The safe removal of personal protective equipment (PPE) can limit transmission of serious communicable diseases, but this process poses challenges to healthcare workers (HCWs).We observed 41 HCWs across 4 Ebola treatment centers in Georgia doffing PPE for simulated patients with serious communicable diseases. Using human factors methodologies, we obtained the details, sequences, and durations of doffing steps; identified the ways each step can fail (failure modes [FMs]); quantified the riskiness of FMs; and characterized the workload of doffing steps.Eight doffing steps were common to all hospitals—removal of boot covers, gloves (outer and inner pairs), the outermost garment, the powered air purifying respirator (PAPR) hood, and the PAPR helmet assembly; repeated hand hygiene ; and a final handwashing with soap and water. Across hospitals, we identified 256 FMs during the common doffing steps, 61 of which comprised 19 common FMs. Most of these common FMs were above average in their riskiness at each hospital. At all hospitals, hand hygiene, removal of the outermost garment, and removal of boot covers were above average in their overall riskiness. Measurements of workload revealed that doffing steps were often mentally demanding, and this facet of workload correlated most strongly with the effortfulness of a doffing step.We systematically identified common points of concern in protocols for doffing high-level PPE. Addressing FMs related to hand hygiene and the removal of the outermost garment, boot covers, and PAPR hood could improve HCW safety when doffing high-level PPE.We identified ways that doffing protocols for high-level personal protective equipment may fail to protect healthcare workers. Hand hygiene, removing the outermost garment, boot covers, and respirator hood harbored the greatest risk and failed in similar ways across different hospitals. Healthcare workers at 4 Ebola treatment centers were observed doffing personal protective equipment for simulated patients with serious communicable diseases. Using human factors methodologies, we identified common points of concern and conclude with potential interventions to alleviate these issues. Prompted by the 2013–2016 outbreak of Ebola virus disease and its consequences in the United States, the Centers for Disease Control and Prevention (CDC) intensified efforts to deliver safer care for patients with suspected or known serious communicable diseases . These dWe performed 41 simulations across the 4 state-designated Ebola treatment centers in Georgia. During each simulation, a single HCW donned high-level PPE for serious communicable diseases in his or her biocontainment unit, performed a standardized task (changing a urinary catheter bag on a mannequin), and then doffed according to his or her institutional protocol. There were 10 simulations at each site (11 at site A) with a different individual performing the role of the HCW each time. Most HCWs were nurses (90%), with the remainder comprising paramedics (5%) and HCWs with other roles (5%). All simulations involved a trained observer (TO); at 2 sites, the same individual served as TO for all or nearly all (90%) of the simulations. Six of the 10 simulations at site B occurred in a high-fidelity mockup of their biocontainment unit.We recorded each simulation using 1 handheld and between 2 and 5 stationary cameras. Using these recordings (and the details of each site’s doffing protocol), we determined the duration of doffing steps, the different ways each step can fail to accomplish its goal(s) , and thFor each site, 2 raters independently tallied the frequency at which each FM occurred in the simulations using the Observer XT version 12.5 . Raw frequencies were transformed into a 5-point frequency scale. At each site, human factors and subject matter experts  independently rated the severity of the effect(s) of each FM using a 5-point scale that ranged from 1 (negligible) to 5 (catastrophic) . For eacImmediately after doffing, half of the HCWs at each site (n = 5) completed the NASA Task Load Index (NASA-TLX)  for the All doffing protocols involved removing boot covers, gloves (outer and inner pairs), the outermost garment , the powered air purifying respirator (PAPR) hood, the PAPR helmet/battery/belt, as well as repeated hand hygiene  and handwashing with soap and water. These 8 common doffing steps are the focus of our analyses.Across the sites, there were commonalities and notable variations in the order of doffing steps and the areas where steps occurred, the items of PPE used and how those items were removed, how hand hygiene was performed after PPE items were removed, and the role of the TO. The order of doffing steps  followedAt all sites, HCWs wore washable shoes, disposable scrubs (except site C), and different colored pairs of gloves, with the outer pair having extended cuffs . Only siFor hand hygiene, sites typically used ABHR; however, site C used disinfecting wipes . Site D was the only site to use automated ABHR dispensers exclusively and to enforce the duration of hand hygiene by having the HCW and the TO sing “Happy Birthday” aloud during each hand hygiene instance and 6 times during handwashing.The role of the TO typically involved observing and verbally guiding the HCW with a written checklist. However, site C divided the observation of the HCW and reading the checklist between 2 observers. At site D, the (fully donned) TO helped remove the HCW’s PPE .The median duration of complete doffing varied between sites, ranging from 6.9 to 22.2 minutes. The median duration of hand hygiene using ABHR between doffing steps  was 16.3 seconds . However, site D (where duration was enforced by singing a song) had a substantially larger percentage of hand hygiene instances that met the World Health Organization’s (WHO’s) recommendation  of 20 seExcluding hand hygiene and handwashing, the median duration of the common doffing steps was 25.2 seconds , whereas PAPR helmet/battery/belt removal was always in the bottom 2 at every site.The overall riskiness of a doffing step is indicated by the sum of the risk indices (ΣRIs) of the FMs associated with that doffing step . Across Summing across the common doffing steps at each site yielded 256 FMs . Of these 256 FMs, 61 (24%) were judged to be occurrences of 1 of 19 common FMs identified at either most or all of the sites. Agreement on these common FMs was 84%, with differences resolved through discussion. r, 0.76) than their physical  or temporal demand . However, this occasionally varied by doffing step. For example, the effortful steps of removing outer gloves had greater mental  than physical demand , but removing boot covers and the outermost garment had greater physical  than mental demand , except at site C. Although temporal demand was almost never the highest subscale for any step, it was the subscale most strongly correlated with frustration . Finally, some steps were almost always greater in workload  than others , regardless of site and workload subscale.The overall workload of the common doffing steps at each site was low , with no step at any site rated as extremely effortful or mentally, physically, or temporally demanding. The majority of the common doffing steps were rated as more mentally demanding  than physically  or temporally demanding , except at site D. The effortfulness of doffing steps was more strongly correlated with their mental demand . Of the 3 sites that primarily used ABHR for hand hygiene , only site D enforced the duration of hand hygiene by singing “Happy Birthday” aloud and had a substantially larger percentage of hand hygiene instances that met WHO duration standards. Thus, the duration of hand hygiene may be improved through standardization, such as using an auditory tool . Other opportunities for improving hand hygiene at most, if not all, sites were also apparent. No site ensured that all surfaces of the HCW’s hands were covered, nor were their hands consistently rubbed until dry. There were also instances of incidental contact with potentially contaminated PPE, such as the outside of the PAPR hood shroud, during hand hygiene, . HCWs atRemoval of the outermost garment  was always among the riskiest doffing steps, requiring the most time and being rated as one of the most effortful steps. The primary task demands tended to be physical rather than mental or temporal, particularly for coveralls. For sites that used coveralls, the HCW’s lower back occasionally became exposed during removal, but this was not observed at site C, where HCWs wore surgical gowns. During removal, coveralls were also difficult to keep within the confines of the disinfectant mat. These FMs were not eliminated by having the TO remove the HCW’s coveralls (site D), which was also associated with the longest doffing duration of all the sites. The decision to use a surgical gown or coverall as the outermost garment should consider these findings, as well as HCW comfort and material durability.Outer glove removal was effortful (except at site B), mentally demanding, and, compared with inner glove removal, took longer to perform (except at site D) and was greater in workload. Each site had a common high-risk FM associated with their glove removal protocol, such as whipping gloves  or snapping gloves . However, the method of glove removal (beaking vs glove-in-glove) did not appear to impact the duration of doffing, workload, or pattern of FMs. These FMs may instead be related to other factors, such as the physical characteristics of gloves . BePAPR hood removal was risky except at site D. Self-removal of the PAPR hood led HCWs to touch the outside of the hood excessively with their inner gloves, remove the hood by pulling from the front (rather than from the back), and touch the face shield with their inner gloves. Having the TO remove the PAPR hood (site D) appeared to mitigate excessive touching and improper removal. However, the decision to have the TO vs HCW remove the PAPR hood should consider the risk(s) to both the TO and HCW.At each site, removal of boot covers was a moderately risky doffing step that was the most physically demanding , with each site seeing HCWs touch their boot covers excessively as well as instances of other common FMs, such as whipping off boot covers. Design strategies may ameliorate the physical demands of this (and other doffing steps) by using the physical environment to promote easier doffing .Additionally, some common problems may also benefit from similar interventions not observed in the present study. Having the TO provide greater supervision during hand hygiene, such as asking, “Are your hands dry?” at the end of each hand hygiene instance, could address common high-risk components of doffing. Other interventions that target the immediate environment, such as installing a mirror for the HCW, may mitigate touching PPE  inappropriately , 18. FinBecause doffing protocols are complex, and our observations may also reflect variables that we did not assess directly , we cannot extrapolate a single “correct” approach to the entire process nor even for an individual step(s). Since any change may lead to unintended consequences downstream in the doffing process, potential modifications spurred by our findings should consider the doffing process carefully and holistically.The common points of concern that we identified, particularly those that have also emerged in related research efforts , unders


We also discovered that the lower frequencies are cluster vibrations that may overlap with acoustic phonons. Whale et al. found in ice XV that some intramolecular vibrational modes include many isolated-molecule stretches of only one O–H bond, whereas the other O–H bond does not vibrate. This phenomenon is very common in ice II, and we attribute it to local tetrahedral deformation. The pathway of combining normal mode analysis with experimental spectra leads to scientific assignments.The vibrational spectrum of ice II was investigated using the CASTEP code based on first-principles density functional theory (DFT). Based on good agreement with inelastic neutron scattering (INS), infrared (IR), and Raman experimental data, we discuss the translation, libration, bending, and stretching band using normal modes analysis method. In the translation band, we found that the four-bond and two-bond molecular vibration modes constitute three main peaks in accordance with INS ranging from 117 to 318 cm These vibrations can be divided into in-phase and out-of-phase modes. The vibrations at 1659 and 1723 cm−1 that possess the lowest and highest energy in this band all present out-of-phase modes, as shown in −1, in which all molecules vibrate in-phase. Two peaks at 1690 and 1748 cm−1 were detected by IR absorption [−1, according to the calculated IR intensities. For the Raman spectroscopy experiment, no peaks were detected in the bending area due to the weak intensity. The INS experiment detected a peak at 1669 cm−1 that corresponds to 1664 and 1699 cm−1 in PDOS [The H–O–H bending band has 12 normal modes from 1659 to 1723 cmsorption . These c in PDOS .−1 is from intramolecular O–H stretching, which contains 24 normal modes. There are two vibrational modes for each molecule, known as symmetric and asymmetric stretching. In the modes of 3193 and 3198 cm−1, only symmetric stretching occurs. In the mode of 3393 cm−1, all molecules show asymmetric stretching. In the stretching band, many molecules present only one vibrating O–H bond, while the other keeps static, as shown in the 3483 cm−1 mode in −1 in The band ranging from 3193 to 3483 cm−1.Bertie et al. reported that six peaks of the IR spectrum  and seveIn summary, using the first-principles density functional theory method, we present the theoretical Raman scattering, IR absorption, and INS spectra (PDOS) of ice II. The PDOS spectrum was used to assign the characteristic peaks in the INS spectrum because INS collects signals throughout the BZ without selection. The 105 optic normal modes in the BZ center could be compared with the Raman and IR spectra. Under this condition, the peaks recorded from photon scattering/absorption could be assigned individually according to the normal vibration mode.The most valuable result was the identification of H-bond vibration modes in the translation band. Inspired by previous studies, we concluded that ice II also has two H-bond vibration modes, even though they show three main peaks, mainly due to the structural deformation of a local regular tetrahedron under pressure, similar to ice XIV. The lower energy regions also have cluster vibrational modes, which may overlap with acoustic phonons, as seen in the INS spectrum. We discussed the “isolated stretching mode” first discovered by Whale et al. in ice XV. In our work, such isolated stretching was found to be common in many vibrational modes. The tetrahedral structure in ice II showed great deformation, in that the bond length of the two H-bonds of one molecule in the six-ring cluster was 1.815 Å, whereas the other two were 1.955 and 2.057 Å. We attributed this phenomenon to local tetrahedral deformation.


