersham Pharmacia Biotech) and binding of calpain-1-CT or calpain-2-CT fusion protein was visualized using enhanced chemiluminescence.Fusion proteins were purified and overlays of the PDZ domain array were performed as previously describedhttp://calpain.org/predict.rb?cls=substrate). SVM RBF prediction model was used. The query sequence (1000 aa maximum) was aa1001–1999 of PTPN13 [Mus musculus] (NP_035334.2). This region was selected because it includes the epitope of PTPN13 antibody LS-C148268 which is aa1279–1883. The top three predicted cleavage sites in this region were 1488, 1023 and 1965. The molecular weights (MWs) of the fragments generated by the cleavage at 1488 are 170 kDa  and 105 kDa . The MWs of the fragments generated by 1023 are 125 kDa  and 150 kDa . The MWs of the fragments generated by 1965 are 210 kDa  and 65 kDa . Among them, the 170 kDa fragment generated by cleavage at 1488 and 210 kDa generated by cleavage at 1965 should be recognized by PTPN13 antibody and fit the MWs of the breakdown products detected in WB  in PTPN13 was performed using the online cleavage site predictor  were grown in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen). For transient expression, cells were transfected with the respective DNA constructs by Lipofectamine 2000 (Invitrogen).55. Mice  were anesthetized using isoflurane and fixed in a stereotaxic frame with a gas anesthesia mask. A heating pad was placed beneath the body to maintain body temperature around 33–35 °C. The head was positioned in the horizontal plane. The top of the skull was exposed, and a 5-mm craniotomy was made using a micro drill lateral to the sagittal suture, and centered between Bregma and Lambda. The skull at the craniotomy site was carefully removed without damaging the dura. The exposed cortex was hit by a pneumatically controlled impactor device . The impactor tip diameter was 3 mm, the impact velocity was 3 m/sec, and the depth of cortical deformation was 0.5 mm. After the impact, the injured region was sutured using tissue adhesive (3 M) and the mice were placed in a 37 °C incubator until they recovered from anesthesia. For sham surgery, mice were sutured after craniotomy was performed.CCI model was established in mice following the protocol described in previous publicationsMice were anesthetized and perfused intracardially with freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer . After perfusion, brains were removed and immersed in 4% paraformaldehyde at 4 °C for post-fixation, then in 15% and 30% sucrose at 4 °C for cryoprotection. Coronal frozen sections at indicated positions were prepared. Sections were first blocked in 0.1M PBS containing 5% goat or donkey serum and 0.3% Triton X-100 (blocking solution) for 1 h, and then incubated with anti-PTPN13 , anti-calpain-1 , anti-calpain-2 , Phospho-c-Abl Tyr245  and/or anti-Tau oligomer , prepared in blocking solution overnight at 4 °C. Sections were washed 3 times in PBS (10 min each) and incubated in Alexa Fluor 488 goat anti-rabbit IgG and/or Alexa Fluor 594 goat anti-mouse IgG  prepared in blocking solution for 2 h at room temperature. After three washes, sections were mounted with mounting medium containing DAPI (Vector Laboratories).56 and Prism. Before analyzing using the JaCOP plugin, each image’s quality and reduction of background increased with noise despeckling, removal of bright pixel outliers with a radius of 2.0 and a threshold of 50, and with a standard increase of gamma (1.25). After which, two images of either PTPN13 and Calpain-2 or PTPN13 and Calpain-1 were compared of the same section and area of interest. Criteria looked at for the quantification of co-localization was the Pearson’s Coefficient. Image acquisition and quantification were done by two persons in a blind fashion.For analysis of PTPN13 and calpain-1/calpain-2 co-localization, sections were visualized under 100X using a confocal (Nikon) and analyzed for co-localization of the CA1 hippocampal region using ImageJ JaCOP pluginFor quantification of