Impulsivity generally refers to a deficit in inhibition, with a focus on understanding the neural circuits which constitute the “brake” on actions and gratification. It is likely that increased impulsivity can arise not only from reduced inhibition, but also from a heightened or exaggerated excitatory “drive.” For example, an action which has more vigor, or is fueled by either increased incentive salience or a stronger action-outcome association, may be harder to inhibit. From this perspective, this review focuses on impulse control as a competition over behavioral output between an initially learned response-reward outcome association, and a subsequently acquired opposing inhibitory association. Our goal is to present a synthesis of research from humans and animal models that supports this dual-systems approach to understanding the behavioral and neural substrates that contribute to impulsivity, with a focus on the neuromodulatory role of serotonin. We review evidence for the role of serotonin signaling in mediating the balance of the “drive” and “brake” circuits. Additionally, we consider parallels of these competing instrumental systems in impulsivity within classical conditioning processes  in order to point us to potential behavioral and neural mechanisms that may modulate the competing instrumental associations. Finally, we consider how the balance of these competing associations might contribute to, or be extracted from, our experimental assessments of impulsivity. A careful understanding of the underlying behavioral and circuit level contributions to impulsivity is important for understanding the pathogenesis of increased impulsivity present in a number of psychiatric disorders. Pathological levels of impulsivity in such disorders are likely subserved by deficits in the balance of motivational and inhibitory processes. Impulsivity is generally conceived of as a deficit in inhibitory control, resulting in unwanted actions. However, impulsive behavior has many diverse presentations and complex neurobiological underpinnings . Many liDysfunction in different nodes of these “drive” and “brake” neural circuits could result in the heterogeneity of phenotypic presentations of impulsivity. Therefore, careful dissection of the underlying behavioral and circuit level contributions to impulsivity is important for understanding the pathogenesis of increased impulsivity. This idea is highlighted in the dual systems and imbalance models of adolescent impulsivity which consider disproportionate development and changes in communication for brain areas involved in reward/motivation and inhibitory control . ConsideThis dual-systems perspective is also relevant to clinical populations with disorders in which impulsivity is dysregulated. For example, attention deficit hyperactivity disorder (ADHD) is characterized by inhibitory deficits, including increased impulsive action . ImpulsiAssuming the presence of competing drive and brake processes in impulsivity, we can examine the behavioral/cognitive components and the underlying neural mechanisms of each of these components. This sets up the possibility to arrive at the endpoint of increased impulsive behavior via a number of different paths and combinations of intermediate phenotypes . For exaand mediating behavioral inhibition, and is poised to modulate the balance of reward-based approach and adaptive inhibition of action. Manipulation of serotonin neuron activity in preclinical models clearly show that serotonin is involved in waiting and inhibiting behavioral responses  has been strongly implicated in encoding reward esponses , 2020. Sted cues . A largeted cues . Given tThe first goal of this review is to synthesize studies, especially in preclinical animal models, that parse excitatory and inhibitory behavioral substrates that contribute to impulsive action. Next, we review potential circuit level mechanisms that underlie the interaction of these opposing learned associations in the generation of impulsivity. We focus on serotoninergic modulation of the underlying neural circuitry of both reward processing and inhibitory control, and also potentially in determining the balance of these competing systems to generate the output impulsive behavior. Finally, we discuss future research questions which examine the relative contributions of initial response-reward associations and subsequently learned inhibitory associations to increased impulsivity in terms of both behavioral substrates and the underlying neural circuits.Impulsivity is innately tied to reward processing, with excitatory drive being a key aspect of motivated behavior. Importantly, before being able to consider an inhibitory process, a motivated behavior needs to exist. This commonly includes a learned cue-reward or action-outcome association. In other words, we first learn to respond to obtain rewarding outcomes, prior to learning to avoid responding in certain circumstances (an innate or learned propensity to “go”). Alterations in appetitive associations may change the strength of the drive for reward. This could include differences in the intrinsic value/pleasurability of a reward (liking), and/or changes in the motivational value of the reward/reward paired cues (wanting). Though these are experimentally separable , they arThough impulsivity is defined in terms of operant behavior, in which impulsive behavior is characterized by actions that have unwanted consequences, the processes that underlie impulsive behavior may also be measurable at the level of Pavlovian tasks if they include changes to reward processing. In other words, if an instance of increased impulsivity was due to a change in the drive process, it may be able to be seen in altered appetitive classical conditioning, when outcomes are independent of action. For example, changes to the magnitude/value of an unconditioned stimulus influences the associative strength of conditioned cues, resulting in enhanced conditioned responding . For appMultiple neural substrates have been implicated in assigning value to an outcome or cue and incentive motivation. Dysregulation of any number of highly interconnected implicated brain regions could therefore result in altered reward related behavior. Several regions appear to represent or integrate reward value, including the nucleus accumbens (NAc), ventral pallidum (VP), basolateral amygdala (BLA), and regions of the prefrontal cortex including the orbitofrontal cortex (OFC) . In partMany brain regions involved in reward encoding and classical conditioning are innervated by the serotonin system, rendering serotonin as a well-positioned modulator of reward processes . Seroton1A receptors (thought to increase synaptic serotonin by antagonizing autoreceptors) or genetic knockout of serotonin transporters , 5-choice serial reaction time test , stop signal reaction time test , and differential reinforcement of low rate responding .Importantly, an increased impulsive action phenotype may influence behavioral readouts in other operant paradigms testing motivation, such as random ratio and progressive ratio. Changes in excitatory responding , for example the vigor of responding, which are subserved by changes in reward circuitry , 2018 maApproaches to dissect the underlying neural circuits of co-occurring reward process and inhibitory dysfunction can determine if the neural circuit dysregulation is subserved by convergent mechanisms. Many of the same brain areas noted to be involved in reward and motivation have also been implicated in impulsive action. In particular, the NAc and its core and shell subregions have been extensively studied for their individual roles in impulsive action through reaction time tests, with pharmacological manipulations and deep brain stimulation of the shell subregion causing elevated premature responding . OptogenThe alterations in reward-related behavior in impulsivity could also be the result of impaired action selection supported by the dorsal striatum, which is important when there is an instrumental contingency between response and reward, as in many tests of impulsive action , 2010. PGiven that serotonin signaling is involved in many aspects of reward processing, it is relevant to consider how the influence of serotonin on reward contributes to its effect on impulsivity. As discussed in prior sections, most, if not all, rodent assays of action impulsivity involve approach toward appetitive cues and outcomes, and are therefore confounded by reward processing. As such, the effects of manipulations to serotonin signaling on tests of impulsive action may in some cases arise from effects on reward responsivity. Directly assessing the effects of experimental manipulations of serotonin signaling on reward related alterations is helpful to accurately interpret the effects in traditional tests of impulsivity.Serotonin enables appropriate waiting for reward , and actSerotoninergic modulation of prospective reward encoding within the OFC is heterogeneous , as are 1B (by gene knockout) or 5-HT2C receptors (by receptor antagonist) impairs response inhibition while also increasing mesolimbic DA release . In this case, the conditioning is specific to the trained set of cues, and the inhibitor does not usually transfer to a different excitatory cue . AdolescThough serotonin is strongly implicated in behavioral inhibition in instrumental conditioning, it is also involved in the acquisition and expression of inhibitory learning in these classical conditioning experiments. operant responding for reward). In fear conditioning, knockout of the serotonin transporter impairs extinction recall, though here the effect may be through the retention or expression, rather than the acquisition, of the new inhibitory association  regions implicated in behavioral inhibition , and siginsensitive to serotonergic manipulation. In the other direction, stimulating serotonergic neurons enhances the ability to wait for reward delivery in “patience” based tasks, but is not, itself, rewarding (Whereas serotonin’s contributions to reward can be assessed directly outside of tests of impulsivity (as discussed), its role in behavioral inhibition is harder to extricate since tests of impulsive action are most commonly tests of behavioral restraint. Nevertheless, both behavioral evidence and a review of areas in which serotonin acts on behavioral restraint support a role for serotonin in “brake” processes. Global 5-HT depletion increases premature responding in the 5CSRT task  and