Pedicle screw loosening is one of the main complications after pedicle screw fixation. However, there are few reliable measures for prediction of screw loosening. The current study was carried out to find an effective method to use preoperative CT scanning as a predictor of screw loosening in the elderly patients and provide guidance for preoperative surgical planning.  Patients who were treated with lumbar pedicle screw fixation procedure in our department for degenerative lumbar disorders between January 2015 and January 2021 were retrospectively included in the current study. CT scan attenuation of each vertebra was measured with Hounsfield units (HU). Screw loosening was determined in postoperatively X-ray tests. One-way analysis of variance (ANOVA) and receiver operating characteristic (ROC) curve analysis were carried out with IBMSPSS 24.00 software. P > 0.05). The average HU value of lumbar vertebra was 122.4 ± 32.8 HU in the screw loosening group and 142.4 ± 38.2 HU in the control group, and the difference was significant (P < 0.01). ROC curve analysis revealed that the average HU value of L1-L5 has a relatively larger area under the curve (AUC) of 0.689 (95% CI: 0.605-0.773). With the sensitivity of 68% and specificity of 57%, a HU cut-off value of ≤124 HU is a plausible cut-off point to predict screw loosening.  Screw loosening was observed in 44 of 215 patients  during a mean follow-up time of 19.0 ± 11.2 months (range 12-32 months). No significant differences were found among the patients concerning patient gender, BMI, habit of smoking, and whether or not the patient had diabetes or suffered from spondylolisthesis ( A prospective CT scan HU value-based prediction can be used to decide whether or not to use screw augmentation methods. A cut-off L1-L5 average HU value of 124 HU can be used as an independent risk factor for screw loosening in instrumented lumbar vertebra. More predictive indexes should be involved to achieve higher sensitivity and specificity in future clinical practice. Treatment of various degenerative spinal diseases such as degenerative scoliosis, lumbar spinal stenosis, and spondylolisthesis involves surgical intervention, and pedicle screw fixation is the current standard technique to achieve adequate stabilization after removing a significant part of the spinal structure , 2. AftePrevious investigations have demonstrated that patients with osteoporosis are at much higher risk of screw loosening than patients with normal bone mineral density because of the significantly reduced integration of the screws at the screw-bone interface –10. As aAll the procedures were approved by the ethical committee of the Sixth Affiliated Hospital of Xinjiang Medical University, and written consent was achieved from the patients before the treatment. Patients who were treated in our hospital with pedicle screw-based lumbar spine surgeries between January 2015 and January 2021 were retrospectively included in the current study. Other indications for inclusion are as follows: had pre- and postoperative CT scans, followed up for at least 12 months; patients with previous history of spinal surgery, patients with congenital scoliosis, spinal fracture, spine tumor, and ankylosing spondylitis; patients with other conditions that may affect bone metabolism such as long-term steroid application; patients with insufficient screw anchorage; and patients who received revision surgery within a year due to infection, hematoma, neural injury, and adjacent level disk degeneration.All CT scans were performed in our Hospital's Department of Radiology. All the patients received a CT scan . Voltage of the Scantron was 120 kV. Two independent examiners used PACS system  to measure the HU value. Besides radiological parameters, basic patient demographic data including patient age, height, and weight were recorded, and the body mass index (BMI) was calculated.A region of interest (ROI) within the cortex of vertebra was determined on a horizontal plane of at the level of pedicle, from which attenuation in HU was computationally generated .All the patients received X-ray scans during and at the end of follow-up visits. Two senior spine surgeons independently evaluated screw loosening and bone fusion. More than 1 mm of clear zone around screw was used as a reference to diagnose screw loosening. Nonfusion was diagnosed when anterior translation was greater than 3 mm, rotation more than 5°, or there was no continued trabecular bone in sagittal flexion-extension X-ray film.X2 and Fisher's exact test. ROC curve analysis was carried out to evaluate the diagnostic value of CT scans.Statistical calculations were performed using SPSS 24.0 . Intraclass correlation coefficient (ICC) was used to test if the measured HU values are similar between the two independent researchers. Normal distribution was reviewed prior to statistical analyses. Continuous variables were recorded as mean ± standard deviation. Quantitative measures were recorded as percentage. One-way ANOVA was applied to compare the variables between groups. Quantitative data was analyzed by P > 0.05) (P < 0.001).A total of 215  patients were included in the final analysis. Indication for surgery includes spondylolisthesis, spondylodiscitis, degenerative scoliosis, or spinal stenosis. The mean duration of the interval was 19.0 ± 11.2 months (range 12–32 months). No significant differences were found among the patients concerning patient gender, BMI, habit of smoking, and whether or not the patient had diabetes or suffered from spondylolisthesis ( > 0.05) . ICC of Among potential risk factors of screw loosening, the incidence of pseudoarthrosis and the difference between the HU values of the lumbar vertebrae were the most significant. Considering that pseudoarthrosis could be the result of screw loosening, and that pseudoarthrosis can only be found during follow-up after surgery, and the purpose of the current study is to find the predictive factors for screw loosening, here we carried out ROC curve analysis on the HU values of the lumbar vertebrae to assess their diagnostic value for screw loosening. ROC curve analysis revealed that the average HU value of L1-L5 has relatively larger area under the curve (AUC) of 0.689 (95% CI: 0.605-0.773) .However, ROC curve analysis failed to provide a cut-off HU value with high sensitivity and specificity to predict the incidence of screw loosening. Although pseudoarthrosis may not be the cause of screw loosening, it is correlated with clinical symptoms and the necessity for a revision surgery. Therefore, we chose specific HU value regions and, respectively, calculated the incidence of screw loosening and pseudoarthrosis in those regions. With the sensitivity of 68% and specificity of 57%, a HU cut-off value of ≤124 HU was a plausible cut-off point to predict screw loosening.Pedicle screw loosening is one of the main complications associated with internal fixation procedure in spine surgeries. It is responsible for failure of bone fusion, consistent pain, and reduced mobility. In some severe cases, a revision surgery is needed to alleviate those symptoms caused by pedicle screw loosening . In prevCement augmentation amid to increase screw-bone integration was used for screw anchorage for more than two decades. Although this method could increase the pull-out forces by twice as much as standard options, it may cause some fatal complications such as pulmonary cement embolism. Therefore, it is only rational to apply this technique only when the patient is in under high risk of screw loosening and its complications. The goal of our current research was to find a high sensitivity predictive reference from conventional computed tomography scans for postoperative screw loosening, and to help with the preoperative planning for measures for screw augmentation without increasing the medical bills or radiation foe patients. The previous research has already shown the correlations between bone mineral density and the incidence of screw loosening and the correlation of HU values in opportunistic CT scans with the results of qCT and dual X-ray absorptiometry (DEXA) tests . The meaAlthough there are no widely accepted methods to evaluate screw stability, several authors chose more than 1 mm of clear zone around screw to use as a reference to diagnose screw loosening , 22. SanThe ratio of screw loosening was reported to be between 0 and 54.7 percent –28. HoweDifferent cut-off values of HU were proposed by various authors to predict a high likelihood of screw loosening. In the study of Bokov , when thRegions for HU measurement were chosen manually by two independent observers in our study. This could raise concerns over the repeatability of those results. However, high intraclass correlation coefficients (ICCs) regarding interobserver reliability indicate high repeatability of this method.Screw loosening is considered a negative influencing factor for successful bone fusion. However, loosened screws do not necessarily lead to spinal instability that requires revision surgery. In our study, while the ratio of successful bone fusion was significantly higher in nonscrew loosening group than in the screw loosening group, the number of patients who needed revision surgery is not significantly different between the two groups. A larger sample size maybe needed to further verify the reliability of those cut-off points. While the current consensus on the pullout strength of pedicle screws is that approximately 80% of the caudocephalad stiffness and 60% of the pullout strength of the pedicle screw depended on the pedicle rather than the vertebral body , and thaA prospective CT scan HU value-based prediction can be used to decide whether or not to use screw augmentation methods. A cut-off L1-L5 average HU value of 124 HU can be used as independent risk factor for screw loosening in instrumented lumbar vertebra.


