IntroductionPedicle screw insertion is the mainstay of thoracic and lumbosacral posterior spinal instrumentation. However, it may be associated with complications such as screw malpositioning. The purpose of this study was to develop a pilot study to compare the accuracy of robot-guided screw insertion versus hand-guided screw placement for spinal instrumentation. The hand-guided screws were placed with assistance from computerized tomography (CT) stealth guidance or fluoroscopy.Materials and methodsA retrospective analysis of medical records was done for all patients that had pedicle screw insertion for instrumentation between the dates of December 2013 and January 2016 with post-screw placement CT imaging. The analysis was conducted on screw accuracy between the two categories based on the Gertzbein-Robbins classification.ResultsA total of 49 screws were analyzed for accuracy in six patients. There was no statistically significant difference between the accuracy of hand-placed pedicle screws versus the robotically placed screws (p = 0.311). There was no statistically significant difference in blood loss (p = 0.616), length of procedure (p = 0.192), or post-operative length of stay (p = 0.587).ConclusionThe findings of our pilot study agree with most prior studies that there was no statistically significant difference in the accuracy of pedicle screw placement between the two methods of screw placement. Therefore, the techniques are equivocal in accuracy. The new technology (robotic-guidance) is as safe as conventional techniques for screw placement. Just like in any surgery, the technique preference should remain surgeon dependent. The results are only from a small sample size in the development of a pilot study so a strong reliance on the data would not be suggested. The study was a preliminary study that will be used as a template and learning process to create a future prospective study to investigate CT stealth and robotically guided screw placement versus “free hand” guided screws. Pedicle screw insertion of the spine is one of the main procedural steps of thoracic and lumbosacral posterior instrumentation and has gone through substantial advancement over the last couple of decades -2. An arIn recent years, image guidance with navigation and intraoperative imaging have been used in order to improve the accuracy and safety of pedicle screw placement. In addition to these navigational systems, a robotic spine surgery system has also been recently added to the arsenal in order to increase the accuracy of pedicle screw trajectories -6. WhileThe main purpose of this pilot study is to compare the accuracy of robot-guided screw insertion in thoracolumbar and sacral surgeries at our center with a cohort of patients who underwent hand-guided screw placement via fluoroscopy-guided or computed tomography (CT) stealth-guided spinal instrumentation. It is hypothesized that accuracies will remain similar between the two cohorts without a significant difference, making the robotic technique equivocally safe to use in spinal surgery. The secondary purpose was to use this pilot study to design a prospective multicenter study with a much larger cohort to validate the findings of present and prior studies and the question regarding screw placement precision superiority.PatientsA retrospective analysis of medical records was performed for all patients that had pedicle screw insertion for instrumentation between December 2013 and January 2016 with post-screw placement CT imaging. The data was extracted via Crimson Continuum of Care system at Desert Regional Medical Center, in Palm Springs, California. The patients who were over 18 years of age and underwent spinal fusion surgery, except cervical spine, and having post-operative CT scan were included.Robot and implantsBone-mounted or table-mounted, miniature robotic spine surgery system named the Renaissance system by Mazor was used.Assessment of screw positionThe primary objective was to compare the accuracy of screw placement with fluoroscopy-guided hand-placed screw versus robotic-placed screws. This was done by measuring the screw accuracy with the Gertzbein and Robbins Scale as described below . A blindGertzbein and Robbins scaleThe Gertzbein and Robbins scale is one of two grading scales currently used to describe pedicle screw placement . In thisCollection of other clinical dataOther parameters that were recorded from the patient charts included operative time, blood loss, and hospital length of stay (LOS) from the day of surgery. The analysis was based on intraoperative and postoperative medical documentation. All information was extracted from the patients’ charts and CT scans.Statistical analysisThe Pearson Correlation test was used for statistical analysis.There were in total six patients who underwent pedicle screw insertion for thoracolumbar/sacral pathology or trauma during the aforementioned period. Four patients had fluoroscopy-guided or CT stealth-guided hand-placed screws and two patients had robotically placed screws. Average patient age was 52.5 years in the robotic group (Group A) and 59.5 years in the hand-placed group (Group B). Patient demographic has been summarized in the table  were in the hand-placed group (Group B) Figure  and 16 . The amo% were inThere was no statistically significant difference in blood loss (p = 0.616) with an average blood loss of 1625 ml for hand placed and 975 ml blood loss for robotic Table . The durAs pedicle screw fixation was first described by Boucher in the 1950s, used more extensively and further examined by Roy-Camille later in the 1960s and 1970s, and then down-classified from an FDA Class III to Class II device in 1998, pedicle screw fixation has evolved and become increasingly popular among spine surgeons -13. OrigIn addition to offering an overall increased construct rigidity, the pedicle screws have several advantages over the traditional hook and rod constructs as they allow the stability essential for spinal arthrodesis and improve deformity correction due to its three-column control over the spinal elements. Pedicle screw fixation promotes multidimensional control and may provide greater fusion rates, making it the mainstay of thoracic and lumbosacral posterior spine instrumentation , 16-18. Robotics, already adopted in other surgical specialties, has also become an innovative development in spine surgery to further improve accuracy and safety -6, 19-22In the past, there have been studies comparing robot-assisted placed screws with fluoroscopy-guided hand placed screws with mix results -28. TherIn this study, six patients were evaluated having 49 screws inserted in total. Out of these, 33 screws were inserted by hand placement and 16 were robotically guided. The accuracy of pedicle screw placement in cohorts of robotic-guided and conventionally placed pedicle screws was assessed. Assessment of pedicle screw position by a single investigator blinded to the insertion technique was performed in order to minimalize the effect of investigator-dependent errors. The investigator also is a neurosurgery trained doctor with experience in image analysis. The long-term results  were not included in our study as it can be anticipated that these would be similar for all pedicle screws regardless of the surgical approach applied. Even more difficult would be finding patients that received serial follow-up imaging post-operatively. The finding of this study showed that there is no statistically significant difference between the accuracy of the two methods for the guidance of pedicle screw placements (p = 0.311).Pechlivanis, et al. and Kantelhardt, et al. reported 98.5% and 98.9% accuracy (Grade A and B) with robot-assisted group , 26. SchSince minor deviations rarely become symptomatic, many clinicians accept deviations up to 2 or 3 mm. In a meta-analysis of 4,790 screws conducted by Lonstein, et al. A total of 5.1% screws were reported to breach the cortical bone and only 0.2% of these caused neurological symptoms . HoweverThe comparative results for operative time and hospital stay between the two groups were statistically insignificant  in our study and were consistent with the earlier studies , 23. KanThe choice of approach between robotic versus the fluoroscopy-guided or CT stealth-guided hand-placed screws is more on the preference of the operating surgeon since there is no significant difference in either of the methods in terms of accuracy and patient safety. In fact, there would be hesitation to strongly rely on the results of this trial due to the limitations of low sample size. The main limitation of this study is low sample size. Although only six patients, there were a large number of pedicle screws to examine. The reason for low patient volume was due to the institution not having regular postoperative CT scans, which severely limited the number of patients included in this study. These factors will be taken into account on the upcoming multicenter prospective study.This study was a preliminary pilot study for the development of a future study of pedicle screw accuracy assessment. The upcoming trial with a much larger cohort and large number of participating surgeons will have improved study protocol which will examine two groups once again but instead of robotic versus other types of screw placement, it will be CT or stealth-guided placement versus placement with fluoroscopy. Fluoroscopic placement is when the surgeon relies on fluoroscopic X-Ray for the location or trajectory of the screws. A study like that would provide readers with information on whether advancements in the real-time image technology will show better accuracy which is predicted. This pilot study was able to provide practice and education to better conduct the upcoming study.Our findings agree with most prior studies that there was no statistically significant difference in the accuracy of pedicle screw placement between the two methods of screw placement. Therefore, the techniques are equivocal in accuracy. The new technology (robotic-guidance) is as safe as conventional fluoroscopy assisted techniques and CT-guided stealth techniques for screw placement, but does not supersede the conventional free-hand method, though the literature seems to be more optimistic. Just like in any surgery, the technique preference should remain surgeon dependent. It should be emphasized that the study was a pilot study to learn how to conduct future studies on pedicle screw placement. The results from this study cannot hold a strong conclusion from the results due to low sample size. In the future study, CT stealth guided and robotically guided screw placements will be compared with fluoroscopy guided placement of screws for accuracy. The increase in sample size will be obtained by regularly performing postoperative CT scans on patients with thoracolumbar-sacral pedicle screw instrumentation and making it a multicenter study to involve more surgeons to eliminate any result bias.