Arctic warming was more pronounced than warming in midlatitudes in the last decades making this region a hotspot of climate change. Associated with this, a rapid decline of sea-ice extent and a decrease of its thickness has been observed. Sea-ice retreat allows for an increased transport of heat and momentum from the ocean up to the tropo- and stratosphere by enhanced upward propagation of planetary-scale atmospheric waves. In the upper atmosphere, these waves deposit the momentum transported, disturbing the stratospheric polar vortex, which can lead to a breakdown of this circulation with the potential to also significantly impact the troposphere in mid- to late-winter and early spring. Therefore, an accurate representation of stratospheric processes in climate models is necessary to improve the understanding of the impact of retreating sea ice on the atmospheric circulation. By modeling the atmospheric response to a prescribed decline in Arctic sea ice, we show that including interactive stratospheric ozone chemistry in atmospheric model calculations leads to an improvement in tropo-stratospheric interactions compared to simulations without interactive chemistry. This suggests that stratospheric ozone chemistry is important for the understanding of sea ice related impacts on atmospheric dynamics. This phenomenon called Arctic Amplification can lead to a weakening of the temperature gradient between the Arctic and the midlatitudes. A weakened temperature gradient is related to a meridionalisation of the atmospheric flow and increased advection of warm air into the Arctic6. In addition, dry cold polar air is transported into the midlatitudes, which can result in cold air outbreaks in Eurasia and North America. Advection of warm air leads to a reduction of Arctic sea ice and therefore an increased transport of heat and momentum into the atmosphere in fall and winter followed by an increase in wave propagation from the tropo- into the stratosphere and weakening of the stratospheric polar vortex8, which then affects the tropospheric circulation in the midlatitudes in subsequent months11. To understand this tropo- stratospheric interaction, an improvement of climate models physical mechanisms is essential13. Recent studies also have shown an impact of Arctic stratospheric ozone on the El-Nino Southern Oscillation through a link to the North Pacific Oscillation16. The purpose of this study is to evaluate the extent to which stratospheric ozone chemistry is important to climate linkages between the Arctic and midlatitudes. Therefor we implement a computationally fast but accurate interactive stratospheric ozone chemistry module into an atmospheric general circulation model, which allows to perform a large number of ensemble simulations.In recent decades, Arctic winter temperature has risen at more than double the rate of lower latitudes17 version 6.3 with a spectral horizontal resolution of T63 (~1.875° longitude by 1.875° latitude on a Gaussian grid) and 95 vertical levels up to 0.01 hPa (~80 km). To isolate the impact of Arctic sea-ice retreat we perform sensitivity experiments only varying the sea-ice concentration (SIC). To simulate high-ice conditions (HICE) we use the average 1979 to 1983 and for low-ice conditions (LICE) the average 2005 to 2009 SICs from the Merged Hadley-National Oceanic and Atmospheric Administration/Optimum Interpolation sea surface temperature (SST) and SIC data set18. For all experiments we use the SSTs of the HICE period. We also reduce the sea-ice thickness (SIT) from 2 m in the HICE experiments to 0.5 m in the LICE setup, since changes in SIT in the Arctic can also contribute to atmospheric circulation changes20.Our study employs the atmospheric general circulation model (AGCM) ECHAM621 and repeat the model experiments with HICE and LICE conditions. Each of these four perpetual model simulations is integrated over 120 years, excluding the first 20 years as a spin-up period from the analysis. Compared to ECHAM6 the newly coupled ECHAM6-SWIFT does not apply ozone climatologies in the stratosphere but solves a set of coupled differential equations, which simulate the polar vortex-averaged mixing ratios of the chemical key species involved in polar ozone depletion on each discrete model level that is located in the range between 20 hPa and 80 hPa. The stratospheric polar vortex edge is assumed to be located at the 36PVU contour line of the modified potential vorticity field22 at the respective model level. Compared to classic chemistry climate models this method can be applied to obtain very large sample sizes while being computationally feasible.To investigate the interactions between ozone, sea ice and the atmospheric circulation we couple ECHAM6 with the fast but accurate interactive ozone chemistry scheme SWIFT23 allows an attribution of changes of atmospheric dynamics to changed sea-ice conditions through the stratospheric pathway, when combined with the interpretation of model results24. To enable comparisons between the ECHAM6 model and reanalysis data, we select two time periods of the ERA-Interim data set representing high (winters 1979/80 to 1999/00) and low (winters 2000/01 to 2015/16) Arctic sea-ice conditions8.As already discussed in recent literature, the ERA-Interim reanalysis dataset26. In the ECHAM6 model without the chemistry module, besides surface warming, a statistically significant response of the atmosphere due to sea-ice reduction is absent in the difference between the LICE and HICE experiments  in this region is an indicator for the momentum transported from the troposphere into the stratosphere by planetary waves28. Here, Fz is integrated over the two months preceeding the month showing the maximum temperature signal29 of each dataset, respectively. Its correlation with the stratospheric temperature is shown in Fig. Fz are related to higher stratospheric temperatures in the following months. All correlations are statistically significant at the 95% confidence level. Also in ERA-Interim the mean of the distributions of Fz at 100 hPa and temperature at 50 hPa are significantly different at the 95% confidence level when comparing the LICE and HICE conditions. In LICE Fz tends to be higher, indicating an enhanced propagation of planetary waves into the stratosphere. By the momentum deposited this way, the stratospheric circulation is disturbed, resulting in higher temperatures. While the LICE and HICE experiments for ECHAM6 show no statistically significant difference in the mean of these distributions, in the ECHAM6-SWIFT LICE and HICE experiments both, Fz and temperature, are significantly different. In contrast to ECHAM6, ECHAM6-SWIFT is able to simulate a qualitatively improved impact of Arctic sea-ice loss on the stratospheric winter circulation. This suggests that wintertime stratospheric dynamics and its impact on tropospheric wave behaviour30 can be improved by the implementation of interactive stratospheric ozone chemistry.To understand how the stratospheric temperature signals are generated, we investigate the mean vertical transport of momentum by atmospheric waves through the tropopause region (100 hPa) between 45°N and 75°N before the maximum monthly mean polar cap temperature signal in 50 hPa occurs. The vertical component of the Eliassen-Palm (EP) flux vector (Fz) at 100 hPa between 45°N and 75°N and its connection to polar-cap stratospheric ozone in the following spring is shown. Momentum is transported through the tropopause to high latitudes where it is deposited. This disturbs the stratospheric polar vortex, leads to warming of the polar cap and to poleward and downward transport of ozone with the amplified residual circulation, resulting in the positive correlation between Fz and ozone31 in Fig. Fz result in lower polar cap temperatures which allows the formation of more Polar Stratospheric Clouds (PSC) leading to activation of chlorine species through heterogeneous reactions. With the return of sunlight this results in chemical destruction of ozone, further steepening the relation between polar cap ozone and Fz, particularly for low values of Fz on the left hand side of Fig. One reason for the improved response is the interaction of propagating planetary waves with the stratospheric polar vortex and its dynamical relation to stratospheric ozone. In Fig. 34. In ECHAM6-SWIFT Arctic sea-ice reduction leads to a reduction of the zonal wind component in February/March (FM) at 10 hPa in the polar cap region of up to 3 m/s, which is of the magnitude of the induced forcing needed to significantly change the interaction between planetary waves and the mean flow9, whereas such a signal is absent in the ECHAM6 experiments without interactive stratospheric ozone chemistry. This change in the stratospheric circulation also leads to tropospheric anomalies in the following months, due to alterations in upward wave propagation. This can be seen in Fig. Several studies have shown that a reduction of Arctic sea ice leads to a negative phase shift of the North Atlantic Oscillation (NAO) in late winter35 for the period of the wind anomalies shown in Fig. 36 of the combined HICE and LICE datasets for the reanalysis data and each of the two model configurations. The corresponding NAO index for each dataset is defined as the leading EOFs mean PC over the respective dataset (e.g. ECHAM6-SWIFT HICE). Changes in NAO indices between LICE and HICE are represented by differences of this mean PCs. For ERA-Interim a strong negative shift in the phase of the NAO is found (PC difference LICE minus HICE: −0.42). Consistent with the zonal wind anomalies ECHAM6 shows no clear changes in the phase of the NAO, even tending to a positive shift (+0.07), while ECHAM6-SWIFT shows a clear tendency towards a negative NAO (−0.29). This shows that changes in Arctic sea ice can lead to a shifted circulation which increases the potential for cold air outbreaks over Europe, in agreement with the reanalysis.To quantify changes in the tropospheric NAO we perform an Empirical Orthogonal Function (EOF) analysis for the fields of 500 hPa Geopotential Height from 90°W to 40°E and 20°N to 80°NDuring LICE conditions ERA-Interim reanalysis data shows enhanced upward wave propagation from the troposphere into the stratosphere that causes a disturbance of the stratospheric polar vortex, which leads to downward propagating signals in the dynamical atmospheric variables. This process influences tropospheric circulation patterns like the NAO, impacting the daily weather patterns of the midlatitudes. This mechanism can not be fully reproduced by the AGCM ECHAM6, which does not respond with a negative phase shift of the NAO during LICE conditions. Coupling ECHAM6 to the fast but accurate interactive ozone chemistry scheme SWIFT improves the interaction between stratospheric chemistry and dynamics due to a more realistic representation of stratospheric processes. These improvements in the stratosphere also lead to a change in tropospheric teleconnection patterns resulting in a negative NAO response that is comparable to changes seen in reanalysis data.Our results imply that interactive stratospheric ozone chemistry in winter and spring is important to understand changes in tropospheric teleconnection patterns caused by decreasing Arctic sea ice via the stratospheric pathway. Being computationally feasible, we also suggest the implementation of the fast ozone chemistry scheme SWIFT into other AGCMs enabling very large ensemble sizes compared to classic Chemistry Climate Models.