The propagation of ocean surface waves within the marginal ice zone (MIZ) is a defining phenomenon of this dynamic zone. Over decades of study, a variety of methods have been developed to observe and model wave propagation in the MIZ, with a common focus of determining the attenuation of waves with increasing distance into the MIZ. More recently, studies have begun to explore the consequences of wave attenuation and the coupled processes in the air–ice–ocean–land system. Understanding these coupled processes and effects is essential for accurate high-latitude forecasts. As waves attenuate, their momentum and energy are transferred to the sea ice and upper ocean. This may compact or expand the MIZ, depending on the conditions, while simultaneously modulating the wind work on the system. Wave attenuation is also a key process in coastal dynamics, where land–fast ice has historically protected both natural coasts and coastal infrastructure. With observed trends of increasing wave activity and retreating seasonal ice coverage, the propagation of waves within the MIZ is increasingly important to regional and global climate trends.This article is part of the theme issue ‘Theory, modelling and observations of marginal ice zone dynamics: multidisciplinary perspectives and outlooks’. Functionally, these definitions are similar, because ocean waves typically propagate into sea ice from open water through a region that is distinct from the main pack ice. MIZs exist year-round at the edges of both Arctic and Antarctic sea ice, though the locations and extents are heavily modulated by seasonal cycles.Decades of research on wave propagation in the MIZ have produced a large knowledge base, much of which is reviewed and advanced in this theme issue. The theoretical bases are wide ranging, as are the field data with which to evaluate the various theories . LaboratAt fixed observing locations, the seasonal evolution is a defining feature of the MIZ.  (i)summarize present knowledge regarding wave propagation in the MIZ; (ii)connect wave propagation in the MIZ to the larger air–ice–ocean–land system; and (iii)describe key feedback mechanisms relating MIZ wave propagation to regional and global climate trends.Wave and ice data from the BGOS moorings are used throughout to demonstrate the scales and magnitudes of these processes. This has an obvious bias of presenting signals most relevant to the western Arctic. Where possible, this is balanced by literature citations and information from the Antarctic and the eastern Arctic.The intent of this review is to. 2The canonical model for wave propagation in the MIZ is an exponential decay of spectral wave energy  decades ,10, incl decades –13. (a)The attenuation rate y, α=afb . The pra the MIZ ,15. In t the MIZ –19.e region ,20, and d 100 km .Figure Obscured by the bulk The lack of energy at high frequencies far within the MIZ has been a measurement challenge for decades, with long-running debates regarding the inference of a ‘roll-over’ in the spectral attenuation rate. Recent re-analysis of several published datasets indicates that most high-frequency measurements far within the MIZ are contaminated by poor signal-to-noise ratios, such that reported roll-overs are spurious . Direct Although less-well constrained than the frequency dependence, attenuation rates also have a strong dependence on ice type, ice coverage, ice thickness and floe size distribution. Ice concentration is the most common input to models , though  (b)There is a long-standing debate over the physical mechanisms of wave attenuation in sea ice and their proper mathematical representation –30. The Recent observational work suggests that dissipation is the dominant mechanism, in which wave energy is lost by the breaking up of sea ice  and by tThe distinction between dissipation and scattering mechanisms is important, even though either process can result in attenuation. When waves are dissipated, energy and momentum are released from the waves and cause dramatic changes to the ocean–ice system. In observations, this is difficult to discern from wave measurements alone, but measurements of ocean turbulence and ice motion can provide proxy evidence for energy and momentum transfers . Fully c (c)In addition to processes that decrease (attenuate) wave energy in the MIZ, wind forcing may increase wave energy locally. This complicates interpretation of attenuation rates, which are typically estimated by comparing observations of en water . At presen water . Anotheren water ,41.The concept of effective fetch can be recast as an efficiency of wind work at the surface, relative to open-water efficiency . Figure Additional analysis of the BGOS observations in this theme issue demonstrates the importance of wind-wave generation in partial ice coverage and leads within the MIZ . Noting . 3A fundamental and universal treatment of wave propagation in the MIZ must include conservation of energy and momentum. The details for the wave physics depend on the relative importance of dissipation and scattering; the details for the rest of the air–ice–ocean–land system depend on what happens to that energy and momentum after being released from the waves. This section explores the processes that are driven by wave propagation into the MIZ. These processes are distinct from the local wind work discussed above; this section is concerned with the energy and momentum that waves carry to the MIZ from open water . A simple analogy is to think of the MIZ as a surf zone, in which the energy and momentum carried by waves are released to other processes. (a)Waves carry a flux of momentum, usually referred to as the ‘radiation stress’,  the MIZ –47. The The magnitude and direction of this effective stress are dependent on the relative alignment of the wave propagation and the axis of the gradient, because the radiation stress is a tensor with four elements, (the MIZ) . In case sea ice . In eith sea ice . (b)Waves carry a flux energy, and gradients in this energy flux are the rates at which energy is transferred from the waves to the ice and ocean of the MIZ. This can be described as a rate of work done, 2 m s−1 . The wavRecent works suggest that significant wave energy is lost in the form of turbulence in the upper ocean ,37, thoue. wind) ,43. The e. wind) . This tue. wind) . There ae. wind) . The proe. wind) .. 4As the planet warms and sea ice losses accelerate, we must consider whether and how wave propagation in the MIZ impacts large-scale, long-term trends. Here, we examine how the processes described above manifest in the climate system and their associated impacts. We look for trends in how the sea ice affects the waves, how the waves affect the ice, and how these then impact the coasts. (a)In the Arctic, it is now well documented that seasonal wave activity is increasing as a direct result of reduced sea ice and increased open-water fetch distances –60. ThisThe increasing wave activity means that more momentum and energy are transferred into an MIZ that is probably growing in extent . Assumin (b)Given that the increase in open-water fetch distances increases wave activity, there is hypothetical feedback mechanism in which waves cause ice retreat that continually increases the fetch (causing larger waves that cause further ice retreat). Although it is clear that storms can cause dramatic ice loss events ,67, attrThere are isolated examples of wave influences on ice loss, such as with Antarctic ice sheets  and in l (c)In coastal regions, any waves not fully attenuated by sea ice may reach the shore. In this case, the analogy of the MIZ as a surf zone becomes explicit. The gradients in wave energy and momentum drive coastal processes, mostly notably near-shore circulation and sediment transport. Wave energy dissipated as turbulence in the surf zone may also increase water temperatures , which iMost Arctic coasts are eroding rapidly ,77, and . 5 —What is the dependence of the attenuation rate on ice type and thickness? —How does heterogeneity in the ice affect wave attenuation? —What nonlinear wave mechanics, if any, are important to attenuation? —What controls the relative amount of wave energy lost to ice fracture/collisions versus to ocean turbulence? —What balances the wave momentum lost to ice motion? —How do local winds generate waves in partial ice coverage? Answers to these process-level questions will support the continued development of fully coupled wave–ice–ocean–atmosphere models, which are needed for operational and climate forecasts in polar regions. The prior decades of work combining theory, experiments, observations and models provide productive examples to follow. The future holds more tools for observations, more realistic experiments and more computational power to incorporate this knowledge into the forecasts. Time to get back to work.Attenuation of waves within the MIZ is a defining feature of the transition from open water to complete ice coverage. There is consensus that attenuation is a spectral process (i.e. per wavelength or frequency), and there are sufficient parametrizations of this process to be useful in wave forecasting. There is also an emerging consensus that wave energy and momentum are lost (i.e. dissipated) to the surrounding ice and ocean, rather than simply redirected (i.e. scattered). Much remains to be learned, including answers to the following questions.


Antrodia cinnamomea, a Basidiomycota fungus, is a medicinal mushroom indigenous to Taiwan and a known prolific producer of bioactive terpenoids, but little is known regarding the enzymes involved in the biosynthetic pathways. Here, we applied a genome mining approach against A. cinnamomea and discovered two non-canonical UbiA-type TPSs that both synthesize (+)--α-bisabolene (1). It was determined that two tailoring enzymes, a P450 monooxygenase and a methyltransferase, install a C14-methyl ester on the bisabolene scaffold. In addition, four new bisabolene derivatives, 2 and 4–6, were characterized from heterologous reconstitution in Saccharomyces cerevisiae. Our study uncovered enzymatic tools to generate structurally diverse bisabolene NPs.Terpenoids represent the largest structural family of natural products (NPs) and have various applications in the pharmaceutical, food and fragrance industries. Their diverse scaffolds are generated via a multi-step cyclization cascade of linear isoprene substrates catalysed by terpene synthases (TPSs). Bisabolene NPs, which are sesquiterpenes (C15), have wide applications in medicines and biofuels and serve as bioactive substances in ecology. Despite the discovery of some canonical class I TPSs that synthesize bisabolenes from plants, bacteria and insects, it remained unknown whether any bisabolene synthases from fungi could produce bisabolenes as a main product. This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’. They are close to human life and have various applications as flavours (e.g. menthol), food colourants (e.g. carotenoids) and medicines  . Terpeno5n isoprene substrates by terpene synthases (TPSs) . He. He15), a dulcis . In addis A H3N2 .Figure α-, β- and γ-bisabolene (b). The bisabolane scaffold is generated from C15 farnesyl diphosphate (FPP) catalysed by class I TPSs, which involve an ionization cyclization reaction -1[ and then led to the formation of 13C6]-2[ and 13C6]-3-3[ was further purified from the 3 l yeast culture fed with 1-13C-sodium acetate after concentration 100 times. The 13C-NMR spectrum of 13C6]-3[ showed that the C1, C3, C5, C7, C9 and C11 signals were increased compared to those of 3, indicating the position of carbons that were isotope labelled (1 proceeds through C1–C6 bond formation (b). This