Although antibodies that effectively neutralize a broad set of influenza viruses exist in the human antibody repertoire, they are rare. We used a single-cell screening technology to identify rare monoclonal antibodies (MAbs) that recognized a broad set of influenza B viruses (IBV). The screen yielded 23 MAbs with diverse germ line origins that recognized hemagglutinins (HAs) derived from influenza strains of both the Yamagata and Victoria lineages of IBV. Of the 23 MAbs, 3 exhibited low expression in a transient-transfection system, 4 were neutralizers that bound to the HA head region, 11 were stalk-binding nonneutralizers, and 5 were stalk-binding neutralizers, with 4 of these 5 having unique antibody sequences. Of these four unique stalk-binding neutralizing MAbs, all were broadly reactive and neutralizing against a panel of multiple strains spanning both IBV lineages as well as highly effective in treating lethal IBV infections in mice at both 24 and 72 h postinfection. The MAbs in this group were thermostable and bound different epitopes in the highly conserved HA stalk region. These characteristics suggest that these MAbs are suitable for consideration as candidates for clinical studies to address their effectiveness in the treatment of IBV-infected patients. Influenza B viruses (IBV) have two antigenically differentiated lineages, termed Yamagata and Victoria , 2, whic4–0 primary protein must first be activated by proteolytic cleavage into the HA1 and HA2 subunits, which remain coupled by disulfide bonds, thereby facilitating the conformational change of HA necessary for fusion of the viral and cellular membranes  surface glycoprotein of influenza viruses have been made for vaccine candidates or therapeutic monoclonal antibodies (MAbs)  high potency in vivo (as assessed in a murine IBV infection model), (iv) suitable for human use, and (v) structurally stable.Our aim was to discover anti-IBV MAbs that have the following qualities: (i) broadly reactive against circulating IBV strains by targeting the conserved HA stalk region, (ii) high-affinity binding +/CD27+) abundance was measured and adjusted to equal levels in each screen. In the primary screen, for most blood samples the average frequency at which memory B cells secreted a MAb against IBV HA was 2 in 10,000, which was about 10-fold higher than what we previously found when screening for MAbs against IAV HAs. Of these anti-IBV MAbs, about half showed cross-reactivity to HAs from both the B/Florida/04/2006 and B/Malaysia/2506/2004 strains. For further characterization, 23 MAbs were expressed in HEK293 cells and confirmed as being double positive for both IBV HAs.To achieve these goals, we applied the CellSpot technology to anonymized human blood samples. To identify broadly reactive anti-IBV MAbs, we used HA antigens from representative members of the two major lineages of IBV strains: B/Florida/04/2006 (Yamagata lineage) and B/Malaysia/2506/2004 (Victoria lineage) . Eight bOf the 23 initial antibody clones examined, 20 were produced at sufficiently high levels (at least 20 mg/liter) in a mammalian HEK293 cell transient expression system to warrant further investigation. The germ line origins for the sequences of these 20 MAbs are diverse, indicating that the CellSpot process surveys a wide sampling of the natural human repertoire . All of For IAVs, it is generally more difficult to generate escape mutants against MAbs that bind the conserved stalk region of HA than to generate escape mutants against MAbs that bind the head region . TherefoOf the remaining final five lead candidate MAbs , most have unique heavy and light chain sequences and likely arose from independent B cell lineages. The exceptions were MAbs TRL845 and TRL847 (derived from the same donor), which were closely related at the sequence level . IdentifDK) values of ≤200 pM. The broad reactivities of the five MAbs against different IBV strains were illustrated by binding to HAs from three lineage Yamagata strains and three lineage Victoria strains  for all of the MAbs was 57°C or above. Interestingly, even though TRL845 and TRL847 are derived from the same germ line sequences and share a high degree of overall sequence identity, TRL847 is significantly more thermostable. The biphasic melting curve seen for most of the MAbs is a common phenomenon, with heavy and light chains differing in their stability  were determined using the chemical linkage of peptides onto scaffolds (CLIPS) technology , comprisTo investigate the extent of HA sequence conservation for each MAb epitope, the entropy scores at each position were determined and mapped against an alignment of all currently available full-length IBV HA sequences isolated from human subjects  . The epi54H55F56A57N58L59K60). In this case, the high-entropy position is HA sequence position 55 (score = 100), where there is either a histidine or a phenylalanine for Victoria and Yamagata strains, respectively. Since TRL848 binds tightly to strains from both lineages  is the first epitope of MAb TRL848  in a plaque formation assay performed on MDCK cells  and that all treatments resulted in 100% survival . In contrast, CR9114 . All MAb-treated mice survived . Mice treated intraperitoneally with TRL849 at 1.0 mg/kg at 24 h postinfection were fully protected and showed a <10% transient weight loss, while mice receiving 0.1 mg/kg experienced >20% weight loss before recovering. Intranasal administration improved the potency, with 0.1 mg/kg at 24 h providing full protection from lethality and <10% weight loss. The increased potency of intranasal administration of neutralizing MAbs can be attributed to the increased levels of virus neutralization locally at the apical side of the site of infection, resulting in a lower viral burden  were assayed following stimulation to proliferate and differentiate using a cocktail of mitogens and cytokines as previously described . Peripheral blood mononuclear cells (PBMCs) were prepared by standard methods, and individual memory B cells , from Protein Sciences for B/Malaysia/2506/2004 , and from Immune Tech for B/Brisbane/60/2008 , B/Victoria/2/1987 , B/Massachusetts/02/2012 , and B/Wisconsin/1/2010 . On the next day, the plates were blocked with 3% bovine serum albumin (BSA) in PBS and then incubated for 1 h with serial dilutions of the MAb starting at 5 μg/ml (30 nM) in 0.5% BSA in PBS-Tween 20 (PBS-T). After washing with PBS-T, horseradish peroxidase-conjugated anti-human immunoglobulin kappa chain was added for 45 min. The plates were then washed with PBS-T and developed with SureBlue tetramethylbenzidine substrate .50) values in a 72-h plaque formation assay on MDCK cells  as described previously –5% fetal bovine serum–1% l-glutamine. Virus stocks whose titers were predetermined were rapidly thawed and diluted in MEM–1% l-glutamine and 1 μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK) trypsin , vortexed, and kept on ice. Antibodies were added to the virus at concentrations ranging from 0 to 20 μg/ml, vortexed, and incubated at 37°C for 1 h. Cell monolayers were washed 3 times with PBS, and 100 μl MEM–1% l-glutamine and 1 μg/ml TPCK trypsin was added to each well before 200 μl of the preincubated virus-antibody preparation was added. After 2 h at 37°C, the supernatant was aspirated, the monolayer was washed once with PBS, and a 1.2% Avicel RC-581  overlay containing 1 μg/ml TPCK trypsin in MEM, 20 mM HEPES, 2 mM l-glutamine, 0.075% NaHCO3, 100 units/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B was applied for 72 h at 37°C in 5% CO2. The plaques were visualized by fixing and staining with crystal violet. First, the Avicel overlay was aspirated and the monolayers were gently washed with PBS. The monolayers were then fixed by applying 1 ml of ice-cold 80% methanol–20% acetone for 10 min. Stock solutions of 0.13% (wt/vol) crystal violet were prepared in deionized water containing 5% methanol and 11% formaldehyde. Working stocks of crystal violet were made by diluting the stock solution 1:2 in PBS. After monolayer fixation, enough crystal violet working solution was added to a well to just cover the cells, and the cells were allowed to stain for 10 to 20 min. The wells were rinsed in distilled deionized water, and the average number of plaques from duplicate wells was counted and expressed as a percentage of the plaques in the uninhibited (100% infected) control wells. IC50s were calculated using the log (inhibitor) versus response nonlinear regression analysis method within the Prism (version 7) software package .The IBV-neutralizing activity of the MAbs was titrated to determine 50% inhibitory concentration  assay was performed as described previously  using stmT) of the MAbs were determined by differential scanning fluorimetry . mT values were determined from the average of 3 or more experiments, each of which was run in duplicate.The melting temperatures (orimetry . ReactioDK by standard methods (A stock solution of MAb at 600 nM was diluted to 5.0 and 0.5 nM in PBS (pH 7.4), and affinities were measured with a ForteBio Octet biosensor . Streptavidin-coated sensors were dipped into biotinylated HA antigen  after a wash step with the MAbs at a concentration of 250 nM. The resulting on and off rates measured yielded the  methods .4NRL).Using the chemical linkage of peptides onto scaffolds (CLIPS) technology , a set of >6,500 peptides covering linear, discontinuous, and conformational epitopes  was designed and synthesized as previously reported  using HAhttp://www.fludb.org) were obtained . Using the IDB set of analysis tools (http://www.viprbrc.org), the HA sequences were aligned and an analysis was performed to determine a positional entropy score (related to the Shannon entropy) at each aligned sequence position. The entropy scores at each HA residue position range from zero (100% conservation) to 232 . These values were used to assess the conservation at each of the IBV HA residue positions making up the epitopes recognized by the anti-IBV MAbs.All unique human-derived full-length IBV HA sequences deposited as of 31 December 2017 in the Influenza Research Database  were anesthetized with a ketamine-xylazine mixture and subsequently infected intranasally using 50 μl of PBS containing 3 times the 50% lethal dose (LD50) of either B/Florida/04/2006  or B/Malaysia/2506/2004 . The anesthetized mice were treated at 24 or 72 h postinfection with 0.1 or 1.0 mg/kg body weight of MAb in PBS delivered as a 0.1-ml bolus by intraperitoneal injection or as 0.05 ml by intranasal instillation. The mice were monitored for death, morbidity, and weight loss, and the date of death was recorded for 14 days.Female BALB/c mice were ordered from The Jackson Laboratory and were 6 to 8 weeks old at the time of the experiments. Mice  software . Area-under-the-curve (AUC) analysis was used to assess the statistical relationship between the percent weight loss profiles of the different treatments. Areas under the weight loss curves were measured using the AUC functionality of Prism (version 7) software. The statistical relationship associated with any two weight loss curves was assessed by calculating the mean AUC, the standard error, and the number of degrees of freedom for each pair and performing the unpaired