We describe covalently binding modulators of the activity of human prolyl hydroxylase domain 2 (PHD2) and studies towards a strategy for photocapture of PHD2 substrates. We describe covalently binding modulators of the activity of human prolyl hydroxylase domain 2 (PHD2) and studies towards a strategy for photocapture of PHD2 substrates. Reversible active site binding of electrophile bearing compounds enables susbsequent covalent reaction with a lysine residue (K408) in the flexible C-terminal region of PHD2 to give a modified protein that retains catalytic activity. PHD inhibitors are in clinical trials for anaemia treatment because erythropoietin (EPO) is a HIF target gene. Clinical candidate PHD inhibitors, like most reported 2OG oxygenase inhibitors, are 2OG competitors coordinating to the active site Fe(ii).8The hypoxia inducible factor (HIF) prolyl hydroxylases PHD1-3) play a central role in cellular responses to hypoxia in humans and other animals.-3 play ae.g. anaerobic glycolysis is a HIF-1α target, whereas vascular endothelial growth factor (VEGF) is a HIF-2α target. We envisaged covalent modification of a non-catalytically essential PHD component may alter the oxygen dependence of the PHDs, ultimately enabling safer medicines than those working by complete inhibition. Covalent inhibition may provide a strategy for selective inhibition by reacting with non-conserved residues. The ability to install a reactive functional group near the active site, without inhibition, may also enable substrate capture. Here we communicate initial findings demonstrating the feasibility of selective covalent modification of human PHD2.Inspired by the mode of action of aspirin in modulating, but not ablating, cyclooxygenase activity by covalent modification,Covalent conjugation of small molecules to proteins is often achieved by reaction of an electrophile with a nucleophilic residue.N-oxalylglycine (NOG)  , indicatthesised .181–426 (hereafter PHD2) was examined by non-denaturing ESI-MS, using cone voltage variation to distinguish covalent and non-covalent complexes .1 and 2 were incubated with equimolar PHD2 and Fe(ii) . After 20 min, analysis at 200 V cone voltage indicated 1/2 react with PHD2 to give a peak with mass of PHD2 + 1/2 – H2O, suggesting condensation , neither covalent nor non-covalent adducts were observed, implying covalent conjugation involves active site Fe(ii) binding, with subsequent condensation. Consistent with this, preincubation with the reversible iron-binding inhibitor FG2216 (IC50 < 1 μM),1/2 did not manifest covalent adducts. No covalent modification was observed with the 5-formyl derivative (S1) or an analogue lacking the glycine sidechain (S2)  and with a pyridinyl analogue (S4), but to a lesser extent than for 1/2 ), suggesting the covalently-linked inhibitors can dissociate from the active site so enabling FG2216 binding, while maintaining the covalent crosslink (1/2 (to maintain concentration of the inhibitor on dilution and so limiting hydrolysis). Controls were carried out without 1/2 and with FG2216 (30 μM)  . CODD hy3); we envisaged this may irreversibly react with a lysine residue by Michael addition . Compound 4, without the amide, was synthesised as a negative control  and analysed by non-denaturing MS. After 5 min, spectra obtained at 50 V indicated the presence of PHD2·Fe·3 and PHD2·Fe·4, which were assigned as non-covalent adducts by analysis at 200 V . The crosslinking could be fitted by a one-site decay function , consistent with a quasi intramolecular reaction of the PHD2·Fe·3 non-covalent species to give a covalent adduct  prior to assays. The extent of crosslinking was determined to be 0%, 50%, 66% and 90%, respectively  associates the active site, and can leave to allow the CODD substrate to bind.To investigate whether the covalent PHD2·Fe·1, 2 and 3 was measured using an antibody based assay to obtain IC50 values , with 3 not reaching 50% inhibition at up to 2 mM. Interestingly, the IC50 values are >10 fold higher than the 30 μM used for covalent conjugation. IC50 values for 1 and 2 were found to drop by ∼2 fold on preincubation, possibly reflecting inhibition of non-covalently versus covalently modified PHD2.PHD2 inhibition by 3 was investigated using protein variants. First, 3 was incubated with C-terminal truncated PHD2 (PHD2181–402) and a loop deletion variant . Crosslinking was maintained with the loop truncate, but near completely absent in C-terminally truncated PHD2, suggesting a Lys-residue  in the C-terminal region is involved in crosslinking  and 414 (PHD2181–414) truncated proteins were covalently modified by 3, suggesting K423 and K416 are not involved in crosslinking . High concentrations of 5 (>250 μM) induced more pronounced HIF-1α upregulation, which may involve inhibition of  PHD2 by the free 1, consistent with IC50 values for inhibition of isolated PHD2·Fe·1 by 1  for activity-based profiling, or as a photoreactive aryl azide for substrate capture. 7 covalently modified PHD2 at a similar rate to 3  was added to the modified protein, causing near complete conversion of covalent PHD2·Fe·7 to biotinylated PHD2·Fe·7.8  of PHD2, and more generally 2OG oxygenases, by covalent reaction of an enzyme region involved in substrate binding. Azide functionalisation of one of the covalently reacting molecules enabled subsequent conjugation with biotin, offering application in activity-based protein profiling. This azide was also found to be photoreactive, suggesting a strategy for covalent capture of PHD2 substrates.Our work was supported by the Newton-Abraham Fund (J. M.), the British Heart Foundation (R. K. L.), the Wellcome Trust (WT 106244/Z/14/Z) and Cancer Research UK (C8717/A18245).There are no conflicts to declare.Supplementary informationClick here for additional data file.


Ecklonia cava (E. cava) were evaluated. Based on the in vitro study, all tested compounds showed potent inhibitory effects on BACE1 and AChE. In particular, 8,8′-bieckol demonstrated the best inhibitory effect against BACE1 and AChE, with IC50 values of 1.62 ± 0.14 and 4.59 ± 0.32 µM, respectively. Overall, kinetic studies demonstrated that all the tested compounds acted as dual BACE1 and AChE inhibitors in a non-competitive or competitive fashion, respectively. In silico docking analysis exhibited that the lowest binding energies of all compounds were negative, and specifically different residues of each target enzyme interacted with hydroxyl groups of phlorotannins. The present study suggested that major phlorotannins derived from E. cava possess significant potential as drug candidates for therapeutic agents against AD.Alzheimer′s disease (AD) is one of the most common neurodegenerative diseases with a multifactorial nature. β-Secretase (BACE1) and acetylcholinesterase (AChE), which are required for the production of neurotoxic β-amyloid (Aβ) and the promotion of Aβ fibril formation, respectively, are considered as prime therapeutic targets for AD. In our efforts towards the development of potent multi-target, directed agents for AD treatment, major phlorotannins such as eckol, dieckol, and 8,8′-bieckol from  Alzheimer′s disease (AD) is a progressive and irreversible neurodegenerative disorder with characteristic features of cognitive dysfunction, memory impairment, and behavior disturbances. The neuropathological hallmarks of AD patients are the presence of extracellular deposits of amyloid plaques and intracellular filamentous neurofibrillary tangles in the brain . AmyloidAβ is generated by the sequential proteolytic cleavage of two aspartic proteases, β- and γ–secretase, in the amyloidogenic pathway. β-Secretase (BACE1) initially cleaves amyloid precursor protein (APP) at the N-terminus of the Aβ peptide domain, which is followed by the cleavage of γ–secretase in the transmembrane region of APP, leading to the production of Aβ peptide . TherefoIn the past, it has been found that acetylcholinesterase (AChE) is involved in the degradation of the neurotransmitter. The observation of a significant loss of cholinergic neurons in AD patients is the major correlate of cognitive impairment. Cholinesterase inhibitors can increase acetylcholine (ACh) levels in the synaptic cleft and partially ameliorate cognitive symptoms for patients with mild to severe AD . Recent Current AD therapies are mainly palliative and temporarily slow cognitive decline, and treatments based on the underlying pathologic mechanisms of AD are totally limited . SeveralEcklonia cava (E. cava) is an edible brown seaweed which is distributed in Japan and the southern coast of Korea, and it is recognized as a rich source of bioactive derivatives, containing 3.1% crude phlorotannins , dieckol oxy-3,5-dihydroxyp henoxy]dibenzo-p-dioxin-1,3,6,8-tetrol], and 8,8′-bieckol dibenzo-p-dioxin-1,3,6,8-tetrol] were shown in 50 values of 5.69 ± 0.42 and 4.59 ± 0.32 µM, respectively) and about twofold greater than the eckol .Three phlorotannins displayed high potencies as AChE inhibitors . Both diTo demonstrate the specificity of the targeted enzymes, all compounds were tested against tumor necrosis-converting enzyme (TACE), which is a candidate for α-secretase, and other serine proteases, including trypsin, chymotrypsin, and elastase . With seKi values of eckol, dieckol, and 8,8′-bieckol were 31.2, 20.1, and 13.9 µM, respectively. On the other hand, our tested compounds were competitive inhibitors of AChE, where the Lineweaver– Burk plots intersected a common point on the y-axis  in Nelumbo nucifera (IC50 in the 6.4–28.5 µM range) have been proven to be efficient BACE1 inhibitors. Umbelliferone, isoscopoletin, 7-methoxy coumarin, esculetin, and daphnetin from Angelica decursiva with IC50 ranging from 7.7–172.3 µM were identified as BACE1 inhibitors. Compared with those plant-derived BACE1 inhibitors, our compounds demonstrated predominantly inhibitory properties against BACE1 [In the current study, three effective phlorotannins—eckol, dieckol, and 8,8′-bieckol—were studied for their inhibitory properties on BACE1 and AChE. These compounds exhibited powerful inhibitory activities on BACE1 with ICst BACE1 ,37,39.Eisenia bicyclis, one of the brown algae, were investigated for their BACE1 inhibitory effects, the result that dieckol was stronger than eckol was similar to that of our present study [Interestingly, the difference in inhibitory properties among phlorotannins is related to the number of hydroxyl groups present. In our new findings, it was shown that 8,8′-bieckol containing 11 OH groups had the highest inhibitory efficacy against BACE1 when compared to dieckol (10 OH groups) and eckol (6 OH groups). When phlorotannins from nt study . Consistnt study . This ob50 of 40 µM, than dieckol and eckol  [Among three phlorotannins, 8,8′-bieckol showed the most potent inhibitory activity against AChE. Similar results regarding the correlation between the molecular size of phlorotannins and enzyme inhibitory efficacy was revealed in a previous study. 