Paracetamol is one of the most commonly used analgesic and antipyretic agents worldwide, attributed in part to its excellent safety profile when administered at recommended doses. Paracetamol allergy is not common, and the majority of the reactions are related to the pharmacological action of cyclooxygenase 1 inhibition. Selective and Immunoglobulin E (IgE)-mediated hypersensitivity reactions are rare. In this article, the authors report two cases of paracetamol allergy in which the mechanism of IgE-mediated hypersensitivity was demonstrated by positive skin tests and basophil activation tests. We highlight the relevance of identifying the mechanism underlying the reaction since patients with IgE-mediated paracetamol allergies will be able to tolerate non-steroidal anti-inflammatory drugs. Paracetamol is one of the most commonly used analgesic and antipyretic agents worldwide, in part due to the paucity of its adverse effects when administered at recommended doses . At therIn this article, we present two rare cases of paracetamol allergy in which the mechanism of IgE-mediated hypersensitivity has been demonstrated and supported by in vivo and in vitro tests.This data has been presented as a poster at the 40th annual meeting of the Sociedade Portuguesa de Alergologia e Imunologia Clínica in October 2019.The first case is a 30-year-old female with no history of drug reactions who presented her first episode of generalized urticaria and oropharyngeal tightness starting 10-20 minutes after oral administration of 1000mg of paracetamol (Ben-u-ron®) as a symptomatic treatment for headaches. A few months later, she reported a second and third episode of generalized urticaria within 10-15 minutes after oral administration of 250 mg of paracetamol, 20 mg of pyrilamine maleate, and 30 mg of caffeine (Antigrippine®) and 1000 mg of paracetamol (Ben-u-ron®), respectively, for symptomatic relief of headaches and coryza. There were no gastrointestinal, pulmonary, or cardiovascular symptoms in any episode. After the third episode, the patient was referred to an Immunoallergology appointment with no instructions to avoid any particular drug. The patient described prior tolerance to acetylsalicylic acid (ASA), ibuprofen, and nimesulide, which she maintained after the three episodes. The investigation of suspected immediate hypersensitivity to paracetamol was performed with skin prick tests (SPT) with injectable paracetamol solution (10 mg/ml) , with thThe second case is of a 26-year-old female who described her first episode of generalized urticaria after oral intake of 1000 mg of paracetamol (Doliprane®) and a second episode characterized by urticaria, and eyelid angioedema 10 minutes after oral administration of 1000 mg of paracetamol (Ben-u-ron®). A third episode of palmar and plantar itching, urticaria, and conjunctivitis started within 10 minutes after oral administration of 500 mg of paracetamol and 65 mg of caffeine (Ben-u-ron Caff®) for headaches. There were no signs or symptoms suggestive of pulmonary, gastrointestinal, or cardiovascular involvement in any episode. During the third episode, she was observed in the emergency department and treated with antihistamines and corticosteroids. She was referred to an outpatient Immunoallergology clinic with an indication to avoid paracetamol. After these three episodes, she tolerated ibuprofen, metamizole, and ASA. The immunoallergic research included SPT and intradermal tests with an intravenous paracetamol solution. The intradermal test at a concentration of 0.1 mg/ml (a non-irritant concentration as recommended by the European Network on Drug Allergy ) showed Written informed consent was obtained from both patients, and they were treated according to the ethical standards established in the Declaration of Helsinki.Hypersensitivity reactions to paracetamol are rare. Paracetamol is a weak COX inhibitor, and, therefore, inhibition of prostaglandin synthesis may be a mechanism to explain paracetamol reactions, similar to NSAIDs mechanisms -4. HowevIn the few cases described of selective hypersensitivity to paracetamol, the diagnosis is mostly based on the oral challenge test, which does not elucidate the underlying pathophysiological mechanism -8. The IThe authors describe two clinical cases of IgE-mediated hypersensitivity to paracetamol. Both are clinically characterized by the occurrence of several immediate-onset reactions 10-15 minutes after paracetamol administration, reproducible with repeated exposure, with no involvement of other drugs or allergen ingestion.Both patients tolerate several NSAIDs, including ASA, demonstrating that COX-1 inhibition is not involved in these reactions. Although it is a limitation of the study, we considered that the benefit/risk ratio did not justify performing oral challenge tests to paracetamol in these patients because despite being the diagnostic gold standard of drug allergy, they are time- and resource-intensive and expose the patient to the risk of severe reactions. NSAIDs are effective antipyretic and analgesic drugs, making them a valid alternative to paracetamol.The underlying specific IgE-mediated mechanism was demonstrated in both patients using paracetamol-positive skin tests. We also performed a BAT, which was positive in both patients. This basophil activation pattern confirmed the significant in vitro basophil degranulation specifically induced by paracetamol, which in these patients suggested an IgE-mediated mechanism -20. SeruThe authors describe two cases of immediate hypersensitivity reactions to oral paracetamol, reproducible with repeated exposure, and tolerance to several NSAIDs, including strong COX-1 inhibitors. The presence of positive skin tests allowed us to presume an IgE-mediated mechanism. Additionally, it was possible to observe in vitro a significant basophilic degranulation specifically induced by paracetamol, which in these patients supports the assumed mechanism.We propose that skin tests should be performed before oral challenge as paracetamol-specific IgE may play a role in this type of allergy, and those patients with an IgE-mediated paracetamol allergy will be able to tolerate NSAIDs.


However, these drugs prolong action potential duration throughout the heart and may trigger polymorphic VT by prolonging repolarization in areas where it already is long. Here we investigate whether site-specific prolongation of repolarization in myocardium adjacent to the infarcted myocardium mitigates reentry and prevents postinfarction VT.We generated myocardial infarction in a 2-year-old male pig by 90-minute occlusion of the left anterior descending coronary artery. Six weeks later, we performed epicardial mapping during Langendorff perfusion as reported previously.Next, we used a digital twin heart model from a patient with postmyocardial infarction arrhythmias D–1F baseOur findings show the potential of local prolongation of repolarization as a novel antiarrhythmic therapy in postmyocardial infarction patients. In the isolated heart and in the virtual patient heart, local prolongation of repolarization in myocardium with fractionated potentials or containing the VT exit site was effective in preventing VT. If multiple exit sites had been present, an unsuccessful intervention would have been inconclusive because the arrhythmias could have been sustained by a different exit site. When multiple exit sites are present, multiple regions need to be selectively targeted.Site-specific therapy may prevent off-target effects of systemic therapy or unnecessary ablation of viable myocardium or the induction of new scars. Local delivery of repolarization prolonging drugs could be achieved by materials that support drug release, such as bioresorbable adhesive devices.in silico observation. Thus, it can be argued that the outcome of the experiment was coincidental. However, we have shown that a pig heart is very stable during Langendorff perfusion and that the heart can serve as its own control for interventions such as infusion of sotalol. Second, we showed that the effect of sotalol was mediated by repolarization prolongation. Also, the results are confirmed by the patient-specific simulations, in which confounding variability is absent, providing translational value. Thus, a single experiment can be valuable to establish proof of concept. Future studies should test our intervention in the setting of more complex reentrant pathways with multiple exit sites.Our study is limited by the results of a single experiment and a single In conclusion, site-specific targeted antiarrhythmic therapy carries promise for nondestructive, mechanism-based treatment of arrhythmic substrates, to a degree not attained with drugs or ablation and without their side effects.