Phospho-c-Abl Tyr245 levels, 3 coronal brain sections at Bregma −0.58 mm, −1.58 mm and −1.94 mm of each brain were immunostained and analyzed. In each section, three 637 µm × 637 µm areas in both ipsilateral and contralateral sides were imaged using a confocal microscope (Nikon). Mean fluorescence intensity (MFI) in each of the three areas was measured in ImageJ and averaged. Then, MFI in the contralateral side  was subtracted from MFI in the ipsilateral side. Data in all three sections from the same brain were averaged. Image acquisition and quantification were done by two persons in a blind fashion.cortex and MFICA1 in three sections from the same brain were averaged. Image acquisition and quantification were done by two persons in a blind fashion.For quantification of Tau oligomers, 3 coronal brain sections at Bregma −1.58 mm, −1.94 mm and −2.30 mm of each brain were immunostained and analyzed. In each section, two 424 µm × 424 µm areas in ipsilateral cortex and two in ipsilateral CA1 were imaged using a confocal microscope . Mean fluorescence intensity (MFI) in each area was measured in ImageJ. MFIMouse brain was homogenized in ice-cold PBS plus protease inhibitor cocktail (Thermo Fisher Scientific). 96-well ELISA plates (Nunc MaxiSorp™) were coated with 15 µg of brain homogenate using 0.05 M sodium bicarbonate, pH 9.6 as coating buffer, followed by overnight incubation at 4 °C. The plates were washed three times with Tris-buffered saline with 0.01% Tween (TBST), then blocked for 3 h at room temperature with 10% nonfat dry milk in TBST. The plates were then washed with TBST and incubated with anti-tau oligomer  or anti-tau  antibody in 5% nonfat milk in TBST for 1 h at room temperature. The plates were washed three times with TBST and incubated with goat anti-rabbit IgG Horseradish-peroxidase (HRP) or goat anti-mouse IgG HRP (1:3000) in 5% nonfat milk in TBST for 1 h at room temperature. Plates were then washed three times with TBST and developed with 1-Step TMB (Thermo Fisher Scientific). The reaction was stopped with 2 M sulfuric acid and the plates were read at 450 nm using a POLARstar OMEGA plate reader (BMG Labtech).2+ treatment or in IP lysis/wash buffer plus protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) for immunoprecipitation.Mouse brain was isolated and placed in ice-cold homogenization buffer containing the following: 320 mM sucrose, 10 mM HEPES, pH 7.4, 2 mM EDTA and protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Brain tissue was homogenized using a glass homogenizer. The homogenate was centrifuged at 1000 × g for 10 min, and the resultant supernatant was collected and centrifuged at 14,000 × g for 20 min. The resultant pellet (P2 membrane fraction) was centrifuged again at 14,000 × g for 20 min to eliminate protease inhibitors. P2 pellets were then resuspended in Tris-acetate buffer  for CaImmunoprecipitation was performed using the Pierce Classic IP Kit  following the instruction. The antibodies used for IP were PTPN13 (LS-C148268), normal rabbit IgG , GFP , c-Abl , tau (AHB0042) and normal mouse IgG .3, 10 glucose, 3 KCl, 1.25 KH2PO4, 5 MgSO4, and 3.4 CaCl2. Hippocampal transversal slices (400 μm-thick) were prepared using a McIlwain-type tissue chopper and transferred to a recovery chamber with a modified aCSF medium, containing (in mM): 124 NaCl, 2.5 KCl, 2.5 CaCl2, 1.5 MgSO4, 1.25 NaH2PO4, 24 NaHCO3, 10 D-glucose, and saturated with 95% O2/5% CO2 for 1 h at 37 °C. After 1 h recovery, hippocampal slices were then transferred into screw-cap microfuge tubes (2–3 slices each tube) containing 2 ml of freshly oxygenated aCSF medium with various drugs and were incubated at 37 °C. At the indicated time points, slices were rapidly frozen on dry ice. Slices were then lysed and protein concentrations were measured using BCA protein assay kit (Thermo Fisher). Equivalent amounts of proteins were processed for SDS-PAGE and western blot.Calpain-1 KO mice at postnatal day 10 were anesthetized with isoflurane and decapitated. Brains were quickly removed and transferred to oxygenated, ice-cold cutting medium (in mM): 124 NaCl, 26 NaHCOData are means ± SEM in all figures. To compute p values, unpaired two-tailed Student’s t-tests and one- or two-way ANOVA with Bonferroni post-tests were used, as indicated in figure legends.The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.Supplementary Data