8,8′-bieckol showed more potent activity against hyaluronidase, with an ICctively) . In addimax is reduced, but Km is unaffected. In BACE1 inhibitory activity, phlorotannins decreased the Vmax values without affecting the affinity of BACE1 toward the Km, which demonstrated that these compounds exhibited non-competitive inhibition against BACE1. However, AChE kinetics results exhibited that our tested compounds are competitive inhibitors with unchanged Vmax and increased Km. In other words, these compounds interacted directly with the catalytic site of AChE instead of with other allosteric pockets.The BACE1 inhibition kinetics indicated that the tested compounds act as non-competitive inhibitors, which means that these compounds can bind either another regulatory site or to the subsite of BACE1. The inhibition level is dependent on the concentration of the inhibitor but is not reduced by increasing concentrations of substrate. Because of this, VIn silico docking analysis is a valuable drug discovery tool and can be used to discover prospective, biologically active molecules from natural product databases. The results of the molecular docking score were provided to evaluate the capacity of different protein–ligand complex interactions and to compare the biological activities and the inhibition mode. In the BACE1 docking simulation, multiple hydrogen interactions were observed in the BACE1–phlorotannins complexes. Eckol interacted with both GLY34 and SER36 of BACE1, and dieckol bounded to TRP76, THR232, and LYS321. In addition, 8,8′-bieckol formed four hydrogen bonds with BACE1 residues, including LYS107, GLY230, THR231, and SER325. These docking results showed that hydrogen bonds between phlorotannins and allosteric residues of BACE1 play an important role in enzyme inhibition. AChE docking analysis provides insight into the mechanism underlying active site binding interaction. The hydroxyl group of eckol formed five hydrogen bonds with THR83, TRP86, TYR124, and SER125 of AChE. In particular, the choline-binding site residue (TRP86) of AChE was involved in hydrogen bond interaction with eckol. Dieckol showed four hydrogen-bond interactions with ASN233, THR238, ARG296, and HIS405, whereas 8,8′-bieckol made one hydrogen bond with ARG296 located in the active site of AChE. These docking results from the in silico study were in agreement with our in vitro experimental data.25–35 toxicity through the regulation of the apoptotic signal and the NF-kB/MAPKs pathway [2O2-induced oxidative stress in murine hippocampus neuronal cells [To date, few studies have investigated the neuroprotective property of phlorotannins. Our previous study demonstrated that phlorotannins ameliorated Aβ pathway . Eckol aal cells . Moreoveal cells . E. Cava extract was shown to be safe for use in food supplements at a maximum daily intake level of 263 mg/day for adults [Nagayama and coworkers demonstrated no significant toxic effects in the oral administration of up to 1,500 mg/kg of phlorotannins for 14 days in male and female Institute of Cancer Research (ICR) mice . In a hur adults . CollectE. Cava are safe, potent, and selective natural dual inhibitors against BACE1 and AChE that can be used for the multi-target, directed agents of AD.Bioavailability parameters such as biotransformation and conjugation during absorption from the GI tract are principle factors influencing in vivo biological activity. Lipinski′s rule of five is a widespread strategy to define bioavailability predictions of drug molecules. According to this predictive model, a compound needs to exhibit optimum GI absorption with a molecular weight of < 500 Da, no more than five hydrogen bond donors, no more than ten hydrogen bond acceptors, and a calculated partition coefficient (LogP) that is no more than five . FortunaElectrophorus electricus (electric eel), 5,5′-dithiobis-(2 nitrobenzoic acid) (DTNB), resveratrol, galantamine, trypsin, chymotrypsin, elastase, and their substrates, including N-benzoyl-l-Arg-pNA, N-benzoyl-l-Tyr-pNA, and N-succinyl-Ala-Ala-Ala-p NA, were from Sigma-Aldrich .Fluorescence and optical density were measured by Bio-TEK ELISA fluorescence reader FLx 800 and Bio-TEK ELx 808, respectively . Eckol (>95%), dieckol (>95%), and 8,8′-bieckol (>95%), were bought from National Development Institute of Korean Medicine . The BACE1 assay kit was purchased from Invitrogen . TACE and substrate were bought from R&D Systems . AChE from C was the fluorescence of control  after 60 min of incubation, C0 was the fluorescence of control at time 0, S was the fluorescence of tested samples  after 60 min of incubation, and S0 was the fluorescence of the tested samples at time 0.Fluorometric assays with a recombinant human BACE1 or TACE were conducted according to manufacturer instructions. Briefly, reaction mixtures containing human recombinant BACE1 (1.0 U/mL), the substrate (75 μM in 50 mM ammonium bicarbonate), and phlorotannins dissolved in an assay buffer  were incubated in darkness for 60 min at 25 °C in well plates. The increase in fluorescence intensity produced by substrate hydrolysis was observed on a fluorescence microplate reader with excitation and emission wavelengths of 545 and 590 nm, respectively. The inhibition ratio was obtained using the following equation:A human recombinant TACE (0.1 ppm in 25 mM Tris buffer), the substrate (APP peptide YEVHHQKLV using EDANS/DABCYL), and phlorotannins were dissolved in an assay buffer, which were then combined and incubated for 60 min in the dark at 25 °C. The increase in fluorescence intensity produced by substrate hydrolysis was observed on a fluorescence microplate reader with excitation and emission wavelengths of 320 and 405 nm, respectively.The colorimetric assays, including AChE, trypsin, chymotrypsin, and elastase were assayed according to previously described methods . The hydN-benzoyl-l-Arg-pNA, N-benzoyl-l-Tyr-pNA, and N-succinyl-Ala-Ala-Ala-pNA were used as substrates to assay the inhibition of trypsin, chymotrypsin, and elastase, respectively. Enzyme, Tris-HCl buffer , and phlorotannins were incubated for 10 min at 25 °C; then, substrate was added for 30 min at 37 °C. The absorbance was recorded at 410 nm. The inhibition ratio was obtained using the following equation:Ki, Vmax, and Km values were calculated by Sigma Plot 12.3 To evaluate the kinetic mechanisms of phlorotannins towards BACE1 and AChE, Dixon and Lineweaver–Burk plots were conducted by various concentrations of substrate and inhibitors. Kinetic parameters such as http://www.rcsb.org/). Three-dimensional (3D) structures of eckol, dieckol, and 8,8′-bieckol were obtained from PubChem with compound identification number (CID) of 145937, 3008868, and 3008867, respectively. The Autodock Vina software version 1.1.2  was used to conduct molecular docking analysis. The dimensions of the grid were 30 × 30 × 30 Å, the cluster radius was 1 Å, and the Cα coordinates in each selected backbone binding residue of the protein receptor was used for the center of docking space. Other options for docking simulations were used as defaults. The atomic coordinates of the ligands were drawn and displayed using Marvin sketch .X-ray crystal structures of human BACE1 (PDB code: 2WJO) and AChE (PDB code: 4PQE) were retrieved from the Protein Data Bank  version 9.3 .E. cava, especially 8,8′-bieckol, have noteworthy potential for the possible development as treatments and/or preventative agents against AD.The integration of enzyme activity, kinetics, and in silico docking studies provided principle insights into the molecular basis underlying ligand binding affinity and BACE1 and AChE inhibition. Accordingly, these results suggested that phlorotannins from


Pedicle screws can be augmented with polymethylmethacrylate (PMMA) cement through cannulated and fenestrated pedicle screws to improve screw anchorage. To overcome the drawbacks of PMMA, a modified augmentation technique applying a self-curing elastomeric material into a balloon-created cavity prior to screw insertion was developed and evaluated. The aim of the study was to compare the effect of the established and novel augmentation technique on pedicle screw anchorage in a biomechanical in vitro experiment.In ten lumbar vertebral bodies, the right pedicles were instrumented with monoaxial cannulated and fenestrated pedicle screws and augmented in situ with 2 ml PMMA. The left pedicles were instrumented with monoaxial cannulated pedicle screws. Prior to left screw insertion, a balloon cavity was created and filled with 3 ml of self-curing elastomer (silicone). Each screw was subjected to a cranio-caudal cyclic load starting from − 50 to 50 N while the upper load was increased by 5 N every 100 load cycles until loosening or 11,000 cycles (600 N). After cyclic loading, a pullout test of the screws was conducted.P = 0.012). The post-cycling pullout test of the loosened screws showed differences in the failure mode and failure load, with predominantly pedicle/vertebrae fractures in the PMMA group (1188.6 N ± 288.1) and screw pullout through the pedicle (671.3 N ± 332.1) in the elastomer group.The mean cycles to screw loosening were 9824 ± 1982 and 7401 ± 1644 for the elastomer and PMMA group, respectively (The modified pedicle screw augmentation technique involving a balloon cavity creation and a self-curing elastomeric silicone resulted in a significantly improved pedicle screw anchorage under cyclic cranio-caudal loading when compared to conventional in situ PMMA augmentation. Posterior implant systems are used for stabilization and fixation of the human thoracic and lumbar spine if the load-bearing capacity is lost by injury, disease, or degeneration . In seleAlthough polymethylmethacrylate (PMMA) has been used for many years—at the beginning for reconstructive surgery, more recently as augmentation material in several conditions—the application can have negative side effects. Due to its exothermic curing behavior, the temperature at the interface increases substantially, and concerns for thermal bone necrosis were raised –13. OtheConsequently, the emphasis was put on the development of alternative augmentation materials—for example, calcium-phosphate cement, calcium-sulfate cement, or silicone. Calcium-based cements are osteoconductive and osteoinductive  but haveWith the development of a medical grade, injectable, self-curing elastomeric polymer with silicone basis, a new alternative material is available for vertebral augmentation. The silicone is intended to be osteoconductive and non-hazardous to the surrounding tissue, showing a non-exothermic curing . AlthougThe purpose of the present study was to compare the fixation strength of in situ PMMA-augmented fenestrated pedicle screws with that of standard pedicle screws inserted in a balloon-created cavity filled with an injectable silicone-based polymer. We hypothesized that the number of cranio-caudal load cycles until screw loosening does not differ between in situ PMMA-augmented screws and balloon cavity-augmented silicone screws.3. Specimens were thawed overnight at 6 °C, prepared, and implanted at room temperature. Monoaxial pedicle screws of identical size and thread geometry were used for the left to right comparison . For in situ PMMA augmentation, the screws were cannulated and fenestrated, while for balloon cavity augmentation, the screws were only cannulated  from human donors were used for biomechanical testing. The bodies were donated by people who had given their informed consent for their use for scientific and educational purposes prior to death –34. The ted Fig. .Fig. 1ScLeft pedicle screws were augmented