could be enabled by the initial isomerization of FPP to give nerolidyl diphosphate (NPP) [S-bisabolyl carbocation intermediate is generated by Tps1A, which therefore determines the stereochemistry of C6 on 1. Deprotonation at C8 generates the cis-configuration of 1 (To examine the cyclization mechanism of labelled . The resormation b. This cte (NPP) –42. Follion of 1 . The res (f)4 and 5, different combinations of genes on tps1 were transformed into S. cerevisiae. As the family of P450 monooxygenases are usually capable of performing C-H oxidation on the terpene scaffold and coded next to a terpene cyclase gene, we coexpressed tps1A/G/H with the possible post-modifying enzymes encoded by tps1B/C/D/E and tps1J/K/L, respectively. LC-MS analysis of the tps1A/G/H with tps1B/C/D/E strain showed no difference compared to the strain expressing tps1A only, while the tps1A/G/H with tps1B/C/D/E or D/E strain both showed the production of 5 and 6 . Next, upon coexpression of tps1A/D/G and tps1A/D/H in yeast, only the tps1A/D/H strain yielded 5 and 6 (b). The above results indicated that the minimal set of genes in the biosynthesis of 5 is terpene cyclase (Tps1A), P450 monooxygenase (Tps1H) and methyltransferase (Tps1D).To understand the function of genes that biosynthesize  5 and 6 b. The ab (g)S. cerevisiae strain that overexpressed tps1H were prepared for in vitro characterization [1 with nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) yielded 5, while no product was observed with the yeast microsomal extract control (a). The results demonstrated that Tps1H catalyses six-electron oxidation at C-14 to form the carboxylic group on 5. To verify the function of methyltransferase Tps1D, recombinant Tps1D with a His6-tag from S. cerevisiae was purified into a soluble form . Incubating 5 with Tps1D and SAM led to the formation of 6 (b). The results confirmed the role of Tps1D in installing a methyl group on the 14-carboxylic group of 5 to form a methyl ester on 6. Furthermore, the incubation of 1 with Tps1H and Tps1D together with SAM and NADPH led to the production of 6 . These results demonstrated the enzymatic synthesis of 5 from 1 in a one-pot reaction.To verify the function of the P450 monooxygenase Tps1H, microsome extracts from the rization . The inc control a. The reion of 6 b. The re. 4α-(E)-bisabolene synthase (AgBIS), are soluble enzymes that contain conserved DDXXD and  DXXXXXE metal-binding motifs, which are known to bind to divalent metal ions and trigger the cyclization of FPP through an ionization-dependent mechanism. Unlike canonical TPSs, the (+)--α-bisabolene synthases Tps1A and Tps2A that we characterized here are membrane-bound UbiA-type TPSs. Sequence alignment of Tps1A and Tps2A to fungal β-bergamotene synthase (Fma-TC) and (–)-cyatha-3,12-diene synthase (EriG) showed that the NXXX(G/A)XXXD and QDXXDXXXD motifs are the conserved catalytic domains in these UbiA-type TPSs, and this was different from the NDXXDXXXD and DXXXD motifs, which are conserved in the UbiA prenyltransferase superfamily (a). Phylogenetic analysis of Tps1A and Tps2A with other UbiA prenyltransferases  from diAbies are known to suppress insect growth and reproduction , and some reductases , which may participate to convert 1 into juvabione-like derivatives with Tps1H and Tps1D. The absence of further downstream products when coexpressed may be owing to the lack of other pathway enzymes or may be because some enzymes were not functional in S. cerevisiae. It is intriguing if the end pathway products of tps1 BGC may participate in any mushroom–insect interaction. Alternative heterologous expression systems using other genus-closed strains deserve further investigation.Oxygenated bisabolane sesquiterpenes, todomatuic acid and its methylester juvabione from the genus oduction  [9,46,47S,Z)-α-bisabolene (1). Four new bisabolene derivatives 2 and 4–6 were characterized from heterologous reconstitution in S. cerevisiae. In addition, two enzymatic machineries, a P450 monooxygenase (Tps1H) and a methyltransferase (Tps1D), were discovered and demonstrated to modify C14 of the bisabolene skeleton. Our study will provide opportunities to generate the chemical and structural diversity of bisabolene NPs using enzymatic approaches.In conclusion, we identified two intramembrane UbiA-type sesquiterpene synthases, Tps1A and Tps2A, that convert FPP into (+)-(


Luxeptinib (LUX) is a novel oral kinase inhibitor that inhibits FLT3 and also interferes with signaling from the BCR and cell surface TLRs, as well as activation of the NLRP3 inflammasome. Ongoing clinical trials are testing its activity in patients with lymphoma and AML. This study sought to refine understanding of how LUX modulates the earliest steps downstream of the BCR following its activation by anti-IgM in lymphoma cells in comparison to ibrutinib (IB). LUX decreased anti-IgM-induced phosphorylation of BTK at Y551 and Y223 but its ability to reduce phosphorylation of kinases further upstream suggests that BTK is not the primary target. LUX was more effective than IB at reducing both steady state and anti-IgM-induced phosphorylation of LYN and SYK. LUX decreased phosphorylation of SYK (Y525/Y526) and BLNK (Y96) which are necessary regulators of BTK activation. Further upstream, LUX blunted the anti-IgM-induced phosphorylation of LYN (Y397) whose activation is required for phosphorylation of SYK and BLNK. These results indicate that LUX is targeting autophosphorylation of LYN or a step further upstream of LYN in the cascade of signal generated by BCR and that it does so more effectively than IB. The fact that LUX has activity at or upstream of LYN is important because LYN is an essential signaling intermediate in multiple cellular signaling processes that regulate growth, differentiation, apoptosis, immunoregulation, migration and EMT in normal and cancer cells. The B-cell receptor (BCR) signaling pathway plays a key role in B-cell development . Apart fin vitro screens, LUX also inhibits recombinant wild type and C481S mutant BTK at 8.4–27 nM and 2.5–13.1 nM [The BCR signalosome has a central role in regulating cellular functions in B-cells , 13, 14.–13.1 nM . However–13.1 nM  but the Luxeptinib was provided by Aptose Biosciences. Ibrutinib (#HY-10997) was purchased from MedChem Express. Goat anti-human IgM (#109-005-043) was purchased from Jackson ImmunoResearch. Human lymphoma cell lines SU-DHL-6 (#CRL-2959), JeKo-1 (#CRL-3006), RL (#CRL-2261) and Fetal Bovine Serum (#30–2020) were obtained from ATCC. SU-DHL-6 is a human lymphoblast-like cell line, JeKo-1 is a mantle cell lymphoma cell line and RL is a human non-Hodgkin’s lymphoma B cell line. RPMI-1640 medium (#11875–093) and penicillin/streptomycin (#15070063) were purchased from Thermo Fisher Scientific. Antibodies against p-BTK (Y223) (#87141), BTK (#56044), p-PLCγ2 (#3871), p-SYK (#2710), SYK (#80460), p-BLNK (#3601), BLNK (#36438), p-CD79A (#5173), p-LYN/LCK/HCK/BLK (#70926), p-LYN (Y507) (#2731), LYN , pSrc family (#6943) and GAPDH (#2118) were purchased from Cell Signaling Technology. Phospho-BTK (Y551) (#ab40770) was purchased from Abcam. PLCγ2 (#sc-5283) and CD79A (#sc-20064) were purchased from Santa Cruz Biotechnology. All other reagents and chemicals used were of analytical grade and were obtained from Sigma-Aldrich or Thermo Fisher Scientific.2 and 37°C. For BCR crosslinking with anti-IgM, cells (1 x 106 cells/ml) were left untreated or pretreated with the indicated concentration of LUX or IB for 2 h followed by stimulation with anti-human IgM (10 μg/ml) for 15 min. Cells were then collected by centrifugation , washed with ice-cold PBS, lysed and analyzed by Western blotting.SU-DHL-6, JeKo-1 and RL cells were maintained in RPMI-1640 media supplemented with 10% FBS, 1X penicillin/streptomycin in a humidified incubator maintained at 5% COAfter the indicated treatments, the media was removed, cells were washed with ice-cold PBS and then lysed using cell lysis buffer . After a 5 min incubation in lysis buffer, the lysates were sonicated and cleared by centrifugation at 14,000 x g for 10 min at 4°C. Equal amounts of lysates were incubated with the primary antibody overnight at 4°C following manufacturer’s recommended dilution. Next day, Protein A/G agarose beads  were added and incubated with rotation at 4°C for 4 h. The beads were pelleted by centrifugation, washed five times with cell lysis buffer and analyzed by Western blotting. Immunoprecipitation specificity was confirmed by using appropriate isotype controls.After the indicated treatments, cells were washed with ice-cold PBS and lysed in RIPA buffer  or cell lysis buffer  containing protease and phosphatase inhibitors. Equal amounts of protein quantified by Bio-Rad Protein Assay Dye (#5000006) were separated by SDS-PAGE, transferred to nitrocellulose membrane and probed with the designated antibodies (1:1000 dilution). GAPDH (1:5000 dilution) was used as a loading control. The membranes were scanned using a LI-COR Odyssey IR imaging system capable of detecting antigen-antibody complexes labelled using fluorescent goat anti-rabbit  or goat anti-mouse . Phosphoproteins were probed first, followed by stripping and re-probing the same blot with the total antibody and loading control. Quantification of bands was done using Image Studio Lite software provided with the LI-COR imaging instrument or Image J. Phosphoprotein band intensities detected by Western blotting were normalized to the levels of total protein detected by respective antibodies. All others were normalized to GAPDH. Experiments were repeated at least 3 times and a representative Western blot from one experiment is shown in the figures.2 incubator and then absorbance was recorded at 450 nm using a plate reader. OD450 values at 72 h were normalized to OD450 values at 0 h for each cell line. Percent viability was calculated from the DMSO control and IC50 values were determined using GraphPad Prism Software.Cells were plated in 96-well plates and exposed to the indicated concentrations of drugs for 72 h. Cell viability was measured using a CCK8 kit . After 72 h exposure to drug, 10 μl CCK8 was added to the wells after which plates were incubated for 3 h in a COt-test was used for comparison between two groups. Ordinary one-way ANOVA was used for comparison between multiple groups. P < 0.05 was considered statistically significant. Quantification of Western blot data is mean ± SEM of 2 independent experiments unless otherwise stated.All analysis was performed using GraphPad Prism software. Unpaired Student’s The ability of LUX and IB to reduce the phosphorylation of BTK at Y551 was examined in SU-DHL-6 large cell lymphoma cells with and without activation of BCR by a 15 min exposure to anti-IgM. Cells were exposed to LUX and IB at concentrations ranging from 0.1–1 μM for 2 h prior to anti-IgM treatment. Stimulation with anti-IgM alone for 15 min did not change the phosphorylation levels of BTK at Y551. However, a 2 h treatment with LUX produced a concentration-dependent decrease in the levels of phosphorylated BTK (Y551), both in the absence and in presence of anti-IgM stimulation . A minimPLCγ2 is a well-documented substrate for activated BTK. When lysates were probed for phosphorylated PLCγ2, IB was found to be 10-fold more potent than LUX at inhibiting anti-IgM-induced phosphorylation of PLCγ2 at Y1217 . Thus, wBLNK is an adaptor protein that is phosphorylated in response to BCR stimulation and plays an important role in BTK activation. SYK-mediated phosphorylation of five canonical YXXP tyrosine residues  in the acidic and proline-rich domains of BLNK induces its association with SH2 domain-containing proteins such as