We use supersonic molecular beam techniques and rovibrationally state-resolved excitation to determine whether this vibration affects condensation of gas phase CO2 to its ice. We detect sticking and CO2 ice formation using RAIRS and quantify the sticking probability using the King and Wells method with modulation of the vibrational excitation and Fourier transform based detection. We find that the influence of this vibration on the structure of the formed ice and on the sticking probability is negligible under our conditions. Based on our detection limit, we quantify the weighted average sticking probability at approximately 0.9 and the difference between the state-resolved and weighted average sticking probability as below 0.03%.Internal vibrations may affect the adsorption, scattering, and reactions of molecules impinging onto a surface. The energy of the  In astronomy and astrochemistry, CO2 occurs, a. o., in the interstellar medium (ISM)  , both inum (ISM) , and in um (ISM) . In the methanol . Collisict sense . Molecul6 and CCl4 had a significant impact on the adsorption and reflection of these gas-phase molecules onto their bulk solids at low collision energies. In precursor-mediated reaction of CH4 on Ir (111) (4 on Si(100)   and SiH4 Si(100) , interna Si(100) .2, a study by Weida et al.  is well below the desorption barrier, the sublimation energy being approximately 26 kJ/mol. The ν3 fundamental vibrational mode  slightly exceeds the latter. A more recent study by Ioppolo et al. (2 ice after irradiation with resonant IR light (ν2 and ν3). For crystalline ice, they found no restructuring, but possible desorption. Their results were inconclusive on vibrational effects to desorption.For COa et al.  investigo et al.  found reν3) on the adsorption of CO2 onto CO2 ice grown on a Cu(111) surface at 80 K. We excite the ν3 asymmetric stretch of CO2 molecules in a molecular beam with light from a tunable continuous wave (cw)-infrared (IR) laser. We look for any changes in the sticking probability of the CO2 by modulating the excitation and using a Fourier transform based detection scheme. An effect of vibrational excitation on either the adsorption or the desorption rate will change the effective sticking probability. We also monitor the surface with Reflection Absorption IR Spectroscopy (RAIRS) and look for any structural differences in the spectra of the resulting CO2 ice layer with and without the additional vibrational excitation. At 80 K, the ice will be crystalline. Ioppolo et al. found no restructuring after IR irradiation of crystalline CO2 ice. However, our situation may be different, as they irradiated the CO2 ice after deposition, while we resonantly excite vibrations in the gaseous CO2 molecules prior to adsorption.Here, we study the effect of vibrational excitation  single crystal in the UHV chamber.Our experiments are performed using a home-built ultra-high vacuum (UHV) system, that has been used for non-state-resolved reaction dynamics measurements before . It cons2  at 300 K. A flow controller feeds the gas at 4 bar into a vacuum chamber  spectrometry ) and the ionizer of the QMS used for TOF measurements.On the beam axis in the analysis chamber, the edge of a disc can be rotated by an UHV-compatible stepper motor. The disc’s edge is manufactured to have two identical ‘open’ and two ‘closed’ sections, each of 90°. The closed sections have a slanted edge as not to reflect the molecular beam back into the last of the differentially pumped chambers. Combined with the beam stopper in the differential stage, this disc allows us to accurately perform King and Wells-type sticking probability measurements , but usiθ manipulator. The crystal is cooled by pouring liquid nitrogen (LN2) into the tube. It is kept at a constant temperature of 80 ± 1 K during experiments, as measured by a K-type thermocouple laser welded to the crystal’s edge. Adsorption of CO2 onto the sample is monitored using an FTIR (Bruker Vertex 70) with an external LN2-cooled MCT detector to perform RAIRS during the experiments. The IR beam paths outside of the UHV chamber, including internal parts of the FTIR, are purged continuously with gaseous dry N2 from an in-house system fueled by the exhaust from the local NMR facility.The Cu single crystal is suspended from a copper block at the end of a vertical ‘cold finger’, i.e., a hollow tube protruding through an x, y, z, 2ν3 antisymmetric stretch vibration is excited in the molecular beam with laser light from an OPO , which we will from now on simply refer to as the laser. Its output can be tuned and is stabilized on a CO2 absorption frequency using the lamb dip measured in a reference cell. The laser light crosses the molecular beam a few cm after the initial expansion in the apparatus and 37 cm before molecules hit the Cu crystal. For this experiment, we tune the laser to the R (4) transition at 4,249.99 nm, where CO2 molecules are excited from the v = 0, J = 4 state to the v = 1, J = 5 state. This transition was chosen because J = 4 has the highest population at the rotational temperature of the molecular beam . A cylieam [see , resulti2, CO and H2O. As we do not regularly perform extensive sputtering and annealing cycles for these experiments, the surface should not be considered an atomically clean Cu(111) surface. It is most likely partially oxidized, as studied previously (2 is dosed from the molecular beam. The CO2 flux is estimated to be 0.15 ML/s as referenced to the Cu atom surface density for Cu(111). The experiment is repeated multiple times with the laser tuned to the CO2 excitation frequency and several times tuned to a non-resonant frequency as a control experiment. Between each experiment, the crystal is flashed to 300 K.At the start of an experiment, the Cu crystal is flashed to 300 K to remove any buildup of COeviously . When th2 by analyzing IR peak integrals was unsuccessful. The integrals increase with the CO2 dose time but did not allow us to detect variations in ‘laser on’ and’ laser off’ experiments. Hence, we measure the sticking probability of CO2 onto CO2 ice at 80 K with a modulated version of the King & Wells method  surface. −1 and the Longitudinal Optical mode. We assign this peak at 2,350 cm−1 to the CO2 physisorbed directly on the (uncleaned) Cu(111) surface, as it is the first peak to appear and it becomes masked after dosing many layers of CO2. A much narrower and redshifted peak appears in the spectrum taken after dosing for 5 min. This ν3 CO2 ice absorbance and the other absorbances are listed and assigned in −1 for (oxidized) Cu(111)  experiments, we studied CO and H2O desorption and found that water contamination was even less than CO contamination on the sample’s surface. The absorbance suddenly appearing in the final spectrum (blue) at 2,320 cm−1 is an artifact. See the Supplementary Information for more detail.We first study the RAIR spectra of the first few layers of physisorbed CO Cu(111) , that co2 prior to impinging onto the surface may have two effects. Firstly, as discussed before, it may affect the effective sticking probability of CO2. Secondly, it may affect the structure of the resulting CO2 ice. The ice is known to grow both in amorphous and crystalline phases  and 3,708 cm−1 (ν1 + ν3). These are sharp, isolated peaks that can be integrated accurately and identify two different subsets of molecules. The former is an isotopologue and cannot have been affected by laser excitation. The latter does results from 12CO2 and may have been affected. 2 in the vacuum chamber to contribute to the ice growth significantly, as the flux of the molecular beam is approximately two orders of magnitude larger than the background flux. Regardless of the (dominant) origin, the variation in time-dependent absorbance is, apparently, significant in comparison to any potential effect of vibrational excitation on the net sticking.We examine the integrals of two peaks in the spectra as a function of time to address a possible influence on the effective sticking probability. We use the peaks at 2,383 cm2 ice structure, we study the shape and position of the peaks in the RAIR spectra. −1 for the four measurements at three points in time. The visible features here are the peak of CO2 on Cu(111) at 2,350 cm−1, the ν3 ice peak at 2,343 cm−1, the broad peaks at 2,360–2,385 cm−1 and the fano line shape at 2,385–2,420 cm−1. As expected from the previous discussion, we find minor variations in peak height between the different measurements. When looking for any spectral change, such as a peak or shoulder that is only present in the data sets where the laser was on resonance, or a shifted peak position, we do not find any such differences. We also looked at changes in the other absorbances outside of the displayed wavenumber range also show no differences between laser ‘on’ and ‘off’ experiments. We conclude that vibrational excitation of the incident molecule does not measurably affect the ice’s IR absorbances, hence the crystallinity of growth at 80 K, nor does the ν3 vibrational excitation affect the rate of CO2 ice growth on the Cu(111) surface significantly.To address a potential effect of vibrational excitation on the grown CO2 adsorption on CO2 ice at 80 K after the beam has been on for more than 1 hour. The results are normalized to the partial pressure of the incoming molecular beam, as this pressure change corresponds to a sticking probability of 1. If all molecules from the beam are adsorbed, the partial pressure would be the same as when the beam does not enter the vacuum chamber at all. CO2 sticking occurs when the second flag is opened, which is approximately between 35 and 110 s. We use the ratios of the first pressure drop and the initial pressure rise and the second pressure rise and final pressure drop to calculate the overall sticking probability, shown in 2 as evident from the curvature in the time-dependence after opening and closing flags. The effective pumping speed varies as a consequence of the changing CO2 partial pressure in the UHV chamber and the cold finger acting as a cyrogenic pump, similar to earlier studies performed on NO sticking  of both the measured partial pressure and the laser modulation signal. The data is not always measured at regular intervals. Since the FFT calculation requires a regular sampling rate, we first interpolate the data to a sampling rate of 100 Hz. The absolute values of the FFT are normalized such that they reflect the quantity of interest: for the laser modulation data, this is the amplitude of the measured square wave . For the partial pressure data, it is the amplitude of the square wave normalized to reflect the change in sticking probability. This means that a peak in the sticking probability FFT spectrum with an integral of 1 would correspond to a change in sticking probability of 1 due to laser excitation, or an integral of 0.05 of the peak would correspond to a change in sticking probability of 0.05, −5 whereas the sticking probability itself is near unity.We can quantify this detection limit to calculate an upper limit for the effect of vibrational excitation of the sticking probability. Due to the finite (and slightly irregular) sampling rate of the measurement, the peak in the FFT spectrum is broadened. Therefore we take the integral of the peak between 2.998 and 3.002 Hz, which is shown as a filled gray area in 2 in the beam is vibrationally excited by our laser. Its linewidth allows only excitation of molecules from a particular initial rotational state, and only a fraction of all CO2 in the molecular beam is in that initial rotational state. Furthermore, due to limited laser power, less than 100% of the CO2 in a particular initial state is excited, even when using rapid adiabatic passage. Finally, spontaneous emission during the 37 cm flight path to the sample after excitation region, also lowers the vibrationally excited fraction of the molecular beam. We have determined the rotational state distribution of the molecular beam and the excited population of the R (4) transition. The methods and results are described elsewhere  → T energy transfer is not affecting the effective sticking probability through enhanced direct scattering or more efficient desorption, it may be of relevance at considerably lower collision energies. In that case, a difference may be more readily detected by monitoring the scattered fraction as it constitutes the minority, as done by Sibener and Lee.In comparison to the earlier study on the condensation of CCler & Lee  our resu2 on CO2 ice and the structure of the resulting ice on a cryogenically cooled Cu sample. We find no evidence of an influence of the ν3 mode on either possible effect within our detection limits at an incident velocity of 587 m/s. With regards to the ice structure are our results consistent with the results of Ioppolo et al. (2 ice. Neither vibrational excitation of molecules prior to impact nor when already in the ice seems to affect the ice structure significantly. With regards to the sticking probability we find that our detection limit supports that, in comparison to the measured average sticking probability for all states present in the beam, it is safe to assume that a single quantum in the ν3 mode is of no relevant influence to the effective sticking probability. This may result from a fortuitous cancellation of effects on the adsorption vs. direct scattering and desorption rates for the excited state, but this seems unlikely. The result, therefore, expands upon the conclusion from an earlier study that used state-resolved detection of CO2 molecules desorbing from its ice: even vibrational energies exceeding the desorption energy seem not relevant to the growth or evaporation of CO2 ice.Using a combination of molecular beam techniques, state-resolved excitation, and RAIRS detection, we have investigated the possible influences of vibrational excitation on both the sticking of COo et al.  for crys