impaewarding . Furtherewarding . Specifiewarding . Becauseewarding , the selewarding  and reguewarding , 2019. Sewarding , as wellewarding .The mPFC receives strong serotonin innervation from the DRN and is critical for behavioral control through both action selection and timing. Paradoxically, though elevated tonic extracellular levels of 5-HT in mPFC correlate with higher impulsive action in the 5CSRTT , directl1B receptor binding within the ACC is strongly associated with inhibiting responses to stimuli in an emotional Go/No-go task  had no effect on impulsivity  Serotonin receptors are positioned to regulate cortical microcircuits and projection neurons. (2) Serotonergic manipulations within prefrontal cortices and striatal outputs alter impulsive behavior. (3) The described functions of these cortical regions align best with “brake” processes in behavioral restraint rather than “drive” processes. To build a model of serotonergic impulsivity-regulation within cortical circuits, these ideas would ideally be integrated in a cohesive framework. For example, future work could characterize the role and impact of serotonergic signaling on the activity of functionally discrete cortical subpopulations e.g., . CausallThrough this review, we have provided an overview of the behavioral and neural systems underlying impulsive action. Dysregulations of either reward or inhibition can create an imbalance of the neural systems responsible for impulse control. Neurally, we suggest that widespread DRN serotonergic projections  place seAdolescence is an interesting case which allows us to probe the role of these two processes and how serotonin influences the balance. Specifically, adolescence is a developmental period characterized by increased impulsivity, risky decision making, and hyper reward-sensitivity. In the dimension of impulsive action, compared to children and adults, teenagers have more false alarms for no-go cues in the Go/No-go test . This he1B receptor. This suggests that some traditional measures of impulsive action are intrinsically tied to reward related behaviors. However, we highlight the idea that additional approaches to measuring impulsivity in the absence of learned appetitive motivators may allow the dissociation of reward processes from behavioral inhibition , to include specific cognitive and behavioral substrates. Studies of disordered impulse control, both in human and animal models, would ideally use multiple and varied behavioral tests to determine the underlying component processes and tease apart influences of reward from inhibitory control. The understanding of the contributions of these processes can provide tractable targets for pursuing neural circuit mechanisms and potentially more individualized treatment approaches for pathological impulsivity. In particular, we propose that serotonin signaling is an important mechanism to explore in this context to understand the behavioral and neural bases of the control of impulsive action.SD, MS, and KN contributed to the ideas, wrote the manuscript, and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Using solution NMR and UV-vis spectroscopy, we found that Cd2+ spontaneously replaced Zn2+ in both structural sites of the C1B domain, with the formation of all-Cd and mixed Zn/Cd protein species. The Cd2+ substitution for Zn2+ preserved the C1B fold and function, as probed by its ability to interact with a potent tumor-promoting agent. Both Cys3His metal-ion sites of C1B have higher affinity to Cd2+ than Zn2+, but are thermodynamically and kinetically inequivalent with respect to the metal ion replacement, despite the identical coordination spheres. We find that even in the presence of the oxygen-rich sites presented by the neighboring peripheral membrane-binding C2 domain, the thiol-rich sites can successfully compete for the available Cd2+. Our results indicate that Cd2+ can target the entire membrane-binding regulatory region of PKCs, and that the competition between the thiol- and oxygen-rich sites will likely determine the activation pattern of PKCs.Conserved homology 1 (C1) domains are peripheral zinc finger domains that are responsible for recruiting their host signaling proteins, including Protein Kinase C (PKC) isoenzymes, to diacylglycerol-containing lipid membranes. In this work, we investigated the reactivity of the C1 structural zinc sites, using the cysteine-rich C1B regulatory region of the PKCα isoform as a paradigm. The choice of Cd By serving as the key node in the phosphoinositide signaling pathway, PKCs regulate cell growth and differentiation  PKC isoforms consists of three peripheral membrane binding modules: the tandem C1A and C1B domains that penetrate the membrane in response to binding a signaling lipid, diacylglycerol, and the C2 domain that binds to anionic phospholipids in a Ca2+ dependent manner  that comprises C1B and the neighboring C2 connected by the native linker region . We found that Cd2+ readily displaces Zn2+ at both structural sites in C1B, and that this process successfully competes with the Cd2+ interactions with the oxygen-rich C2 sites. Furthermore, despite the identical coordination spheres, the two Zn2+ sites show different thermodynamics and kinetics of Cd2+ binding. C1 and C2 domains are the basic building blocks of more than 100 proteins involved in signal transduction. Hence, the knowledge gained from this study will be applicable to other C1- and C2-containing proteins . From the spectral overlay, it is evident that the N-H resonances of many C1B residues, particularly those coordinating Zn1 and Zn2, experience large chemical shift perturbations upon C1B binding Cd2+.While the UV-vis data show that Cdous work . Upon adZn, there are three other possible Cd/Zn protein states: C1BCd, C1BZn/Cd, and C1BCd/Zn that can co-exist in solution. The N-H groups of three residues in C1B: His140, Ile145, and Val147 show four cross-peaks each  relative Cd2+ affinity for either site does not depend on the chemical identity of the metal ion, Cd2+ or Zn2+, that occupies the other site . We conclude that both thiol-rich coordination sites in C1B are reactive with respect to Cd2+ substituting for the native Zn2+ ion.In addition to native C1Baks each  and serv2+ that is released as a result of the signaling events preceding PKCα activation. The Ca2+ binding site is formed by the Ca2+- and membrane-binding loops or CMBLs  through the loop regions  that report on Cd2+ binding to either site 1 or site 2. The kinetics data shown in To investigate the site-specific kinetics of Zn2+, reaching the Cd2+-bound population of 53% within the first 15 min of the experiment. This exceeds the equilibrium value by ∼10%, and the site 1 population by 17%. As shown on the 3D structure of the C1B domain in 2+ at site 2 brings the termini of C1B together by coordinating His102 at the N-terminus and Cys151 at the C-terminus. This part of the protein has a relatively high degree of solvent exposure and is therefore readily accessible to Cd2+. Another distinct feature of site 2 is the presence of a reactive Cys residue, Cys151, which serves as the entry point for the reactive oxygen species that activate PKCα in a process involving Zn2+ release. The structural dynamics of site 2, associated with the loss of Cys151 coordination bond with Zn2+  and ∼2-fold (site 1) higher affinity to Cd2+ relative to Zn2+ (2+ being a softer Lewis acid (larger ionic radius and polarizability) than Zn2+ and therefore forming stronger interactions with thiolate ligands 2·4H2O  in the appropriate buffer. Unless indicated otherwise, the experiments were conducted in the “MES buffer” comprising 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) at pH 6.0 in HPLC-grade water (Avantor), 150 mM KCl, and 1 mM tris(2-carboxyethyl) phosphine (TCEP). The buffers were passed through the Chelex® 100 (Sigma-Aldrich) column to remove residual divalent metal ions.The CdM. musculus for C1B-C2 and C1B; R. Norvegicus for C2) were amplified by PCR using the cDNA clone of PKCα (Open Biosystems) as a template and cloned into the pET-SUMO vector (Invitrogen). Isolated C1B, C2, and C1B-C2 were expressed and purified as described previously , isolated C1B (residues 100–152) or C2 (residues 155–293) of PKCα  solution or MES buffer  were placed in the sample cuvette; the reference cuvette always contained metal ion-free MES buffer. Cd15N]-enriched C1B was dissolved in 6 M guanidine hydrochloride (Acros Organics) and the “refolding buffer” comprising 20 mM MES at pH 6.0 and 1 mM TCEP. The final protein concentration was between 15 and 35 μM during the denaturation step. The refolding was conducted in three dialysis steps, all of them carried out in the refolding buffer: (1) against 8 M urea at room temperature, for 8 h; (2) against 1.5 M urea and 100 μM Cd(II) nitrate at 4°C, overnight; and (3) against urea-free buffer at 4°C, for 3 days to ensure complete removal of urea. The refolded protein was concentrated in a Vivaspin® spin concentrator with a 3 kDa cut-off and subsequently exchanged into an “NMR buffer”  D2O using a Midi-Trap G25 desalting column . DMSO (Cambridge Isotopes) and added to the sample containing 94 μM of [U-15N] enriched C1BCd in the presence of 10 mM mixed micelles. Mixed micelles comprising [2H38] dodecylphosphocholine,  and 2-dihexanoyl-sn-glycero-3-[phospho-l-serine]  at a molar ratio of seven to three were prepared as previously described (All proteins were concentrated and buffer exchanged using 10 kDa (C1B-C2), 3 kDa (C1B) and 5 kDa (C2) cut-off Vivaspin® 15R concentrators into an NMR buffer. The experiments were carried out on Avance III HD NMR spectrometers (Bruker Biospin), operating at the  NMRPipe . The cro NMRPipe . SequencHN(CO)CA  experimed HNCACB  spectra lexed C2  domains.escribed . The finZn , C1BCd , C1BZn/Cd (Zn2+ at site 1 and Cd2+ at site 2), and C1BCd/Zn (Cd2+ at site 1 and Zn2+ at site 2). The fractional populations of those protein species can be defined as:I is the intensity of the corresponding N-H cross peaks in the 15N-1H HSQC spectra for H140, I145, and V147. The concentrations of free Cd2+ ([Cd2+]) and Zn2+ ([Zn2+]) can be calculated from the following mass balance equations:0, [Cd2+]0, and [Zn2+]0 = 2×P0 are the total concentrations of protein, Cd2+, and Zn2+, respectively. It is convenient to define the affinities of metal ions to C1B in terms of individual sites. For the single metal-ion bound species, we use the M [n] notation, where M = Zn or Cd, and n = 1 or 2. For example, C1BZn[2] defines C1B with site 2 populated by Zn2+ and a vacant site 1, and KaZn[1] defines the association constant for the binding of Zn2+ to site 1 when site 2 is already populated by Zn2+. The following equilibria describe the binding processes and the associated Ka values:The four possible Zn/Cd metallated protein states are identified using the following nomenclature: C1B2+ and Zn2+ to sites 1 and 2 can then be defined as the ratio of the association constants:The relative affinity of Cd2+ and Zn2+ to a given site C1B site when Zn2+ populates the other. A similar set of equilibria can be constructed to obtain the relative Cd2+ and Zn2+ affinities when Cd2+ populates the other site:The χ[n] (n = 1 or 2) values report on the relative affinities of Cd2+, C1B, and Zn2+ in the system  equipped with a room temperature probe. The first time point started 12 min post Cd2+ addition, and each SOFAST HMQC experiment took 15 min. Because the inter-conversion between Zn2+- and Cd2+-complexed states is in the slow exchange regime, at any time point their fractional population can be determined from the intensities of the corresponding amide cross-peaks in the SOFAST-HMQC spectra. We used the N-H resonances of His102, Cys132, Thr134, Cys135, and Leu150 as the reporters of Cd2+ binding to site 1; and Phe114, Cys115, His117, Cys118, Gly119, Ser120, Tyr123, Lys141, and Cys143 as the reporters of Cd2+ binding to site 2.To monitor the kinetics of Cd