Vav, BTK and PLCγ2 with the phosphorylated tyrosines on BLNK leading to their activation , 27, 28.Further upstream of BLNK in the BCR signaling cascade, spleen tyrosine kinase (SYK) is another important component of the BCR signaling pathway that is phosphorylated after stimulation of the BCR with anti-IgM. A 2 h treatment with LUX produced a concentration-dependent decrease in the levels of phosphorylated SYK (Y525/Y526) in both the absence and presence of anti-IgM stimulation in SU-DHL-6 cells . The abiLYN is a member of the Src kinase family that is a key upstream regulator of the BCR signaling pathway. Following activation, proteasome-mediated degradation of LYN is a necessary event in the regulation of BCR signaling . We obseUpon antigen stimulation of BCR, an activated form of LYN phosphorylates the cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs) of the immunoglobulin α (Ig-α) and Ig-β domains (CD79A/CD79B) of BCR leading to its activation and the activation of other downstream components such as SYK. When the lysates were probed for phospho-CD79A, it was found that the anti-IgM-induced phosphorylation of CD79A (Y182) was inhibited by LUX in a concentration-dependent manner, whereas IB had no effect . This prThe activation of LYN involves phosphorylation on Y397 and dephosphorylation of the inhibitory Y507 site , 31. We To specifically determine the effect on phosphorylation of LYN (Y397), the effect of LUX and IB on phosphorylation of LYN (Y397) was further confirmed by immunoprecipitating LYN and probing the LYN immunoprecipitates with an antibody for phospho-Src family kinase (Y416). LYN (Y397) is located in the catalytic domain and is structurally equivalent to Y416 of the Src family kinases. Phosphorylation at Y397 correlates with enzyme activation of LYN , 31. ProDephosphorylation of the inhibitory tyrosine Y507 in the C-terminal domain of LYN is a necessary event for LYN activation. When lysates were probed for phospho LYN (Y507), no effect of either LUX or IB in the absence or presence of anti-IgM stimulation was observed in SU-DHL-6 cells, JeKo-1 or RL cells . This suAlthough both LUX and IB are potent inhibitors of recombinant forms of BTK, and of cells engineered to express wild type and some mutant forms of this enzyme, they have different efficacy profiles as observed in studies of LUX in xenograft models and in initial clinical trials in patients with CLL and lymphomas , 35. In Following BCR activation, BTK is phosphorylated at Y551 and Y223. Y551 is located in the activation loop and can be either trans-autophosphorylated or phosphorylated by some upstream kinases. Y223 is located in the ligand binding pocket and regulates BTK kinase activity , 37. PhoAmong the kinases upstream of BTK, SYK and members of the Src family kinase such as LYN and the adaptor protein BLNK, play important roles in the BCR signalosome , 38. Pho+2 flux and other downstream effectors such as ERKs, JNK, NF-κB etc. leading to full effect of BCR signaling [50 of 59 nM (Carna Biosciences) and Kd of 180 nM (Discover X) against recombinant SYK [When activated, SYK interacts with and phosphorylates BLNK which has binding sites for BTK and PLCγ2. The association of BLNK with BTK and PLCγ2 brings both BTK and PLCγ2 into proximity and this further potentiates BTK signaling. A 5-fold increase in BTK activity was reported when BTK and PLCγ2 are bound to BLNK , 10, 28.ignaling , 44. Thunant SYK .The kinase screens also highlighted the ability of LUX to target other members of Src family kinase . The SrcA necessary step in the activation of LYN, as well as other members of the Src kinase family, is the dephosphorylation of inhibitory tyrosine residue Y507 in the C-terminal domain which is carried out by protein tyrosine phosphatase CD45. This induces unfolding and exposure of Y397  within the activation loop of LYN leading to transphosphorylation of Y397 and full activation of LYN , 48. LYNEvidence that the effect of LUX on the phosphorylation of LYN resulted in reduced activity of the enzyme was provided by the observation that anti-IgM-induced phosphorylation of CD79A (Y182) was inhibited by LUX in a concentration-dependent manner. The fact that IB had no effect provided further evidence of a differential effect of the two drugs on the activity of LYN. Activation of BCR was associated with reduction in the level of total LYN consistent with anti-IgM-induced degradation as previously reported . InteresThe question of whether the unique differences in the effect of LUX and IB on phosphorylation of kinases in the BCR pathway detectable with a 2 h exposure to drug are linked to differences in cytotoxic potency is of interest. When exposed for 72 h, LUX was found to be between 18- and 1,272-fold more potent at inhibiting growth than IB across the 3 cell lines  suggestiIn summary, in human lymphoma cells LUX and IB have quite distinct mechanistic effects on the phosphorylation of BTK and its activity, and on the upstream kinases SYK and LYN, and the adaptor protein BLNK against which LUX is very potent and IB had little effect. The BCR pathway kinases whose phosphorylation is modulated by LUX are highlighted in S1 File(PDF)Click here for additional data file.S1 FigCells were pre-treated for 2 h with either LUX or IB and then stimulated with anti-human IgM or left unstimulated. Whole cell lysates were analyzed by Western blotting for the indicated phosphoproteins in (A) JeKo-1 and (B) RL cells. Representative blots are shown.(TIF)Click here for additional data file.S2 FigCells were left untreated or pre-treated with 1 μM LUX or IB for 2 h followed by stimulation with anti-IgM. Representative blots showing BLNK immunoprecipitates probed for BTK and BLNK in (A) JeKo-1 and (B) RL cells sampled at 5 min. Histograms show quantification of co-precipitated protein relative to that of the protein targeted by the precipitating antibody. Bars are mean ± SEM of data from 2 independent experiments.(TIF)Click here for additional data file.S3 FigCells were pre-treated with vehicle or 1 μM LUX or IB for 2 h followed by stimulation with anti-human IgM for 5 min. Western blot analysis of LYN immunoprecipitates using antibodies for phospho-Src (Y416) and LYN in (A) JeKo-1 and (B) RL cells. Histograms show quantification of phospho-Src (Y416) band intensity relative to that LYN. Data is mean ± SEM of data from 2 independent experiments. Representative blots are shown. **p<0.001.(TIF)Click here for additional data file.S1 Table50 values for LUX and IB in SU-DHL-6, JeKo-1 and RL cells. Data is mean ± SEM of 3-independent experiments.Table showing IC(DOCX)Click here for additional data file.


While standard chemotherapy is able to target rapidly proliferating immature blasts, it fails to eradicate slowly proliferating Leukemic Stem Cells. Therefore, new therapeutic strategies that efficiently target LSCs are urgently needed. Recent studies suggest that LSCs have particular metabolic vulnerabilities, which would open the possibility of a therapeutic window with limited off‐target effects on the normal hematopoietic system. In this issue of EMBO Molecular Medicine, So and colleagues investigate the mechanism of action of AG636, a new potent inhibitor of  et al (in this issue of EMBO Mol Med) that investigates the mechanism of action of AG636, a new potent inhibitor of de novo pyrimidine synthesis, in the context of acute myeloid leukemia. E. Donato and A. Trumpp discuss the study by So  AML is the most aggressive blood cancer with a 5‐year survival rate lower than 20% for patients older than 65 years. While the majority of patients initially reach complete remission after standard chemotherapy, the rate of mortality remains very high due to recurrent disease. The formation of AML relapse is ascribed to the persistence of chemotherapy‐resistant LSCs with long‐term self‐renewal activity able to regenerate the disease. Previous studies have demonstrated that AMLs show a hierarchical organization with quiescent/low cycling LSCs at the top of the tree giving rise to fast proliferating immature myeloid blasts unable to generate terminally differentiated mature cells , an essential enzyme localized in the mitochondrial inner membrane and catalyzing the fourth step of the et al, de novo pyrimidine synthesis is not limited to MLL rearranged AMLs, since also AML1‐ETO or IDH1R132H/DNMT3AR882H/NrasG12D mouse models were susceptible to DHODH inhibition. Even though AG636 treatment was associated with reduction of normal B cells, myeloid and megakaryocyte‐erythroid progenitors as well as LT‐HSCs, this effect was transient and normal hematopoiesis returned to baseline after drug discontinuation. Transcriptomic analyses of enriched LSCs (cKithigh CD11blow) identify genes associated with protein synthesis as strongly deregulated after DHODH inhibition. This observation was further validated by polysome profiling and L‐Azidohomoalanine incorporation, functionally demonstrating that AG636 treatment leads to the inhibition of the protein translation machinery. By chromatin accessibility analysis on enriched LSCs (cKithigh CD11blow) and data mining using publically available datasets, the authors identified YY1 as a potential transcriptional regulator of genes controlling protein translation. This observation was particularly interesting since YY1 is post‐translationally modified by the addition of O‐GlcNAc: a post‐translational modification dependent on pyrimidine biosynthesis. The crucial role of O‐GlcNAc modification was confirmed by rescue experiments with an inhibitor of O‐GlcNAcase (PUGNAc). Thus, de novo pyrimidine synthesis inhibition by AG636 is altering YY1 stability by preventing O‐GlcNAc modification with consequent downregulation of its target genes (Fig In this issue of EMBO Molecular Medicine, So and colleagues report findings from a preclinical study assessing the novel potent DHODH inhibitor AG636 (So Finally, by performing CRISPR‐Cas9 KO screens using a focused library of epigenetic regulators, the authors identified CDK5 and members of the INO80 chromatin remodeling complex as sensitizer to DHODH inhibition. Transcriptomic analysis of CDK5 knockout cells treated with AG636 revealed numerous genes involved in OxPHOS and the Electron Transport Chain as being strongly downregulated, suggesting that the synthetic lethality could be explained by a dual synergistic attack on mitochondria Fig .de novo pyrimidine synthesis for survival and cell fate decision, respectively. Interestingly, in LSCs, pyrimidine starvation is controlling protein modification and stability of key transcription regulators and YY1 was identified as a novel regulator of protein translation in AML. Finally, synthetic lethality was observed when DHODH inhibition was concomitant with depletion of CDK5. Indeed, the authors showed for the first time that CDK5 regulates the expression of ETC complex I and II components (Fig et al will certainly guide future investigations dissecting the full spectrum of the roles of pyrimidines in AML.In summary, So and colleagues shed new light on the mechanism of action of DHODH inhibition, further proving LSC and blasts dependency on