This comparison reveals mismatches between the annual mean response to obliquity and precession in models versus the signals within Antarctic ice cores. Weighting the model-based reconstruction toward austral winter or spring reduces these discrepancies, providing evidence for a seasonal bias in ice cores.Much of the global annual mean temperature change over Quaternary glacial cycles can be attributed to slow ice sheet and greenhouse gas feedbacks, but analysis of the short-term response to orbital forcings has the potential to reveal key relationships in the climate system. In particular, obliquity and precession both produce highly seasonal temperature responses at high latitudes. Here, idealized single-forcing model experiments are used to quantify Earth’s response to obliquity, precession, CO Periodic changes in the tilt of the Earth’s axis alter the distribution of incoming solar radiation. Here, the authors show that the temperature response to this forcing seemingly differs in models and Antarctic ice cores, with a better agreement reached if ice cores are recording a seasonally weighted signal. Primarily, this paper focuses on the climate response to obliquity, or the axial tilt of the earth, which varies by up to ~2.5° with a period of 40 ka.Slow temperature variations over Quaternary glacial-interglacial cycles have been dictated by changes in Earth’s orbital obliquity, precession, and eccentricity, as well as slow feedbacks involving atmospheric composition and ice sheets. Although questions remain surrounding the interaction between orbital forcing and greenhouse gas and ice sheet dynamics7). These anomalies amplify the annual mean equator-to-pole insolation gradient and diminish the seasonal insolation cycle in both hemispheres. Unlike precession, obliquity’s seasonally-varying insolation anomalies do not locally sum to zero in the annual mean.Changes in Earth’s obliquity affect the latitudinal and seasonal distribution of insolation. Because this forcing is well known, it presents a good target to explore the climate system’s response. Reduced obliquity results in positive annual mean insolation anomalies in the tropics while higher latitudes have increased insolation in winter and strongly decreased insolation in summer  Climate Model 2.1 (CM2.1) and the National Center for Atmospheric Research (NCAR) Community Earth System Model (CESM) version 1.2. The temperature response to orbital forcing is also explored in the Hadley Centre Coupled Model version 3 (HadCM3). For CM2.1 and CESM, single-forcing equilibrium simulations are conducted at low (22.079°) and high (24.480°) obliquity, the extreme values of the past 900 ka10. All other forcings, such as greenhouse gases, ice sheets, precession, and eccentricity, are prescribed at preindustrial values. The models lack dynamic ice sheet and carbon cycle components, so orbital experiments capture the short-term climate response, but exclude slow feedbacks related to ice sheets and the carbon cycle. Additional single-forcing experiments isolate the climate response to changes in precession, CO2, and ice sheets  response and a high obliquity (24.480°) response.Two-coupled atmosphere-ocean general circulation models (GCMs) are used to model the climate response to changes in obliquity, precession, CO−2 for CM2.1 and +0.2 W m−2 for CESM). Despite this mildly positive radiative forcing, cooling is much more widespread than warming, with all three models producing negative global mean temperature change. Global mean temperature anomalies are −0.54 °C for CM2.1, −1.07 °C for CESM, and −0.48 °C for HadCM3. The level of agreement between the models over most latitudes suggests that these results are fairly robust. The primary difference between models occurs in the Northern Hemisphere high latitudes, where CESM produces considerably more cooling than the other models, associated with widespread Arctic albedo anomalies in summer and fall due to changes in snow cover and sea ice.In all three models, reduced obliquity produces pronounced cooling in the mid and high latitudes of both hemispheres and some areas of mild warming near the equator Fig. . Obliqui−2 (radiative forcing increases by 2.4 W m−2). Analysis of energy transports and radiative feedbacks helps explain the lack of a tropical temperature response to obliquity forcing. In part, the increased radiative forcing is balanced by increased poleward heat transport in both the atmosphere and ocean. In the atmosphere, the tropical portion of this modified heat transport is associated with changes in the Hadley circulation6  Dome C22, but there is a consistent mismatch in magnitude between the short-term obliquity response in models and that inferred from Antarctic proxies. Multiple long ice cores show similar responses in Antarctica, so the following analysis focuses on that region rather than Greenland. The Antarctic ice cores support only 24–33% of the fast, direct response to obliquity simulated by CM2.1 , so a large temperature response seems reasonable, provided there is no error in the calculated strength of the orbital cycles. Dust is one possible source of error, as changes of dust23 are not included in this methodology. Some GCM simulations suggest that dust has little effect on Antarctic temperatures25. However, dust may have considerable indirect effects on clouds26, so the potential impact of dust in the present study is unclear. The mismatch could also stem from differences in the obliquity temperature response at the surface and higher in the atmosphere, where the snow preserved in ice cores forms, but the model results do not seem to support this. However, the discrete and course nature of the models’ vertical grids makes this possibility difficult to evaluate.A second possible explanation for the mismatch is that the models may be too sensitive to obliquity forcing in the Antarctic. This could happen, for instance, if the modeled surface albedo or lapse rate feedbacks are too strong in that region, a negative feedback is absent, or if the southward heat transport from lower latitudes is too weak. The role of heat transport in dampening the Antarctic response to obliquity has been explored in past work using the LOVECLIM Earth system model of intermediate complexity and the MIROC GCM27 suggests that the climate response to obliquity is not heavily dependent on ice sheet extent or greenhouse gas concentrations  would be very distorted by a seasonal bias in the recording mechanism due to the large seasonality of these responses  5a–e and 7 Fig. -8. A sealts Fig. , with th31. Another study showed that a seasonal bias could explain why the precession signal in Antarctic ice cores appears to be out of phase with local insolation30. That research focused on the timing of precession variability, and here we show that this explanation helps improve the fit of the obliquity signal as well. Performing the analysis by applying seasonal weighting to the obliquity or precession components alone shows that mismatch is generally reduced in both cases , observations from Vostok, Mizuho, and Dome Fuji stations, as well as data from the European Centre for Medium-Range Weather Forecasts (ECMWF) suggest that Antarctic snowfall is reduced during Antarctic summer, in line with this explanation. This snowfall seasonality is supported by some modeling results33, but not by others. In general, it is unclear whether models can simulate Antarctic precipitation accurately enough to be useful for this purpose34. The importance of seasonal precipitation to the interpretation of ice cores has been previously argued35, as well as the potential importance of seasonal sublimation36. If precipitation is reduced during Antarctic winter instead, it would be evidence against the seasonality argument presented in this paper.A seasonal bias in ice cores could result from several factors. In particular, the mean isotopic signature could be biased by preferential snowfall during a particular part of the year. As compiled in previous work  ice sheets44 and NCAR CESM uses the Paleoclimate Modelling Intercomparison Project Phase III (PMIP3) LGM ice sheets, which are a blend of three ice sheet reconstructions, with added ice shelves in the western Labrador Sea45. Simulations were run for 500 years or longer. These experiments are here referred to as 'fingerprint' experiments, since they quantify the distinct climate response patterns to individual forcings. A different experimental design was employed for the HadCM3 simulations used in Fig. To quantify the climate responses produced by changes in obliquity, precession, CO46. To account for this complicating factor, all