with medical silicone  using the kyphoplasty technique  to create a cavity which was filled with 3 ml of silicone prior to screw placement. After this, a cannulated pedicle screw was inserted into the cured silicone.Into the right pedicles, cannulated and fenestrated pedicle screws were implanted and in situ augmented with 2 ml of PMMA cement .Isolated single vertebral bodies were embedded in PMMA  for fixation purpose in the testing machine, and an axial X-ray was taken to document the cement distribution.x-y plane-bearing table, and a straight rod (Ø 5.5 mm) was connected to the pedicle screw. Axial loading was conducted with a lever arm of 15 mm to the pedicle screw head [Cyclic loading in cranio-caudal direction was conducted in a servohydraulic biaxial material testing machine . Specimens were fixed on an rew head . A 3D moEach pedicle screw was initially cycled with 50 N in tension and 50 N in compression (speed 5 mm/s), with an increase of compressive load by 5 N every 100th cycle for a total of 11,000 cycles (600 N compressive loading). The cyclic protocol was terminated after 10 mm axial displacement of the machine crosshead.For post-test data analysis, the motion of the screw head relative to the vertebra was evaluated. Screw loosening was defined as an absolute angular tilt of more than 8° or an increase in angular motion of more than 1° within one load step (100 cycles).After cyclic loading, each pedicle screw was subjected to a pullout test in the direction of the screw axis. Pullout testing was conducted in a material testing machine  with a speed of 10 mm/min. During all testing, the displacement and force at the actuator were recorded with 100 Hz.t tests were applied for comparisons between the two augmentation techniques (screws inserted into the left pedicles (silicone) vs. screws inserted into the right pedicles (PMMA)). Level of significance was set to a value of P < 0.05.Statistical analysis was carried out using SPSS software package . Data are given as mean ± SD. Paired One in situ augmented pedicle screw placed in an L1 vertebra breached the pedicle after implantation. Therefore, this vertebra was excluded from the data analysis resulting in a total of nine vertebrae with left-right comparisons of the two augmentation techniques.All in situ PMMA-augmented screws loosened by caudal screw cutout through the pedicle. In the balloon cavity silicone-augmented group, only two screws loosened by caudal screw cutout through the pedicle. Two screws showed a loosening of the screw inside the intact pedicle, and five screws did not reach the predefined failure criteria.P = 0.012). With the stepwise increasing load magnitude, these cycle numbers correspond to a mean load level of 420 ± 82 N for in situ PMMA-augmented screws and 542 ± 99 N for balloon cavity silicone-augmented screws  .The mean maximum pullout force after loosening was 1189 ± 288 N for in situ PMMA-augmented screws and 671 ± 332 N for balloon cavity silicone-augmented screws (03) Fig. . The meaFor balloon cavity silicone-augmented screws, the main failure mode (seven of nine) during the pullout test was axial pullout of the screw with the silicone cloud still bonded to the trabecular structure while only two specimens failed by pedicle fracture. For in situ PMMA-augmented screws, the main failure mode (eight of nine) during pullout testing was pedicle fracture with the PMMA cement cloud still attached to the screw while only one specimen failed by axial screw pullout.Several biomechanical studies have shown that, compared to standard non-augmented pedicle screws, augmentation of the screw with PMMA cement can significantly improve screw anchorage in patients with reduced bone quality, independent of the augmentation technique , 35, 36.The test setup and loading protocol used in the present study consisted of a cyclic cranio-caudal loading with a superimposed bending moment and thus resembles the physiological loading of pedicle screws reported in “in vivo” measurements of patients with an instrumented internal fixator –42 much For “in vivo” measurements with an instrumented internal fixator, Rohlmann et al. showed that pedicle screws are mainly loaded axially in cranio-caudal direction and can be superimposed with a small bending moment. In daily activities of the patients, the peak axial forces were reported to range from 100 to 250 N , 42. UsiThe post-cycling pullout force of silicone-augmented screws was lower than that of PMMA-augmented screws but was still of higher magnitude than the values reported by Liu et al. for non-augmented and non-cycled screws in osteoporotic (528 N) and severely osteoporotic (358 N) vertebrae . The posA limitation of this study is that two different augmentation techniques and two different augmentation materials were used. Therefore, it is not possible to decisively attribute the better performance of the balloon cavity silicone augmentation to the material or the technique. Most likely, it is a combination of both. Another limitation is that the study was conducted on cadaver specimens and therefore cannot take account of any biological factors such as bone remodeling and osteointegration, as well as PMMA aging and volumetric shrinkage.In a comparative study on PMMA screw augmentation techniques, Becker et al. reported no difference in screw anchorage between the augmentation techniques, vertebroplasty, kyphoplasty, and in situ augmentation technique . The prePedicle screw augmentation with balloon cavity creation and self-curing elastomeric silicone represents a valuable alternative to PMMA augmentation and resulted in superior pedicle screw anchorage under cyclic cranio-caudal loading. Pullout forces after cyclic loading were higher for in situ PMMA-augmented screws and showed a different failure mode. Using an alternative silicone-based augmentation material, however, might also necessitate a modification of the conventional in situ augmentation technique.


The mid-Pliocene warm period provides a natural laboratory to investigate the long-term response of the Earth’s ice-sheets and sea level in a warmer-than-present-day world. Proxy data suggest that during the warm Pliocene, portions of the Antarctic ice-sheets, including West Antarctica could have been lost. Ice-sheet modelling forced by Pliocene climate model outputs is an essential way to improve our understanding of ice-sheets during the Pliocene. However, uncertainty exists regarding the degree to which results are model-dependent. Using climatological forcing from an international climate modelling intercomparison project, we demonstrate the high dependency of Antarctic ice-sheet volume predictions on the climate model-based forcing used. In addition, the collapse of the vulnerable marine basins of Antarctica is dependent on the ice-sheet model used. These results demonstrate that great caution is required in order to avoid making unsound statements about the nature of the Pliocene Antarctic ice-sheet based on model results that do not account for structural uncertainty in both the climate and ice sheet models. Ice sheet models forced by climate model output indicate ice-sheet retreat during the Pliocene, yet concerns remain regarding potential model bias. Here, the authors present results from the Pliocene Ice-sheet Modelling Intercomparison Project, and show that results are highly dependent on the model forcing used. Estimates of sea level change between 10 and 30 m above present day8 have been reconstructed based on geological evidence, suggesting that a significant contribution to sea level from both the Greenland and Antarctic Ice-sheets (AIS) is required at certain intervals within the mPWP. This makes the mPWP an important interval to understand in terms of future climate change and ice-sheet stability9.A warm interval during the Late Pliocene  offers the most recent time in Earth history where global annual mean temperatures were on average 1.8–3.6 °C higher10. Marine records from the margin of East Antarctica also have been interpreted to suggest a dynamic EAIS margin at this time13. A more recent study used a combination of cosmogenic nuclide exposure ages alongside ice-sheet modelling to suggest that the EAIS continental interior could have been up to 600 m higher than present14. Such limited direct proxy evidence means that ice-sheet modelling is particularly important in understanding more about the behaviour and dynamics of ice-sheets during the Pliocene.The extent and nature of the AIS during the mPWP remains uncertain. Sedimentological evidence from the ANDRILL core suggests that there were periods where no ice was present on West Antarctica (WAIS) during the Pliocene15. However, models are not in agreement as to what degree the EAIS could vary during the warmer intervals of the Pliocene16. Transient simulations with ice-sheet models (ISMs) have not shown significant ice retreat in the areas that proxy data suggests18. However, studies using climate forcings derived from climate models with imposed Pliocene boundary conditions  to force shallow ice approximation (SIA) ISMs have shown the potential for ice-sheet reduction20. More recent studies have advocated the inclusion of alternative physics within ISMs in order to facilitate the simulation of different extents of the Pliocene AIS23, and these have shown the potential for marine incursions into the Wilkes and Aurora subglacial basins (SGB). Nevertheless, it is difficult to perform a true comparison between such previous studies due to the very different models and modelling frameworks that have been adopted.Previous modelling studies have supported the geological inferences and shown the potential for significant ice-sheet collapse events during the Pliocene in West Antarctica24 offers a unique opportunity to test the dependency of ice-sheet simulations to the climate model forcing that is implemented. The models used in PlioMIP1 are similar in terms of complexity  and are all models that were represented in the IPCC 5th Assessment report25. All of the PlioMIP1 models implemented PRISM3 boundary conditions for their mid-Pliocene experiment, which included a reduced AIS26 (see Methods). It has been shown previously, that whilst the PlioMIP1 model results have commonalities in terms of the large-scale features of Pliocene climate, there is substantial regional variation in the sensitivity of models to the implementation of the Pliocene boundary conditions1. Therefore, it is useful to assess the impact of any simulated regional climate differences over Antarctica on the prediction of the Pliocene.The Pliocene Model Intercomparison Project 27, which was initiated in order to understand the extent to which using different climate and ISMs changes the predicted ice-sheet configuration for the warm Pliocene. We use output from the climate modelling intercomparison project, PlioMIP24, to force ice-sheet simulations of the mPWP. The significance of using different ISMs has been demonstrated previously28 and therefore, here we force three different ISMs  and simulate equilibrium ice-sheets following the initial PLISMIP experimental set-up27 (see Methods). Using our ensemble of simulations, we demonstrate that there is a high level of climate model dependency in the reconstruction of the present-day AIS as well as the