However, by 2100 (SSP5-8.5 scenario), radiative fluxes in the ablation zone will become more than twice as sensitive (±0.39 W m−2) to changes in cloudiness due to reduced surface albedo. Accurate representation of clouds will therefore become increasingly important for forecasting the Greenland Ice Sheet’s contribution to global sea-level rise.Clouds regulate the Greenland Ice Sheet’s surface energy balance through the competing effects of shortwave radiation shading and longwave radiation trapping. However, the relative importance of these effects within Greenland’s narrow ablation zone, where nearly all meltwater runoff is produced, remains poorly quantified. Here we use machine learning to merge MODIS, CloudSat, and CALIPSO satellite observations to produce a high-resolution cloud radiative effect product. For the period 2003–2020, we find that a 1% change in cloudiness has little effect (±0.16 W m Here the authors use remote sensing observations and machine learning to show that clouds will become increasingly important for determining the Greenland Ice Sheet’s contribution to global sea levels due to decreasing albedo in the ablation zone. This recent mass loss is primarily due to enhanced surface meltwater runoff, most of which derives from the narrow ablation zone where bare, dark glacial ice is exposed each summer3. There is a growing realization that clouds play an important role in determining the amount of meltwater runoff generated in the ablation zone7. Clouds are known to regulate surface energy balance through the competing effects of shortwave radiation shading (i.e. cooling) and longwave radiation trapping (i.e. warming)9. However, the precise radiative effects of clouds in the ablation zone remains uncertain due to a reliance on modeled radiative fluxes which are known to contain biases11. Therefore, it remains largely unknown how changes in cloudiness, such as those which occur due to atmospheric blocking events12, alter the rate of meltwater production from the ablation zone both currently and in the future.The Greenland Ice Sheet has been losing mass since the 1990s and is currently the single largest cryospheric contributor to observed global sea-level rise16. Satellite remote sensing therefore provides the only practicable technique for observing CRE across the whole ice sheet. A few studies have used CloudSat and Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observation (CALIPSO) to characterize cloud properties and their radiative effects over Greenland and the Arctic18. However, the active remote sensing instruments employed on these satellites have sparse spatiotemporal sampling, requiring aggregation into very coarse grid cells (e.g. 2 × 2° or 2 × 1°) to ensure a sufficient number of samples within each grid cell. Such products are therefore too coarse to resolve Greenland’s narrow ablation zone11, preventing characterization of CRE for the very part of the ice sheet that generates runoff. Optical remote sensing instruments such as the MODerate Imaging Spectroradiometer (MODIS) onboard Terra and Aqua offer much finer spatial and temporal resolution19. Yet visible-infrared wavelengths cannot provide a complete vertical profile of cloud structure, meaning that MODIS-derived cloud properties and associated radiative effects are inherently uncertain. The production of high-quality CRE observations at high spatial resolution therefore requires intelligent integration of cloud profiling with optical imagery, to combine the strengths of these different sensor technologies while overcoming their individual limitations.While in situ observations of cloud radiative effects (CRE) have been acquired at research stations, they are sparsely distributed and mainly constrained to the accumulation zone  and MODIS , enabling accurate, daily quantification of CRE over the Greenland Ice Sheet during summer for the 2003–2020 period (see Methods). We then exploit the unprecedented spatial resolution of our product (1 × 1 km grid cell size) to resolve fine spatial gradients and interannual variations of CRE within the narrow ice sheet ablation zone, where nearly all meltwater runoff is produced. We also use our product to investigate the impacts of anticyclonic circulation regimes (i.e. blocking events) on clouds and radiative fluxes in the ablation zone. Finally, we combine our product with projections from the CMIP6 experiment to assess the radiative effects of clouds and atmospheric blocking events on Greenland Ice Sheet surface energy balance in a future, warmer climate.th percentile) in the Northeast Greenland to 71% (95th percentile) in South Greenland  than the ablation zone (+11.4 ± 3.1 W m−2), a finding that corroborates previous studies16 , indicating that the ablation zone will darken in a warmer climate  during our 2003–2020 study period, we find substantial interannual variability (std. dev. = ±1.6%) in summer “cloudiness” Fig. a and S4,ate Fig. .−2 while net shortwave radiation changes by only 0.83 W m−2   on surface energy balance and associated meltwater production from the Greenland Ice Sheet. This finding is supported by the fact that intense melt years  were not significantly cloudier/clearer than low melt years   Fig. . The com15) Fig. .25. Blocking events have been attributed to negative phases of the North Atlantic Oscillation (NAO) and have significantly increased in frequency over the past twenty years27. Ward et al. (2020)25 find that summer blocking occurred on 205 days (15%) during the 2003–2018 period using used from Modern-Era Retrospective Analysis for Research and Applications, version 2 (MERRA-2) reanalysis data. The net radiative effects of summer blocking in the ablation zone, however, have yet to be directly observed.Since MODIS has daily temporal resolution, our high-resolution product enables investigation of the radiative effects of short-lived atmospheric circulation features occurring for days-to-weeks over Greenland. One of the most important of these for ice sheet surface melt is anticyclonic blocking events, which enhance ice sheet melt through cloud suppression and warm air advection25 who locate blocking events into four quadrants  based on local maximum 500 hPa geopotential height anomalies. Depending on where the blocking is centered, we find that blocking events reduce cloudiness in the ablation zone by 0.2–6.4% percentage points ts Table . When blly Table ; Fig. 3,nes Fig. . General−2 . By applying our model to a range of projected future air temperatures from CMIP6 global climate model simulations, we find that summer surface albedo in the ablation zone will darken by ~0.02–0.14 depending on the emissions scenario Fig. . Interan−2 respectively, relative to mean 2100 conditions (Table −2 by 2100 in SSP5-8.5), together with enhanced sensible heat fluxes from warm air advection25, will make these anomalous circulation events extremely potent for ice sheet melt in the future. It is therefore critical that global climate models used to forecast future ice mass loss accurately represent the frequency and duration of atmospheric blocking events over the Greenland Ice Sheet.This increasing sensitivity of Greenland ablation zone energy balance to cloudiness implies that, in a warmer climate, atmospheric blocking events will enhance ice sheet melt even more than they do currently. By 2100, in an SSP5-8.5 scenario, we compute that the same blocking events centered over Northwest, Northeast, and Southwest Greenland will increase net radiation fluxes in the ablation zone by +15.7 ± 4.2, +6.6 ± 1.8, and +5.3 ± 1.4 W mns Table . An overTo conclude, we used machine learning to merge accurate Cloudsat/CALIPSO cloud profiling retrievals with high temporal and spatial resolution MODIS visible-infrared imagery, enabling retrieval and investigation of fine-scale CRE variations across the Greenland Ice Sheet surface from 2003 to 2020. The high spatial resolution (1 × 1 km) of our CRE product represents a major advance for understanding interactions between clouds and specific areas of the ice sheet surface. Previous observational studies of CRE were limited to either very coarsely gridded satellite products (e.g. 2 × 2°) or sparsely located in situ instruments, most of which are located in the accumulation zone. Our high-resolution spatially-continuous product overcomes these limitations, thus enabling systematic assessment of radiative fluxes over the entire Greenland Ice Sheet, including the ablation zone where the vast majority of ice sheet meltwater runoff is produced.2, the shortwave shading effect of clouds will become increasingly critical for determining meltwater production and associated ice sheet contributions to global sea-level rise. By the century’s end, we project that surface energy balance in the Greenland Ice Sheet ablation zone will be twice as sensitive to clouds than it is today and that atmospheric circulation patterns that suppress clouds (i.e. blocking events) will enhance meltwater production ~34% more than they already do.Analysis of our fine-scale CRE product reveals an important emerging role for clouds on future meltwater production on the Greenland Ice Sheet surface. During our 2003−2020 study period, the competing radiative impacts of clouds (i.e. longwave warming vs. shortwave cooling) approximately compensated over the ablation zone, signifying that clouds currently have a small regulatory role on meltwater runoff production. But as the ablation zone darkens28:↓all−sky and LW↓all−sky are the downward shortwave and longwave fluxes at the surface during all-sky conditions, SW↓clear−sky and LW↓clear−sky are the downward shortwave and longwave fluxes that would occur at the surface in the absence of clouds, and α is the surface albedo. A positive CRENET (in W m−2) indicates net cloud warming at the surface, whereas a negative CRENET indicates net cloud cooling.Our general approach for investigating the impacts of clouds on Greenland Ice Sheet mass balance from satellite remote sensing observations and machine learning is outlined here with further details provided in the section “Prediction of cloud enhancement factors”. Central to our approach is the computation of CRE which describes the instantaneous effect of clouds on the surface energy budget relative to clear skiesNET using high-quality all-sky and clear-sky radiative flux observations from CloudSat/CALIPSO and surface albedo observations from MODIS. Yet, while this approach may be accurate, CloudSat/CALIPSO has a relatively narrow ground footprint (~1.4 km), long repeat interval (16 days), and short timespan (2006−2010) which limits its usefulness of for investigating CRE at fine spatiotemporal resolution over long time periods18. To overcome this, we train a machine learning model, using CloudSat/CALIPSO observations, to predict CRE from passive visible-infrared satellite observations (i.e. from MODIS). This approach involves computation of a cloud enhancement factor (F) from CloudSat/CALIPSO given as:We first derive CRE7. This value depends upon cloud optical thickness, phase, temperature, and height, as well as solar zenith angle for shortwave fluxes and near-surface air temperature for longwave fluxes29. Our supervised machine learning algorithm is trained to predict FSW and FLW from cloud properties derived from passive visible-infrared sensors such as MODIS. Since MODIS has much wider spatial coverage and shorter repeat intervals (i.e. sub-daily) than CloudSat/CALIPSO, this approach enables computation of observational based CRE at finer spatial resolution and longer time-scales (2003−2020 vs. 2006–2010) than was previously possible. Further details on our approach are provided in the section “Prediction of cloud enhancement factors”.The cloud enhancement factor represents the change in downward shortwave or longwave radiation due to clouds relative to clear skies31. This product incorporates (1) cloud and aerosol observations from CloudSat/CALIPSO; (2) ancillary temperature and humidity profiles from ECMWF atmospheric re-analyses; and (3) International Geosphere-Biosphere Programme surface albedo/emissivity data to constrain broadband radiative fluxes using the two-stream radiative transfer model “BugsRad”31. Since our study is