Peumus boldus). It acts as a free radical scavenger and controls glycemia in diabetic rats. Various mechanisms have been proposed for this effect, including inhibited glucose absorption, stimulated insulin secretion, and increased expression of genes involved in glycemic control. Direct effects on glucose synthesis and degradation were not yet measured. To fill this gap, the present study is aimed at ensuring several metabolic pathways linked to glucose metabolism  in the isolated perfused rat liver. In order to address mechanistic issues, energy transduction in isolated mitochondria and activities of gluconeogenic key enzymes in tissue preparations were also measured. Boldine diminished mitochondrial ROS generation, with no effect on energy transduction in isolated mitochondria. It inhibited, however, at least three enzymes of the gluconeogenic pathway, namely, phosphoenolpyruvate carboxykinase, fructose-bisphosphatase-1, and glucose 6-phosphatase, starting at concentrations below 50 μM. Consistently, in the perfused liver, boldine decreased lactate-, alanine-, and fructose-driven gluconeogenesis with IC50 values of 71.9, 85.2, and 83.6 μM, respectively. Conversely, the compound also increased glycolysis from glycogen-derived glucosyl units. The hepatic ATP content was not affected by boldine. It is proposed that the direct inhibition of hepatic gluconeogenesis by boldine, combined with the increase of glycolysis, could be an important event behind the diminished hyperglycemia observed in boldine-treated diabetic rats.The alkaloid boldine occurs in the Chilean boldo tree ( Peumus boldus)  mainly found in the leaves and bark of the Chilean boldo tree . Boldine-based and boldo-containing pharmaceutical preparations have been used for decades in the treatment of a variety of liver ailments . The hepOther studies demonstrated that boldine presents antihyperglycemic action in animals. Jang et al.  reportedBoldine , enzymes, and coenzymes were purchased from Sigma-Aldrich Co. . All other chemicals were from the best available grade (98–99.8% purity).ad libitum with a standard laboratory diet . Depending on the protocol, rats were fed or starved for 18 h before the experiments. For all the experiments, the rats were anesthetized by intraperitoneal injection of ketamine (70 mg/kg)+xylazine (7 mg/kg) before removal of the liver. All experiments were done in accordance with the worldwide accepted ethical guidelines for animal experimentation and were previously approved by the Ethics Committee of Animal Experimentation of the State University of Maringá (protocol number 6416011220).Male Wistar rats (200–230 g) were used in all experiments. Animals were fed μM). It is already amply documented that dimethylsulfoxide does not significantly affect liver metabolism, at least not when infused at rates up to 32 μL/min NaHCO3 into components of the tricarboxylic acid cycle. After 10 minutes of incubation at 37°C, the reaction was stopped by adding 0.5 volume of 2 N perchloric acid. Sequentially, the excess [14C]NaHCO3 was removed by bubbling CO2 for 5 minutes, and aliquots were collected to quantify the incorporated radioactivity into acid-stable compounds [14C per minute per milligram protein.The pyruvate carboxylase (PC) activity of intact mitochondria was determined by quantifying the incorporation of ompounds . The enzp ≤ 0.05) using the GraphPad Prism software (version 5.0). Half-maximal effect concentrations (IC50) were computed by means of numerical interpolation with Stineman's formula using the Scientist software from MicroMath Scientific Software .Statistical analysis was evaluated by variance analysis (ANOVA) with post hoc testing according to Student-Newman-Keuls  in the presence of lactate (2 mM) as substrate. After preperfusion with the substrate-free Krebs-Henseleit buffer , lactate infusion produced rapid increases in glucose and pyruvate productions and in oxygen uptake, with subsequent stabilization at about 34 minutes of perfusion time. The infusion of boldine at 36 minutes of perfusion time resulted in a progressive decrease in glucose production, achieving complete inhibition  at 56 minutes of perfusion time. This inhibition was partially reversed upon cessation of boldine infusion. Oxygen uptake experienced a slight diminution, but the significance is doubtful because the variations do not exceed the corresponding standard errors. Pyruvate production, on the other hand, was progressively elevated by boldine, an elevation that was completely abolished upon cessation of the boldine infusion. The same protocol was followed with two other concentrations, and the effects versus concentration relationship are illustrated in μM. Pyruvate production, on the other hand, was elevated at 100 𝜇M and maintained until 200 𝜇M. For oxygen uptake, on the other hand, there was a tendency toward diminution when the boldine concentration was increased. However, no statistical significance was found.Livers from 18 h fasted rats were perfused to ensure low glycogen levels. Under this condition, glucose output reflects mainly the rate of gluconeogenesis .  FigureIn the liver, fructose metabolism can undergo both transformation into glucose (anabolic pathway) and catabolic degradation into pyruvate and lactate (fructolysis). Fructose transformation into glucose uses only a fraction of the enzymes needed for lactate gluconeogenesis. The experimental protocol with fructose as substrate was similar to that one already described for the experiments with lactate. Here again, the livers from fasted rats were used in order to avoid interference by endogenous glycogen , 35.  FiμM. Oxygen uptake was not significantly modified by the various boldine concentrations the same being valid for pyruvate production. In the case of lactate production, however, a concentration-dependent inhibition was found with an IC50 of 128.5 μM.The same protocol was repeated with two other boldine concentrations, and the new steady states induced by each boldine concentration are represented in In order to focus further the investigations on limited portions of the gluconeogenic pathway, the action of boldine on glycerol-driven glucose production was measured. The experimental protocol was the same as that one used with other gluconeogenic substrates and is illustrated in μM. Boldine elevated pyruvate production, an effect that was significant only at the 200 𝜇M concentration. Lactate production was inhibited by almost 50% at the 200 𝜇M concentration. Nitrogen catabolism (ammonia and urea productions) was practically not affected by boldine, the same being valid for oxygen uptake.In order to investigate the possible effects of boldine on nitrogen metabolism, alanine was infused. This procedure allows determining simultaneously ureagenesis and ammoniagenesis in addition to gluconeogenesis. The experiments were conducted with the livers from fasted rats, and the results are shown in μM. The fructose 1,6-bisphosphatase concentration dependence suggests that inhibition is of the incomplete type, as no substantial increment was found when the boldine concentration was increased from 100 to 200 μM. Pyruvate carboxylase, finally, was not inhibited by boldine.In order to investigate a possible direct effect of boldine on rate-limiting enzymes of gluconeogenesis, several assays were performed in cellular fractions of the liver. μM and achieved 84% at 200 μM. Interpolation predicts that 50% inhibition can be expected to occur at the concentration of 84.8 μM. The adenine nucleotide content of the liver in the presence of boldine is listed in The results of the present work reveal that boldine is able to modify several metabolic pathways linked to carbohydrate metabolism in the perfused liver. The major findings were (1) increased glycolysis from liver glycogen, (2) substantial inhibition of gluconeogenesis from several substrates, and (3) inhibition of fructolysis. All these effects were not associated with significant modifications in the hepatic ATP content nor with impaired mitochondrial respiration. Additionally, boldine diminished ROS production in isolated liver mitochondria. Modifications in metabolic fluxes result from multiple actions. Most of them are summarized in Stimulation of the glycolytic pathway was demonstrated when the livers from fed rats were perfused. Under this condition, boldine expressively stimulated lactate and pyruvate productions from endogenous glycogen. This is an important finding because the enhancement of glycolysis is generally regarded as an efficient way of lowering plasma glucose and consequently controlling hyperglycemia . Stimula50). Half-maximal inhibition of lactate and alanine gluconeogenesis occurred at boldine concentrations of 71.8 μM and 85.2 μM, respectively. These are similar concentrations. The first step of lactate and alanine metabolization in the liver is their conversion into pyruvate, catalyzed by two different enzymes, lactate dehydrogenase, and alanine aminotransferase, respectively. Inhibition of the net flux through these two enzymes at almost exactly the same degree by boldine is not a very likely event. Furthermore, these enzymes operate under near-equilibrium conditions in the liver, requiring, thus, very high inhibition degrees for exerting a significant influence on the gluconeogenic pathway. It is important to note that when the livers were supplied with lactate or alanine, pyruvate overflow was slightly increased at the highest boldine concentration. The phenomenon suggests that a step situated downstream to pyruvate generation is more likely to be inhibited by boldine. One such step could be the reaction catalyzed by the phosphoenolpyruvate carboxykinase (PEPCK), which our experiments revealed indeed to be inhibited by boldine. PEPCK catalyzes the conversion of oxaloacetate into phosphoenolpyruvate. Although the metabolic control of this enzyme over gluconeogenesis is low under normal conditions [The hypothesis of an increase in fructose 2,6-phosphate, however, would not completely explain the observed inhibition of gluconeogenesis, since it has been demonstrated that alterations in this metabolite do not have dramatic effects on gluconeogenesis from lactate . Therefonditions , inhibitnditions .50 of 83.62 μM, which is comparable to that one found for alanine or lactate, and the pathway that leads to its transformation into glucose is situated downstream to the step catalyzed by PEPCK. Furthermore, glycerol gluconeogenesis, which uses in part the same pathway as fructose gluconeogenesis was also inhibited, though to a much lesser extent . This combination of observations suggests not only that there must be other steps that can be inhibited by boldine in addition to the PEPCK step but also that there must be a step whose inhibition specifically affects fructose metabolism. Additional enzymatic steps sensitive to boldine were indeed found in the present work, more specifically FBPase-1 and G6Pase. G6Pase, an enzyme found mainly in the liver, plays the important role of providing glucose during starvation. G6Pase catalyzes the last step of gluconeogenesis, and its inhibition could partly explain the reduction of gluconeogenesis. However, as already demonstrated [Even so, it is unlikely that the inhibition of PEPCK is the sole responsible for the inhibition of gluconeogenesis. Fructose transformation into glucose was also substantially inhibited with an ICnstrated , even a In addition, we cannot discard a possible diversion of glucose 6-phosphate for boldine transformation. Boldine's hepatic biotransformation occurs mainly by O-conjugation with glucuronate and sulphate and, to a lesser extent, via N-demethylation . The latin vitro studies have revealed that boldine is a very efficient scavenger of the hydroxyl radical (HO•) and peroxyl radical [μM, our results showed significant inhibition starting at 50 μM. These differences can possibly be attributed to the distinct assays used for measuring mitochondrial ROS generation. Jang et al. [Direct inhibition of ROS generation in mitochondria by boldine is certainly a beneficial effect, corroborating previous publications . Several radical , 10, 45.g et al. , who shog et al. .μM. Therefore, the fact that boldine inhibits gluconeogenesis at relatively low concentrations, besides not modifying mitochondrial energy transduction, makes it a potential molecule for being used in the therapy of diabetes.It is worth mentioning that metformin, a classical drug for glycemic control, also reduced hepatic gluconeogenesis in the perfused liver . DiffereCmax of 31 μM is reached after the administration of 20 mg/kg (intravenously) in rats [μM (that were those used in the present study) are easily achieved, or even surpassed in hepatocytes. Available data in isolated cells and various animal models point out that boldine presents low toxicity [Absorption of boldine into plasma is rapid, and an average  in rats . Howevertoxicity , effectiIn conclusion, the inhibition of gluconeogenesis by boldine in the perfused liver is probably the consequence of the inhibition of several rate-limiting enzymes with a possible minor contribution of the deviation of glucosyl units for the glucuronidation reactions. The capacity of boldine to modulate glucose metabolism in the liver by inhibiting gluconeogenesis and stimulating glycolysis and inhibiting mitochondrial ROS generation, associated with the lack of interference with mitochondrial respiration, makes this alkaloid a potential therapeutic tool for glycemic control.