mPWP AIS. This is especially so in terms of the reconstructed ice volume. For the Pliocene, we use both PRISM3 and present-day AIS configurations to initialise our ISMs. We show that in general when using PRISM3 initial conditions in the ISMs, there is a consistent reduction in the broad extent of the WAIS in the Pliocene. However, some ensemble members retain ice over the West Antarctic Islands, while others lose this ice completely. When using present-day ice-sheet initial conditions different outcomes arise. One model predicts a WAIS with an extent equivalent to modern, whereas the remaining ice-sheet simulations reduce the size of WAIS. Over East Antarctica, the areas of ice reduction in the Pliocene (when compared to modern) differ according to which climate forcing but also importantly which of the ISMs is employed. It is worthy of note that there is more consistency in the results when present-day initial conditions are used within the ISMs. We conclude that it is very important to acknowledge the likely biases in Pliocene ice-sheet simulation based on a single model or even combination of models. Whilst it is useful to show the plausible range in Pliocene AIS reconstructions as a result of choosing different GCMs or ISMs, it is difficult to use the results presented here to directly inform us about the exact configuration of the AIS during the warmest intervals of the Pliocene due to insufficient geological data constraints on the ice-sheet itself.Here, we present results from the Pliocene Ice-sheet Modelling Intercomparison Project (PLISMIP)32 as used in ref. 28, COSMOS33, CCSM434, IPSLCM5A35, MIROC4m36, MRI-CGCM2.337 and NorESM-L38; Supplementary Table 1 and the differences in model resolution are shown in Supplementary Table 26 (see Methods). Near surface temperature differences over the AIS (including over ice shelves) simulated by the PlioMIP1 models range from +3.9 to +10.5 °C  from PlioMIP Phase 1 , and is generally larger than the Bedmap2 reconstruction ; ref.39; Fig. A visual comparison of the simulated modern ice-sheets highlights some GCM–ISM combinations that result in a poor reconstruction of the present day ice-sheet. The final simulated modern AIS grounded ice volume varies considerably between the models . This was done in order to allow for the assessment of model dependency of the results . We have shown that five out of the seven GCMs lead to a reasonable representation of present-day Antarctica. This gives us confidence when using the same model set-up to assess the climate model dependency of mid-Pliocene Antarctic simulations. For clarity, we have excluded two of the GCM–ISM combinations from our Pliocene analysis, however, the results from these GCM–ISMs are shown in Supplementary Figs. Ice-PRISM3; Fig. 24. Fifteen ensemble members were run, using temperature, precipitation and ocean temperatures from the five remaining PlioMIP climate models to force three ISMs. In general, there is a greater degree of variation between the individual ISM reconstructions for the mPWP in terms of ice presence than has been shown in the modern experiments  to 33.18 × 106 km3 , which is a likely contributor to broad differences in ice-sheet thickness in the centre of East Antarctica.A final observation for the Pliocene26. It can be argued that such a prescribed retreat in the climate modelling simulations will lead to the ISMs predicting a greater level of ice reduction than may have occurred in the warmest intervals of the Pliocene. However, to have prescribed a modern AIS in the PlioMIP1 GCMs would have also been incorrect, as this would have been incompatible with various lines of proxy evidence (e.g. sea level data). Ultimately, in the absence of reliable geological reconstructions of mPWP ice-sheets a choice must be made of how to initialise climate model simulations. It is nevertheless useful to test the sensitivity of our mid-Pliocene simulations to the prescribed PRISM3 ice-sheet in the climate models. It is not possible for each GCM to repeat their PlioMIP1 experiment with a different AIS boundary condition (e.g. modern), therefore we have analysed whether or not the initial conditions prescribed in the ISM impact upon the predicted result. We ran a suite of simulations where each ISM experiment was initialised from the Bedmap2 modern ice-sheet configuration .In terms of ice-sheet extent, most of the three ISMs showed a similar pattern of large-scale response, in that the Plioceneon Figs.  and 6. IIce-PD that are relatively close to their predicted PlioceneIce-PRISM3 volume  and 2.43 ± 3.53 m s.e. )42.Our results indicate a high level of climate model dependency in reconstructing the Antarctic ice-sheet volume Fig.  and resuIce-PRISM3 ensemble  over West Antarctica relative to the other GCMs helping to promote cooling. In the PlioceneIce-PRISM3 simulation, the precipitation rates over West Antarctica are not sufficient to create a strong positive SMB . Using just the ANICE ISM, the range in sea level change predictions is 7.04 m s.e. for the PlioceneIce-PRISM3 simulations, and for the SICOPOLIS ISM the range is slightly larger . Following a similar experimental design to that presented here and initialising from a PRISM3 ice-sheet, ref. 28 predicted a range in mean sea level rise for equilibrium Pliocene ice-sheets of 5.83 m s.e. (7.96–13.79 m s.e.) when considering six different SIA-SSA ISMs but only one climate model forcing (HadCM3). The comparable range shown here incorporating five different GCMs is 12.33 m s.e., supporting the conclusion that climate model dependency on AIS predictions for the Pliocene is high.Focussing primarily on studies employing SIA-SSA models, Table 43. They simulated AIS configurations ranging from −4.2 to 6.6 m s.e. volume (causing a 10.8 m s.e. variation due to parameter uncertainty). In the same study, temperature and precipitation were also increased in steps of 1 °C for temperature and 5.1% increase in precipitation per degree of temperature change in order to explore the influence of uncertainty in the Pliocene climate forcing on the modelled AIS. Considering this additional warming, the mass loss of the AIS relates to a global mean sea level rise of 6.2–13.0 m relative to present day . The impact of uncertainties in dynamic topography44 and Antarctic bedrock topography45 have also been considered in a Pliocene ISM framework, however, these mechanisms only cause a small variation in predicted AIS volume and hence mean sea level change  factors for ice and snow, enhancement flow factors for the SIA and SSA velocities and the sub-shelf melt parameter) using the Parallel ISM (PISM) driven by climatological forcing from the NorESM climate model46. Although in general all data suggest a higher-than-present sea level indicative of a reduced Greenland ice-sheet and retreated AIS12, estimates range from between 10 and 30 m above present-day8, based on a range of global proxy records , for which the largest uncertainties arise from considering processes such as mantle convection and glacial isostatic adjustment47. Recent studies inferring sea level changes from oxygen isotopes have offered a more constrained reconstruction of Pliocene sea level, suggesting that high-stands were between 9 and 13.5 m above modern, implying that the EAIS may be less sensitive during the Pliocene than previously considered48. A further study using a novel approach to constrain mPWP sea level high stands by assessing whether or not particular AIS configurations are consistent with the oxygen isotope record have suggested that AIS contribution to mPWP sea level rise was at most ~13 m46. However, a recent review49 of the sources of uncertainty associated with using marine geochemical proxies to infer past changes in sea level (e.g. refs. 48), has shown that diagenesis and potential long-term changes in seawater chemistry likely have large and poorly constrained effects on interpretations of such records. This may limit their ability to make meaningful Pliocene sea level estimates at the present time49.While ideally proxy-based estimates of global mean sea level change for the mPWP could be used to constrain which of the modelled AIS scenarios is more or less likely, significant uncertainty remains in the estimates of Pliocene sea level high stands19). Our results show that in around 40% of the simulations for the PlioceneIce-PRISM3 experiment, substantial ice-sheet reduction is exhibited in the Wilkes and Aurora SGB and around Wilkes Land . Nevertheless, the results presented here also demonstrate that, while the total AIS volume is most heavily influenced by the choice of climate model, detailed predictions of areal extent are more likely to be influenced by the ISM used and ISM initial conditions  resolution. DeConto and Pollard also employ a parameterisation of ice-shelf hydrofracturing and ice-cliff collapse of marine-terminating ice margins, which is not included in any of the ISMs here and this could potentially have a large impact on predictions of sea level change for the Pliocene22  can have a significant impact on the predicted equilibrium state ice-sheet for the mPWP. When compared against other sources of uncertainty (e.g. ISM parameterisations and the choice of ISM), the atmospheric forcing field used brings in the largest source of uncertainty in terms of the global mean sea level change associated with volumetric changes in the ice-sheet Table . We also27. Briefly it involved the implementation of PRISM3 boundary conditions26 in IPCC AR4 class general circulation models (GCMs). PRISM3 boundary conditions included increased atmospheric CO2 concentrations, an altered palaeogeography, including changes to the land-sea mask (LSM), a changed vegetation cover55 and altered Pliocene ice-sheet configurations. In PRISM3 West Antarctica is deglaciated and East Antarctica is retreated beyond the marine portions of the Wilkes and Aurora SGB56. Where possible, modelling groups were asked to alter their LSM to allow for a West Antarctic Seaway. More details of the seven GCMs that have been used in this study and their implementation of the PRISM3 boundary conditions can be found in Supplementary Table Surface temperature, precipitation, ocean temperature and topography , which approximates the stress balance by only taking into account horizontal shear stress and lead to velocities that are dependent on depth59. For the 3-D ocean temperatures, we use a distance weighting scheme to extrapolate temperatures to underneath the ice shelves, where ocean temperatures are largely unresolved in the GCMs.All ice-sheet models are run on a 40 km by 40 km grid of 141 × 141 grid points. All climate fields from the GCMs, i.e. surface-air temperature, precipitation, ocean temperature and reference topography, are interpolated on the ice-sheet rectangular grid using OBLIMAP v2.0. A polar stereographic projection is employed, using a central longitude of 0°E and a standard parallel of 24.7°, i.e. a latitude of true scale of 65.3°S39  ice-sheet model package. The model we use here is the same version as has been used in previous publications61. Sub-shelf melting underneath the ice shelves is included as a heat-transfer equation63:The surface mass balance is determined with monthly fields of precipitation, temperature and insolation at the top