Schisandra chinensis has been widely used as a traditional herbal medicine to treat chronic coughs, fatigue, night sweats, and insomnia. Numerous bioactive components including lignans have been identified in this plant. Lignans with a dibenzocyclooctadiene moiety have been known to possess anti-cancer, anti-inflammatory, and hepatoprotective activity. Fragmentary studies have reported the ability of some lignans to modulate some cytochrome P450 (P450) enzymes. Herein, we investigated the drug interaction potential of six dibenzocyclooctadiene lignans  on nine P450 enzymes  and six uridine 5′-diphosphoglucuronosyl transferase (UGT) enzymes  using human liver microsomes. We found that lignans with one or two methylenedioxyphenyl groups inhibited CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2E1 activities in a time- and concentration-dependent like their CYP3A inhibition. In comparison, these lignans do not induce time-dependent inhibition of CYP1A2, CYP2A6, and CYP2D6. The time-dependent inhibition of gomisin A against CYP2C8, CYP2C19, and CYP3A4 was also elucidated using glutathione as a trapping reagent of reactive carbene metabolites given that gomisin A strongly inhibits these P450 enzymes in a time-dependent manner. A glutathione conjugate of gomisin A was generated in reactions with human recombinant CYP2C8, CYP2C19, and CYP3A4. This suggests that the time-dependent inhibition of gomisin A against CYP2C8, CYP2C9, and CYP3A4 is due to the production of carbene reactive metabolite. Six of the lignans we tested inhibited the activities of six UGT to a limited extent (IC50 > 15 μM). This information may aid the prediction of possible drug interactions between Schisandra lignans and any co-administered drugs which are mainly metabolized by P450s. Schisandra + at m/z 710.2576 (mass error < 2 ppm), 293 Da higher than that of gomisin A. This suggests that gomisin A first loses [-CH2] before it is conjugated with one molecule of GSH [m/z 710.2576 through collision gave the characteristic daughter ions at m/z 692.2443, 581.2149, and 435.1485, suggesting the loss of a water molecule (−18 Da), the loss of a pyroglutamate residue (−129 Da) of GSH, and cleavage of the cysteinyl C-S bond (−275 Da)  . The fra−275 Da) .Schisandra extracts and lignans were found to alter the pharmacokinetics of drugs which are substrates of CYP3A. Animal studies show that oral Wuzhi capsules  significantly increase blood tacrolimus concentration through CYP3A inhibition [Schisandra chinensis alcoholic extracts  with tacrolimus exert a greater increase on tacrolimus’s Cmax and AUC values than when used alone, thereby indicating inhibition of CYP3A, a major tacrolimus-metabolizing enzyme [In previous studies, hibition . Wang etg enzyme . Deoxyshg enzyme . The magg enzyme .Schisandra lignans based on each of the inhibitory potential. Gomisin A inhibited CYP2C8 activity with an IC50 value of 2.8 μM, similar to CYP3A inhibition (IC50 = 0.77~1.2 μM) in a time- and concentration-dependent manner. Considering that gomisin A participates in the pharmacokinetic intervention of cyclophosphamide by blocking CYP3A-mediated metabolism and reducing chloroacetaldehyde production in rats [50 = 3.5 μM) is similar to CYP3A inhibition (IC50 = 1.7 μM). In vivo studies are required to determine the clinical relevance of potential herb drug interactions between typical doses of Schisandra extracts, including lignans, and CYP2C8 or CYP2C19 substrate drugs.In contrast to the extensive studies on drug interactions with CYP3A substrates, there is a paucity of data detailing drug interactions with other P450s. We predicted the clinical herb drug interaction risk induced by  in rats , gomisin in rats , paclita in rats , and rep in rats . In rats in rats . Gomisin in rats  and omep in rats , because50 > 15 μM). Moreover, our data show that gomisin A inhibits CYP2C8, CYP2C19, and CYP3A4 enzymes in a time-dependent manner by forming carbene reactive metabolites in a similar fashion to gomisin C-induced inhibition of CYP3A4. Additionally, these lignans may result in clinically relevant pharmacokinetic interactions with other co-administered drugs biotransformed by CYP2B6, CYP2C8, CYP2C9, CYP2C19, and/or CYP2E1.In conclusion, we report that lignans containing one or two methylenedioxyphenyl groups inhibit CYP2B6, CYP2C8, CYP2C9, CYP2C19, and/or CYP2E1 in a time- and concentration-dependent manner which is similar to CYP3A inhibition. We found that six lignans inhibit six UGTs to a limited extent (IC


Gynostemma pentaphyllum) is a traditional Chinese medicinal herb that has been widely used in food and supplemental products. In the last 20 years, extensive research has been conducted to investigate the medicinal prospects of jiaogulan, and in this regard, more than 200 compounds have been isolated with various medicinal properties such as anticancer, anti‐obesity, anti‐inflammation, and antioxidation. In respect of potential benefits, jiaogulan market is likely growing, and various food items comprised of jiaogulan  have been commercialized in the United States of America, China, and other Asian countries. More recently, there has been growing interest in the prebiotic potential of jiaogulan, especially at the interface of the gut microbiota. This review focuses on the prebiotic and therapeutic aspects of saponins and polysaccharides of jiaogulan tea by summarizing the literature on cancer, obesity, antioxidant activity, and immune‐modulatory properties.Jiaogulan ‐modulating properties of various compounds from jiaogulan. This review highlights the therapeutic effect of jiaogulan on the diversity and composition of GM. For centuries, the herbal tea made from the aerial part (including stems and leaves) of Gp has been consumed in China as a general tonic. Today, it is progressively popularizing around the world for lowering serum lipid and cholesterol levels  at the interface of the GM. The study particularly focuses on the anticancer, anti‐obesity, and antidiabetic properties of jiaogulan.2G. pentaphyllum saponin (GpS) was reported for the anticancer properties by upregulating Prdx1 and Prdx2 expression and suppressing Ras, RAF/MEK/ERK/STAT, PI3K/AKT/mTOR signaling  producing bacteria. However, the growth of potential pathogens, for example, Dysgonomonas spp., Helicobacter spp., sulfate‐reducing bacteria, were suppressed after GpS introduction to mouse gut  to a cancer preclinical mouse model and noticing anticancer effects, we proved that the anticancer effect of GpS is partly through stimulating the growth of beneficial bacteria in the gut  polysaccharides. It was observed that GpS and polysaccharides from lucidum can greatly improve the inflamed gut barrier of ApcMin/+ mice by inhibiting polyp formation, changing colonic M1 to M2 macrophages, stopping the oncogenic signaling molecules, and increasing the E‐cadherin/N‐cadherin ratio  and in the large intestine, metabolize into lactic acid and SCFAs that improve host physiology, particularly gastrointestinal health  also be reported for anticancer properties. Polysaccharides are potential prebiotic polymers that have been extensively studied. They promote the growth of certain beneficial bacteria  and GpS could equally suppress the growth of prostate cancer PC‐3 cells, with IC3Fissicatena and Akkermansia, which are enriched in high‐fat and high‐cholesterol‐induced NAFLD mice  and inhibited potential pathogens  and Toll‐like receptor 4 (TLR4) are significantly increased during the progression of nonalcoholic fatty liver disease (NAFLD). A report showed that GpS improved NAFLD induced by high‐fat diet induced through regulating LPS/TLR4 signaling pathway .The authors declare no competing financial interest.Not applicable.