Although it is well known that active and inactive BRAF have differential phosphorylation sites that play a crucial role in regulating BRAF, key details are still missing. In this study, we report the characterization of a novel phosphorylation site, BRAFS732 , located in proximity to the C-terminus binding motif for the 14-3-3 scaffolding protein. At the C terminus, 14-3-3 binds to BRAFpS729 (CRAFpS621) and enhances RAF dimerization. We conducted mutational analysis of BRAFS732A/E and CRAFS624A/E and revealed that the phosphomimetic S→E mutant decreases 14-3-3 association and RAF dimerization. In normal cell signaling, dimerized RAF phosphorylates MEK1/2, which is observed in the phospho-deficient S→A mutant. Our results suggest that phosphorylation and dephosphorylation of this site fine-tune the association of 14-3-3 and RAF dimerization, ultimately impacting MEK phosphorylation. We further characterized the BRAF homodimer and BRAF:CRAF heterodimer and identified a correlation between phosphorylation of this site with drug sensitivity. Our work reveals a novel negative regulatory role for phosphorylation of BRAFS732 and CRAFS624 in decreasing 14-3-3 association, dimerization, and MEK phosphorylation. These findings provide insight into the regulation of the MAPK pathway and may have implications for cancers driven by mutations in the pathway.Rapidly accelerated fibrosarcoma  kinase is central to the MAPK pathway (RAS–RAF–MEK–ERK). Inactive RAF kinase is believed to be monomeric, autoinhibited, and cytosolic, while activated RAF is recruited to the membrane  RAF kinases are essential components of the MAPK (RAS–RAF–MEK–ERK) pathway, which governs key cellular processes including cell growth, differentiation, proliferation, and migration , 2. AmonpS365) within a RSXpSXP motif recognized by the 14-3-3 scaffolding protein; CR2 interacts with 14-3-3 to lock BRAF in its autoinhibitory conformation (The three isoforms of RAF share three conserved regions (CR1/2/3). CR1 contains the RAS-binding domain and the cysteine-rich domain (CRD). The RAS-binding domain interacts with canonical activated RAS-GTP at the plasma membrane to initiate RAF activation . In inacormation . CR3) coormation . The BRAormation . DimerizS729/CRAFS621) that is recognized by the 14-3-3 scaffolding protein. Phosphorylation of this C-terminus site has been suggested by activated protein kinase, PKA, and autophosphorylation of the activation loop (BRAFpT599)  , 11. The99 , 11. activity , 13. It V600E/K  data supports that phosphomimetic mutants decrease 14-3-3 binding at the C terminus. Additionally, we found that the phosphomimetic mutant BRAFS732E displays decreased activation loop phosphorylation, whereas the phospho-deficient BRAFS732A exhibits the opposite effect, promoting activation loop phosphorylation. Furthermore, the phosphomimetic BRAFS732E reduces BRAF homodimerization and BRAF:CRAF heterodimerization. Concurrently, the homologous CRAF phospho-deficientS624A mutant displays the similar behavior. Collectively, our data suggest that the conserved serine in BRAFS732 and CRAFS624 acts as a negative regulator of BRAF and CRAF, providing further complexity to a tightly regulated system.Phosphorylation and dephosphorylation tightly regulate the pathway output of RAF kinase. In this study, we report the characterization of BRAFing site . We hypopS732 was identified in both populations along with other reported known and characterized phosphorylation sites , and this serine is well conserved among the three human RAFs  . Phosphorylation sites were searched against available literature and databases. The PhosphoSite database reports that BRAF pS732 has been previously annotated by three internal PhosphoSite database curation sets: 12,432, 9879, and 9883  to be present in the 14-3-3 binding groove (beige) (B and C), although BRAFS732 is located outside of the C-terminus BRAFS729 (green) 14-3-3 recognition motif (RSXpSXP)  . C-termiRSXpSXP) . PhosphoS365 to dissociate 14-3-3 . BRAFS732E was chosen as the representative phosphomimetic mutant since it can mimic the phosphate oxygens of a phosphorylated-serine due to the longer side chain length when compared to an aspartic acid (A–C). Further experiments were carried out to investigate the local effects of 14-3-3 binding at the C terminus of BRAF, using a mutant lacking the N-terminal 14-3-3 (BRAFpS365) binding region . Notably, BRAFS732E displayed a highly significant decrease in C-terminal region 14-3-3 association (D and E). Full-length BRAFS732 WT, A, and E mutants were expressed and immunoprecipitated from HELA and HCT116 cells . We. WeS365 A mutant , A–C. BRtic acid . Both phtic acid , A–C. Fuociation , D and E16 cells . HELA ceRAFS732A . TogetheS732 WT/A/E-FLAG as protomer 1 and BRAFS732 WT/A/E-V5 as protomer 2; protomer 2 was coimmunoprecipitated using the FLAG epitope tag of protomer 1 (A and B). Phosphomimicry of both protomers reduced BRAF homodimerization and MEK1/2 phosphorylation (B–D). Dimerization is a key regulatory element of BRAF kinase activity. In immunoprecipitated BRAF-FLAG, phospho-deficient BRAFS732A-FLAG is observed to have a relatively higher activation loop phosphorylation (BRAFpT599) than BRAFS732E (E and F). The increased activation loop phosphorylation is also observed in HELA cells in the BRAFS732A relative to BRAFS732E (A and C). In an in vitro Western blot–based kinase assay, Escherichia coli expressed GST-MEK1 was added to immunoprecipitated BRAF S732WT/A/E-FLAG. Similar to the endogenous MEK1/2 phosphorylation in HEK293 cells, in vitro immunoprecipitated phosphomimetic BRAFS732E phosphorylated MEK1 less relative to the phospho-deficient A mutant (H and I). Phosphomimicry decreases local C-terminal region 14-3-3 scaffolding protein association and reduces BRAF homodimerization with observed reduced activation loop phosphorylation and downstream MEK1/2 phosphorylation.C-terminal region 14-3-3 has been shown to facilitate the dimerization of two RAF protomers . To modeotomer 1 , A and Brylation , B–D. DiRAFS732E , E and FRAFS732E , A and CA mutant , H and IV600E/K mutants . In contrast, BRAFS732E required a higher concentration than BRAFS732A to induce paradoxical activation, displaying enhanced MEK and ERK phosphorylation consistently at 1 μM dabrafenib treatment (C and D). Phosphomimicry of BRAFS732 reduces homodimer formation, narrowing the range of paradoxical activation to 1 μM unlike phospho-deficient BRAFS732A, which has paradoxical activation observed at 0.1 to 1 μM. Phosphomimicry of BRAFS732 does not abolish paradoxical activation but rather, paradoxical activation requires a higher dosage to occur, likely due to the reduced homodimer formation in BRAFS732E  have been successful in targeting metastatic melanoma with BRAF mutants , 15; how mutants . Paradox mutants . Several mutants . These mμM range . Short-treatment , C and DRAFS732E .Figure 4S732 phosphoregulatory site is conserved across the RAF family kinases in humans, with CRAFS624 being the conserved site (A). Phosphorylation of the CRAFS624 site has been previously annotated in the PhosphoSite database (S624E to mimic phosphorylation and CRAFS624A to abolish phosphorylation (A and B). Consistent with the BRAF experiments, phospho-deficient CRAFS624A displayed enhanced association with the 14-3-3 scaffolding protein (B and E). To further probe the regulation of CRAF, we examined the phosphorylation status (CRAFpS338) of immunoprecipitated CRAF. CRAF is regulated by similar but distinct mechanisms compared with BRAF, with a CRAF SSYY motif (residues 338–341) instead of the BRAF SSDD motif (residues 446–449), the N-terminal acidic region . In CRAF, there is no significance in the phosphorylation of MEK1/2, unlike BRAF (B and F), suggesting that CRAF homodimer is less sensitive to the phospho-status of Ser624.The BRAFved site A. Phosphdatabase , 23, 28.rylation , A and B protein , B and Ec region , 30. Phorization , 30. We E mutant , B and Dike BRAF , B and FC, G–I). The CRAF homodimer alone does not affect the phosphorylation of MEK but combined with BRAF in the heterodimer, the decrease in MEK1/2 phosphorylation for the phosphomimetic mutants is highly significant (F and I). This observation is consistent with the BC heterodimer having the highest MAPK pathway activity and that CRAF alone has low-basal activity.Since the CRAF homodimer is not the major RAF dimer to regulate MAPK pathway, we investigated how this phosphorylation modification impacts BRAF:CRAF heterodimer, the most potent MAPK pathway output dimers . The BRAnificant , F and IS732F is reported by the COSMIC database as a missense somatic mutation in carcinoma and malignant melanoma with an unknown effect . BRAFS732F decreases homodimerization in a similar manner to the phosphomimetic model, which further emphasized by the low MEK1/2 activity in HEK293 cells (E–G). The 14-3-3 association is not significant in HEK293 cells, but mutant phenylalanine decreases BRAF homodimerization and MEK activity in a similar fashion to the phosphomimetic E model (E and F). Based on decreased dimerization and MEK activity, we rationalized that mutant BRAFS732F would respond to dabrafenib treatment similar to the phosphomimetic E model (S732F and no significance was observed between the comparison of relative pMEK1/2 and pERK1/2 between the mutant F and E model (D and E). BRAFS732F behaves more similarly to the decreased MEK activity and decreased BRAF homodimerization observed in the phosphomimetic BRAFS732E model. These observations suggest that dysregulated BRAFS732F does not solely follow the phosphomimetic nor phospho-deficient model of regulation. BRAFS732F is not significantly affected by 14-3-3 association nor activation loop phosphorylation compared to the A model; however, this mutant significantly decreases MEK phosphorylation and BRAF homodimerization. Mutant BRAFS732F is dysregulated as it increases 14-3-3 association, while decreasing BRAF homodimerization and downstream MEK1/2 activity. The characterization of mutant phenylalanine further emphasizes that dysregulated RAF does not follow a clear model.While the focus of our analysis relied heavily on WT, phospho-deficient, and phosphomimetic BRAF and CRAF, many oncogenic cell lines contain mutations that dysregulate the RAF–MAPK pathway. BRAFn effect , 20. Simrylation  and S1. rylation . Both cerylation , E and F93 cells , E–G. Th E model , E and F E model . Both mo E model , D and EpS729/CRAFpS621 is widely accepted to be constitutively phosphorylated and associated with 14-3-3 . During upstream stimulation via RAS-GTP, 14-3-3 association is enhanced, which increases homodimer and heterodimer formation, activation loop phosphorylation, and subsequently MEK activation (B). Subsequent phosphorylation of BRAFpS732 dislodges the association of the 14-3-3 scaffolding protein at the C terminus, which decreases dimerization, activation loop phosphorylation, and attenuation of MEK1/2 phosphorylation (C and D). While CRAF is sensitive to the C-terminal region 14-3-3 association, no downstream consequences are observed. However, CRAF coupled with BRAF to form the BC heterodimer induces similar effects observed in the BRAF homodimer (C and I). This data underscores the importance of the proper positioning of the two dimerized protomers of the RAF kinase domain with the 14-3-3 scaffolding protein as disruption has been previously demonstrated to disrupt RAF activity . How exactly RAF inhibitors affect the RAF:14-3-3 