of the atmosphere. The precipitation is adjusted with surface temperature. Surface melting is calculated with an insolation temperature melt model, for which a linear relation is used for temperature combined with the net incoming short-wave radiation, using an internally calculated albedon = 1. Here, Toc is the ocean temperatures of each GCM that are vertically interpolated to the depth of the ice shelves and Tf is the freezing temperature63:SO is the mean value for the salinity of the ocean and zb is the bottom of the ice-shelf below sea level. The different parameters are described in Supplementary Table −1 are included, respectively. No additional calving law is applied.The different parameters are described in Supplementary Table 29. On land, the model utilises a hybrid combination of the SIA and the SSA  as described in ref. 30, following ref. 62.SICOPOLIS  is a 3-D thermo-mechanical finite difference ice-sheet-shelf model64. The distribution of basal sliding coefficients used in all runs is obtained from a calibration run using an iterative technique30.Basal sliding is described by a Weertman-type power law with a cubic dependence on basal shear stress, allowing sub-melt sliding following ref. 65. Surface melting is parameterised by a PDD method67. PDD factors are 8 and 3 mm day−1 °C−1 for ice and snow, respectively.For the surface mass balance, precipitation is converted to snow accumulation with a linear function of surface temperatures68, with n = 2. For the control simulations, a tuning melt factor of Fmelt = 0.2 m s−1 K−1 is applied at the floating side of some grounding lines (northwest of Ronne Ice Shelf and south of Amery Ice shelf) to better represent their modern positions. At each time step, the calving front is iteratively checked for grid points with a thickness below 50 m, which are calved out, until no more calving occurs.Ice-shelf basal melting is computed using Eq. 68, with 56. Basal sliding is not included in the model. Here, the model is free to grow to the extent of the modern grounding line.BASISM (British Antarctic Survey Ice-sheet Model) is a finite difference thermomechanical 3-D ice-sheet model, only including land-based ice using the SIA66, which shows a high level of correlation between warmest month temperatures and observations of present-day melt56. This assumes that melt is determined by temperature variations alone and uses separate PDD factors for ice and snow melt and a maximum fractional refreezing rate. Here, standard parameters are set to 8 and 3 mm day−1 °C−1 for ice and snow melt, respectively69.An exponential function is used to convert temperatures into the number of PDDs70 and therefore provides a frame of reference against which the more complex SSA–SIA hybrid models can be compared.BASISM has been used in a number of previous mid-Pliocene modelling studiesData available on request from the authors.Supplementary Information


Bothrops lanceolatus snake venom causes systemic thrombotic syndrome but also local inflammation involving extensive oedema, pain, and haemorrhage. Systemic thrombotic syndrome may lead to fatal pulmonary embolism and myocardial and cerebral infarction. Here, we investigated the ability of B. lanceolatus venom to activate the Complement system (C) in order to improve the understanding of venom-induced local inflammation. Data presented show that B. lanceolatus venom is able to activate all C-pathways. In human serum, the venom strongly induced the generation of anaphylatoxins, such as C5a and C4a, and the Terminal Complement complex. The venom also induced cleavage of purified human components C3, C4, and C5, with the production of biologically active C5a. Furthermore, the venom enzymatically inactivated the soluble C-regulator and the C1-inhibitor (C1-INH), and significantly increased the expression of bound C-regulators, such as MCP and CD59, on the endothelial cell membrane. Our observations that B. lanceolatus venom activates the three Complement activation pathways, resulting in anaphylatoxins generation, may suggest that this could play an important role in local inflammatory reaction and systemic thrombosis caused by the venom. Inactivation of C1-INH, which is also an important inhibitor of several coagulation proteins, may also contribute to inflammation and thrombosis. Thus, further in vivo studies may support the idea that therapeutic management of systemic B. lanceolatus envenomation could include the use of Complement inhibitors as adjunct therapy. Bothrops are responsible for the majority of venomous ophidian accidents in South and Central America [Snakes from the genus  America . They in America . Dermone America , 4.Bothrops lanceolatus, commonly named Martinique lancehead (“Fer-de-lance”), is a native species confined to the Caribbean island of Martinique. Systemic clinical symptoms of B. lanceolatus envenomation differ from envenomation by other Bothrops species as it is characterized by a predominant prothrombotic profile and is rarely haemorrhagic [B. lanceolatus and B. carribeus, a Bothrops species from the neighbouring island of Saint Lucia. This can lead to death due to myocardial and cerebral infarction or pulmonary oedema [B. lanceolatus venom , can prevent the development of systemic thrombosis, which if untreated can result in death [orrhagic . In appry oedema . Local ein death , 6. But Bothrops snake venoms are complex mixtures of bioactive organic and inorganic components, such as proteins, peptides, carbohydrates, lipids, and mineral salts. These venoms display a wide range of interspecies variations both in composition and biological activities [Bothrops venoms. They play a central part in the local and systemic development of the pathophysiology of envenomation, respectively, by inducing haemorrhage, myonecrosis, inflammation, cutaneous lesions, and haemostasis disturbances [tivities –12. Metaurbances .The Complement (C) system, a complex group of more than 50 blood-circulating and cell-surface-expressed and intracellular proteins, is an important effector mechanism of innate and adaptive immunity . AmplifiBothrops species from South and Central America [Bothrops venoms triggered the C-cascade by one or several activation pathways, generating high quantities of anaphylatoxins by directly cleaving C3 and C5 or by inactivating the regulator C1-INH. These events involved both metalloproteases and serine proteases. No modifications of the membrane regulators, DAF, CR1, and CD59, were observed in case of Bothrops venoms exposure [Bothrops venoms may constitute an important event in human bothropic envenomation pathophysiology.Inappropriate activation of the Complement cascade can be harmful to the host and can lead to inflammation and thrombosis . Numerou America , 29. In exposure , 30–32. B. lanceolatus venom is antigenically similar to other Bothrops venoms but differs from other Bothrops venoms in that it does not induce coagulation of human plasma, has very low hyaluronidase activity, and their proteases have different substrate specificities [B. lanceolatus venom contains glycosylated proteins that could potentially trigger the lectin Complement pathway [B. lanceolatus antivenom Bothrofav is not always 100% percent effective at preventing local and systemic events [B. lanceolatus to activate the Complement cascade in vitro, and interact with its proteins, regulators, and receptors. Here, we show that B. lanceolatus venom activates Complement cascade, affecting major C-components and soluble/membrane-bound inhibitors, and inducing the production of proinflammatory mediators.ficities , 33. The pathway , 34. Conc events , supplemortho-phenylenediamine (OPD), phenylmethanesulfonyl fluoride (PMSF), 1,10-phenantroline, mannan, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich . Purified human Complement components C3, C4, C5, and C1-INH, as well as goat anti-human C4 antibody were purchased from Quidel Corporation . Fluo-4 AM was from Life Technologies . The following murine antibodies against human CD46 (clone E4.3), CD55 (clone IA10), and CD59 (clone p282-H19) were purchased from BD Pharmingen . Polyclonal goat anti-mouse IgG-PE, polyclonal rabbit anti-goat IgG labelled with alkaline phosphatase, and goat anti-human C1-INH antibodies were obtained from Sigma-Aldrich . Foetal bovine serum (FBS) was from Cultilab . Dulbecco's modified Eagle's medium (DMEM), penicillin, and streptomycin were from Gibco/Thermo Fisher Scientific . Buffers include saline solution (150 mM NaCl), PBS , Alsever's solution , Veronal-Buffered Saline , AP buffer , BVB2+ buffer , BSS buffer , Krebs buffer , and FACS buffer .Tween-20, Dextran 70, ethylene diamine tetraacetic acid (EDTA), ethylene glycol-bis-(beta-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), barbituric acid, sodium barbital, Bothrops lanceolatus (B. lanceolatus) was obtained from Latoxan . Stock solution was prepared in sterile saline solution at 5 mg/mL and stored at −80°C.Venom from Human blood was obtained by signed consent from healthy donors. Blood was collected by venepuncture without anticoagulant and refrigerated for 4 h. After clot formation, blood was centrifuged at 400 ×g for 15 min at 4°C, serum was collected, aliquoted, and stored at −80°C. This study was approved by the Human Research Ethics Committee (HREC) from the Institute of Biomedical Sciences at the University of São Paulo, São Paulo, Brazil . Sheep and rabbit erythrocytes were collected in Alsever's. All the procedures involving animals were in accordance with the ethical principles in animal research adopted by the Brazilian Society of Animal Science and the National Brazilian Legislation number 11.794/08. Protocols were approved by the Institutional Animal Care and Use Committee .B. lanceolatus venom in the appropriate buffer , at 37°C for 30 min, to assess effects on the Complement system. Samples were tested for the remaining Complement activity or detection of the activation by-product. The role of metalloproteases in the venom was investigated by coincubation with 1,10-phenantroline (10 mM).Normal human serum (NHS) was incubated with the same volume of S) for the CP [R) for the AP [50 and AP50 were calculated for each venom concentration. Venom concentrations able to inhibit 50% (IC50) of the activity of each pathway and their respective 95% confidence intervals (CI95%) were determined from the curves of CH50 and AP50 plotted as a function of the logarithm of venom concentration by nonlinear regression using GraphPad Prism 6.0 software  [Haemolytic activity was assessed using sensitized sheep erythrocytes  , 35.50 was calculated for each venom concentration. Venom concentrations able to inhibit 50% (IC50) of the activity of LP and its 95% confidence interval (CI95%) were determined from the values of LP50 for each venom concentration (see LP activity was assessed using mannan-coated microtiter plates and measurement of C4 deposition as previously described . LP50 wation see  by nonliμg/mL) were determined by linear regression from the standard curve. TCC (in its soluble form SC5b-9) was determined using the MicroVue™ SC5b-9 Plus EIA Kit , according to the manufacturer's instructions. TCC concentration (μg/mL) in the samples was calculated from a linear regression of the standard curve.C3a/C3a-desArg, C4a/C4a-desArg, and