focused on the summer, we only use 2B-FLXHR-LIDAR data acquired during June, July, and August. We use Release 05 version of the 2B-FLXHR-LIDAR product which provides a more robust partitioning of ice-only and liquid-bearing clouds than Release 047. The radiative fluxes of the 2B-FLXHR-LIDAR algorithm have been extensively evaluated31 and previously used to study cloud impacts on surface energy budgets in the polar regions18.We derived high-quality downward all-sky and clear-sky radiative fluxes at the surface from the CloudSat/CALIPSO Level-2 ‘Fluxes and Heating Rates’ (2B-FLXHR-LIDAR) product which was the first to use active remotely sensed cloud observations to retrieve surface radiative fluxes at a global scale19. These parameters are available at a spatial resolution of either 1 km or 5 km resolution (at nadir). Since NASA’s Aqua satellite orbits within two to three minutes of CloudSat/CALIPSO in the A-Train constellation, cloud properties from the MYD06_L2 product are near-coincident with the 2B-FLXHR-LIDAR product. We also derived surface albedo from the MODIS daily snow cover product, MOD10A1 Collection 6.1.2. These data were averaged to provide mean summer albedo for the Greenland Ice Sheet at 1 × 1 km grid cell resolution. Since satellite measurements of albedo are acquired under clear-sky conditions and snow/ice albedo is known to be higher under cloud cover, we applied a correction to our clear-sky albedo climatologies following Key et al.32. To this, we multiplied the mean cloud effect (+0.05) with the cloud fraction of each grid cell , cloud optical thickness, and effective particle radiusell Fig.  and addeWe predicted cloud enhancement factors for each MODIS pixel using a Random Forests regression algorithm trained from CloudSat/CALIPSO radiative flux observations. The approach was introduced in the section “General approach” but the specific steps are described here:SW and FLW) from all CloudSat/CALIPSO downward all-sky and clear-sky radiative flux observations acquired over the Greenland Ice Sheet between 13 June 2006 and 18 August 2010 , we acquired coincident cloud properties from the MYD06_L2 product and near-surface temperatures from ERA5 climate reanalysis using a nearest neighbors approach (k = 1). Cloud properties used to predict FSW include cloud optical thickness, cloud top pressure, cloud effective radius, cloud top temperature, cloud top height, and cloud water path from MODIS, and near-surface air temperature from ERA5. Cloud properties used to predict FLW include cloud optical thickness, cloud top pressure, cloud effective radius, cloud top temperature, cloud top height, cloud water path, and cloud phase from MODIS and near-surface air temperature from ERA5. Since downward shortwave all-sky radiative fluxes are highly dependent on solar zenith angle, we grouped the training data into 5° bins (between 35 and 80°) before statistical modeling.For each CloudSat/CALIPSO cloud enhancement factor (SW and one group for FLW), we fitted a Random Forests regression model with 100 trees to a random sample containing 50% of the training data . We then used the regression models (which depend on solar zenith angle for the FSW model) to predict FSW and FLW for every valid pixel in every MYD06_L2 product acquired over the Greenland Ice Sheet during the 2003–2020 period. The importance of each variable for predicting FSW and FLW is provided in Table For each group of training data  to compute downward all-sky fluxes 92 to com−2 2. The ablation zone was defined as all areas of the ice sheet where the bare ice is present for more than 10% of the record-setting melt summer of 2012 with the rest of the ice sheet classified as the accumulation zone. The ablation zone represents 14.8% of the ice sheet area. We average our sub-daily CRE products for each summer  during the 2003–2020 period, providing an 18-year record of cloud conditions and radiative effects over the Greenland Ice Sheet.We bilinearly resampled our products to a standard 1 × 1 km study grid used by the Greenland Surface Mass Balance Intercomparison Project (GrSMBMIP)SW and FLW) and clear-sky radiative fluxes  that were not used to train the machine learning algorithm. We found a mean absolute error (MAE) of ±0.075 (±11.7%) for predicted FSW  between clear-sky shortwave fluxes  for the clear-sky longwave fluxes 13 observed a CRENET of +35.3 W m−2 during the summer which is very similar to what we find over the same time period (+32.3 ± 8.7 W m−2).We carried out an independent check on our satellite-derived product by comparing it to high-quality CRE observations acquired by in situ instruments at Summit Station 36. Regional lapse rates were calculated from all ERA5 grid points within 100 km distance from the target grid point. The obtained lapse rates were used to remove the elevation gradient from the ERA5 grid points. We then interpolated the residuals over the 1 km target DEM by inverse distance weighting using a scale of 1 km, and the power value alpha set to 1. Finally, the locally calculated lapse rate is used again to add the elevation gradient to the 1 km DEM to the interpolated grid.While we find no evidence that cloudiness in the ablation zone will substantially change as the climate warms, the albedo of the ablation zone will likely reduce in a warmer climate due to migrating snowlines, algal growth, and enhanced meltwater production. To forecast future albedo in the ablation zone, we developed an empirical model that predicts albedo for each grid cell based on a reference albedo grid (2015–2020) and near-surface air temperatures from ERA5 climate reanalysis and 26 models from the CMIP6 climate model experiment. We first linearly regressed mean summer albedo from MODIS with downscaled air temperature from ERA5 for each 1 × 1 km grid cell for the 2003–2020 period. ERA5 summer air temperatures were downscaled onto the target 1 km DEM by using the Local Inverse Distance Weighting (IDW) Lapse Algorithm from the MeteoIO libraryR2 = 0.83) with an accuracy of ±0.04  and unrealistic relationships (i.e. positive correlations) were removed from analysis. We then computed the mean slope of these linear relationships within nine temperature bands Fig. . This me.04 Fig. .net [i.e. SW↓all−sky * (1 – α)] based on the forecasted albedo in the ablation zone. This enabled us to assess the sensitivity of ice sheet meltwater production to changing cloudiness in a future, warmer climate  . We used 26 models, namely: ACCESS-CM2, ACCESS-ESM1-5, CESM2-WACCM, CESM2, CMCC-CM2-SR5, CNRM-CM6-1-HR, CNRM-CM6-1, CNRM-ESM2-1, CanESM5-CanOE, CanESM5, EC-Earth3-Veg, EC-Earth3, GFDL-ESM4, GISS-E2-1-G, INM-CM4-8, INM-CM5-0, IPSL-CM6A-LR, KACE-1-0-G, MIROC-ES2L, MIROC6, MPI-ESM1−2-HR, MPI-ESM1-2-LR, MRI-ESM2-0, NorESM2-LM, NorESM2-MM, and UKESM1-0-LL. This ensemble has a size and breadth of models that is comparable to the ensembles used in parts of the IPCC AR6 WG1 report. For each model we resampled near-surface air temperatures onto a common study grid using bilinear interpolation. For each model we also normalized air temperature by subtracting the mean summer ice sheet air temperature to account for any temperature bias. We then derived albedo for each grid cell based on the temperature deviation from a 2015–2020 reference period and the empirical albedo scheme described above. During this process, grid cell albedo is prevented from going below 0.3, below which would be an unrealistically low albedo for bare ice Fig. . FinallySupplementary Information


Topological phases of spin liquids with constrained disorder can host a kinetics of fractionalized excitations. However, spin-liquid phases with distinct kinetic regimes have proven difficult to observe experimentally. Here we present a realization of kagome spin ice in the superconducting qubits of a quantum annealer, and use it to demonstrate a field-induced kinetic crossover between spin-liquid phases. Employing fine control over local magnetic fields, we show evidence of both the Ice-I phase and an unconventional field-induced Ice-II phase. In the latter, a charge-ordered yet spin-disordered topological phase, the kinetics proceeds via pair creation and annihilation of strongly correlated, charge conserving, fractionalized excitations. As these kinetic regimes have resisted characterization in other artificial spin ice realizations, our results demonstrate the utility of quantum-driven kinetics in advancing the study of topological phases of spin liquids. A kagome lattice spin-ice system is created with the superconducting qubits of a quantum annealer, and shown to exhibit a field-induced kinetic crossover between spin-liquid phases. Specifically, kinetics within both the Ice-I phase and the unconventional field-induced Ice-II phase are  presented. One might expect the kinetics of disordered systems to be naturally free. But in spin liquids, where disorder is present but constrained, kinetics often also proceeds through defects or excitations endowed with a conserved topological charge2. For instance, frustrated spin systems, such as pyrochlore4 or square7 spin ices, remain disordered at low temperatures, leading to a Pauling residual entropy, and their disorder is constrained by the so-called ice rule8. There, kinetics consists of creation/annihilation and walks of localized violations of the ice rule, in the form of emergent magnetic monopoles9 that conserve a topological charge.Dynamics in crystals typically proceed via the motion of topological defects such as dislocation gliding18 has been widely studied because it mimics a remarkable variety of natural and artificial systems, from rare-earth pyrochlores3, to nanomagnetic fabrications19, gravitationally trapped colloids15, as well as  many others28. Kagome spin ice can in principle manifest unusual phases15, but the large energy scales of artificial implementations pose an experimental challenge; thorough measurements of these phases and the physical conditions driving the phase-to-phase transition are scarce, and an experimental description of their kinetics is entirely lacking.Among spin ices, the kagome ice modelHere we present a kagome qubit ice realized in a superconducting quantum annealer. Using this experimental platform, we study its field-induced spin-liquid phases and quantum-activated kinetics. We experimentally establish that topological constraints affecting the dynamics proceed via charge-conserving fractionalized excitations. Using thousands of programmable external magnetic fields, we detune the system from its more common ice-rule-obeying “Ice-I” phase into a field-induced “Ice-II” phase, which exhibits charge order while remaining spin-disordered. We then report on the essential differences in quantum-activated kinetics between the two phases.q = −3, −1, 1, 3  to another Fig. a. We canfrustrated. The ground state is therefore an extensively degenerate ensemble of disordered spins obeying the (pseudo-) ice rule: frustration is minimized when each vertex has two spins pointing in and one pointing out, or vice-versa. This ice manifold is often called the Ice-I phase and can be thought of as a spin liquid forming an overall neutral plasma of disordered ±1 magnetic charges. In the Ice-II phase29, disordered spins still obey the ice rule but charges are ordered in an ionic lattice33.The simplest magnetic kagome model includes interactions only among spins impinging on the same vertex. Since not all pairs of spins at a vertex can simultaneously assume an energy-minimizing head-to-tail configuration, the system is hi are per-qubit programmable longitudinal fields34, and Jij is programmable two-qubit couplers. Γ is a transverse field entangling the Pauli matrices and thus controls quantum fluctuations.In this work, we realize kagome spin ice in a quantum annealer. Its superconducting flux qubits are described by the transverse field Ising Hamiltonian35, and therefore to the Hamiltonian of Eq.  