complex is not well defined and requires further understanding. This cell-based data provides an additional therapeutic target for reducing BRAF dimerization and activity. Two independent groups identify targeting the 14-3-3 interaction with BRAF as a site of therapeutic intervention as the scaffolding protein is important as a molecular switch  detection  was purchased from Invitrogen. Anti-HA (3724), anti-pMEKS217/S222 (9154), anti-MEK (9154), anti-pERKT202/Y204 (4370), ERK1/2 (4696), anti-β-Actin (3700), anti-pan 14-3-3 monoclonal (8312), anti-pan 14-3-3 polyclonal , anti-pCRAFS338 (9427) was purchased from Cell Signaling. Anti-GST was purchased from Santa-Cruz (SC-138). Anti-rabbit (926–32211) and anti-mouse (926–68070) secondary antibodies were purchased from LICOR.Anti-Flag (F1804) was purchased from Sigma. Anti-pBRAFvia PCR, following the manufacturer’s instructions, Q5 site–directed mutagenesis (NEB E0554). Mini preps were carried using the Qiagen  mini prep kit according to the manufacturer’s protocols. Midi preps for transfection were carried out using the ZymoPURE (D4201) midi prep purification kit according to the manufacturer’s protocols. DNA elutions were carried in water.All expression vectors are in the transient expression vector, pcDNA4/TO (Invitrogen V102020). BRAF-FLAG was cloned into the pcDNA4/TO as previously described . The pri2 until 70 to 90% confluency and passaged until passage 16 and discarded. Cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with 1% L-Glutamate, 1% penicillin and streptomycin, and 10% fetal bovine serum.HEK293 cells  were maintained at 37 °C, 5% CO2 before harvest using cold PBS. Cells were lysed using a modified radioimmunoprecipitation assay buffer. Cells were lysed using a modified radioimmunoprecipitation assay buffer ) at 4 °C, 2 h rotation. Immunoprecipitation was carried according to manufacturer’s protocols for the immunoprecipitation of FLAG-tagged proteins (Sigma M8823) and quenched in 4× Laemmli loading dye for 5 min at 95 °C)For co-IP experiments, cells were seeded with 5 million cells per 100-mm dish and transfected at 60% confluency with a 1:3 ratio of DNA:transfectant . Single transfections were carried with 10 μg in a 100-mm dish. The BRAF homodimer was carried using a 1:1 ratio of BRAF-FLAG: BRAF-V5. The BC heterodimer was carried using a 1:2 ratio of BRAF:CRAF due to the low expression of CRAF Cells 40 to 60% confluency before transfection. Transfections were cultured for 48 h at 37 °C, 5% COSingle transfections were carried with 2 μg/well in a 6-well plate (1 million per well) for the dabrafenib experiments. Following 48 h of protein expression, the cells were washed with PBS and replenished with 2 ml of fresh media. Dabrafenib (Selleckchem S2807) was diluted to a final concentration of 0.1 to 10 μM, using a 100 μl treatment volume. The negative control 0 μM contained the same volume of dimethyl sulfoxide as the 10 μM treatment. The 0 control contains the highest dilution volume of dabrafenib to account for effects of DMSO, all under 1% DMSO. Cells were incubated at 37 °C, 1 h prior to cell harvest. Cells were washed with PBS twice and lysed immediately with 4% SDS and subsequently applied to a homogenizer column.Whole-cell lysate protein quantification was carried using a Pearce bicinchoninic acid assay and a Clariostar plate reader (BMG). Samples were diluted in 4× Laemmli loading dye and boiled for 5 min. Samples were visualized using 10% SDS-PAGE electrophoresis or 4 to 15% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad 4561084). Membranes were incubated in primary antibodies, overnight in 4 °C and secondary antibody 2 h, at room temperature. Nitrocellulose membranes were visualized using a LICOR scanner, and images were cropped to highlight the molecular weight marker, which is not visible on each scanning channel. For these channels, the molecular weight is placed next to the expected band size. If there are multiple bands beyond the expected molecular weight, a black arrow was placed to the right of the blot to indicate the correct band(s). Each reported experiment was performed three or more times, as indicated.https://imagej.nih.gov/ij/download.html) (NIH) through manual selection of the area under the curve (AUC) of the density of pixels. The AUC was normalized relative to the A mutant or the 0 μM treatment. For example, relative 14-3-3 to A mutant: AUC of sample/AUC of A mutant. This was chosen to highlight the dichotomy between the A and the E mutant. The following readouts use the same relative to A mutant method: 14-3-3, BRAF pT599, CRAF pS338, dimer readouts (BRAF-V5 relative to BRAFS732A-V5 of the BRAF homodimer and CRAF-HA relative to CRAFS624A-HA of the BRAF-CRAF heterodimer), and the in vitro kinase assay. For the kinase assay, relative phosphorylated MEK was relative to the phospho-deficient A mutant to reflect kinase activity between mutants. For the BRAF homodimer, CRAF homodimer, and the BRAF:CRAF heterodimer, endogenous phosphorylated MEK1/2 was normalized to the total MEK1/2 protein to reflect the population of MEK that is phosphorylated in the cells. Relative pMEK: phosphorylated MEK1/2/total MEK1/2. Dabrafenib treatments were normalized to the 0 μM treatment of the phosphorylated MEK. Previous reports  (2023b v 10.05) software. Data points are within the bar graphs; bars are represented as mean ±SD and statistical significance as: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. A p-value of <0.05 was considered statistically significant. All ANOVA values are reported in Supporting information All experiments were at least three independent biological replicates. Experimental differences were tested for statistical significance using one-way ANOVA followed by the post hoc Tukey’s honest significant difference test for co-IP experiments. Two-way ANOVA was used for the dabrafenib experiments with mutant BRAF and dabrafenib as the factors followed by the post hoc Holm–Bonferroni test for multiple comparisons . StatistQ02750) was adapted from a previous report  and mechanical lysis via French press. Lysed supernatant of 6×HIS-kinase dead MEK1 was purified using buffer equilibrated nickel resin . The resin was washed in sequential alternating low- and high-salt buffers to remove nonspecific binding .Purification of recombinant kinase dead MEK1 . The reaction incubated at 30 °C for 3.5 min was immediately quenched in 4× loading dye. The kinase reaction was visualized using the phosphorylated-MEKS217/S22antibody  and anti-GST (Santa Cruz SC-138) for the recombinant 6×HIS-GST-MEK1K97M.The kinase reaction was carried out using an adapted protocol as previously described . Immunopvia SpeedVac and loaded into a reverse phase C18 column. Samples were directly eluted into the mass spectrometer using a two-buffer gradient elution (buffers A and B) at a flow rate of 450 nL/min. Buffer A was 0.1% formic acid and buffer B was 99.9% acetonitrile in 0.1% formic acid. Elution was carried using 0% buffer B (2 minutes), 30% buffer B (50 minutes), 50% buffer B (10 minutes), and finally 95% buffer B (10 minutes). The data-dependent acquisition method was used to acquire MS and tandem mass spectrometry (MS/MS) data  through the Xcalibur software  and the UniProt database. Peptides were identified via Proteome Discoverer (Thermo Fisher Scientific). The data is available in Excised bands of basal, cytosolic BRAF, and membrane recruited, activated BRAF were excised and sent to the Proteomics and Mass Spectrometry Facility, University of Georgia for post translational modification identification (Thermo Fisher Scientific LTQ Orbitrap Elite MS with a Proxeon Easy NanoLC system). Briefly, gel bands were followed by an in-gel trypsin digestion. Extracted peptides were dried The data that support the findings of this study are available on request from the corresponding author, Zhihong Wang, upon reasonable request.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


It is often assumed that α-subunit phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) complex is just a mechanism to control protein synthesis. However, eIF2α phosphorylation induced by multiple kinases can recognize various intracellular and extracellular stress conditions, and it is involved in various other cellular processes beyond protein synthesis. This review introduces the roles of eIF2α phosphorylation in translational regulation, the generation of reactive oxygen species, changes in mitochondria structure and shape, and mitochondrial retrograde signaling pathways in response to diverse stress conditions. Most prAll the transcripts described above have short upstream open reading frames (uORFs) located in the 5'-untranslated region (5'-UTR) of the mRNA. They are preferentially translated through uORF-mediated mechanisms during diverse stressful conditions in which eIF2α phosphorylation is induced by four eIF2α kinases . The uORAlthough the biochemical mechanism of translation inhibition mediated by eIF2α phosphorylation is well understood, its downstream physiological consequences are less clear at the cellular level. Upon ER stress, viral infection, and other cellular stresses, four eIF2α kinases phosphorylate eIF2α, which leads to transient attenuation of global protein synthesis and transcriptional induction through preferential translation of selected transcription factor genes   MEFs, lower average levels of glutathione (GSH), a tripeptide antioxidant that contains L-cysteine, L-glutamic acid, and glycine, can be expected, compared with wild-type  MEFs. Reduced GSH levels in A/A cells are related to impaired glutathione metabolism because of decreased eIF2α phosphorylation–dependent ATF4 translation and subsequent downregulation of the light chain, xCT (encoded by the Slc7a11 gene), of the Xc- cystine/glutamate exchanger , and cystathionine γ-lyase (Cth)  .in vitro studies, basal eIF2α phosphorylation is required to protect specialized secretory cells, including pancreatic β cells, from oxidative stress in vivo  called supercomplexes (SCs) , mitophagy, and the ISR pathway are known. However, many experimental paradigms indicate that the UPRmt can be cross-regulated with ISR , which signal mitochondrial dysfunction to the cytosol or nucleus and then induce anterograde mechanisms to protect mitochondria and restore cellular homeostasis . In mammwith ISR . This reet al. suggest that PKR is a responsible enzyme that can be activated by cellular double-stranded RNAs (dsRNAs) during mitosis  in response to the accumulation of a deleted mutant form of the mitochondrial matrix protein, ornithine transcarbamylase (OTC) (ER) is not involved in the CHOP-mediated gene expression of the UPRmt. Nevertheless, eIF2α-phosphorylation dependent ISR pathway reportedly can interact with multiple points of the UPRmt  of the mitochondrial mutant protein prel/ups1p induces dendritic regression of Drosophila peripheral sensory neurons by mitochondrial dysfunction , is expressed in an ATF4-dependent manner in response to mitochondria stresses induced by mETC inhibition, mitophagy defects, and defects in mitochondrial dynamics . During hibition .Mammalian cells are equipped with four eIF2α kinases to sense and control diverse intracellular and extracellular stresses. The cells activate a common adaptive pathway, the ISR, through eIF2α phosphorylation, to restore cellular homeostasis, and eIF2α phosphorylation is involved in multiple biological processes. In protein synthesis, eIF2α phosphorylation