monthly output from CM2.1 and CESM were processed onto a common fixed-angular calendar, in which every 'month' corresponds to a 30° arc of Earth’s orbit. This calculation was done using the method described in47. No monthly results from HadCM3 are presented, so no calendar correction is needed.For CM2.1 and CESM monthly output, an adjustment was typically made to the calendar. When the timing of perihelion is modified, as in some of the simulations described above, the speed at which Earth travels through different parts of its orbit is altered according to Kepler’s second law. This means that calendar months in different simulations will correspond to different parts of Earth’s orbit, complicating the interpretation of monthly anomalies. This 'calendar effect' has been described in past research17: anomalies from orbital experiments  are scaled by orbital parameters48; Anomalies from the CO2 experiment are scaled by the radiative forcing of CO2 from a combined CO2 record from EPICA Dome C and Vostok49 as well as the radiative forcing of CH4 from EPICA Dome C50, computed according to the equations in Table 6.2 of the Intergovernmental Panel on Climate Change (IPCC) Third Assessment Report51; Anomalies from the ice sheet experiment are scaled by sea level changes, which is used as an analog for ice sheet volume. The sea level record is a stack of seven different sea level records computed using different methods52. Potentially, uncertainties in the magnitude of obliquity variability in the sea level record could affect the results of this paper, especially if obliquity variations unrelated to sea level are also being recorded in the sea level record. However, the use of a multi-record stack rather than an individual sea level record should help reduce the impact of potential uncertainties in any particular method. It should also be noted that the climate response to ice sheets may relate to both ice sheet area and volume, but a linear scaling with sea level has been used here for simplicity. An additional source of uncertainty concerns the timing of individual ice sheet changes relative to the global mean sea level record. In the present work, the linear reconstruction assumes that all ice sheets vary synchronously with the global mean sea level record. However, evidence suggests that after the LGM, at least, Antarctic ice melt may have lagged considerably behind the global sea level changes44. Differences between the timing or characteristics of Antarctic ice sheet changes and global sea level anomalies could affect results of this analysis, especially considering that orbital-scale variations are present in the sea level curve  are left on their published age models.To estimate the effects of each forcing in the past, and to allow for comparison with proxy time series data, a linear reconstruction methodology is employed. Model output from the single-forcing experiments is scaled by past forcings using an updated version of the method described in the appendix of17 primarily in four ways: a slightly different data set of orbital parameters is used here 48, greenhouse gas values from EPICA Dome C/Vostok are used instead of those from Vostok alone, a sea level stack is used rather than a single record, and linear reconstruction anomalies are computed relative to the preindustrial simulation rather than a zero eccentricity state.The computed 'linear reconstructions' estimate the contribution of individual forcings in producing past climate change, and can be summed together to compute a model-based estimate of total temperature change at any location. The linear reconstruction methodology used here has been updated from previous work15, Dome Fuji19, Vostok20, and NGRIP53 were analyzed. For other regions, many proxy records which had been compiled for a global temperature stack18 were used. The records in the temperature stack18 were aligned to the LR04 benthic stack by that study’s authors, generally using δ18O to align records. The NGRIP ice core was left on its published ages53, with a shift so that ages are relative to year 1950 instead of 2000. The Antarctic ice core records were aligned to the annual mean linear reconstructions, with these alignments done separately for the two models used in the analysis (GFDL CM2.1 and NCAR CESM). The alignment was done using matching software55. If no alignment is done, offsets in the timing of deglaciations affect the results. To test that the analysis is not overly sensitive to the details of this alignment, an alternate approach was also taken: no alignment was performed on the ice core age models, but periods of deglaciation prior to the most recent one were omitted from the analysis. This alternate analysis yields results similar to the main results presented in this paper, suggesting that the details of this alignment do not overly affect the results. Not all records were well suited to the present analysis, so two selection criteria were applied. Records that span multiple orbital cycles are preferred, so any records which contained less than 100 ka of overlap with the linear reconstructions were rejected. Additionally, some records exhibit temporal variability distinctly different from the typical 'sawtooth' shape observed in the greenhouse gas and sea level forcing records, potentially because of uncertainties in the proxy temperature relationship. To remove records with unusual variability as well as records with apparent age model mismatch, records which do not match the unfitted annual mean linear reconstruction of either model with at least a coefficient of efficiency of 0.5 were excluded. This selects records, which already match the linear reconstruction somewhat well, a qualification which should be kept in mind when evaluating results.To explore climate signals preserved in proxies, long proxy temperature records were selected from multiple sources. For polar records, EPICA Dome CTo conduct the model–data comparison, proxy records were compared to the linear reconstruction at the location of the proxy record, using modeled surface temperature for sediment cores and NGRIP and surface (2 m) air temperature for the Antarctic ice cores. To determine how much of the modeled annual mean obliquity signal was reflected in the proxy records Fig. , the annTo determine if a seasonal-weighted linear reconstruction better matched the Antarctic proxy records, RMSE was calculated between each proxy record and a set of linear temperature reconstructions computed using different monthly weightings. As opposed to the calculation described above, no aspects of the linear reconstruction were directly scaled to be larger or smaller. Instead, climate signals only changed as a result of different monthly averages. Averaging windows were centered on each month and spanned either 1, 3, 5, 7, 9, or 11 months, for a total of 72 monthly weighting combinations plus the 12-month mean Fig. . A boxcaCode from this study is available from the corresponding author upon reasonable request.Model output from this study is available on Zenodo (zenodo.org) for both GFDL CM2.1 (doi:10.5281/zenodo.1194480) and NCAR CESM (doi:10.5281/zenodo.1194490) data. Annual and monthly climatologies are stored. Monthly data on a fixed-angular calendar is not stored on Zenodo, but is available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


Fixation with a dynamic hip screw (DHS) is one of the most common methods for stabilizing intertrochanteric fractures, except for unstable and reverse oblique fracture types. However, failure is often observed in osteoporotic patients whereby the lag screw effectively ‘cuts out’ through the weak bone. Novel anti-migration blades have been developed to be used in combination with a lag screw (‘Blade Screw’) to improve the fixation strength in osteoporotic intertrochanteric fractures. An in-vitro biomechanical study and a retrospective clinical study were performed to evaluate lag screw migration when using the novel Blade Screw and a traditional threaded DHS. The biomechanical study showed both the Blade Screw and DHS displayed excessive migration (≥10 mm) before reaching 20,000 loading cycles in mild osteoporotic bone, but overall migration of the Blade Screw was significantly less (p ≤ 0.03). Among the patients implanted with a Blade Screw in the clinical study, there was no significant variation in screw migration at 3-months follow-up (P = 0.12). However, the patient’s implanted with a DHS did display significantly greater migration (P<0.001) than those implanted with the Blade Screw. In conclusion, the Blade Screw stabilizes the bone fragments during dynamic loading so as to provide significantly greater resistance to screw migration in patients with mild osteoporosis. Intertrochanteric fractures are common injuries in the elderly and are often a consequence of sudden impact to an osteoporotic hip, such as that experienced during a fall . DynamicInternal fixation of intertrochanteric fractures in osteoporotic bone is a challenge for orthopaedic and trauma surgeons. Osteoporosis reduces the thickness of cortical bone and increases the porosity of cortical and trabecular structures. This can subsequently lead to implant failure and joint collapse, as one the primary indicators of the mechanical stability of implants is bone quality. Various techniques and implants have been developed to enhance implant stability in osteoporotic bones and reduce lag screw cut-out. With the use of cement-augmented lag screws, Szpalski et al.  reportedin vitro gait simulation, and 2) to identify signs of early lag screw migration on two-dimensional radiographs of intertrochanteric fractures treated using the Blade Screw and a DHS.To improve the fixation strength in intertrochanteric fractures, a bladed screw design was developed which combines a conventional lag screw shape with two anti-migration blades running along the length of the screw barrel   represented the homogeneous and uniform