Cellular uptake analysis demonstrated higher intracellular concentrations of 5a‐2 (408.3 nm) compared with CX‐4945 (119.3 nm). This finding clarifies the comparable effects of both compounds on the intracellular CK2 activity despite their different inhibitory potency in vitro [IC50 = 25 nm (5a‐2) and 3.7 nm (CX‐4945)]. Examination of the effects of both CK2 inhibitors on cancer cells using live‐cell imaging revealed notable differences. Whereas CX‐4945 showed a stronger pro‐apoptotic effect on tumor cells, 5a‐2 was more effective in inhibiting tumor cell migration. Our results showed that 49% of intracellular CX‐4945 was localized in the nuclear fraction, whereas 71% of 5a‐2 was detectable in the cytoplasm. The different subcellular distribution, and thus the site of CK2 inhibition, provides a possible explanation for the different cellular effects. Our study indicates that investigating CK2 inhibition‐mediated cellular effects in relation to the subcellular sites of CK2 inhibition may help to improve our understanding of the preferential roles of CK2 within different cancer cell compartments.Highly pleiotropic and constitutively active protein kinase CK2 is a key target in cancer therapy, but only one small‐molecule inhibitor has reached clinical trials—CX‐4945. In this study, we present the indenoindole, 5a‐2, is shown to inhibit CK2 activity in cancer cells equivalently to the well‐known inhibitor CX‐4945. Whereas CX‐4945 shows strong pro‐apoptotic and anti‐proliferative effects, we demonstrate a strong anti‐migratory effect for 5a‐2. We conclude that subcellular localization might be critical for the differential cellular effects of CK2 inhibitors.In this study, a 4,5,7‐trisubstituted indeno[1,2‐ AKTprotein kinase BCEcapillary electrophoresisHPLChigh‐performance liquid chromatographyLIFlaser‐induced fluorescenceMOPS3‐(N‐morpholino)propane sulfonic acidMSmass spectrometrySARstructure–activity relationshipHuman protein kinase CK2 is a constitutively active serine/threonine kinase . Today, CK2 is a well‐established therapeutic target in cancer , 14, 15.b]indole derivatives inhibiting this protein kinase have already been described [b]indoles as CK2 inhibitor is the compound 4p (5‐isopropyl‐4‐(3‐methylbut‐2‐enyloxy)‐5,6,7,8‐tetrahydroindenoindole‐9,10‐dione), which inhibited the activity of the protein kinase in vitro with an IC50 value of 25 nm. Furthermore, 4p was shown to inhibit intracellular CK2 activity and to exhibit anti‐cancer effects on breast, prostate, skin, and lung cancer cells indole scaffold, as well as an alkyl group in position 7 and an alkoxy group in position 4, increased the inhibitory potential of the compounds indoles were identified as potent, ATP‐competitive compounds. Their flat and tetracyclic structure designed to address the ATP‐binding pocket of CK2 allows a diverse derivatization of the ring system. Therefore, numerous indenoindole derivatives, which showed strong inhibitory effects against protein kinase CK2 in vitro. Using CX‐4945 as a reference compound, the most potent CK2 inhibitor 5a‐2 was examined for its effects on intracellular CK2 activity in three different tumor cell lines. In addition, the cellular uptake of 5a‐2 was analyzed to estimate the amount of the compound that could interact with the target in the cell. Subsequently, we examined the changes in cell proliferation, apoptosis, and migration mediated by this indenoindole as compared to those induced by CX‐4945. To help clarify the observed preferential anti‐cancer effects for the investigated compound, we compared its subcellular distribution with that of CX‐4945. Through linking the cellular effects mediated by CK2 inhibition with subcellular distribution of the investigated compounds, this study helps to provide hints for the impact of differential CK2 inhibition in cell compartments on several biological processes.Based on our knowledge from the SAR analysis, we developed new 4,5,7‐trisubstituted indenoindole‐9,10‐diones 5a,b were synthesized according to previously reported methods  , and anti‐CK2α‐specific antiserum . α‐Tubum for 24 h. The cells were detached from the cell culture plates with cell scrapers, sedimented via centrifugation at 700 × g and 4 °C for 5 min, and washed with PBS. Afterward, cells were lysed by suspending in 100 µL lysis buffer (50 mm Tris/HCl (pH 7.5), 0.15 mm NaCl, 1 mm Na3VO4, 0.5 mm NaF, 0.8 µm aprotinin, 0.01 mm pepstatin A, 0.02 mm leupeptin 0.1 mm PMSF, 1 mm benzamidine, 0.5% sodium deoxycholate, and 1% Triton X‐100). Cell debris was removed by centrifugation at 10,000 × g and 4 °C for 10 min. Protein concentrations were determined via Bradford analysis . 80 µL kinase buffer  containing 90 µg protein per sample was preincubated at 37 °C for 10 min. Afterward, 120 µL assay buffer  were added. The samples were incubated at 37 °C for 15 min. The reaction was stopped by transferring the samples on ice and adding 5 mm EDTA. Intracellular CK2 activities were determined via CE analysis.To analyze the influence of CK2 inhibitors on the intracellular CK2 activity, an adapted experimental procedure described by Schneider et al.  combined2 in 6‐well plates. Afterward, cells were treated with either 1 or 3 µm inhibitors for 1, 5, and 12 h. Cells were washed with PBS twice and detached using trypsin/EDTA. The number of cells in each sample was determined with an automated cell counter . After centrifugation at 700 × g and 4 °C for 5 min, cells were washed multiple times with PBS followed by lysis in 100 µL homogenization buffer (50 mm Tris/HCl (pH 7.5), 1 mm EDTA, 1 mm Na3VO4, 0.5 mm NaF, 0.1 mm PMSF, 1 mm benzamidine, and 1% Triton X‐100). Proteins were precipitated by adding 500 µL ice‐cold ACN/MeOH 1/1 (v/v). Samples were centrifuged 30 min at 20,000 × g and 4 °C. Supernatants were evaporated to dryness using a vacuum centrifuge maintained at 50 °C under full vacuum for 2 h. Residues were resolved in 100 µL ACN/H2O 1/1 (v/v) and analyzed via HPLC‐MS/MS. Intracellular inhibitor concentrations were calculated according to Rahnel et al. indoles . Thus, the latter two compounds were 200‐ to 350‐fold more potent with respect to inhibition of CK2 activity in vitro. These results indicate that a methoxy group in position 4 of the indenoindole scaffold  indole inhibits CK2 more effectively within the cell than CX‐4945. The observed weaker CK2 activity inhibition by 5b‐2 compared to 5a‐2 is consistent with its lower potency toward CK2 inhibition as mentioned before in Table 50 = 47 nm). In further studies, we therefore focused on 5a‐2 as the more potent CK2 inhibitor.To directly analyze the interaction of d et al. . As show5a‐2 and CX‐4945 in vitro. To estimate the ability of the indenoindole and the reference compound to penetrate cells, their polarities were determined based on n‐octanol/water partition coefficients (LogD7.4) according to Galla et al. indole.Since several studies reported that a decrease of CK2 activity in cancer cells leads to their apoptosis , we inve5a‐2 was investigated in this study. For this analysis, cells were cultured in 96‐well ImageLock plates (Sartorius) to a confluence of 100%. Afterward, a scratch wound was created in the cell layer of each well using an IncuCyte® WoundMaker tool (Sartorius). Then, cells were treated with 5a‐2 or CX‐4945 at 1 or 10 µm. The cell migration into the wound was determined using an IncuCyte® S3 Live Cell Imaging system (Sartorius). The results of this experiment . Also in this case, 5a‐2 had a stronger effect than CX‐4945, further confirming the strong anti‐migratory effect of the indenoindole.Cellular migration is a process involving CK2 that plays a role in tumor metastasis , 55. Theent Fig.  showed a5a‐2 or apoptosis for CX‐4945.These observations raise the question whether variable degrees of inhibition of CK2 in cellular compartments by the studied inhibitors may be responsible for their preferential interference with cellular processes, that is, migration for 5a‐2 and CX‐4945 clearly differ in their pro‐apoptotic . This could be shown by the reduction of the CK2‐specific Akt phosphorylation in LNCaP treated with 5a‐2 and CX‐4945 as well as by the inhibition of intracellular CK2 activity in A431, A549, and LNCaP cells by both CK2 inhibitors. Since intracellular target inhibition is highly dependent on drug concentration within the eukaryotic cell indole. Thus, these higher concentrations may explain why 5a‐2 and CX‐4945 affected intracellular CK2 activity to the same extent, despite their different inhibitory potential in vitro. Furthermore, we were able to show that the uptake of 5a‐2 was mainly driven by passive diffusion, while active transporters appeared to play a major role in the uptake of CX‐4945. This observation correlated with the lower polarity of the indenoindole, which was determined here from the LogD7.4 values of the compounds. Consistent with our observations, a correlation between LogP values and passive diffusion of drugs was previously reported indole suppressed HUVEC proliferation with an EC50 value of 30.8 µm, while the reference compound exhibited an EC50 value of 10.7 µm. This anti‐proliferative effect of CX‐4945 on non‐cancer cells has been observed in previous studies indole. Recently, Wells et al. described a potent, highly selective CK2 inhibitor, which did not suppress tumor cell proliferation to the same extent of CX‐4945. This observation led Wells et al. to hypothesize that the potent anti‐proliferative effect of CX‐4945 may not be due to inhibition of CK2 alone but rather to off‐target effects. This hypothesis was supported by the inhibitory effect of CX‐4945 on the activity of other kinases indole must also be tested in future studies.Consideration of the cellular effects of  studies , 59. The kinases . Off‐tar5a‐2 also induced apoptosis, but to a much weaker extent. On the other hand, 5a‐2 reduced the migration of A549 and A431 cells more potently than CX‐4945. As part of tumor metastasis, migration is a cellular process that has previously been described as sensitive to CX‐4945 treatment indole over the reference compound in the treatment of systemic cancers indole as potent new CK2 inhibitor based on the results obtained in this study. Most notably, 5a‐2 was shown to inhibit the intracellular CK2 activity equivalent to CX‐4945, despite a lower inhibitory potential in vitro. Interestingly, 5a‐2 and CX‐4945 differed in the extent of their cellular effects and subcellular distribution. This led us to the theory that these effects are dependent on the site of action of CK2 inhibition. With this study, we present an approach to elucidate biological and pharmacological processes based on comprehensive investigation of effects of CK2 inhibitors on cancer cells. On this basis, insights could be gained to better understand the different roles of CK2 in cancer cell biology and ultimately help to develop new anti‐cancer drugs.In conclusion, we identified a 4,5,7‐trisubstituted indeno[1,2 The authors declare no conflict of interest. The authors alone are responsible for the content and writing of this article.RB and EE planned experiments; RB, LB, FA, LE, and CG performed experiments; RB, EE, and DA analyzed data; MLB supervised the chemical synthesis part of the project; RB and EE wrote the paper; JJ supervised and coordinated the project, critically analyzed the data, and designed the concept of the manuscript. All authors read and approved the final manuscript submitted for publication.Scheme S1. Chemical synthesis reagents and conditions.Fig. S1. 1H NMR of compound 5a‐1 – CDCl3.Fig. S2. 13C NMR of compound 5a‐1 – CDCl3.Fig. S3. 1H NMR of compound 5a‐2 – CDCl3.Fig. S4. 13C NMR of compound 5a‐2 – CDCl3.Fig. S5. 1H NMR of compound 5b‐1 – CDCl3.Fig. S6. 13C NMR of compound 5b‐1 – CDCl3.Fig. S7. 1H NMR of compound 5b‐2 – CDCl3.Fig. S8. 13C NMR of compound 5b‐2 – CDCl3.Click here for additional data file.