suppresses translational initiation of general mRNAs and activates it from specific mRNAs in response to stress. However, regulation of protein synthesis by eIF2α phosphorylation does not always work as intended. In effect, too much is as bad as too little. Hyper and prolonged phosphorylation of eIF2α can increase expression of proapoptotic genes through downstream transcription factors, which can make matters worse, and sustained translational suppression can disrupt cellular homeostasis through deficiency of cell type–specific proteins or important cellular function–maintaining proteins. Beyond translation regulation, both hyper- and hypo-phosphorylation of eIF2α cause ROS damage because of both translational dysregulation of general mRNAs and the absence or overexpression of its downstream target genes. In addition, eIF2α phosphorylation can modulate mitochondrial structure and shape to maintain homeostasis and preserve its integrity through the PERK-eIF2α axis during ER stress. Mitochondria appear to use eIF2α phosphorylation to communicate with the cytosol and nucleus and protect and restore mitochondria homeostasis. Mitochondrial RNAs are recognized by PKR to regulate cytosolic translation through eIF2α phosphorylation during the mitotic phase and mitochondrial stress conditions. In mitochondria-nuclear communication, diverse mitochondrial dysfunctions, except for accumulation of a misfolded mitochondrial protein, activate eIF2α phosphorylation–dependent ISR pathways, which can be divided into ATF4-dependent and -independent pathways. However, only further study can shed light on what induces eIF2α phosphorylation during mitochondrial dysfunctions. MRSP induced by eIF2α phosphorylation does not always protect or ameliorate but can deteriorate mitochondria stress. The level of eIF2α phosphorylation should be in a balanced range to promote successful adaptation to cellular stress.


These epitopes may have the potential to contribute towards a broader cross-protective IBV vaccine.The avian coronavirus, infectious bronchitis virus (IBV), is an economically important infectious disease affecting chickens, with a diverse range of serotypes found globally. The major surface protein, spike (S), has high diversity between serotypes, and amino acid differences in the S1 sub-unit are thought to be responsible for poor cross-protection afforded by vaccination. Here, we attempt to address this, by using epitope mapping technology to identify shared and serotype-specific immunogenic epitopes of the S glycoprotein of three major circulating strains of IBV, M41, QX, and 4/91, via CLIPS peptide arrays based on peptides from the S1 sub-units. The arrays were screened with sera from chickens immunised with recombinant IBV, based on Beau-R backbone expressing heterologous S, generated in two independent vaccination/challenge trials. The screening of sera from rIBV vaccination experiments led to the identification of 52 immunogenic epitopes on the S1 of M41, QX, and 4/91. The epitopes were assigned into six overlapping epitope binding regions. Based on accessibility and location in the hypervariable regions of S, three sequences,  The avian coronavirus, infectious bronchitis virus (IBV), is an endemic poultry pathogen, causing a highly contagious respiratory disease . EconomiThe issue of limited cross-protection between IBV serotypes is often associated with the variance in the major surface protein, spike (S), a type 1 glycoprotein which oligomerises to form trimers ,14,15. TSeveral studies have identified neutralising epitopes present on the S glycoprotein of other coronaviruses, including severe acute respiratory syndrome (SARS) coronavirus (CoV) ,36, SARSThe aim of this study was to identify shared and unique immunogenic epitopes of the S glycoprotein of three major circulating strains of IBV. We used CLIPS arrays based on the peptides from the S1 of IBV serotypes, QX, 4/91, and M41. The screening of the peptide arrays was performed with sera from chickens immunised with recombinant IBV, generated in two independent homologous and heterologous vaccination/challenge trials ,59. Due All animal experimental protocols referenced here were carried out in strict accordance with the UK Home Office guidelines and under license granted for experiments involving regulated procedures on animals protected under the UK Animals (Scientific Procedures) Act 1986. The experiments were performed in The Pirbright Institute Home Office licensed (X24684464) experimental animal house facilities, and were approved by the animal welfare and ethical review committee under the terms of reference HO-ERP-01-1.The chicken sera used to screen the synthetic CLIPS peptide arrays were generated from two independent in vivo vaccination/challenge experiments of specific-pathogen-free (SPF) Rhode Island Red chickens with recombinant IBV (based on Beau-R backbone), as described in ,59. BrieThe CLIPS peptide arrays were synthesised by Pepscan Presto BV . Three different arrays were generated based on the S1 sequence of three economically important strains of IBV, M41-CK (Massachusetts serotype), 4/91 , and QX-like (L1138). The 15-mer peptides, derived from the target S1 sequences, were positioned on the arrays, with the secondary structure and CLIPS technology used to inform positioning to simulate both linear and conformational epitopes. The linear 15-mer peptides were offset by one residue and additionally chemically constrained via the CLIPS technology into loop, helical, or coil constructs. The three IBV S1 peptide arrays contained helical (1564), coil (1553), linear (1574), and loop (1574) constructs with one array per serotype .The CLIPS peptide arrays were incubated with chicken serum or S-specific mouse monoclonal antibodies (mAbs) . Each inTo validate the specificity of the synthesized CLIPS peptide arrays, expressing IBV peptides, two negative mAbs, CVI-TGEV-57.9 specific for the S protein of TGEV  and HercIntensity profiles were examined for each individual chicken serum sample, tested on all three  IBV S1 peptide arrays, and screened against all four types of constructs: linear, helical, coil, and loop. The level of non-specific background staining was determined on an individual sample level, with a common range of 0–200 AU. Peptides associated with a peak in the absorbance traces were recorded. The intensity of the antibody binding peaks in the absorbance traces varied at an individual sample level. Consequently, the levels of non-specific background staining displayed by individual serum samples were taken into consideration, and clear discernible peaks in intensity were evident. The levels of conservancy of peptides selected by antibody binding across serum samples were assessed. The epitopes were classified as “linear” or “conformational”, depending on the type of peptide construct associated with the peak in absorbance. The peptide binding profiles of serum samples were compared to identify regions of similarity and differential binding. Heat map profiles of the binding intensity were constructed using the ‘pheatmap’ function (pheatmap package) in R version 3.4.1. Dendrograms between serum samples were calculated using the Pearson correlation clustering algorithm, based on the average pairwise distance between all points of each data set and scaled to the intensity of rows.2SO4 and read at 450 nm on a microplate reader.To validate the antibody binding of the three selected peptide sequences on an individual capacity, the recognition of linear peptides was assessed in a synthetic peptide ELISA. Biotinylated synthetic peptides were synthesized  based on the binding specificity of the chicken sera with the peptide arrays. The three selected peptides were diluted to 10 µg/mL, and incubated on streptavidin pre-coated plates  overnight at 4 °C. The plates were then washed four times with PBST (0.05% Tween-20). Serum samples were diluted 1:10 in PBS, applied to the plates, and incubated for 2 h at 37 °C. The plates were re-washed with PBST and mouse anti-chicken IgY (Clone G-1) , diluted 1:1000, was applied and incubated for 1 h at 37 °C. The plates were washed with PBST and HRP-conjugated goat anti-mouse IgG1 tertiary antibody (Southern Biotech), diluted 1:2000, was applied and incubated for 1 h at 37 °C. Finally, the plates were washed and developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Thermo Fisher), and the reaction was stopped with 2 M HA bioinformatic analysis to assess the conservation of the identified epitopes amongst IBV serotypes was performed. Sequence logos were used to determine the sequence conservation of the identified epitopes, amongst the three IBV strains studied here, QX, 4/91, and M41, using AliView v1.25 . A sequeConstrained linked peptide screening (CLIPS) oligopeptides were synthesised using various scaffold combinations on a solid surface to produce three individual peptide arrays to permit the probing of antibody binding in a high-throughput epitope mapping system. These CLIPS were used to produce three peptide arrays based on the known S1 sequence of IBV strains; M41-CK, 4/91 (UK), and QX (L1148), containing 2093, 2090, and 2082 oligopeptides, respectively. Using the BLOSUM62 similarity matrix, the predicted S1 amino acid sequences of M41-CK and 4/91 (UK) are 81.7% identical and 89% similar, those of M41-CK and QX (L1148) are 80.8% identical and 89% similar, and those of 4/91 (UK) and QX (L1148) are 83.9% identical and 91.2% similar . These cAs a validation step to assess the specificity of the three peptide arrays based on the S1 sequence of IBV strains, M41, QX, and 4/91, the arrays were screened using eight mAbs, which had been raised against IBV strain D207 , and seln = 5)   also boun = 5) d. One can = 5) a–c. Monon = 5) a and CVIn = 5) b recognin = 5) b.The recognition site for CVI-IBV-69.3 was conserved across all three IBV S1 peptide arrays a; howeven = 20). There were two main aims in these screening experiments: (1) assess the antibody specificity of the polyclonal serum for CLIPS-peptides, and (2) identify if there are serotype-specific epitopes recognised on the S1 of the three IBV strains under investigation. In addition to the sera from homologous vaccinated/challenged chickens, the three IBV S1 peptide arrays were also screened with pools of sera, collected at 14 dpc, from non-vaccinated/non-challenged chickens (“Mock/Mock”) (pool of n = 10 samples) and non-vaccinated/challenged chickens for both viruses, (Mock/QX or Mock/M41) . Intensity profiles for all homologous serum samples were recorded against each type of construct displayed on the three IBV S1 peptide arrays following screening with pooled Mock/Mock and Mock/challenge only sera  showed very little background reactivity of the CLIPS peptides a. In comCollectively, there were 36 epitope sequences recognised by polyclonal sera from the homologous vaccinated/challenged chickens a, with 1n = 38). In a similar manner to the homologous serum screening, two additional pools of age-matched serum controls were used (n = 10 birds per pool): (1) non-vaccinated/non-challenged group and (2) non-vaccinated/QX challenge only nge only . Epitopenge only b. The hinge only b. As allnge only b.Collectively, a total of 78 epitope sequences were identified through screening the IBV S1 peptide arrays with the panel of polyclonal sera from both the homologous and heterologous vaccination/challenge experiments a,b.All of the sera samples from vaccinated chickens  were