An exceptional case of Nos-independent regulation by Pum has been described—repression of maternal bicoid (bcd) mRNA at the anterior pole of the early embryo, dependent on both Pum and conserved Pum binding sites in the 3’-UTR of the mRNA. We have re-investigated regulation of bcd in the early embryo; our experiments reveal no evidence of a role for Pum or its conserved binding sites in regulation of the perdurance of bcd mRNA or protein. Instead, we find that Pum and Nos control the accumulation of bcd mRNA in testes.Drosophila Pumilio (Pum) is a founding member of the conserved Puf domain class of RNA-binding translational regulators. Pum binds with high specificity, contacting eight nucleotides, one with each of the repeats in its RNA-binding domain. In general, Pum is thought to block translation in collaboration with Nanos (Nos), which exhibits no binding specificity in isolation but is recruited jointly to regulatory sequences containing a Pum binding site in the 3’-UTRs of target mRNAs. Unlike Pum, which is ubiquitous in the early embryo, Nos is tightly restricted to the posterior, ensuring that repression of its best-characterized target, maternal  Mated females were returned to the appropriate vial and allowed to lay eggs until the next generation of flies was about to emerge, when the mated females were discarded. After incubating the vials for a further 5 days  , and pum pumET3 ; nosBN  , nosL7 /Exel6183 ; bcd6 // bcd 12 ; tud1 /k #7554) ; wild ty females . The bcdbcd and eve mRNAs were detected by standard in situ hybridization methods using digoxigenin-labeled cDNA probes and visualized with a Zeiss Axiophot microscope using Nomarski optics and a Q Imaging digital camera. For immunohistochemical experiments, embryos were fixed in 4% formaldehyde and incubated with rabbit anti-Bcd sc-66818  and the 2B8 anti-grasshopper Eve (at 1:50) developed by Kai Zinn obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa. Secondary antibodies (1:200) were from Jackson ImmunoResearch Laboratories. Embryos were visualized on a Zeiss 510 confocal microscope. The timing of embryonic development was assessed both by morphology [eve mRNA pattern [rphology  and evol pattern , with Bo pattern . An apprbcd+ genomic DNA that includes the entire transcription unit, 4 kb of 5’-flanking DNA (and thus a portion of the adjacent Ama gene), 1 kb of 3’-flanking DNA, and that fully rescues the maternal bcd phenotype, as described [bcd constructs were inserted into pCasper4 and germline transformants generated by standard methods. The DNA Strider program was used to analyze sequences [We used a 9 kb fragment of escribed . The fraequences .bcd (assay DM02148187_g1) or nos mRNAs (DM02134535_g1) relative to the level of Rps2 mRNA (DM02361142_s1). Measurement of the relative level of bcd mRNA was assayed in three independent samples prepared from pools of 50 males for the experiment of nos mRNA in nosBN / Df males referred to in the text was assayed in four independent samples from pools of 50 males. In each case statistical significance was determined using an unpaired t test. To measure the level of bcd transgene-encoded mRNA, we collected duplicate samples of 0–3 hour embryos from females bearing a single transgene copy and duplicate samples of single-copy transgenic males. Four bcd (NRE)+ transgenes and four bcd (NRE)mut transgenes were analyzed. The average level of bcd mRNA in the 8 embryonic samples was 1.6-fold higher than in wild type, close to the expected value of 1.5; but the average level in the 8 male samples was 76-fold higher than in wild type.Electromobility gel shift experiments were performed essentially as described  using RN