To compare the safety and accuracy of cortical bone trajectory screw placement between the robot-assisted and fluoroscopy-assisted approaches., whereas conventional fluoroscopy-guided screw placement was used in the fluoroscopy-assisted group. The accuracy of screw placement and rate of superior facet joint violation were assessed using postoperative computed tomography (CT). The time of single screw placement, intraoperative blood loss, and radiation exposure to the surgical team were also recorded. The χ2 test and Student’s t-test were used to analyze the significance of the variables (P < 0.05).This retrospective study was conducted between November 2018 and June 2020, including 81 patients who underwent cortical bone trajectory (CBT) surgery for degenerative lumbar spine disease. CBT was performed by the same team of experienced surgeons. The patients were randomly divided into two groups—the fluoroscopy-assisted group  and the robot-assisted group . Robots for orthopedic surgery were used in the robot-assisted groupP = 0.003). The RA group had a lower violation of the superior facet joint than the FA group. The number of screws reaching grade 0 in the RA group  was more than that in the FA group  (P = 0.041). Screw placement time was longer in the FA group (7.25 ± 0.84 min) than in the RA group . The FA group had more intraoperative bleeding (273.41 ± 118.20 ml) than the RA group . The radiation time of the FA group (0.43 ± 0.07 min) was longer than the RA group . Furthermore, the overall learning curve tended to decrease.A total of 376 screws were inserted in 81 patients, including 172 screws in the robot-assisted group and 204 pedicle screws in the fluoroscopy-assisted group. Screw placement accuracy was higher in the RA group  than in the FA group  (Robot-assisted screw placement improves screw placement accuracy, shortens screw placement time, effectively improves surgical safety and efficiency, and reduces radiation exposure to the surgical team. In addition, the learning curve of robot-assisted screw placement is smooth and easy to operate. In 2009, Santoni et al. introduced a new method for screw insertion called cortical bone trajectory (CBT) . This neCBT has been shown to be a safe and valuable option for screw fixation in spinal surgery. Level 2 and 3 clinical studies have demonstrated equal clinical and radiographic outcomes and lower perioperative complication rates than conventional techniques . As the Navigation or robot-assisted screw placement can be used to improve screw placement accuracy. The accuracy of robot-assisted screw placement has been demonstrated in previous studies , 10. ParThis study reviewed patients who underwent CBT for degenerative lumbar spine disease between November 2018 and June 2020. The inclusion criteria were as follows: (I) age ≤ 85 years, (II) diagnosis of degenerative lumbar disease that required surgery, and (III) need for the cortical trajectory screw technique for screw placement. Meanwhile, the exclusion criteria were as follows: (I) patients with scoliosis > 30°; (II) patients with infection, trauma, or severe psychosis; and (III) patients with vertebral body dysmorphism affecting screw placement, such as severe lumbar degeneration.Patients were placed in the prone position, and the surgical site was determined using fluoroscopy. An approximately 5-cm midline skin incision was made. The muscles were then separated layer by layer to expose the surgical site, and screw trajectories followed the mediolateral and caudal-directed paths. The entry point was located at the junction of the midpoint of the superior facet joint process 1 mm below the lower edge of the transverse process. After confirming correct placement with anteroposterior and lateral fluoroscopy, the guidewire for positioning was used to perform screw placement by hand. Decompression was performed as appropriate after the completion of screw placement.The TiRobot consists of a surgical plan, an optical tracking unit, a control workstation, and a surgical robotic arm that has six degrees of freedom (6-DOF) to allow accurate and stable positioning.Patients were placed prone on a robot-assisted specialized operating table, with the tracker positioned over the spinous process at the level of the operative vertebral body. The calibration device was fixed by the robotic arm on the skin as close to the surgical site as possible. Three-dimensional images of the screw placement area were then acquired using a C-arm scanner  Fig. . The regThe time for single screw placement, intraoperative blood loss, and radiation exposure to the medical team were recorded. The time for single screw placement was defined as the total time for satisfactory screw placement for all screws divided by the total number of screws. Robot-assisted screw placement included screw path planning and screw placement time, whereas fluoroscopy-assisted screw placement included guidewire positioning and screw placement time. The time to adjust for an unsatisfactory trajectory indicated by intraoperative fluoroscopy was also accounted for. Radiation exposure to the medical team was measured using total intraoperative fluoroscopy time.Screw placement accuracy was evaluated using the score proposed by Ding et al.  Their meFacet joint violation was evaluated according to the classification described by Yson et al.  and MoshIn this study, screws with a length of 35 mm and a diameter of 5.5 mm were used in both groups. Patients in both groups routinely underwent CT scanning before and after surgery, and the CT in our center was performed using GE CT machine , with scanning parameters of interval (mm) 0.625, gantry tilt S0.0, kV 120, and mA 450.2 test was used for unquantified data, and Student’s t-test was used to compare differences between groups. Statistical significance was set at P < 0.05.The SPSS version 21.0 was used to analyze the collected data. All values were expressed as means ± standard deviation. The χA total of 81 patients were included in this study, with an average of 4.64 ± 0.94 screws placed per patient. There was no statistically significant difference in the number of screws placed between the two groups. A total of 172 screws were placed in 37 patients who underwent robot-assisted CBT screw placement, whereas 204 screws were placed in 44 patients who underwent fluoroscopy-assisted CBT screw. There were no significant differences in parameters, including sex, age, and body mass index (BMI), between the two groups.P < 0.001). The FA group had more intraoperative bleeding (273.41 ± 118.20 ml) than the RA group . The radiation time of FA group (0.43 ± 0.07 min) was longer than in the RA group . No patients in the RA group underwent revision surgery, while three patients in the FA group underwent revision surgery due to neurological injury caused by screw misplacement. The symptoms of these patients were relieved after surgery. There were also no statistically significant differences in revision and neurological injury complication rates between the two groups. Furthermore, there were four patients who developed infectious complications, with one patient in the FA group and three patients in the RA group, although no statistically significant difference was found (Table Screw placement time was longer in the FA group (7.25 ± 0.84 min) than in the RA group . In the RA group, Grade 1 was achieved in 5% of screws, which was lower than that in the FA group (10%) (p = 0.058). Moreover, four screws (2%) in the RA group achieved Grade 2, which was fewer than the 15 Grade 2 screws (7%) in the FA group (Table P = 0.041). The remaining classifications of the RA group included 12 Grade 1 screws and 4 Grade 2 screws, whereas in the FA group, there were 22 Grade 1 screws and 10 Grade 2 screws  and 169 screws (83%) in the RA and FA groups, respectively, achieved Grade 0 (r screws % in the % and 169P = 0.461). Based on the linear distribution of the scatter diagram, the average screw placement time showed a gradually decreasing trend  was longer than that in the last 50% of cases . In the present study, the screw trajectory was evaluated using the novel method, and 93% of the screws in the robot-assisted group achieved a Grade 0 screw trajectory, as compared with the 83% of the screws in the fluoroscopy-assisted group. The difference between the two was statistically significant (P = 0.003).The results of this study also showed that the trajectory of CBT screws was more accurate with robotic assistance than with fluoroscopy. To maximize cortical bone contact and improve purchase strength, the novel method of CBT relies on an accurate screw trajectory based on the four-point purchase between the dorsal cortex at the insertion site, the medial cortex of the posterior pedicle wall, the lateral cortex of the anterior pedicle wall, and the curvature of the vertebral body wall . TherefoThe mean intraoperative fluoroscopy time was longer in the fluoroscopy-assisted group than in the robot-assisted group. Since intraoperative CT scanning is required when robot-assisted screw placement is used, intraoperative CT can improve the accuracy of screw trajectory, thereby shortening the time required for intraoperative fluoroscopy to obtain the ideal screw placement. This was consistent with the conclusions of previous studies . HoweverP < 0.05) reduce the invasion rate of superior facet joints and reduce the probability of superior facet joint degeneration.In addition, our study found that robot-assisted screw placement had a low rate of superior facet joint violation, that is, the distance between the facet and the screw, and the accuracy of the screw in the pedicle. Facet joint violation is one of the most common complications of posterior lumbar fusion and has attracted increasing attention in recent years. It has been reported to cause symptomatic adjacent segment disease and may affect the fusion rate of fusion surgery, resulting in low back pain . Our stuBased on the learning curve associated with CBT described by Dayani et al. , a 6% meDespite these findings, the study had some limitations. First, the lack of accurate radiation exposure dose data precludes an accurate assessment of the difference in patient radiation doses using robot-assisted or fluoroscopy-assisted screw placement. Second, long-term follow-up of patients with facet joint invasion was lacking. Third, many factors that can influence adjacent degeneration, such as the length of the fixed segment. Lastly, long-term follow-up is needed to confirm if robot-assisted screw placement does reduce the incidence of adjacent facet joint invasion, and therefore, adjacent degeneration.Robot-assisted screw placement can improve screw placement accuracy, shorten surgery time, effectively improve the safety and efficiency of surgery, and reduce radiation exposure to the medical team. Additionally, the difficulty and learning costs in robot-assisted screw placement are low.