subjected to a clustering analysis to investigate if any similarities or patterns between the immune recognition of individual samples were evident .n = 10, homologous vaccination/challenge and n = 15 heterologous vaccination/challenge) was either due to lower affinity for the CLIPS or lack of recognition of the epitope mimics. From the comparison of the epitope binding profiles, it appears that these sera had lower affinity for the CLIPS and did not reach the same intensity as the other sera from vaccinated chickens  and termed “Epitope regions A–F”. Analysis showed that 55% (43/78) of the epitope sequences identified were regions A–C. Epitope regions A and B are located across the S1-NTD (19–237 aa), speculated to contain the receptor binding domain (RBD) and the hypervariable regions HVR1 38–67 aa) and HVR2 (99–141 aa) ,65,66,67 aa and H25YVYYYQSAFRPPNGWHLQGGAYAVVNSTN54 (Epitope 1—QX), 67TVGVIKDVYNQSVASI82 (Epitope 2—QX), and 83AMTVPPAGMSWSVS96 (Epitope 3—4/91). Sequence logos were used to assess the conservation of individual amino acids in each identified epitope, with the height of each letter indicating the proportion of sequences that contain the residue at that site  were shared amongst IBV serotypes QX, 4/91 and M41, but there were amino acid substitutions at positions 25, 37, 38, 43, 50, and 51–54 (83AMTVPPAGMSWSVS96 (Epitope 3) was also shared amongst serotypes, with some degree of variation evident in residues and where substitutions occurred ; there were changes in amino acid hydrophobicity when serine was in these positions (67TVGVIKDVYNQSVASI82 (Epitope 2) appeared to be serotype-specific with a high level of residue variability, evident from the equal weighting of residues across the peptide sequence , with the recognition of the peptide being highly variable within the other vaccinated groups and the majority of serum samples having low to moderate binding. This pattern of low binding was also evident against Epitope 2, with no significant differences detected across the vaccinated groups in comparison to the Mock control  5/17 (CR88) and 5/15 (793B) epitopes, respectively, matched our generated data ,82. In oata sets ,74. Seveata sets ,75,83. Hata sets ; protectata sets .8NCPYVSYGKFCIKPDGSIST27, identified by Ignjatovic and Sapats [Interactions between the S1 and S2 subunits are critical for the conformation and efficient fusion of IBV S to host cells, as a single amino acid change in the S2 may influence the secondary structure . This isd Sapats , was shod Sapats . CollectThe publication of the cryo-EM structure of M41  allows tTowards the rational design of broad cross-protective vaccines against IBV with high efficacy and that can elicit strong neutralising responses in hosts, an appealing strategy would be to target a combination of epitope domains. This approach has been successful against the MERS-CoV, targeting epitopes which were non-RBD S1-specific, and preventing the emergence of escape mutants with amino acid substitutions in the RBD that was reported with the use of a single mAb . One cauGlycans have an important role in post-translation modifications and protein folding, with N-linked glycosylation sites in the IBV S protein differentially affecting spike-membrane fusion, viral infectivity, and replication . The recBased on the known characteristics of IBV S and the epitopes identified here, three sequences were selected based on the following criteria: (1) spatially separated, (2) in the S1-NTD (likely to contain the RBD), and (3) with any proximity to the hypervariable regions . These three sequences were selected for further assessment of antibody recognition by synthetic peptide ELISAs. All selected epitopes were recognised to varying degrees by experimentally derived immune chicken serum from the rIBV vaccinated groups. Variability in host genetics may play a role in the generation of differential activities of species-specific antibodies to same B-cell epitopes . As the In summary, the present study demonstrated the application of screening CLIPS arrays as a useful tool for mapping the antigenic regions of the S1 of IBV serotypes M41, QX, and 4/91, in an attempt to identify epitopes that might be associated with cross-protection. The screening of serum samples from rIBV vaccination experiments resulted in the detection of 52 highly immunogenic epitopes in the S1 sequences. These epitopes were assigned to six overlapping epitope binding regions and mapped onto the structural model of the IBV M41 S. A comparison with existing data on IBV S epitopes showed that some of the epitopes identified here coincided well with sites where neutralising antibodies are experimentally detected to bind. Notably, our study also validated that synthetic peptide mimics of the three epitope sequences selected for further investigation, due to their accessibility, were recognised, to a varying degree, by complex polyclonal serum. However, the amino acid residues responsible for controlling the structural conformation and properties of these epitopes are not yet determined. Therefore, for a true appreciation of the potential of these epitopes to contribute towards the rational design of broad-spectrum IBV vaccines, it is necessary to conduct site-directed mutagenesis to identify changes in residue binding affinity.


Rapid sea level rise due to an ice sheet collapse has the potential to be extremely damaging the coastal communities and infrastructure. Blocking deep warm water with thin flexible buoyant underwater curtains may reduce melting of buttressing ice shelves and thereby slow the rate of sea level rise. Here, we use new multibeam bathymetric datasets, combined with a cost–benefit model, to evaluate potential curtain routes in the Amundsen Sea. We organize potential curtain routes along a “difficulty ladder” representing an implementation pathway that might be followed as technological capabilities improve. The first curtain blocks a single narrow (5 km) submarine choke point that represents the primary warm water inflow route towards western Thwaites Glacier, the most vulnerable part of the most vulnerable glacier in Antarctica. Later curtains cross larger and deeper swaths of seabed, thus increasing their cost, while also protecting more of the ice sheet, increasing their benefit. In our simple cost–benefit analysis, all of the curtain routes achieve their peak value at target blocking depths between 500 and 550 m. The favorable cost–benefit ratios of these curtain routes, along with the trans-generational and societal equity of preserving the ice sheets near their present state, argue for increased research into buoyant curtains as a means of ice sheet preservation, including high-resolution fluid-structural and oceanographic modeling of deep water flow over and through the curtains, and coupled ice-ocean modeling of the dynamic response of the ice sheet. Collapse of the West Antarctic ice sheet is a major risk, with ocean melting having already destabilized the equivalent of a meter of global sea rise, and the Thwaites ice shelf recognized as the most vulnerable sector of the ice sheet. Reducing greenhouse gas emissions cannot stabilize the ice sheet, which needs to re-establish contact and regain buttressing on sea floor highs. We explore siting options for subsea curtain barriers that might be able to block deep warm ocean currents and thus protect Thwaites and other nearby ice shelves. Our preliminary cost–benefit analysis helps to clarify societal options in the event of rapid ice sheet retreat and motivates future work on the glaciological, oceanographic, engineering, ecological, and governance questions raised by these barriers.Ice sheet collapse has long been one of the most feared consequences of climate change, particularly for the West Antarctic ice sheet . This fPreliminary research into targeted glacial geoengineering  aims to To be clear, we do not advocate for deployment of ice sheet interventions in either the short or the medium term. There are many scientific, engineering, and political unknowns that need to be addressed before even small pilot projects could begin, much less large-scale deployment in the Amundsen Sea. What we aim to do here and in Keefer et al. (in press) is to develop an agenda for future research, organized around a schematic proposal with many intermediate steps along the way. Here we hope to make a small contribution forwards using newly released multibeam bathymetry data  to quantWe choose four candidate routes to investigate, three of which can be grouped into a combined proximal route, and one of which is a standalone distal route Fig. . The disWithin the proximal routes, T2 has the best value and the lowest cost. This route is characterized by a single narrow trough less than 800 m deep and 4–5 km wide Fig. a. This iThe next best value of the proximal routes is IB. The IB route crosses multiple narrow troughs nearly 1000 m deep Fig. b. In betThe lowest value proximal route is T3, which protects the secondary CDW inflow pathway towards the western half of Thwaites Glacier. Nearly half of the T3 route is reliant on gravity inversions rather than multibeam data for its bathymetric profile. Because T3 protects much less of the ice sheet, its value ratio is much lower, a clear outlier with respect to the other three routes . The smoother bathymetry of the distal route, by contrast, allows for a curtain that is consistently lower to the seafloor, with a maximum height only slightly larger than the mean height Table . Thus, wThe biggest research priorities needed to evaluate the potential of curtains as a glaciological intervention are (1) high-resolution fluid-structural modeling and tank tests to determine the “leakiness” of the curtain design and (2) coupled ice-ocean modeling to determine circulation changes, melt changes, and ice-dynamic response as a result of curtain installation.The potential for “leakiness” of the curtains is one of the largest unknowns impacting their effectiveness. We envision that the curtains would be composed of separate overlapping panels in order to allow them to deform around impinging icebergs and minimize damage . This design will produce gaps between adjacent panels, through which some water will leak, especially if the curtains experience fluid-structural oscillations. In addition, the literature on bathymetric sills suggests that even a gapless curtain would not be expected to completely block the transport of deep warm water. Water masses can be drawn up from depth and transported over bathymetric sills even when the sill crest is shallower than the origin of the water mass e.g. , 33). In. In33)).In addition, research is also needed on the larger-scale circulation changes curtains might cause, and on whether those changes would help or hinder the effectiveness of the curtains. For example, Gurses et al.  used an Finally, some have objected to research into targeted geoengineering because “the limited resources available should instead be used to address the root causes of accelerating ice loss—namely emissions and human-induced climate change” . This reThere is therefore a strong argument to be made in favor of preserving the ice sheets as close to their present-day configuration as possible, on the basis of both socioeconomic and intergenerational equity. The alternative to ice sheet preservation is not emissions reductions; rather, in the event that the tail risk of rapid ice sheet collapse is realized, the alternative to ice sheet