material properties of severely osteoporotic cancellous bone. A cellular rigid polyurethane foam  was used to simulate mild osteoporotic bone. The polyurethane foams were purchased in blocks and molded to the shape of a femoral head with a radius of 19 mm, height of 20 mm and a base thickness of 10 mm  system was created to evaluate lag screw migration in each femoral head test model after multi-axial loading that simulates walking conditions  12]. Th. Th12]. The test structure was subjected to sinusoidal loading ranging from 5 N to 1,450 N at a frequency of 1 Hz, simulated walking conditions . To simuFour marks were placed 90° apart on the surface rim of the steel shell. Spatial migration of the femoral head test model in relation to the lag screw was continuously recorded using a pure video-based tracking and analysis software  . An indeFrom May 2013 to July 2014, intertrochanteric fracture fixation was performed by the same surgeon on twenty female patients: 11 fixed with a Blade Screw and 9 fixed with a conventional DHS. Inclusion criteria were low bone mineral density (T-score < = -2.0), stable intertrochanteric fractures (AO/OTA 31-A1/2) and a minimum of 3 months follow-up. Additional exclusion criteria were incomplete chart records, radiographs not meeting diagnostic standards and loss to follow-up. Both patient groups presented classifiable intertrochanteric fractures (AO/OTA 31-A1) with a mean patient age of 76 years  for the Blade Screw group and a mean age of 80 years  for the DHS group. No patients presented intraoperative complications, infections or intraoperative or postoperative femoral fracture around the screw tip during a 3-month follow-up period. The bone mineral density (BMD) of all patients was measured and evaluated using a T-score. As lag screw cut-out through the femoral head generally occurs after weight bearing, the anteroposterior (AP) and lateral radiographs of each patient were collected immediately after surgery and at 3 months follow-up. The distance from the tip of the screw to the apex of the subchondral bone  was measured with AutoCAD software to determine the position of the lag screw within the femoral head on the AP and lateral radiographs , 16. TheThe study protocol was approved by the Institutional Review Board (IRB) of National Yang-Ming University, Taiwan on 20 February 2015 (IRB No.: YM103107WE). All patients provided informed written consent prior to their clinical records being accessed and published and prior to being included in this study.In the mild osteoporotic femoral models, both screw types demonstrated excessive migration (≥10 mm displacement) before reaching 20,000 loading cycles . The meaIn the severe osteoporotic bone models, both screw types demonstrated excessive migration before reaching 1,000 loading cycles. The mean number of loading cycles achieved by the Blade Screw and traditional DHS were 642±26 and 125±11 respectively. At the point of excessive migration, the mean DFH of the Blade Screw was 9.44±0.99 mm, which was significantly less than the traditional DHS (11.26±0.77 mm). Because the bone models were destroyed after relatively few loading cycles, the mean DDLS, VC, and RN could not be accurately recorded or analyzed.2 (T score = -2.8) and the mean of traditional DHS group was 0.871±0.2572 g/cm2 (T score = -2.7). The difference in BMD between the two groups was not significant. Also, no significant difference was detected in mean TAD between the two groups immediately after surgery and at 3-months follow-up. In addition, there was no significant intra-group variation in TAD seen in the Blade Screw group either post-surgery or at follow-up (P = 0.12). However, the mean TAD in the traditional DHS group immediately after surgery was significantly larger than at 3-months follow-up (P<0.001). By using a post hoc power analyses based on the repeated-measures differences in TAD values, the results demonstrated 84% power in detecting time differences at a significance level of 0.05 . This indicates a greater resistance to implant migration through the bone. Kouvidis et al. used similar testing protocols to investigate the number of loading cycles to implant failure in single and dual lag screw systems and showed that the average number of cycles leading to cut-out was 6,638 and 10,054, respectively . In the th loading cycle. Therefore, it was not possible to adequately evaluate lag screw migration using the current method. However, the Blade Screw did sustain a greater number of loading cycles before the point of excessive implant migration. Displacement of the femoral head was also less than in the traditional DHS model. The two longitudinal blades on the Blade Screw enlarge the contact surface between the lag screw and bone in the superior-inferior direction, resulting in additional resistance to rotation of the femoral head around the screw.In the severely osteoporotic bone model, both lag screws failed to achieve good bone purchase. Since engagement of the cortex was neglected, the structure of the open-cell rigid polyurethane foam was destroyed before the 1,000In addition to the sawbone implantation models, 20 patients exhibiting intertrochanteric fractures were also treated with lag screws; 11 with a Blade Screw and 9 with a DHS. Immediately after surgery and at 3-months follow-up, the mean TAD of both groups was less than 25 mm, indicating a low risk of lag screw cut-out in all patients –20. AlthThere are some limitations to the methods used in this study that should be noted. First, the femoral head bone samples were synthetic bones made of rigid polyurethane foam with isotropic mechanical properties. Although these models might not fully reproduce the biomechanical properties of human bone, these are standardized sawbones that have been shown to provide consistent material properties similar to a human cancellous bone , 22. SynCompared to the traditional DHS, the Blade Screw provides significantly greater resistance to lag screw migration and cut-out in low density bones. The findings of this study show that the Blade Screw design has the advantage of improving the postoperative fixation in osteoporotic bones by securing the fracture fragments during dynamic loading.


Non-invasive real time evaluation of living cell conditions and functions are increasingly desired in the field of clinical diagnosis. For diagnosis of type I allergy, the identification of antigens that induces activation of mast cells and basophils is crucial to avoid symptoms of allergic diseases. However, conventional tests, such as detection of antigen-specific IgE antibody and skin tests, are either of low reliability or are invasive. To overcome such problems, we hereby applied an impedance sensor for label-free and real-time monitoring of mast cell reactions in response to stimuli. When IgE-sensitized RBL-2H3 cells cultured on the electrodes were stimulated with various concentrations of antigens, dose-dependent cell index (CI) increases were detected. Moreover, we confirmed that the impedance sensor detected morphological changes rather than degranulation as the indicator of cell activation. Furthermore, the CI of human IgE receptor-expressing cells (RBL-48 cells) treated with serum of a sweat allergy-positive patient, but not with serum from a sweat allergy-negative patient, significantly increased in response to purified human sweat antigen. We thus developed a technique to detect the activation of living cells in response to stimuli without any labeling using the impedance sensor. This system may represent a high reliable tool for the diagnosis of type I allergy. Since the cell is the minimum unit of living organisms, non-invasive real time observation and the evaluation of living cell conditions and functions are increasingly desired in the field of life science and for clinical diagnosis. Recently, various kinds of biosensors for living cell analyses, such as the quartz crystal microbalance (QCM) sensor ,2, the f2) into two crucial second messengers: inositol 1,4,5-triphosphate (IP3), a Ca2+ releaser from cellular stores, and diacylglycerol (DAG), an activator of protein kinase C (PKC). IP3 induces the depletion of Ca2+ stores, which in turn activates Ca2+ release-activated Ca2+ (CRAC) channels and causes capacitative Ca2+ entry . . 19]. To confirm the potential of impedance analysis of the cells as a technique for diagnosis of type I allergy, we cultured RBL-48 cells on the electrodes of iCELLigence™ with the sera of various donors. We then stimulated cells with anti-IgE and sweat antigen, QR, which is known as a major antigen associated with atopic dermatitis . The senAs shown in In this study, we established living cell reaction-based diagnostic test of type I allergy with a small sample of conserved patient’s serum (6 µL per well) using an impedance sensor. Conventional tests, such as the histamine release test, require a relatively large amount of blood (>1 mL). However, less than one milliliter of blood (300–500 µL of serum) is enough for testing using the system developed in this study. Specific IgE-antibody detection tests, such as IgE binding assays require relatively small amount of serum, but detect only the presence of IgE molecules that bind to an antigen, regardless of their biological activities. Ishii et al. reported a human monoclonal IgE antibody that binds to MGL-1304 in human sweat, but does not activate human basophils . On the SPR and SPR imaging sensors also detect reactions of living cells without any labeling as the change of RI on the sensor chip (thin gold surface) and may be applied for clinical diagnosis of type I allergy ,27,28,29We developed a technique using an impedance sensor to detect the functional activation of human IgE that binds to and activates FcεRI-expressing cells in response to antigens. The technique may be a useful tool for highly reliable, high-throughput diagnosis testing of type I allergy with a small sample of a patient’s serum.