Despite global warming and Arctic sea-ice loss, on average the Antarctic sea-ice extent has not declined since 1979 when satellite data became available. In contrast, climate model simulations tend to exhibit strong negative sea-ice trends for the same period. This Antarctic sea-ice paradox leads to low confidence in 21st-century sea-ice projections. Here we present multi-resolution climate change projections that account for Southern Ocean mesoscale eddies. The high-resolution configuration simulates stable September Antarctic sea-ice extent that is not projected to decline until the mid-21st century. We argue that one reason for this finding is a more realistic ocean circulation that increases the equatorward heat transport response to global warming. As a result, the ocean becomes more efficient at moderating the anthropogenic warming around Antarctica and hence at delaying sea-ice decline. Our study suggests that explicitly simulating Southern Ocean eddies is necessary for providing Antarctic sea-ice projections with higher confidence. Delayed Antarctic sea-ice decline is linked to Southern Ocean eddies - and their explicit treatment in models is crucial. New multi-resolution climate change projections give a possible reason for low confidence in IPCC’s current 21st-century Antarctic sea-ice projections. This supports the notion of a possible delayed sea-ice decline in the Antarctic compared to that in the Arctic. In the few years from 2016 onwards, there has been significantly lower Antarctic SIE8, which one could consider harbingers of imminent change. However, others9 have argued that SIE is expected to regress to the near-neutral decadal trend in the near future; and indeed data for 2020/2021 seem to support this view8.Climate models that participated in the Coupled Model Intercomparison Project Phase 5 (CMIP5)10 and variability of the Amundsen Sea Low12, dynamic sea-ice transport changes16, recent absence of deep open-ocean convection18, additional meltwater from the Antarctic ice sheet and ice shelves21, and internal variability of tropical origin23. While some of these factors are likely to contribute to the slightly positive real-world trend, here we argue that mesoscale dynamical processes in the ocean play a critical role for explaining a delayed Antarctic sea-ice decline. Due to the contrasting trends between observations and existing climate models  concludes that our confidence in projections of Antarctic sea ice is low28.To explain the lack of long-term Antarctic sea-ice decline since 1979, previous studies have proposed various mechanisms such as decadal wind trends associated with the Southern Annular Mode (SAM)29. In fact, realistically representing the Southern Ocean circulation and its response to global warming is known to be a challenging task in coarse-resolution models30, requiring parameterizations (e.g. for the effect of ocean eddies) that are sensitive to how they are tuned. In particular, changes in forcing can be compensated via eddy compensation and saturation31, resulting in a notably insensitive transport of the ACC through Drake Passage. Both changes in mesoscale transient eddies and changes in meandering flows32 (standing eddies) may contribute to these phenomena, and the degree to which eddy compensation and saturation is realized in simulations thus depends on parameterizations in low-resolution (LR) models. There is some level of arbitrariness involved due to necessary choices being made for individual implementations of parameterizations in climate models. Although the development of eddy parameterizations is still an active field of research, it is increasingly being recognized that explicitly simulating mesoscale processes is often more accurate33. Therefore, an alternative way is to develop and use high-resolution (HR) sea ice-ocean models34 that employ more of the laws of physics by explicitly simulating rather than parameterizing the effects of ocean eddies. Compared to LR simulations for the Southern Ocean, earlier studies using HR ocean components have reported changes in the hydrography35 as well as of the overturning and ocean heat transport in idealized climate change simulations35.It has been suggested that models with a faithful representation of mesoscale ocean eddies in the Antarctic Circumpolar Current (ACC) are needed in order to better represent the behaviour of the Southern OceanHere, we show the impact of explicitly simulating mesoscale processes in the ocean on Antarctic sea-ice trends in comprehensive CMIP-type projections using a high-emission scenario. Our HR climate model configuration simulates stable September Antarctic SIE that is not projected to decline until the mid-twenty-first century. This is in stark contrast to typical CMIP models and our complementary LR configuration. From additional mixed-resolution experiments, in which only the atmosphere or ocean is highly resolved, we attribute this delayed decline to the HR ocean. Our study provides support for the hypothesis that ocean models that permit or resolve eddies are necessary to increase the trustworthiness of climate projections of the Southern Ocean, and it thus constitutes a step towards providing Antarctic sea-ice projections with higher confidence.2 per decade; the coarsest models, on the other hand, show negative trends amounting to as much as −0.8 million km2 per decade  sea-ice trends depend on the resolution of the ocean model used in the Southern Ocean. Even in the low spatial resolution range of 35–200 km available from CMIP5, models with higher resolution tend to show positive or moderate negative SIE trends ≥ −0.2 million km979–2018 40  that has been integrated until the end of the twenty-first century, following the HighResMIP protocol41. AWI-CM employs a sea ice-ocean model formulated on multi-resolution unstructured meshes42. This technique permits ocean eddies over the whole core of the ACC when compared to the local Rossby radius of deformation43 by concentrating grid points in that area 48; a cold blob over the North Atlantic49 is evident at both resolutions that is probably associated with a slowdown of the Atlantic Meridional Overturning Circulation51; and Arctic Amplification, which is strongest in winter, is clearly discernible52. Importantly, large differences between the HR and LR projections can be seen in the Southern Hemisphere , under a representative high-emission concentration pathway , depict typical global warming patterns with some well-known features  compared to 1990–2019, while HR loses ΔV = 1600 km3.Consistent with the temperature changes, projected September SIE in the Antarctic is different between the two experiments Fig. a: The SI010 Fig. a. The So18. Furthermore, the weaker pan-Antarctic fingerprint in HR when compared to LR is also clearly visible when using different baselines for comparing the late twenty-first century changes . The mixed-resolution simulations reveal that the cause for the delayed sea-ice decline is likely to be found in the HR ocean . The upwelled water of Atlantic origin (σ2 ≈ 37.1) extends too far south in the low-resolution experiment, probably in part due to numerical diapycnal mixing, leading to the relatively warm and salty subsurface conditions. Another contribution could be the combined effect of the Gent–McWilliams (GM) parameterization60 for mesoscale eddies, which acts to flatten isopycnals, and isoneutral (Redi) diffusivity. This latter mixing along isopycnals is not well constrained and might lead to diffusive transport of warmer waters. A similar statement as for the upwelled waters holds for lighter waters (σ2 ≈ 36.1–36.4) that reach under the sea ice in LR  the water mass structure and (ii) the resulting meridional overturning circulation. In the following, both aspects will be discussed. Starting with the water mass properties, LR shows a pool of warm subsurface waters southward of 60° S, which is up to 1 °C warmer than observed in the upper 500 m Fig. d, f. The61. This residual circulation is difficult to estimate directly from observations62; therefore its magnitude is still rather uncertain. Estimates also vary considerably across ocean models63. Using salinity as a tracer, observed present-day salinity sections indirectly reveal the structure of the residual circulation , with a marked upwelling branch of saline waters within the region 40–70° S . Observations and previous ocean simulations67 in the upper ~1000 m. The canonical MHT is generally poleward .The shape of the MHT is largely determined by the vertical structure of the ocean circulationThe heat transport associated with the northward residual branch of the upper circulation cell  than might have been anticipated. According to the concept of eddy saturation31, ocean eddies compensate for changes in external forcing. Both transient and standing eddies may contribute to this phenomenon, and the simulated compensation is dependent on parameterizations in low-resolution models. In contrast to HR, our low-resolution climate change experiment shows increased poleward MHT even south of 60° S . Bryan et al.32 explain this difference with increased (approximately doubled) heat transport by standing eddies, the meanders of the ACC, which are more pronounced in the HR configuration. In order to compensate for the change in forcing, the heat flux response by transient eddies can thus be much smaller in HR than in LR (where only transient eddy effects are parameterized), despite similar increases in westerly winds. We suspect that a similar explanation holds for our simulations. Across 60° S, in particular, anomalous equatorward MHT in our HR is larger than in LR by approx. 0.033 PW (1 PW = 1015 W) , which the authors find “a little surprising, given that the zonal wind stress does increase slightly”. The difference in MHT is in principle sufficient to help explaining the delayed onset and reduced rate of September sea-ice decline in our HR experiment (see “Methods”).In a previous modelling study by Bitz and PolvaniIn this study, we show that a HR climate model is capable of simulating a stable Antarctic sea-ice cover over the past few decades in CMIP-type simulations—consistent with available satellite data. This is in stark contrast to the behaviour of relatively coarse-resolution climate models, including those which participated in CMIP5 and CMIP6 in support of IPCC Assessment Reports. In our HR simulations, the decline of Antarctic sea ice is not projected to start before well into the twenty-first century; and this “delayed decline” of the Antarctic sea-ice cover can be traced back to the use of a HR ocean component. As outlined below, the exact reason for this behaviour remains to be determined. However, the faithful representation of ocean eddies by the laws of physics rather than physical parametrizations seems to be one plausible major contributor.72 and some of the results, in particular the sign of the MHT response, may seem contradictory to what is concluded here. However, as detailed below, a meaningful comparison with previous studies is surprisingly difficult to carry out given major differences in model forcing , model tuning , scientific question , and possibly various mechanisms at play70. Furthermore, what is actually known about the impact of resolution is obtained from a rather limited number of coupled models , reflecting the fact that coupled climate modelling with eddy-resolving ocean components is still in its infancy.The impact of ocean resolution has been discussed in previous modelling studies54 at 0.25° resolution, which is eddy-permitting only over parts of the ACC , while AWI-CM-LR with its 133 Sv shows lower transport, which is well within the CMIP5 range of 155 ± 55 Sv74. Both models thus do not suffer from a weak ACC, which is consistent with the fact that they are either lower resolved (AWI-CM-LR with eddy parameterization) or more highly resolved (AWI-CM-HR without paramerization) in that region than typical 0.25° configurations. Fully eddy-resolving models, with local resolutions better than 3–4 km over the ACC, are currently under development, and it remains to be seen how these models will perform in the Southern Ocean.It is important to realize that regular-grid models that are termed “eddy-resolving” based on their characteristics in mid-latitudes, e.g., at 1/10° resolution, stop being eddy-resolving in polar regions due to the latitudinal dependence of the Rossby radius of deformation  in AWI-CM for HR compared to LR. This may be due to differences in the representation of the underlying eddy-induced circulation and the degree to which it can oppose or compensate the changes in the wind-driven overturning. A wind-driven increase of the upper clockwise meridional circulation cell, with the northward branch being its upper representation, may be partly balanced by a compensating counter-clockwise eddy overturning in LR, similar to partial (parameterized) eddy compensation as shown for CMIP5 models LR Fig. . Because32 of climate models with lower-resolution ocean grids could overcome some of the shortcomings discussed in this study. However, current eddy parameterizations, with their individual implementation in climate models, often can not account for the full effect of eddies or may generate ambiguous results78. In fact, while Southern Ocean eddy compensation in a recent coarse-resolution climate simulation77 was shown to cancel the warming within the temperature inversion layer that had been predicted in an earlier study78 (as a response to an increase of the SAM index), the authors mention that their finding is in contrast with Bitz and Polvani71, whose 1/10° coupled model warmed in an ozone-depletion experiment. In their studies with the same coupled model CCSM3.5, Bryan et al.32 also highlight a role for standing eddies, i.e., meanders of the ACC, in balancing the wind-driven overturning. This mechanism of ACC equilibration will need further investigation in future studies, as its effects are fully beyond the scope of what eddy parameterizations in current climate models can achieve.It can be argued that either dedicated tuning or new flow-dependent approaches for how the space and time-dependent GM coefficient is determined in modern eddy parameterizations79 or differing mean states. It has been suggested, for example, that thermodynamic processes may be critical: Thicker sea ice decays faster than thinner ice, which is a basic thermodynamic property of sea ice81. At least for our simulations with AWI-CM, this mean state dependence is not the main explanation for