preservation will be widespread spending on local coastal protection by nations and communities that can afford it, and retreat from the coast for those who cannot. Stopping, or at least slowing, the sea level rise at the source is a harm-reduction measure that benefits vulnerable communities nearly equally. Any construction in the Amundsen Sea would require detailed impact assessments to be done under the framework of the Antarctic Treaty System and especially the Madrid Protocol. The potential impact of curtains on marine life needs to be thoroughly researched before any intervention could begin. While preliminary research can be undertaken within the existing limits of the Treaty, deployment will probably require unanimity amongst the parties, which is a high bar to clear . Nonethelimited evidence and low confidence to describe the possibility that various forms of geoengineering, including localized interventions, might reduce the sea level commitment from MISI  report used the calibrated language rom MISI . We agrerom MISI , it willOur results suggest that trough T2 may be the highest-leverage location for an intervention, where the primary ingress route for CDW into the Thwaites sub-ice cavity may be blocked at 550 m depth with a curtain only 4 km long, and a cost function an order of magnitude smaller than any of the other routes we considered. However, this conclusion depends on the assumption that blocking T2 will not cause water to reroute through T3; if that assumption is violated, then T2 could be combined with IB and T3 to produce a combined proximal route that protects all of PIG and Thwaites, albeit for substantially higher cost. The longer OB route has an easier seabed setting than the proximal route and protects Crosson ice shelf as well. In our simplified cost–benefit analysis, all of the candidate routes reach peak value at target blocking depths of 500–550 m. This finding can serve as a starting point for future experiments with ocean circulation and ice flow models, but it should not be viewed as the final word on blocking depth.Indeed, the primary purpose of this and our companion paper is not to propose a finished design, but rather to make a plausible schematic proposal that can then be the focus of future research. Both fluid-structural modeling and scale model tank tests are needed to quantify mixing of warm water through and over the curtains and to understand curtain deformation during iceberg encounters. Mixing parameterizations derived from these experiments can be used in ocean model experiments to quantify how different blocking heights and curtain routes actually translate into changes in ice shelf basal melt rates. Dynamic ocean models will also be able to evaluate the extent to which transient upward excursions in the thermocline may overtop the curtain, which could potentially push the best-value blocking depths to shallower (and thus more expensive) levels than those calculated with our benefit function based on the mean oceanographic profile. Changes in melt rates can be used as forcing for ice flow models to determine whether an intervention would indeed produce societally beneficial changes in the magnitude, rate, or timing of sea level rise. Coupled ocean circulation and ice flow modeling is needed to produce more robust estimates of the response of the ice-ocean system. Attention is needed from marine biologists to quantify the likely side effects of curtains on local marine ecosystems, both during the initial installation and in the long term. Before any intervention could be attempted in the Amundsen Sea, smaller pilot projects would need to be constructed at more accessible locations elsewhere in the world to demonstrate feasibility, verify models, and of course, to identify unanticipated consequences that may have gone unnoticed in earlier research. Last, but not least, work is needed from political and social scientists to understand how the peoples of the world view deliberate interventions in the ice sheet system, and to devise legitimate decision-making structures that can balance the needs of many different stakeholders within a context of humanity’s collective need for a stable sea level.Keefer et al. (in press) estimate the cost of 80 km long curtain to be about 40–80 billion USD spread over a decade of construction, followed by 1–2 billion per year in maintenance. Costs estimated for deployment of stratospheric aerosol injection are put at 7–70 billion USD/year , while cWe manually pick candidate curtain routes on the bathymetric map Fig. , guided The two fundamental assumptions underlying our method are (1) the dominant factor controlling variance in curtain cost between different routes in the Amundsen Sea will be the bathymetry, which in many cases is known; and (2) the dominant factor controlling curtain effectiveness will be the height of the curtain top within the thermocline, which is also known. Factors that make work in the Amundsen Sea very difficult, such as transportation across rough seas from Punta Arenas, the need for hardening against sea ice, and work stoppages due to iceberg obstruction, are factors shared between all potential routes; the variation between routes should be due to site-specific factors, of which bathymetry is the most important. Thus, by comparing the known oceanography with the known bathymetry, and correcting for the fact that different curtain routes protect different ice sheet catchments, we can estimate the relative value of different curtain routes and thus provide a starting point for future research.Our multi-part cost function reflects the major factors that increase the degree of difficulty in construction and maintenance of underwater curtains. Those factors are (1) curtain height above the seabed, (2) depth of foundation, and (3) likelihood of iceberg impacts to the foundation. Since each component of the cost metric takes the form of an integral along the path of a potential curtain route, we combine them by expressing them all in units of length. The resulting cost function can be regarded as the difficulty weighted length of a particular curtain route for a given blocking depth. Each term has a single free parameter representing a characteristic scaling value of the variable in question.Oceanographic loads on the curtain increase strongly with height above the seabed. Greater loads place greater demands on the entire structure, requiring more tensile strength and greater buoyancy in the curtain panels as well as more robust foundations. Hydrostatic loads scale quadratically with curtain height , while hydrodynamic loads only scale linearly, so we use a quadratic height term in our cost function:CH is the height component of the cost function, H(x) = DB(x) − DT is the height of the curtain above the seabed, and H0 is the characteristic scaling height for static loads. We take H0 = 150 m; this corresponds to a tensile load in the curtain of approximately 3 × 104 N m−1, a density contrast of Δρ = 0.5 kg m−3, and a lean angle of 30° .where Construction in deep water is likely to be harder than construction in shallow water. Therefore, we add a seabed-depth-weighted term to our cost :function,CD is the depth component of the cost function and D0 is a characteristic scaling depth. We choose D0 = 700 m, the base of the thermocline, as the purpose is to block deep warm water. Thus, developing the ability to install curtains in waters about as deep as the thermocline is a common cost, the same for all routes. By contrast, routes that cross troughs much deeper than the thermocline will be penalized, as they may require the development of additional advanced installation techniques not needed for shallower routes.where Shallower curtains will be more impacted by icebergs than deep curtains. Furthermore, we expect that impacts between iceberg keels and the foundation modules will be more damaging than impacts with the flexible part of the curtains, which are designed to deform around the bergs. We therefore construct a cost function that scales inversely with foundation depth:Dberg is a characteristic iceberg keel depth. We take Dberg = 400 m as a representative ice shelf draft. Thwaites’ frontal ice shelf draft is about 300 m, while PIG has a frontal draft of about 500 m  length, and the weights 0.5 and 0.25 have been chosen to provide an illustrative example. We have chosen to emphasize the curtain height term in the overall cost function as increasing oceanographic loads on the curtain, combined with the requirement that the curtain remain roughly upright within the water column, produce cascading increases in the design requirements for every component of the curtain, including tensile strength, lateral reinforcement, buoyancy, and foundation mass; as our design analysis indicated , curtain height above the seabed is likely to be the single biggest determinant of curtain difficulty.where S) . We use the reduction in TF between the bottom of the thermocline, taken to be 700 m, and the curtain top depth as our measure of effectiveness. For the OB curtain route  casts in the World Ocean Database in the Amundsen Sea, binned them by depth, and computed mean profiles of conservative temperature (Θ) and absolute salinity (S) Fig. . We usedute Fig. , the maxute Fig. c, so we R . Scaling the TF reduction by these values allows us to produce a benefit function that is comparable between different routes, and the ratio of the benefit function over the cost function can be used to select a best-value blocking depth for each curtain route.Additionally, different curtains protect different regions of the ice sheet, and some regions are more likely to trigger an unstable collapse than other regions. For example, an intervention that only protected Thwaites would be less likely to prevent an ice sheet collapse than an intervention that protected both PIG and Thwaites. We therefore use the volume above flotation (VAF) of the glaciers protected by each curtain as a proxy for this area-protected effect. We measure VAF totals using the ice thickness map from Ref.  and the While this approach to calculating a benefit function is crude, these weights do capture the general picture of societal risk posed by the different ice-dynamic regions of the Amundsen Sea. A full treatment of this problem would require a coupled ice-ocean model with a large enough ensemble to meaningfully evaluate the change in probability that various societally significant thresholds for sea level rise magnitude, rate, or timing are exceeded when ocean heat flux through key specific choke points is reduced. If a fully coupled model is not available, then melt rates from a standalone ocean model could be used as forcing for a standalone ice sheet model. In either case, the ocean model would either need to be informed by high-resolution fluid-structural modeling that quantified heat flux reduction as a function of curtain height and ambient oceanographic conditions, or else the “leakiness” of the barrier could be treated as a free parameter whose influence could be explored alongside other model parameters. As with any geophysical problem, there is a spectrum of complexity at which the problem of quantifying curtain benefit could be approached. We leave more advanced treatments of the problem for future work. For our purpose, here we aim for the lowest level of complexity that is informed by both the known oceanography and the relative societal importance of different ice sheet sectors.