We propose eIF4B as a mediator between neuronal activity and translation, with relevance in the control of synaptic plasticity.Neuronal physiology requires activity-driven protein translation, a process in which translation initiation factors are key players. We focus on eukaryotic initiation factor 4B (eIF4B), a regulator of protein translation, whose function in neurons is undetermined. We show that neuronal activity affects eIF4B phosphorylation and identify Ser504 as a phosphorylation site regulated by casein kinases and sensitive to the activation of metabotropic glutamate receptors. Ser504 phosphorylation increases eIF4B recruitment to the pre-initiation complex and influences eIF4B localization at synapses. Moreover, Ser504 phosphorylation modulates the translation of protein kinase Mζ. Therefore, by sensing synaptic activity, eIF4B could adjust translation to neuronal needs, promoting adaptive changes in synaptic plasticity. We also show that Ser504 phosphorylation is increased  In particular, eIF4B favors the interaction of the mRNA molecule with the initiation complex, by binding both eIF3 and the 18S ribosomal RNA4, and stimulates the helicase activity of eIF4A7, thereby supporting the translation of mRNAs with highly structured transcript leaders, often coding for proteins involved in vital cellular processes8. eIF4B activity is modulated through phosphorylation by various protein kinases10. Although several phosphorylation sites have been identified or predicted14, only Ser422 and Ser406 have been characterized as regulated sites of phosphorylation in proliferating cells16. Both sites are targeted by ribosomal S6 kinase (RSK), downstream the mitogen-activated protein kinase (MAPK) pathway, and by S6 kinase 1 (S6K1), activated through the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) axis17. Phosphorylation of Ser422 modulates the interaction of eIF4B with eIF317, whereas phosphorylation of Ser406 is important for optimal translational activity of eIF4B16.The eukaryotic translation initiation factor 4B (eIF4B) is part of the eIF4 group of factors, which assist the 43S ribosomal initiation complex in mRNA recruitment and scanning18. It has been reported that the state of phosphorylation – and thus the activity – of eIF4B is modulated by several extracellular stimuli19 and influences functions as important as cell survival and proliferation19. Accordingly, a pro-oncogenic role for eIF4B has been demonstrated in cancer cells, probably because of its ability to positively regulate the translation of pro-survival mRNAs19. Despite its ubiquitous expression, very little is known about the role of eIF4B in neurons. These highly specialized cells require a tight control of general and local protein synthesis to rapidly adjust protein composition to the incoming synaptic activity, and thus to sustain plasticity processes22.The function of eIF4B has been widely investigated at the molecular level, whereas its characterization at the cellular level is still limited and even contradictoryin vivo taking advantage of the pilocarpine-induced model of epilepsy.We investigated how perturbations of synaptic activity modulate the expression, phosphorylation, localization and function of eIF4B in mature hippocampal neurons in culture, and validated our results The expression of eIF4B was detected by Western blotting in both rat neuronal and astroglial primary cultures, but with a distinct pattern of electrophoretic migration: in astrocytes a single sharp band of approximately 80 kDa was visible, whereas neurons displayed two close bands of similar MWs Fig. . Notably24. Three SP sites  are present in eIF4B and have been shown to be phosphorylated according to large-scale mass spectrometry analysis of phospho-peptides27 and data deposited in PhosphoSitePlus28. We focused on Ser504, since a phospho-specific antibody was already available for this SP site29.The electrophoretic shift of eIF4B observed in neurons may be caused by phosphorylation of serine-proline or threonine-proline (SP/TP) sites, which has often been reported to greatly alter the electrophoretic mobility of proteinsInterestingly, we observed that Ser504 is phosphorylated to a significantly higher degree in neurons with respect to astrocytes Fig. . The antin vivo in the brain. Strikingly, Ser504 phosphorylation was virtually absent in the liver but high in the cerebral cortex , a sodium channel blocker that inhibits neuronal firing, or bicuculline , a blocker of the GABAect Fig. . At variect Fig. .Figure 2Since neuronal network activity is mainly sustained by excitatory glutamatergic neurons, we verified the effects exerted by inhibitors of the different glutamate receptors (GluRs) on eIF4B phosphorylation, both at rest and after exposure to 10 μM bicuculline Fig. . More spIn resting conditions, the blockade of AMPA-R or NMDA-R activity did not affect Ser504 phosphorylation, while the mGluR antagonist AIDA decreased it by almost 50% Fig. . StimulaIn contrast Ser422 phosphorylation appears to be governed mainly by the activity of NMDA-Rs, with an additional component related to AMPA-Rs under increased network activity. In fact, only the NMDA-R antagonist reduced the basal level of Ser422 phosphorylation to values close to those observed after TTx treatment, while both NMDA-R and AMPA-R antagonists were able to inhibit the phosphorylation increase induced by bicuculline Fig. . The ant2+ channels (VOCCs), which are known to actively participate in neuronal activity32 and to regulate protein phosphorylation34. BAYK8644  was used to prolong the opening time of the L-type VOCCs35, while two different antagonists were employed together to block the activity of most post-synaptic VOCCs: nimodipine (10 μM) for the L-type and NNC55–0396 (10 μM) for the T-type38. Modulation of VOCC activity by either BAYK8644 or the mix of VOCC blockers had virtually no effects on the levels of Ser504 phosphorylation  for the MAPK kinase pathway; rapamycin (20 nM) for mTOR; and LY294002 (25 μM) for PI3K.In accordance with previous studies in non-neuronal cells, basal Ser422 phosphorylation was significantly affected by both rapamycin and LY294002, while U0126 had no effect Fig. . However39 and Group-based Prediction System (GPS 2.1 software)40. We found that Ser504 is located in putative consensus sequences for the following kinases: MAPKs, cyclin-dependent kinase 5 (CDK5), casein kinase (CK) 1 and 2, and conventional isoforms of PKC (cPKCs). Having already excluded a direct contribution of MAPKs, we used specific inhibitors for the other kinases: 20 uM roscovitine for CDK541; 10 uM D4476 for CK142; 25 uM TBB for CK243 and 2 uM Gö6976 for cPKCs44. While roscovitine was ineffective in modulating eIF4B phosphorylation (not shown), D4476, TBB and Gö6976 were able to significantly reduce the basal levels of Ser504 phosphorylation . Co-localization was quantified in terms of index of correlation (ICorr), a measure of the fraction of positively correlated pixels for the two signals. Similar ICorr values were obtained when the signal for Bassoon was compared with that of total or Ser504-phosphorylated eIF4B  it is rapidly synthesized under conditions of increased synaptic activity leading to LTP induction48. The knock down of endogenous eIF4B by siRNAs significantly reduced PKMζ expression  its mRNA is characterized by a structured 5′UTR; (ii) its translation is reported to be regulated by eIF4B phosphorylationion Fig. . Conversion Fig. , the proion Fig. . This efion Fig. . In addiion Fig. , prevention Fig. .45, we also tested the phosphorylation state of this residue upon bicuculline treatment and CK inhibition. Under these conditions, the phosphorylation levels of Ser406 were not significantly changed  or 50 days after the occurrence of the first spontaneous seizure (chronic epilepsy period). Phosphorylation of Ser504 was significantly increased during latency, i.e. when plastic changes are occurring that lead to brain transformation from normal to epileptic50, but not in the chronic period . We used a rat model in which SE induced by a drug (pilocarpine) is followed, after a latency period of about 10 days, by spontaneous recurrent seizuresiod Fig. . Interesiod Fig. . These d51, modulates protein translation19 and influences cell survival and proliferation in mitotic cells. In this work, we describe a neuron-specific regulation of eIF4B phosphorylation at the previously uncharacterized Ser504 site. Our in vitro results show constitutive phosphorylation of Ser504 in neurons, which is further increased under conditions of synaptic activation induced by bicuculline. We found that also Ser422 phosphorylation, which has been extensively studied in highly proliferating cells18, is controlled by neuronal activity, being decreased by TTx and increased by bicuculline. However, Ser422 phosphorylation is influenced by the activity of the ionotropic glutamate receptors with the contribution of VOCCs, whereas Ser504 phosphorylation appears to be mainly sustained by the tonic activity of the metabotropic glutamate receptors. This differential involvement of glutamate receptors corresponds to the engagement of distinct signaling pathways.It is widely recognized that eIF4B phosphorylation, which increases its association with the pre-initiation ribosomal complex17, the PI3K/mTOR and the MAPK pathways participate in Ser422 phosphorylation in neurons, with the first mainly regulating basal phosphorylation levels and the latter intervening in conditions of increased synaptic activity. The interplay between the MAPK and the PI3K/mTOR pathways has already been recognized to be important in hippocampal neurons for the regulation of protein synthesis, by differentially affecting the phosphorylation of various components of the translation machinery in response to neuronal activity52. Interestingly, these two pathways do not contribute to Ser504 regulation, further highlighting the independent