We use h and J (with no index) to denote the total field on a three-qubit chain and the total coupling between two neighboring chains, respectively, obtaining the kagome qubit ice (KQI) HamiltonianJFM37, i and j are also over three-qubit chains, rather than individual qubits.We then embed the kagome lattice in the graph of available two-body couplers, as shown in Fig. h = 0, the extensively degenerate ground state manifold of HKQI corresponds to that of HI, which is the commonly seen Ice-I phase19. But we can go beyond this regime. In nanoscopic realizations, another phase of lower entropy is possible38. In such systems, it is driven by the long-range nature of the dipolar interactions14. It still has disordered ice-rule obeying spins, but with charges ordered in an ionic lattice where A and B vertices have opposite charges. While the spins remain disordered, though at lower entropy39, their disorder is topologically constrained: it can be mapped to a dimer cover model39 and considered a case of classical topological order41. This is often called the Ice-II phase, and its topological nature should show topologically protected kinetics. .When not gapped: It is possible to flip a single spin—or indeed an extensive number of single spins—without violating the ice rule and thus without creating an excitation  eventually giving way to a polarized state in which the longitudinal field overcomes the ice rule, forming ionic crystals of ±3 charge, and all spins have value si = 1.Figure h/J = 2.5. In Fig. K in the proximity of the Ice-II phase. These peaks correspond to the expected logarithmic divergence of the dipolar correlations12 . They, and the pinch points, follow therefore from the topological properties induced on the phase by the charge ordering. From an implementation point of view, S(q) reveals a highly symmetric system in which the multi-qubit embedding of kagome spins preserves isotropy. This is an important advance over the previous work7.Figure charge order parameter, defined as one-third of the average staggered charge of a vertex. The two broad plateaus at ±1/3 correspond to the Ice-II phases.Figure h/J > ∣4∣ where, from Fig. Figure J), we can also probe the quasi-classical kinetics.These measurements validate the annealer’s effectiveness as an experimental platform for probing phases of the Ising kagome spin ice system near a low-temperature equilibrium. By employing  consecutive output states separated dynamically by a relatively short exposure to a relatively weak transverse field Γ . We see many excitations popping up erratically, and they are charge order violations, due to their small energy cost  begins in an easily prepared superposition ground state and ends in a low-energy state of a classical Ising Hamiltonian. For simulating spin systems, it has proven useful47 to employ a “quantum evolution Monte Carlo” method, in which a chain of classical samples S0, …Sk is generated , then “reverse annealed” back to some intermediate s*, paused at s* to allow equilibration for some time tp, then quickly quenched back to s = 1. Although this method can be used to estimate observables from a transverse field Ising model at s*47, here we just use quantum fluctuations as a driver of mixing dynamics between low energy states in the kagome ice system.Thus a typical “forward anneal”, in which see Fig. . To genek = 128 samples, starting with a random initial state S0. To estimate equilibrium properties  as Monte Carlo burn-in. For dynamics inquiries . When statistical quantities are estimated, we take the average of 200 repeated experimental iterations; each iteration includes a call to the QPU for each value of h probed.In this work, we generate chains of 48, in which a qubit is coupled to up to 15 other qubits. From these available couplers, we select a geometry that represents a kagome graph using three qubits per kagome spin as depicted in Fig. The qubits in the QA processor are intercoupled in a “Pegasus” layoutJij = 0.9 for AFM couplers and Jij = −1.5 for FM couplers. This choice of ferromagnetic coupling is sufficient to guarantee that chains are almost never broken in QPU output, despite the frustration in the system.Since ferromagnetic chains are sometimes broken, they are majority-voted to provide an unambiguous mapping from the qubit system to the kagome system. We run all experiments presented herein with In this application, we perform many experiments on a single programmed lattice, whose classical ground state is highly degenerate. Under such conditions, it is appropriate to refine the general-purpose QA calibration by exploiting symmetries in the system.h = 0 each qubit should have average magnetization h = 0, then use the same flux offsets when h ≠ 0. In this experiment, we are not interested in probing boundary conditions. Rather, we want to simulate the thermodynamic limit of an infinite system. In an infinite system, for any fixed h, the correlation of two neighboring kagome sites h ≠ 0, the magnetization of each qubit should be equal; we fine-tune the per-qubit fields hi to promote this property (maintaining the property that the average h). These calibration refinements are performed before collecting the analyzed data. Figure J = 0.9, h = 0.6, with the magnetizations and correlations achieved, and the programmed values that achieve them.For example, when Supplementary Information


However, the consequences of GSK3β inhibition by alternative Ser389 phosphorylation remain poorly characterized. Here we have examined neuroinflammation in GSK3β Ser389 knock-in (KI) mice, in which the phosphorylation of Ser389 GSK3β is impaired. The number of activated microglia/infiltrated macrophages, astrocytes, and infiltrated neutrophils was significantly higher in these animals compared to C57BL/6J wild-type (WT) counterparts, which suggests that the failure to inactivate GSK3β by Ser389 phosphorylation results in sustained low-grade neuroinflammation. Moreover, glial cell activation and brain infiltration of immune cells in response to lipopolysaccharide (LPS) failed in GSK3β Ser389 KI mice. Such effects were brain-specific, as peripheral immunity was not similarly affected. Additionally, phosphorylation of the IkB kinase complex (IKK) in response to LPS failed in GSK3β Ser389 KI mice, while STAT3 phosphorylation was fully conserved, suggesting that the NF-κB signaling pathway is specifically affected by this GSK3β regulatory pathway. Overall, our findings indicate that GSK3β inactivation by Ser389 phosphorylation controls the brain inflammatory response, raising the need to evaluate its role in the progression of neuroinflammatory pathologies.The inhibition of Glycogen Synthase Kinase 3 β (GSK3β) by Ser Glycogen Synthase Kinase 3 (GSK3) is a serine-threonine kinase with broad specificity. GSK3 regulates processes as diverse as inflammation, cell growth, cell differentiation, and energy metabolism and is implicated in age-related diseases such as diabetes, Alzheimer’s disease, and cancer . Both GS9 and Ser389 [9 is a GSK3β regulatory pathway present in most cell types, and it is the default pathway to limit GSK3β activation and prevent cell death. In contrast, we have found that p38 MAPK-induced Ser389 phosphorylation is restricted to specific tissues such as the brain, thymus, and spleen [389 GSK3β is developmentally regulated [389 is triggered primarily by DNA double-strand breaks response and it is critical to allow DNA repair [9 is the main pathway for the inactivation of cytoplasmic GSK3β, Ser389 phosphorylation is used to inactivate the nuclear pool of GSK3β to increase survival during DNA DSB repair [389 phosphorylation in response to DSB leads to increased cell death by necroptosis [9, the implications of phosphorylation of GSK3β on Ser389 remain poorly characterized.GSK3β is constitutively active, although its activity can be inhibited by sequestration into cytosolic complexes as well as by phosphorylation at residues Serd spleen  and thategulated . The phoA repair ,7. Thus,roptosis ,7. This A number of studies have supported the association between GSK3 and inflammation. Thus, activation of GSK3 by inflammatory stimuli such as stress, infection, or trauma promotes the production of pro-inflammatory cytokines such as IL-6, IL-1β, and IFNγ, while GSK3β inhibition suppresses the synthesis of pro-inflammatory mediators and increases IL-10 production by immune cells ,12. UponNuclear factor kB (NF-kB) mediates the production of pro-inflammatory cytokines and is one of the main transcription factors activated by TLRs. Several authors have confirmed that GSK3 is able to activate NF-kB signaling through phosphorylation of the IkB kinase complex (IKK), which phosphorylates IkB and releases active NF-kB. Another transcription factor phosphorylated by GSK3 is a signal transducer and activator of transcription-3 (STAT3), which mediates neuroinflammation in the context of septic shock . STAT3 pMicroglia cells are resident macrophages in the CNS. Together with infiltrated leukocytes, microglia activation plays a critical role in neuroinflammation. Astrocytes are also glial cells with neuroimmune functions as they crosstalk with microglia, leukocytes, and endothelial cells, thus modulating both innate and adaptive immune responses . Activat9 phosphorylation upregulates the production of anti-inflammatory mediators and downregulates the release of pro-inflammatory cytokines [9 phosphorylation has been related to inflammatory neuropathologies such as Parkinson’s disease [389 GSK3β found in the brain [Phosphorylation is a regulatory mechanism of GSK3 activity with major implications for the control of the immune response. Thus, GSK3 inhibition by classical Serytokines , althougytokines . Within  disease  and schi disease . In conthe brain , it rema389 phosphorylation-mediated GSK3β inactivation in neuroinflammation using GSK3β KI mice in which GSK3β Ser389 was replaced by Ala to prevent the C-terminal phosphorylation of GSK3β [389 under physiological conditions leads to chronic low-grade neuroinflammation as well as the lack of response to inflammatory stimuli such as LPS. This chronic inflammation seems to be specific to the brain since the peripheral immune system was largely unaffected. We also show that the absence of phospho-Ser389 GSK3β in the brain of GSK3β KI mice regulates NF-kB signaling, which is a central event in the control of neuroinflammation. Therefore, our findings provide support for the involvement of GSK3β inactivation by Ser389 phosphorylation in inflammatory neuropathologies. Here, we investigate the possible role of Serof GSK3β ,8. Our r9 has been associated with an anti-inflammatory stage in the brain [389 is important to reduce neuronal death caused by DNA double-strand breaks in the brain, no studies have investigated whether this pathway could also regulate the neuroinflammatory response. We, therefore, used the previously described GSK3β Ser389 KI mice, in which Ser389 is replaced by Ala, to prevent Ser389 phosphorylation. We examined resident and infiltrated immune cells in the whole brain (except cerebellum and olfactory bulbs) from WT and GSK3β Ser389 KI mice by mechanical dissociation of the tissue and Percoll gradient, following a previously optimized protocol to obtain a balanced mixture of resident and infiltrated cells in the brain, including microglia/macrophages, astrocytes, infiltrated neutrophils and lymphocytes [+CD45high and CD11b+CD45low, respectively, whereas