the different sea-ice behaviour (see “Methods”). An open question is why the different mean states arise in the first place. Since the SIE for HR is closer to the observed extent than LR without particular tuning, it is possible that the different sea-ice mean state in LR itself is affected by details of the eddy parameterization and the choice of GM coefficient, which has been shown to determine the pattern of warming south of the ACC70.More generally, there are other possible mechanisms that could explain part of the resolution dependence of Antarctic sea-ice cover, including those that relate to vertical eddy heat fluxesDue to computational constraints in HR climate modelling, the spinup outlined in the HighResMIP protocol is relatively short (see “Methods”). However, we do not think that this has a major impact on our key findings. This is due to the fact that (i) the differences between LR and HR in sea-ice evolution are large ; (ii) differences between HR and LR can be explained physically ; and (iii) the findings are “reproducible” in full vs. mixed-resolution experiments. Having said this, considering a revised version of the HighResMIP protocol to allow for longer spin-ups may be worthwhile for future efforts, especially if faithful simulations of the Southern Ocean are required.82 of these CMIP6 models shows the same structural uncertainties of Antarctic sea-ice cover over the satellite era as found for CMIP5. This is consistent with our results, given that none of the CMIP6 models is eddy-permitting over the entire ACC in the Southern Ocean or even eddy resolving.In our study, we also considered results from the wide range of CMIP5 models, which are generally rather coarse, and found additional evidence for a resolution dependence of the timing of Antarctic sea-ice decline Fig. . When it34 to achieve a more faithful representation of the circulation and Southern Ocean hydrography . More specifically, we used historical and RCP8.5 scenario simulation data available on the German Climate Computing Centre’s (DKRZ) supercomputer Mistral, which is mirrored from the DKRZ node of the Earth System Grid Federation . For models with multiple ensemble members available for both the historical and the scenario period, we computed ensemble means prior to any further analyses  in Fig. p = 0.011; R2 = 0.403). The actual choice of the box width turns out not to be critical, given that significant correlations were also found for wider and smaller widths of ±2 million km2  and ±4 million km2 , respectively. When including all models, that is, also those with more unrealistic SIE of less than half the observed value, the relation is significant at the 10% level only .For the analysis in Fig. To compute the average ocean resolution in the 45–65° S latitudinal band, for every model we located all ocean model cells in that band according to their centre. The average resolution was then determined as the square root of the average grid cell area.2 per decade , similar to that for LR (−0.49 million km2 per decade).Data points for AWI-CM at LR and HR  are also added to the CMIP5 results in Fig. In order to diagnose the location of the sea-ice edge in the AWI-CM simulations Figs. b, e, in 91 (their Section 2) to derive global northward heat transport from the CMIP5 heat flux variables “hfx” and “hfy” in x- and y-direction, thereby accounting for peculiarities of the individual native model grids. The variables “hfx” and “hfy” were available only for a subset of the models listed in Fig. For the CMIP5 MHT analysis Fig. , we impl40 covering 150-yr periods were performed using a slightly modified version of the HighResMIP protocol41 (see below). One set of simulations (AWI-CM-HR) was run at eddy-permitting ocean resolution r over the ACC region , with some further eddy-resolving refinements  to about 8 km over the Agulhas and Brazil-Malvinas Confluence regions . For Fig. R was computed from the PHC climatology92 as z, where f is the Coriolis parameter, H is the ocean floor, and N is the Brunt-Väisälä frequency. The average spatial ocean resolution in the 45–65° S latitude band amounts to about 14 km, and no eddy parameterization was used in this band . HR uses a higher-resolution T127 atmosphere (ECHAM693), corresponding to a resolution of about 1°. These simulations are compared to corresponding low-resolution simulations (AWI-CM-LR) to determine the added value of increased resolution. LR uses a nominal 1° ocean mesh  with average spatial ocean resolution of 63 km in the 45–65° S latitude band (r > 2R). The influence of eddies is therefore fully parameterized. The LR ocean is coupled to a T63 atmosphere, corresponding to a spatial resolution of ≈2°. In order to better understand the origin of the resolution dependence, another set of “mixed-resolution” simulations was carried out comprising a HR ocean and low-resolution atmosphere, and vice versa. Because of the length of the control and scenario simulations, only monthly-mean output was stored for all experiments. Added meltwater or melt scenarios/projections were not accounted for in these simulations, which could provide further means for delaying Antarctic sea-ice decline21.Several control and scenario experiments with the coupled AWI Climate Model (AWI-CM)94 fields . Regarding the spinup of the coupled systems we followed the original HighResMIP protocol41. That is, starting from the initial 50-yr spinup, control simulations with fixed 1950 atmospheric conditions were continued for another 150 years for the HR and LR configurations. In parallel, historical and RCP8.5 scenario simulations of same length have been performed in both configurations for the period 1950–2099. CMIP5 forcing has been used since CMIP6 forcing was not available at the time when the model experiments were carried out. For all projections shown  with fixed 1950 atmospheric conditions was performed with both AWI-CM configurations, following an uncoupled 5-yr ocean spinup initialized from mean 1950–1954 EN4 ocean reanalysisWe estimate unforced variability in the controls to assess the physical significance of the simulated sea-ice concentration and ocean potential temperature changes. To this end, the standard deviation of rolling 30yr-means of sea-ice concentration from the last 80 years of the 1950 control simulations with AWI-CM-LR and AWI-CM-HR are computed  thus requires ΔELR = ρice × Lf × ΔVLR = 1.79*1021 J. The according smaller change in HR (ΔVHR = 1.6 × 1012 m3) requires only ΔEHR = ρice × Lf × ΔVHR = 0.49 × 1021 J. The change in MHT across 60° S until the end of the century is larger in HR than in LR by 0.033 PW = 0.033 × 1015 W = 3.3 × 1013 J/s  × 1021 = 1.3 × 1021 J, thus allowing to moderate the sea-ice decline in the Southern Ocean.Changes in the amplitude of the MHT can be related to the simulated sea-ice changes as follows. The energy Δ32 argue that the stronger sea-ice volume loss in LR compared to HR (CCSM3.5 model) is not because the LR ice extent decreases more than the HR; instead, they attribute this to a basic property of sea-ice thermodynamics, that is, thicker sea ice decays faster than thinner sea ice81. However, this thermodynamic argument is only part of the explanation for the different sea-ice behaviour in our simulations with AWI-CM as explained below.Bryan et al.2, HR extent: 17.84 million km2). Despite the very similar mean states in 2020–2049, the thickness change patterns relative to the 2020–2049 baseline are very different for LR and HR  for their LR compared to their HR configuration. When compared also to typical differences between CMIP models, the mean state differences between AWI-CM-LR and AWI-CM-HR in 2020–2049 of 0.46 million km2 are relatively small. For the 2020–2049 period, the CMIP5 multi-model mean (MMM) SIE amounts to 16.1 ± 4.2 million km2 (mean ± one standard deviation). For sea-ice volume, the MMM for the 2020–2049 period ranges even more widely: its standard deviation amounts to 8.47 × 1000 km3, which is about 58% of the MMM volume (14.49 × 1000 km3). In summary, this shows that a purely thermodynamic argument81 is unlikely to be the sole explanation for the different sea-ice behaviour in our simulations.To put the mean state differences for AWI-CM in 2020–2049 into perspective, it is worth noting that the sea-ice volume of the LR and HR models in Bryan et al.Supplementary InformationPeer Review File


Mast cells are activated upon immunoglobulin E (IgE)-mediated antigen stimulation, and release a wide variety of mediators, including histamine to trigger inflammatory responses. The surface expression levels of Fcε receptor I (FcεRI), a high affinity receptor of IgE, were found to be positively regulated by IgE. IgE could protect murine cultured mast cells from apoptotic cell death induced by the deprivation of interleukin-3 and a certain kind of IgE could activate immature mast cells in the absence of antigens, leading to the release of pro-inflammatory cytokines and a transient increase in histamine synthesis. Histamine synthesis in mast cells was found to be required for the maturation of murine connective tissue-type mast cells, raising the possibility that IgE indirectly modulates local mast cell maturation. Although it remains controversial to what extent this concept of “monomeric IgE effects” could have relevance in the modulation of human mast cell functions, the therapeutic effects of anti-IgE antibodies might be accounted for in terms of the decreased serum IgE concentrations. Because drastic increases in serum IgE concentrations are often observed in patients with atopic dermatitis and chronic urticaria, a close investigation of the roles of IgE in mast cell maturation should contribute to development of novel therapeutic approaches for these inflammatory diseases. Mast cells are distributed in nearly all vascularized tissues, and are involved in the regulation of a wide array of immune responses, including IgE-dependent immediate allergy ,2,3. EarMast cells are generally identified by their surface expression of c-kit, which is the receptor of the stem cell factor , and FcεRI, which is the high affinity receptor of IgE, and these two receptors are closely associated with the maturation and activation of mast cells. Several murine strains that harbor mutated c-kit genes have been used as mast cell-deficient models , indicatThe concept of therapy with an anti-IgE antibody initially targeted the interruption of IgE binding to FcεRI. However, it has been recognized that decreased serum IgE levels and the subsequent downregulation of FcεRI should play critical roles in its therapeutic effects. Serum concentrations of IgE in patients with allergic asthma were found to be significantly decreased when they were treated with omalizumab, a humanized IgG that specifically binds to human IgE ,11. SurfThe high affinity binding of IgE to FcεRI and its slow dissociation indicate that tissue mast cells should be consistently armed with IgE . Murine Early studies indicated a positive correlation between extracellular IgE levels and IgE binding to FcεRI, indicating the presence of a kind of positive feedback loop ,27,28,29Sakanaka et al. made a comparison of the sensitizing effects between two IgE clones, SPE-7 (HC-IgE) and IgE-3 (PC-IgE) . When BMBecause the effects of IgE in the absence of antigens were observed in the buffer solutions containing bovine serum albumin, the possibility that the constituents in the fetal bovine serum could cross-link the FcεRI by binding to the specific IgE clone might be excluded. James et al. demonstrated, using the approach of structural biology, that SPE-7 should have a potential to bind two or more structurally different antigens . This fiN-glycan composition and the potency as HC-IgEs. Because removal of sialic acid leads to the exposure of galactose at the end of N-glycans, galactose-binding lectins, such as galectin-3 and galectin-9, may be involved in the suppression of FcεRI-mediated activation of mast cells. Both galectins were found to suppress FcεRI-mediated activation of mast cells, although a previous study using gene-targeted mice that were lacking galectin-3 suggested that galectin-3 enhances the activation [Shade et al. recently reported that the sialylation of IgE plays a critical role in FcεRI-mediated activation of mast cells . They detivation ,49,50. IThe Fab region-mediated activation has also been explored from different points of view. Histamine-releasing factor (HRF) was identified as a secretagogue of human basophils, and its action was found to be mediated by a certain type of IgE (IgE+)  Figure g. Becaus2+ mobilization and degranulation in the cultured mast cells, prepared from bone marrow of gene-targeted mice lacking Src homology 2-containing inositol phosphatase (SHIP) [2+ mobilization, and HC-IgE-mediated responses depended on Lyn, Syk, and phospholipase C [2+ mobilization was found to be characteristic of IgE-triggered responses in the absence of antigens [2+ mobilization was resistant to La3+ and Gd3+, which could suppress Ca2+ influx induced upon IgE-mediated antigen stimulation [2+ influx induced upon IgE-mediated antigen stimulation is mediated mainly by the STIM1-Orai1 system [2+ channels mediate HC-IgE triggered Ca2+ influx, although several transient receptor potential families were found to be expressed in mast cells [3+ and Gd3+ indicated that the molecular mechanisms of Ca2+ influx are different between stimulation with HC-IgEs and antigen stimulation. HC-IgE-induced activations of mast cells were found to be significantly suppressed by several protein kinase inhibitors, such as Gö6976, which could suppress conventional protein kinase C (PKC) [2+ mobilization in the absence of antigens. These findings suggested that PKCβII is dispensable for antigen-induced Ca2+ influx, but essential for IgE-triggered Ca2+ mobilization. Gonzalez-Espinosa et al. demonstrated that IL-2, IL-4, and several chemokines, such as CCL2, CCL3, and CCL4, were transcriptionally induced in IgE-sensitized BMMCs in the presence of low concentrations of antigen, where no detectable levels of degranulation were found, whereas IL-3, IL-6, and IL-13 were induced only in the presence of high concentrations of the antigen, indicating that weak stimulation of FcεRI could induce cytokine/chemokine induction without degranulation [A series of studies about IgE-mediated activation of cultured mast cells demonstrated that high concentrations of HC-IgEs (1–5 µg/mL) could induce degranulation ,43,45,46e (SHIP) . They sulipase C ,39,40,41antigens ,41,55. Hmulation . Accumul1 system . It remast cells . Distinc C (PKC) ,41. Liu  C (PKC) . P-815 wnulation . Suzuki nulation . These f−/− mice were restored by intravenous injection before hapten sensitization with monoclonal IgE that was unrelated to the hapten. One of the mast cell-specific granule proteases, mouse mast cell protease-6 (MMCP-6), was transcriptionally downregulated in the cutaneous tissues of the IgE−/− mice, but was upregulated by the injection of IgE. These findings strongly suggested that IgE could enhance the potential to trigger inflammatory responses in cutaneous mast cells in an antigen specificity-independent fashion. Because the surface FcεRI in the cutaneous mast cells might be occupied by IgE in the wild type mice, these findings were obtained under the artificial settings. However, deprivation of circulating IgE by an anti-IgE antibody may impair the functions of newly recruited tissue mast cells.Bryce et al. demonstrated that the presence of IgE is required for hapten-induced contact sensitivity in gene-targeted mice lacking IgE . ImpaireL-histidine, which is mediated by histidine decarboxylase (HDC) [1, H2, H3, and H4 [1 subtype [1 receptor antagonists have been developed as antiallergic pharmaceuticals, antihistamines, which are also used as the first-line drug for urticaria. Recent studies suggest that the blockade of H4 receptors is also promising for inflammatory diseases, such as asthma and pruritis [4 subtype was found to be preferentially expressed in leukocytes, and mediates histamine-induced chemotaxis [4 receptor antagonist, JNJ7777120, and an H1 receptor antagonist, olopatadine, showed an excellent therapeutic efficiency in the picryl chloride-induced chronic dermatitis model [Mast cells produce a wide variety of proinflammatory mediators upon various stimuli ,3. Amongse (HDC) . A varie, and H4 . Accumul subtype . Many H1pruritis . The H4 emotaxis . Ohsawa is model .6-dependent decarboxylases, including aromatic-L-amino-acid decarboxylase (dopa decarboxylase). HDC is different from the other decarboxylases in terms of the presence of the carboxyl-terminal extra domain. The cDNA of HDC encodes a protein, of which the molecular weight is 74-kDa, whereas the purified enzyme has been reported to consist of a homodimer of the 53-kDa subunit [HDC is the rate-limiting enzyme for histamine synthesis. It belongs to the family of vitamin B subunit . The 74- subunit . Post-tr subunit . In a ra subunit . These f subunit .2+-mobilizing reagents, such as thapsigargin and a calcium ionophore, A23187, which could also drastically induce histamine synthesis. Only the 74-kDa form of HDC was detected in BMMCs treated with SPE-7, indicating that IgE alone induces histamine synthesis mainly in the cytosol. The finding that only a moderate increase in histamine synthesis was found in rat purified peritoneal mast cells stimulated with SPE-7 [HDC was found to be transcriptionally induced upon various stimuli . The drath SPE-7  raises tHdc−/− strain, but trace amounts of histamine, which originated in the standard diet, might be detected in several tissues. Unexpectedly, mast cells in the cutaneous tissues and peritoneum of the Hdc−/− mice exhibited aberrant granule morphology. Transmission electron microscopy revealed that they had granules with extremely low density. The protein expression levels of MMCP-4, MMCP-5, and carboxypeptidase A were decreased in the Hdc−/− mast cells. The Hdc−/− mice were resistant to IgE-mediated passive cutaneous responses and compound 48/80-induced cutaneous extravasation, and dietary supplemented histamine could partially restore these responses [Hdc−/− mice. Conditional deletion of HDC in neutrophils raised the possibility that neutrophil-derived histamine may partially contribute to IgE-mediated systemic anaphylaxis [Ohtsu et al. first reported the gene targeting of mice lacking HDC . De novoesponses . These fphylaxis .Hdc−/− mice are more susceptible to chemical carcinogenesis [+ cells infiltrated in the tumor tissues as CD11b+Ly6G+ immature myeloid cells, which supported the tumor growth. Exogenously added histamine promoted the maturation of the Hdc−/− CD11b+Ly6G+ immature myeloid cells, which resulted in the loss of their immunosuppression. Recently, Takai et al. demonstrated, through using a bacterial artificial chromosome DNA-directed GFP reporter transgenic mouse model, that CD11b+Ly6G+Ly6Clow myeloid cell population accumulated in the lung during sepsis [+Ly6G+ cells.Yang et al. found that the ogenesis . They idg sepsis . It remaHdc−/− mice, although it remained unknown how histamine is involved in granule formation. Previous studies using the gene-targeted mice lacking heparan sulphate N-deacetylase/N-sulphotransferase 2 (NDST2) demonstrated that a lack of heparan sulphate caused a drastic decrease in the amount of granule proteases and histamine [Ndst2−/− mast cells. A comparison of gene expression profiles of peritoneal mast cells revealed that a series of genes involved in granule formation, such as Ext1, Ndst2, Mcpt4, Mcpt5, Mcpt6, Mcpt7, and Ctsg, were significantly downregulated in the Hdc−/− mice, whereas that of Hdc−/− BMMCs were unchanged [Hdc−/− BMMCs reproduced the impaired granule formation observed in the Hdc−/− mice [4 receptor subtype, a similar aberrant granule formation to that of the Hdc−/− mast cells was not observed in the Hrh4−/− mast cells. Reconstitution analyses, in which BMMCs were injected into the peritoneal cavity of mast cell-deficient KitW/KitW-V mice, indicated that granule maturation is dependent solely on the presence of HDC in BMMCs. These findings strongly suggested that endogenous histamine synthesis is required for intact granule formation [Mast cells with aberrant granules were observed in the istamine ,80. Becanchanged . Co-cultnchanged ,83. This−/− mice . Exogeno−/− mice , and tha−/− mice . However−/− mice , indicatormation .2+ ionophore [2+-dependent manner [αo1, and Gαq, undergo a post-translational modification, in which the glutamine residues are covalently modified by histamine in P-815 cells [These findings might exclude the possibility that receptor-mediated actions or granular localization enhance histamine-induced granule maturation. Schneider et al. demonstrated that organic cation transporter-3 (OCT-3) mediates the bidirectional transport of histamine and regulates the cytosolic histamine levels . Exogenoonophore . Transglt manner . Vowinck15 cells . They obHistamine contents in BMMCs derived from female C57BL/6J mice were found to be higher than those from male mice . HistamiAccumulating evidence indicates that mast cells acquire the potential to produce a specific set of inflammatory mediators in response to changes in the microenvironment. During the process of differentiation into connective tissue-type mast cells, the amount of a large part of granule components were found to be increased. Recent studies demonstrated that granule components are involved in a wide variety of inflammatory responses.Although mast cells were found to undergo degranulation in response to IgE-independent stimuli, it remains unknown how such secretagogues induce degranulation. Recent progress has revealed that Mas-related G protein-coupled receptor (Mrgpr) family accounts for a substantial portion of IgE-independent degranulation. Mrgpr gene family might be a novel therapeutic target of inflammatory diseases, such as chronic urticaria and atopic dermatitis ,92,93.Mast cell granules share many characteristics with lysosomes. The activity of a lysosomal enzyme, β-hexosaminidase, is measured to determine the percentages of degranulation, instead of measuring the amount of histamine.TC, which expresses tryptase and chymase; and MCT, which expresses only tryptase. Murine mast cells could be categorized into connective tissue-type mast cells (CTMCs), a majority of which expresses MMCP-4, -5, -6, and -7; and mucosal mast cells (MMCs), which expresses MMCP-1, and -2. Because tryptase was found to activate proteinase activated receptor 2 (PAR-2) [A, resulted in degranulation of mast cells, leading to the release of carboxypeptidase A3.The expression profiles of granule proteases were found to reflect the heterogeneity of tissue mast cells. Granule proteases are classified into three categories, tryptase, chymase, and carboxypeptidase A3. Human mast cells are categorized into MC (PAR-2) ,95, some (PAR-2) . Cui et  (PAR-2) . The pat (PAR-2) , whereas (PAR-2) ,100. Mas (PAR-2) ,102. TheThe increased granule storage of heparin is the hallmark of mature connective tissue-type mast cells. Oschatz et al. demonstrated that factor XII-mediated formation of bradykinin was triggered in the presence of heparin, and that bradykinin increased vascular permeability by acting on the kinin B2 receptors, raising the possibility that heparin should have pro-inflammatory roles in addition to the maintenance of granule homeostasis .Mast cells have a wide variety of surface receptors, and a potential to respond to a diverse array of environmental changes in addition to IgE-mediated antigen stimulation . Among tIt remains to be fully clarified how chronic cutaneous inflammatory diseases, such as chronic urticaria and atopic dermatitis, are exacerbated. Because MRGPRX2 could be activated by various structurally different agonists, it is plausible that MRGPRX2 plays a critical role in mast cell-dependent chronic cutaneous inflammation . Serhan 2+ concentrations induced by IgE could lead to transglutaminase-mediated histaminylation of G proteins, which may enhance granule-related gene expression. Gu et al. previously suggested that Rac2 is involved in the maturation of BMMCs [Accumulating evidence suggests that mast cell-committed progenitors are infiltrated from bone marrow into tissues upon inflammation, and that they then proliferate in response to environmental factors including c-kit ligand. The process of mast cell maturation in the murine connective tissues could be depicted as follows . Surfaceof BMMCs . HoweverMore attention has been recently paid to the pathophysiological roles of tissue-resident mature mast cells. Previous studies focused mainly on the process of mast cell activation upon IgE-mediated antigen stimulation, using various mast cell lines and immature primary cultured mast cells. This is of great significance to understand the role of mast cells in immediate allergic responses, but is not always applicable to understanding of the pathophysiological roles of local mast cells. The absence of suitable culture models has introduced a delay in the investigation of mature mast cell-related functions, including MRGPRX2-mediated activation. Recent progress in the techniques of mast cell research, such as novel culture systems and gene-targeted mice models, has enabled us to investigate the roles of local mast cell populations. Close investigation of local mast cells will provide us with novel therapeutic approaches for chronic inflammatory diseases.