control of phosphorylation at the two sites.We found that, analogously to what observed in proliferating cells54. In addition, CK-dependent eIF4B phosphorylation has been reported in both rabbit reticulocytes and plants, even though the phosphorylation sites were unknown56. Recent reports suggest a relevant role of CKs in translation regulation, achieved through inhibition of the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1)57, the coordination of the ternary complex formation and eIF4F assembly58, and the modulation of the interaction between eIF4G, eIF4A and eIF4B in the helicase complex59. Here we describe a novel mechanism through which CKs positively modulate the translation initiation of mRNAs: by mediating Ser504 phosphorylation, CKs promote eIF4B recruitment to the initiation complex. In addition, we show that CK-mediated Ser504 phosphorylation modifies eIF4B subcellular localization upon increased neuronal activity, leading to its accumulation at bona fide synaptic sites. This observation points to the possibility that Ser504-phosphorylated eIF4B may participate in the regulation of local translation according to the levels of synaptic activity. Of note, this role can be exerted by modulating not only the interaction of the translation machinery with the mRNA, but also the helicase activity of the eIF4F complex. This latter effect is expected to be particularly relevant for transcripts endowed with structured transcript leaders, which are abundant in the pool of synaptic and stress regulated proteins45. This is the case of PKMζ, a protein with a highly structured transcript, which has been proposed to be involved in the induction and maintenance of long-term synaptic changes. We provide experimental evidence that eIF4B Ser504 phosphorylation exerts a translational control on the expression of endogenous PKMζ. Therefore, our data support a relevant contribution of eIF4B Ser504 phosphorylation on PKMζ expression, in addition to the role recently proposed for Ser406 phosphorylation in this process45. Further studies are needed in order to identify and characterize the full panel of neuronal transcripts whose translation is specifically regulated by eIF4B.We demonstrate that activation of Ser504 phosphorylation is primarily sustained by the constitutive activation of CKs, and is further boosted by cPKCs. While cPKCs, well-know players in neuronal physiology, are expected to exert pleiotropic roles in the phosphorylation of eIF4B, CKs appears to selectively act on the Ser504 site, although the relationship, if any, between cPKCs and CKs remains to be understood. In our experimental conditions, CKs appear to be responsible for the high levels of basal phosphorylation of Ser504 and to contribute to the modulation seen upon mGluR activation. Indeed, CKs, in particular CK1, are characterized by a high constitutive activity that can be further modulated by mGluRs in neuronsin vivo, we decided to employ a rat model of epilepsy, in order to compare the situation of normal brain activity with that of a hyper-excitable system. Interestingly, while Ser504 phosphorylation levels are unchanged in animals in the chronic phase (i.e. in condition of overt epilepsy), they are increased in the latency period, when the epileptic phenotype is not apparent yet, but maladaptive plastic changes are ongoing and eventually lead to spontaneous seizures. These changes in phosphorylation are expected to reflect an alteration of the neuronal activity since it is highly unlikely that they may be ascribed to the presence of a different population of cells. Indeed, if a certain extent of white blood cell infiltration is reported in animal models of epileptogenesis, this is observed immediately after the status epilepticus, while in the late latency phase is minimal; in any extent, even considering their possible contribution, white cells may represent only a tiny percentage of total cells, compared to neurons. Moreover, we did not detect Ser504 phosphorylation in blood-rich tissues such as liver, heart and kidney.To verify whether Ser504 phosphorylation is regulated by neuronal activity also 60. Great attention has been paid in the last years to the understanding of the – still elusive – molecular mechanisms underlying epileptogenesis, in the continuous search for new therapeutic targets able to modify disease progression, given that the available therapies for epilepsy are only symptomatic61. Our findings prompt the provocative hypothesis that modulation of eIF4B phosphorylation  may play a role in the molecular mechanisms of epileptogenesis. This hypothesis, when confirmed by further investigation, may lead to the identification of new therapeutic targets for the prevention of epilepsy development.The epileptogenic process is characterized by profound alterations in neural circuitries, including specific changes in synaptic transmission and strength that depend on the molecular composition of synaptic contacts and on local protein synthesisIn conclusion, we show that eIF4B, through its tightly regulated phosphorylation, appears as a key player in the control of protein translation in neurons. eIF4B is a point of convergence of multiple signaling pathways and may represent a crucial “fine tuner” in the local control of mRNA translation, when active protein synthesis is required. This may be relevant not only in physiological conditions, but also under pathological conditions that depend on stable changes in synaptic transmission and neuronal network activity.Cell culture media and reagents were from Lonza , culture flasks and multiwell plates from Nalge Nunc  and Petri dishes from Falcon BD . DL-2-amino-5-phosphonopentanoic acid (APV), 7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-carbonitrile (CNQX), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), tetrodotoxin (TTx), bicuculline, NNC55-0396, (±)-BAYK8644 were from Tocris . Gö6976 and U0126 were from Merck Millipore . Chymostatin, Leupeptin, Antipain, Pepstatin, Nimodipine, Rapamycin, LY294002, 1,2-dioctanoylglycerol (DiC8), 4--5-pyridin-2-yl-1H-imidazol-2-yl)benzamide (D4476), 4,5,6,7-Tetrabromo-2-azabenzimidazole, 4,5,6,7-Tetrabromobenzotriazole (TBB) and other chemicals for general use were from Sigma-Aldrich .Antibodies against eIF4B , eIF4E, eIF4A, and ribosomal protein S6 were from Cell Signaling Technology . Rabbit monoclonal antibody against phospho-S504 eIF4B was from Abcam . Other mouse monoclonal antibodies were against Bassoon , alpha-tubulin (Sigma-Aldrich), PSD95 . Anti eIF4B C-terminal polyclonal antiserum was raised by Eurogentec , immunizing rabbits against the rat C-terminal sequence of the protein (SVDGEDEDEGDDCTE).Human eIF4B sequence was obtained from pcDNA3-Flag4Bwt (kind gift of J. Hershey). Ser504 mutants were generated by PCR: products obtained with overlapping primers containing the mutation were used as template for a subsequent PCR with external primers and inserted into pcDNA3-Flag. Oligonucleotides used (mutations in lower case) were: 5′-GAG CAG CAG gCC CCT ACA AGT GGT-3′ (forward) and 5′-ACC ACT TGT AGG GGc CTG CTG CTC-3′ (reverse) for eIF4B S504A; 5′-CAG AGC AGC AGg agC CTA CAA GTG GTG G-3′ (forward) and 5′-CCA CCA CTT GTA GGc tcC TGC TGC TCT G-3′ (reverse) for eIF4B S504E; 5′-CCG CTC GAG CTG CTT CTA TCT TTG GAG-3′ (forward) and Reverse: 5′-CCG CTC GAG CGG CCG CCT ATT CGG CA-3′ (reverse) as common external primers. All inserts were sequenced.4 and 0.5 mg/ml DNase IV. Cells were plated onto poly-ornithine coated coverslips and maintained at 37 °C in a 5% CO2 incubator in minimal essential medium with 20 mM glucose, B27 , 2 mM glutamax, 5% fetal clone III  and 5 μM 1-β-D-cytosine-arabinofuranoside (Sigma-Aldrich). Neurons were used between 7 and 15 days after plating.The Institutional Animal Care and Use Committee approved the animal use. Hippocampal neurons were prepared according to ref. 2 flasks with Minimum Essential Medium Eagle with Earle’s balanced salt solution, supplemented with 10% donor horse serum, 33 mM glucose, 2 mM glutamax (Gibco), 50 U/ml penicillin and 50 μg/ml streptomycin, and kept at 37 °C in a 5% CO2 incubator. Two steps of overnight shaking at 230 rpm were performed to induce selective detachment of microglia. After reaching confluence, astrocytes were re-plated onto poly-lysine-coated multiwells and used within 3 days.Cortical astrocytes were obtained from 1–2 day-old Sprague-Dawley rats  according to ref. HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FCIII, 2 mM glutamine, 10 mM Na pyruvate, 50 U/ml penicillin and 50 μg/ml streptomycin.TTx 1 μM, bicuculline 10 μM, BAYK8644 20 μM, nimodipine 10 μM, NNC 55–0396 10 μM, and DiC8 100 μM were used for the indicated times. In some experiments cells were pre-treated before the bicucullin treatment as follows: (i) 100 μM APV, 20 μM CNQX and 1 mM AIDA, 30 min; (ii) 20 nM rapamycin, 10 mM U0126, or 25 μM LY294002, 2 h; (iii) Gö6976 2 μM, 1 h; (iv) D4476 10 μM and TBB 25 μM, 2 h.64.Cells were lysed with 2x SDS sample buffer . After SDS-PAGE proteins were blotted. After blocking, membranes were incubated overnight with the primary antibodies and, after washing, with horseradish peroxidase-conjugated secondary antibodies . For loading controls membranes were stripped in acidic buffer  and re-probed. Proteins were revealed on auto-radiographic films  by Super Signal West Chemiluminescent Substrate  and quantified using ImageJ3VO4) with 30 strokes of a glass-Teflon homogenizer and centrifuged at 15,000 g, 4 °C for 15 min. The protein content was analyzed by BCA (ThermoFisher Scientific).Tissues were homogenized in radio-immunoprecipitation assay (RIPA) buffer  at 4 °C using a Teflon-glass homogenizer, brought to 1.5 M sucrose and divided into two ultracentrifuge tubes (SW41Ti 14 × 89mm). The homogenate was overlaid with 3 ml of 1.25 M sucrose, 3 ml of 1 M sucrose and 3 ml of homogenization solution, and then centrifuged at 100,000 g for 4 h at 4 °C. Synaptosomes were collected at the 1.25 M/1.0 M sucrose interface and centrifuged, after 1:4 dilution with phosphate buffered saline solution (PBS) at 100 000 g for 1 h at 4 °C. The pellet was then solubilized  before SDS-PAGE analysis on 7.5% SDS gels and transfer of proteins to nitrocellulose membranes for Western blotting analysis.Synaptosomes from the cortex of adult rats were prepared as described previously3VO4) and extracts were applied to 18 cm IPG strips pH 3–10 non-linear  for isoelectrofocusing . Second dimension was then performed on 7.5% SDS-PAGE gels and proteins transferred onto nitrocellulose membranes for Western blotting analysis.Cells were lysed with two-dimensional electrophoresis buffer  and bi-distilled water were added to 400 μg of protein to a final volume of 500 μl. The mixture was divided into two aliquots and 600 units of λPPase  were added to one of the aliquots. Aliquots were incubated for 6 h at 30 °C and proteins were acetone-precipitated at −20 °C before SDS-PAGE. Dephosphorylation of HEK293 cell lysate before immunoprecipitation was performed in the same manner, except that the lysate was incubated for 2 h at 30 °C.Neurons were lysed in lysis buffer . 