the lymphocytic population was marked as CD45+CD11b− [+Ly6G+ cells; and astrocytes were defined within the CD45− population by expression of the cell surface antigen-2 (ACSA-2). This marker is used for the detection of developing and adult astrocytes [+ astrocytes in mice brains increases after LPS treatment, further indicating that the level of ACSA-2 expression correlates with astrocyte activation in response to inflammatory stimuli [+ population.Inactivation of GSK3β by phosphorylation on Serhe brain . Althougphocytes . Cells wphocytes ,32,33. A5+CD11b− . Neutroptrocytes . Upregultrocytes . We prev stimuli . Althoug stimuli , these c389 KI mice compared to WT mice  in GSK3β Ser389 KI mice  in the brain was not affected in GSK3β Ser389 KI mice  were also evaluated  of bacterial lipopolysaccharide (LPS). The brain inflammatory response was examined three days later since such treatment has been previously shown to produce low-grade inflammation in mice brains [389 KI mice . the number of Ly6G+ neutrophils (++CD11b++Ly6G− monocytes/macrophages (389 KI mice (+ cells (+ cells (+ cells); and B220+ B cells were unaffected by LPS independently of genotype  and after 4 h, the hippocampus was harvested. Whole-cell extracts were used to examine phospho-IKKα/β and total IKKα/β by Western blotting. As expected, LPS increased IKK phosphorylation in the hippocampus in WT mice . Therefore, we conclude that, in the absence of pro-inflammatory stimuli, the lack of GSK3β Ser389 phosphorylation leads to a degree of neuroinflammation that should be classified as low-grade. One of the key findings in the present work is that the brain of GSK3β Ser389 GSK3β KI mice could be the consequence of neurodegeneration, as previous studies have detected neuronal death in the cortex and hippocampus of these animals [389 phosphorylation as a mechanism to prevent cell death and promote DSB repair [389 phosphorylation is prevented, is not protected against DSB-induced neuronal death. The link between phospho-Ser389 GSK3β expression and cell survival was first proposed in lymphocytes, as a mechanism implicated in the adaptive immune response. Thus, the absence of GSK3β Ser389 phosphorylation in KI mice was found to induce necroptosis in B cells during its late maturation, which is a highly inflammatory type of cell death related to several pathologies [389 phosphorylation but also Ser9 phosphorylation can be triggered to inactivate GSK3β in response to DNA damage. However, Ser389 GSK3β KI mice present a neurodegenerative phenotype [9 GSK3β KI mice [389 phosphorylation plays specific roles in the control of GSK3β activity and that there are scenarios in which Ser9 phosphorylation cannot compensate for the loss of Ser389 phosphorylation. Accordingly, the intriguing link between Ser389 GSK3β deregulation, neuroinflammation, and neurodegeneration unveiled here needs to be further explored to clarify whether altered GSK3β phosphorylation at Ser389 may be a key factor in neuropathologies in which chronic inflammation is distinctive, such as Alzheimer’s disease. Numerous studies support the association between GSK3 hyperactivity and neurodegeneration in neurological disorders . In this animals . This isB repair ,8. In cohenotype  not pres KI mice ,50, indi389 KI mice is consistent with the hypothesis that failure to phosphorylate Ser389 may be associated with immunosenescence. This is an age-related process characterized by a decline in immune functions together with an increase in the expression of pro-inflammatory mediators; which leads to chronic, low-grade inflammation [389 GSK3β KI mice could contribute to the loss of neurons previously reported in these animals [In addition, the mild but persistent activation of neuroimmune cells observed in GSK3β Serammation ,51. Seveammation . It has ammation . Thus, a animals . 389 phosphorylation. According to this observation, extended GSK3β hyper activation in the brain may eventually downregulate the sensitivity to pro-inflammatory insults. It is noteworthy that the defective response to LPS in the mutant mice is compatible with the mechanism of immunotolerance or resistance, a common process in the CNS aimed at reducing glial cell activation and limiting brain damage from excessive inflammation [LPS has been extensively used to induce inflammation in both “in vivo” and “in vitro” models. However, there is evidence that the dose of LPS administered largely determines the type of inflammatory response induced ,55,56. Hammation ,61. In aammation . 389 GSK3β KI animals presented the same profile in unstimulated conditions, as well as a similar response to LPS. This suggests that the GSK3β inactivation pathway dependent on Ser389 phosphorylation plays specific roles in the regulation of neuroinflammation. In agreement with this hypothesis, brain cells show a high expression of phospho-Ser389 GSK3β compared to other tissues, and this expression is tightly regulated during brain development [It is also interesting to consider that the brain effects described in this study show high specificity. Thus, contrarily to the brain, most spleen cell populations evaluated in WT and Serelopment . 389 is specifically mediated by the NF-kB signaling pathway, since STAT3 activation was unaffected in Ser389 GSK3β KI. Many downstream inflammatory mediators are regulated by NF-kB activation, including cytokines, receptors, transcription factors and enzymes. Identifying which of these NF-kB targets are influenced by Ser389 GSK3β phosphorylation is beyond the scope of our work, but it will be an exciting goal for future studies aiming to clarify the mechanisms of neuroinflammation. Finally, according to our findings, the regulation of GSK3β by phosphorylation at Ser389 KI mice, in which the Ser389 residue of GSK3β was replaced by Ala to prevent phosphorylation, were generated as previously described [C57BL/6J Wild-type (WT) mice were purchased from Jackson Laboratories. C57BL/6 GSK3β SerTo induce an inflammatory response, mice received a single intraperitoneal (i.p.) injection of 0.5 mg/kg LPS. Salmonella enterica LPS  was dissolved in sterile, endotoxin-free 0.9% saline at 10 mg/mL. Three days after treatment, mice were euthanized, and the brain and spleen were removed for immunohistochemistry or flow cytometry analysis of glial cells and infiltrated immune cells. For Western blotting analysis, animals were euthanized 4 h after i.p. injection of 0.5 mg/kg LPS, as LPS has previously been reported to upregulate inflammatory proteins in mouse and rat brains 2–4 h after i.p. administration ,64. For flow cytometry analysis of brain tissue and spleen cells, animals were anesthetized with an i.p. injection of tribromoethanol, which has been recommended for acute terminal studies. The low dose of tribromoethanol used (200 mg/kg) provides light anesthesia without serious adverse effects or influence on inflammatory cytokine expression . Animal Mice were intracardially perfused with 50 mL of 0.9% NaCl before tissue collection to remove circulating cells from brain blood vessels. After perfusion, the brains were removed, the meninges and choroid plexus were carefully separated, and the cerebellum and olfactory bulbs were discarded. The remaining tissue was placed in cold Hank’s Balanced Salt Solution, which is calcium chloride and magnesium chloride free . Tissue samples were then finely chopped with a scalpel before digestion.g for 10 min at RT.Chopped tissue was then placed in MACS C tubes  in a total volume of 5 mL, and processed with a mechanic dissociator at 6 rpm for 30 min. Enzymatic dissociation was performed according to our own protocol  using paTM  gradient was used to remove myelin and enrich the homogenate into viable glial cells. For this purpose, a stock solution of isotonic Percoll (SIP) was prepared , and cell pellets were resuspended in 30% (v/v) SIP (in 1× HBSS). The 30% Percoll gradient was carried out by adding 3 mL of SIP to 7 mL of 1× HBSS containing the cell suspension in a 14 mL polypropylene tube. Each gradient was centrifuged without a brake at 300× g for 30 min at 18 °C. After centrifugation, the cells from the bottom layer were collected, washed with 1× HBSS, centrifugated for 10 min at 300× g, and resuspended in 10 mL of 1× red blood cell lysis buffer at 4 °C. After lysis, cell suspension was centrifuged at 300× g for 10 min, and the resulting cell pellet was finally resuspended in 50 μL of blocking buffer before staining for flow cytometry. A 30% PercollSpleens were placed on a Petri dish containing RPMI 1640 and cut into small pieces. The resulting tissue was passed into a 70 μm cell strainer  and broken up in a circular motion with the ribbed top of a syringe’s plunger. RPMI 1640 was then passed through the strainer. The disintegrated tissue was transferred to 50 mL tubes  and centrifuged at 100 g for 10 min. The supernatant containing the cell suspension was then collected and centrifuged again at 300 g for 10 min. The resulting supernatant was removed, and the pellet was resuspended in 10 mL phosphate-buffered saline (PBS) with 1% FBS to count the number of cells under the microscope, after which it was re-centrifuged at 300 g for 5 min. Only 500,000 cells per sample were used. Each sample was then resuspended in 50 µL of blocking buffer before staining for flow cytometry.g. Finally, the cell pellets were resuspended in 500 μL of PBS and samples were immediately used for flow cytometry. Data were acquired using a MACSQuant flow cytometer and analyzed with MACSQuantify software (Miltenyi Biotec). Prior to sample analysis, fluorescent calibration beads (1000 beads/µL) were added to count the absolute number of cells. After excluding cellular debris and aggregates, a minimum of 10,000 events corresponding to individual viable cells were examined in each analysis. The cell number measurements are presented as normalized with respect to their controls. Flow cytometry staining was performed as previously described . To blocw/v). Their brains were collected and post-fixed with 4% paraformaldehyde for 24 h. After post-fixation, they were cryoprotected with 30% sucrose and frozen at −20 °C until sectioning. Tissue sections (20 μm) were cut using a cryostat , thaw-mounted onto Superfrost Plus slides  and finally stored at −20 °C until staining.Mice were deeply anesthetized and intracardially perfused with saline 0.9% . To ensure equal loads, the integrated density obtained for each band was expressed as a relative value with respect to the appropriate housekeeping loading control.Mouse brains were quickly removed, and the hippocampi were immediately dissected and frozen. Tissue was prepared using a hypertonic lysis buffer containing protease inhibitors as previously described . The prin = 4–9). After confirming the normal distribution of data by the Shapiro-Wilk test, statistical significance between two groups was determined by the Student t-test for parametric data. Differences were considered statistically significant when probability (p) values were less than 0.05 (*), 0.01 (**), or 0.001 (***). Figures show representative experiments.Results were statistically analyzed with the GraphPad Prism software . Data are presented as mean ± standard error of the mean (S.E.M) of generally four independent experiments (389 phosphorylation leads to chronic inflammation and endotoxin tolerance and that the inhibition of GSK3β by Ser9 and Ser389 phosphorylation plays complementary and not redundant roles in the neuroimmune response. Therefore, the specific consequences of GSK3β Ser389 phosphorylation for neuropathology and brain aging should be investigated.In summary, our findings indicate that failure to inactivate GSK3β by Ser