C5a/C5a-desArg were measured using the Human Anaphylatoxin Cytometric Bead Array (CBA) , following the manufacturer's instructions. Cytometric analysis was performed using a FACSCanto-II , and the data were analysed with the Flow Cytometric Analysis Program (FCAP) Array 3.0 . Anaphylatoxin concentrations , were incubated with the same quantity of venom for 30 min at 37°C. C1-INH was incubated with venom in the presence or absence of protease inhibitors, PMSF (20 mM), EDTA (20 mM), or 1,10-phenantroline (20 mM). Samples were then submitted to SDS-PAGE (10% separating gel) under reducing conditions and silver-stained to detect cleavage [To evaluate the direct cleavage of the Complement components by the cleavage , 37. In B. lanceolatus venom. As control, saline-treated C1-INH samples were used. Following the assay procedure, samples (diluted 1 : 25) were incubated with biotinylated C1s (venom concentration of 8 μg/mL), and were incubated on avidine-coated microtiter plates. Horseradish peroxidase- (HRP-) conjugated goat anti-human C1-INH was added to each test well and the reaction developed enabling the detection of C1-INH-C1s complexes deposited on the plates. After subtraction of the blank, the residual C1-INH-C1s-complexing activity of each sample was expressed as a percentage of the control sample activity.C1s-binding activity of C1-INH was assessed using the MicroVue™ C1-Inhibitor Plus EIA , according to the manufacturer's procedure. Samples of purified human C1-INH were exposed to μM) at 2 μg/107 cells/mL, at room temperature for 30 min. Cells were washed with BSS and resuspended in Krebs buffer containing 0.1% BSA. Cells (106/well in 200 μL Krebs buffer) were stimulated with venom-treated C5 , in a 96-well plate and the fluorescence variations were measured every 3 sec (λex = 485 nm and λem = 510 nm), using the fluorimeter FLUOstar Omega . As positive control, cells were exposed to purified C5a  and, as negative controls, cells were exposed to C5 or venom incubated with saline solution. For each well, the values of fluorescence were normalized by the initial fluorescence F0, the average fluorescence during 30 sec before stimulation, and then plotted against time.Functional C5a generation was assessed by measuring cytoplasmic calcium release from intracellular stores in leukocytes, of which neutrophils are the main population and the main responders, using the calcium indicator Fluo-4 AM. Leukocytes were separated from platelets and red blood cells by dextran sedimentation from heparinised human blood using standard procedures. Cells were resuspended in Dulbecco's modified Eagle's medium (DMEM), 10% heat-inactivated foetal bovine serum (FBS), and loaded with the fluorescent calcium indicator Fluo-4 AM (1 v/v) heat-inactivated FBS, and the antibiotics, penicillin, and streptomycin (100 IU/mL) at 37°C in humidified air with 5% CO2. Cells were harvested using trypsin/EDTA and incubated at 106 cells/mL with 100 μg/mL of B. lanceolatus venom, for 2 h at 37°C under constant agitation. Cells were centrifuged at 400 ×g and resuspended in FACS buffer. Cells (5 × 104 cells/well) were incubated for 1 h at 4°C with monoclonal antibodies anti-human MCP, DAF, or CD59 (1 μg/mL), followed by incubation with goat anti-mouse IgG-PE (1 : 100), for 1 h at 4°C. Cells were analysed by flow cytometry  using the software FACSDiva .The endothelial cell line EA.hy926 was cultured in DMEM containing 10% . A P value < 0.05 was considered significant.Numerical data were expressed as mean ± SD. Data were analysed statistically by Student's B. lanceolatus venom and residual haemolytic activity was assessed. As shown in Figures 50 of 156.6 μg/mL (CI95%: 147.3–166.6 μg/mL) and the alternative pathway with an IC50 of 294.5 μg/mL (CI95%: 273.2–317.4 μg/mL)  (Human serum (NHS) was incubated with  Figures . The ven0 μg/mL) .B. lanceolatus venom induces a significant production of C4a, C5a, and TCC, thus confirming direct activation of the Complement system by the venom. Surprisingly, the venom caused a significant decrease of C3a in the serum, when compared with the control. These activities were inhibited with the addition of a metalloprotease inhibitor, 1,10-phenantroline, suggesting the participation of B. lanceolatus venom metalloproteases in Complement activation.To establish whether the observed reduction C-activity resulted from inhibition or activation (resulting in consumption) of the Complement cascade by the venom, the presence of anaphylatoxins and terminal Complement complex (TCC) in venom-treated NHS samples was investigated. B. lanceolatus venom, in the presence or absence of proteases inhibitors. SDS-PAGE  [B. lanceolatus venom induces inflammation.The C1-inhibitor, C1-INH, is the only known serine protease plasma inhibitor. It regulates not only the Complement cascade  but is also an important regulator of the coagulation cascade activation and inhibits several fibrinolytic proteins  . It inhinditions . This loma (HAE) . This maLoxosceles spider venom [B. lanceolatus venom did not reduce expression of any of these regulators but, significantly, increased the detection of MCP and CD59 (B. lanceolatus envenomation is unlikely due to inefficient C regulation on the endothelial cells, but endothelial activation in response to excess C5a or TCC generation, as a consequence of the effect of the venom on the Complement system, may contribute. Bothrops species venoms activate the Complement cascade by several pathways and complex mechanisms [B. asper and B. pirajai venoms, class I metalloproteases are involved [Bothrops toxins, B. lanceolatus PI-SVMPs may be involved in B. lanceolatus Complement activation, but this remains to be investigated.As Complement activation on endothelial cells can lead to a more prothrombotic phenotype of these cells , endotheer venom  and thisand CD59 . Thus, ichanisms . In the involved , 32. SinB. lanceolatus venom is a potent trigger of the Complement cascade in vitro. Thus, further in vivo studies may support the idea that therapeutic management of systemic B. lanceolatus envenomation could include the use of Complement inhibitors as adjunct therapy.In conclusion, here we show that, like its continental counterparts,


Objectives: The high-mobility group A protein 1a (HMGA1a) protein is known as a transcription factor that binds to DNA, but recent studies have shown it exerts novel functions through RNA-binding. We were prompted to decipher the mechanism of HMGA1a-induced alternative splicing of the estrogen receptor alpha (ERα) that we recently reported would alter tamoxifen sensitivity in MCF-7 TAMR1 cells.Methods: Endogenous expression of full length ERα66 and its isoform ERα46 were evaluated in MCF-7 breast cancer cells by transient expression of HMGA1a and an RNA decoy (2′-O-methylated RNA of the HMGA1a RNA-binding site) that binds to HMGA1a. RNA-binding of HMGA1a was checked by RNA-EMSA. In vitro splicing assay was performed to check the direct involvement of HMGA1a in splicing regulation. RNA-EMSA assay in the presence of purified U1 snRNP was performed with psoralen UV crosslinking to check complex formation of HMGA1a-U1 snRNP at the upstream pseudo-5′ splice site of exon 1.Results: HMGA1a induced exon skipping of a shortened exon 1 of ERα in in vitro splicing assays that was blocked by the HMGA1a RNA decoy and sequence-specific RNA-binding was confirmed by RNA-EMSA. RNA-EMSA combined with psoralen UV crosslinking showed that HMGA1a trapped purified U1 snRNP at the upstream pseudo-5′ splice site.Conclusions: Regulation of ERα alternative splicing by an HMGA1a-trapped U1 snRNP complex at the upstream 5′ splice site of exon 1 offers novel insight on 5′ splice site regulation by U1 snRNP as well as a promising target in breast cancer therapy where alternative splicing of ERα is involved. We have recently found that high-mobility group A protein 1a (HMGA1a) is involved in alternative splicing of ERα  gene, interacts with U1-70K   and authentic-5′ splice site (αA5′SS) and the ones used for digesting 5′ ends of U1 (αU1) and U2 (αU2) snRNA, as well as the control oligonucleotide (cont), are shown in Table E. coli and microdialyzed in buffer D .All of the linearized plasmids were transcribed with SP6 RNA polymerase in the presence of α-3PUTP. TheEMSA was performed as previously described  was incubated at 30°C for 2 h in a 12.5 μl reaction mixture containing 3 mM ATP, 20 mM creatine phosphate, 20 mM HEPES-NaOH (pH 7.3), 3.5 mM MgCl2, 2% (wt/vol) low-molecular-weight polyvinyl alcohol (Sigma), and 3.5 μl of HeLa cell nuclear extract (CilBiotech). Recombinant protein was added first to the probe prior adding nuclear extract. Where indicated 20 pmol of SRSF1 was added. Psoralen-mediated UV crosslinking assay was performed as previously described fixing the incubation time at 5 min . After passing through a 25 G needle five times, protein was purified from the interphase and organic phase by precipitating with 6 V of a solution for precipitating protein . Twenty micrograms of protein was boiled in sample buffer and separated by 10% sodium dodecyl sulfate-PAGE (SDS-PAGE), transferred to nitrocellulose membrane and analyzed by an ERα antibody (HC-20)  which recognizes the C-terminus of the protein or HMGA1a antibody . Anti-rabbit immunoglobulin G conjugated to alkaline phosphatase (Promega) was used as secondary antibody and detected by BCIP (5-bromo-4-chloro-3-indolylphosphate) (Promega).t-test using Microsoft Excel.Data are presented as mean ± SD. Group differences were analyzed by Students estrogen receptor alpha (ERα) gene. It functioned in MCF-7 cells, induced estrogen-dependent growth in these cells as well as nude mice, and tamoxifen-resistant MCF-7 TAMR1 cells were sensitized to tamoxifen  gene. The 5′ splice site scores of the two 5′ splice sites are comparable both with a MAXENT score of 8.6. In normal expression of the ERα gene the intron-proximal 5′spice site is utilized. Regulation of U1 snRNP binding to alternative 5′ splice sites has been reported to occur during A complex formation, with no difference of U1 snRNP binding to upstream and downstream 5′ splice site in E complex  and there is a strong possibility of secondary structure leading to close enough range of the HMGA1a RNA-binding site and 5′ splice site for HMGA1a-induced U1 snRNP trapping. This also well explains the increased crosslink of U1 snRNP and the authentic 5′ splice site when the upstream 5′ splice site was mutated (Figure However, there is still various limitations in this study. First, if U1 snRNP protection at the upstream 5′ splice site is extended more than U1 snRNP binding in PTB independent conditions (Sharma et al., d Figure , lane 14ERα exon 1, this event consequently induced exon skipping and expression of the isoform ERα46. Using decoy RNA that binded to HMGA1a and inhibited HMGA1a-induced exon skipping of ERα, we observed enhanced estrogen-dependent tumor growth and sensitization of tamoxifen-resistant tumor cells to tamoxifen due to increased expression of full length ERα by correction of alternative splicing (Ohe et al., Besides the precise mechanism of HMGA1a-induced silencing of the downstream authentic 5′ splice site of KO conceived the original idea, conducted experiments Figures , 2, 3, aThe 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.