50 μl of 20 mM MnCl2, 10% glycerol, CLAP). Lysates were treated with λPPase and then immunoprecipitated with Protein A-sheparose (Sigma-Aldrich) conjugated with anti-Flag polyclonal antibody (Sigma-Aldrich) at 4 °C overnight. After three washes with lysis buffer, proteins were eluted with 2x SDS sample buffer and analyzed by Western blotting.Transfected HEK293 cells were lysed in lysis buffer  with lysis buffer  and 400 μg were incubated with 7-methyl GTP Sepharose 4 B beads  in the presence of 100 μM GTP overnight at 4 °C. The beads were washed three times  and eluted with 2x SDS sample buffer.Proteins were extracted from neurons (7–9 days 3VO4), protein content determined with the BCA reagent and then used for immunoprecipitation or Western blotting.HEK293 cells were plated the day before transfection at 50% of confluence in poly-L-Lysine (Sigma-Aldrich) treated 6 well plates and then transfected with 1.8 μg of DNA (1.5 μg of plasmid encoding WT-eIF4B and 300 ng of plasmid encoding GFP) and 4 μl of Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions. After 24 h cells were lysed  were transfected with 100 pmol of siRNA (ON-TARGETplus NON-TARGETING rat POOL and ON-TARGETplus SMART POOL against rat eIF4B - ThermoFisher Scientific) and 4 μl of Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions. At 8 days in vitro neurons were collected, lysed in PBS , total protein content determined with the BCA reagent  and then analysed by Western blotting.Neurons . Antibodies were diluted in goat serum dilution buffer, containing 15% goat serum (Gibco-Life Technologies), 450 mM NaCl, 0.1% Triton X-100, 20 mM phosphate buffer at pH 7.4, as in ref. Neurons were grown on glass coverslips for 8 days Images were acquired with a laser scanning confocal microscope  with a 63x/1.4 NA objective . Z-stacks were collected at 20 μm intervals and maximum projections were obtained using the Leica confocal software.64. Co-localization analysis was performed using the ImageJ Colocalization Colormap Plugin, based on ref. Image analysis was performed with ImageJ programThe Color map was also used to select regions of interest (ROIs) in which the Ser504-eIF4B antibody signal was co-localized  or not co-localized  with the Bassoon signal. A total of 675 red- and 675 green-ROIs were selected for each experimental condition on images obtained from three independent experiments. Normalized fluorescence intensity of the Ser504-eIF4B signal was calculated for each ROI after subtraction of the mean fluorescence of background. Normalized fluorescence intensity values were finally analyzed with Prism software.in vivo experiments. All procedures and animal care were carried out in accordance with European Community, national and local approved guidelines, laws and policies. All protocols were approved by the University of Ferrara Ethical Committee for Animal Experimentation and by the Italian Ministry of Health (authorization number: D.M. 246/2012-B). Experiments were run following the ARRIVE guidelines68 and the NC3Rs recommendations69. Rats were housed under constant temperature (22–24 °C) and humidity (55–65%), 12 h light/dark cycle, free access to food and water. All animals were (i) acclimatized to laboratory conditions for 1 h before the start of the experiment, (ii) used only once during the pilocarpine protocol and (iii) euthanized at different times points by anesthetic overdose.Male Sprague-Dawley rats  were used for All experimental procedures were as described in ref. in vivo experiments. A value of P < 0.05 was considered to be statistically significant.Statistical analysis was performed with Prism software version 5.0 . For biochemical experiments, columns in the graphs represent the mean (±SEM) of at least three independent experiments. For immunofluorescence analyses, columns in the graphs represent the mean (±SEM), obtained by a total of 10 z-stacks per experimental conditions, that were collected, processed and analyzed from three independent experiments. Statistical significance was evaluated  by one-way ANOVA followed by Dunnett post hoc test (for multiple comparisons against a single reference group), Bonferroni post hoc test (for multiple comparisons between groups), two-tailed Student t-test , the nonparametric Mann-Whitney U test for significant differences between experimental groups in the co-localization analysis or the nonparametric Kruskal-Wallis one-way analysis of variance followed by Dunns post test for the analysis of the brain samples from the Supplementary info


We find noticeable shifts from the stoichiometric compositions, expressed as (Scx1−Mx)MO3 with x = 0.05–0.11 and M = Co,  and . The crystal structure of (Sc0.95Co0.05)CoO3 is refined using synchrotron x-ray powder diffraction data: space group Pnma (No. 62), Z = 4 and lattice parameters a = 5.26766(1) Å, b = 7.14027(2) Å and c = 4.92231(1) Å. (Sc0.95Co0.05)CoO3 crystallizes in the GdFeO3-type structure similar to other members of the perovskite cobaltite family, ACoO3 (A3+ = Y and Pr-Lu). There is evidence that (Sc0.95Co0.05)CoO3 has non-magnetic low-spin Co3+ ions at the B site and paramagnetic high-spin Co3+ ions at the A site. In the iron-doped samples (Scx1−Mx)MO3 with M = , Fe3+ ions have a strong preference to occupy the A site of such perovskites at small doping levels.We synthesize ScCoO In the case of the Dh4 site, the LS state of Co3+ will only be realized at α′4 > 27/10JH, [ELS–EHS = (2×(−8/9α′4) + 4×(−4/27α′4) + 2×(−3JH)) –(1×(JH)) < 0]. Considering that α′4 should be smaller than α4 (and with the same JH for Co3+), the above conditions result in the HS state of Co3+ at the Dh4 site. Note that the HS state of Co3+ was experimentally found in BiCoO3 [3+ ions are located in a square pyramidal coordination.The presence of Co) figure  30]. In. In3+ io site 16/7α′4. Morn BiCoO3 , where C0.9CoO2.85, we prepared solid solutions with the total composition of Sc0.9Cox1−FexO2.85 . However, the samples with x = 0.2, 0.4 and 0.6 contained Sc2O3 impurity MO3 with M = Co0.6Fe0.4) was prepared whose Mössbauer spectrum at 300 K is shown on figure 0.95M0.05)MO3 (m)Co3+, mFe3+} in the local surrounding of the Fe3+ ions within the B sublattice. We fitted the experimental spectrum as a superposition of a discrete doublet Fe2 and a distribution p(ΔFe1) of the quadrupole splittings (ΔFe1), assuming a linear relation between ΔFe1 and δFe1 [0.95M0.05)MO3 (p(ΔFe1) distributions are shown in figure δFe1〉 and 〈ΔFe1〉 values for the Fe1 subspectra) and relative intensities (Ii) of the partial spectra are listed in table IFe1/IFe2 (table 0.89M0.11)MO3 (M = Co0.6Fe0.4), Fe3+ ions were distributed almost statistically between the A and B sites %). The resulting distribution p(ΔFe1) for (Sc0.95M0.05)MO3 MO3 compounds are the first example of such behaviour. It should be noted that Fe3+ ions were found by the Mössbauer spectroscopy at the A′ site of A-site ordered perovskites with the general composition of AA′3B4O12, for example, in CaCu3Fe4O12 [3Mn4O12 [3+ ions substitute for Cu2+ or Mn3+ ions—other transition metals—in a special A′ position, whose coordination environment (square-coordinated A′O4) is quite different from a typical coordination of the A site in perovskites (AO8-AO12).The location of small Feu3Fe4O12  and CaMn[3Mn4O12 . However0.9Cox1−CrxO2.85 (x = 0.25 and 0.5). The structural parameters of (Sc0.95M0.05)MO3 (M = Co0.75Cr0.25) are given in tables 0.95M0.05)MO3 (M = Co0.75Cr0.25 and Co0.5Cr0.5) are given on figure χ−1 values were almost field-independent above 70 K for (Sc0.95M0.05)MO3 (M = Co0.75Cr0.25) and above 100 K for (Sc0.95M0.05)MO3 (M = Co0.5Cr0.5) suggesting an impurity-free paramagnetic behaviour. The Curie–Weiss fits of the data corrected for diamagnetic contributions gave μeff = 2.47μB/f.u. and θ = −144 K for (Sc0.95M0.05)MO3 (M = Co0.75Cr0.25) with the expected μeff = 2.20μB/f.u and μeff = 2.99μB/f.u. and θ = −107 K for (Sc0.95M0.05)BO3 (M = Co0.5Cr0.5) with the expected μeff = 2.91μB/f.u. The anomalies at 100 Oe below 70 K in (Sc0.95M0.05)MO3 (M = Co0.75Cr0.25) and below 100 K in (Sc0.95M0.05)MO3 (M = Co0.5Cr0.5) could originate from the onset of short-range or long-range magnetic interactions.We also prepared solid solutions with the total composition of Sc4.3’-based perovskites are formed as non-stoichiometric (Scx1−Mx)MO3 with x = 0.05–0.11 and M = Co,  and  under high pressure (6 GPa) and high temperature (1570 K) conditions. There is evidence that (Sc0.95Co0.05)CoO3 has non-magnetic low-spin Co3+ ions at the B site and paramagnetic high-spin Co3+ ions at the A site. In the iron-doped samples (Scx1−Mx)MO3 with M = , Fe3+ ions have strong preference to occupy the A site of such perovskites that is quite unusual for perovskites.We found that ‘ScCoO