This paper reviews sea level contributions from land ice across the Arctic, including Greenland. We summarize ice loss measurement methods, ice loss mechanisms, and recent observations and projections, and highlight research advances over the last 3–5 years and remaining scientific challenges.Mass loss across the Arctic began to accelerate during the late twentieth century, with projections of continued loss across all future greenhouse gas emission scenarios. Recent research has improved knowledge of ice hydrology and surface processes, influences of atmospheric and oceanic changes on land ice, and boundary conditions such as subglacial topography. New computer models can also more accurately simulate glacier and ice sheet evolution.Rapid Arctic ice loss is underway, and future ice loss and sea level rise are guaranteed. Research continues to better understand and model physical processes and to improve projections of ice loss rates, especially after 2050. ArctiLand ice is a single component of the larger sea level rise budget, but an increasingly important one •, 6. CurMeasuring mass change in glaciers and ice sheets is not straightforward. A variety of techniques are well established, however, and measurements of Arctic ice loss are in good agreement , 11••. TMeasurements of Earth’s changing gravity field via the Gravity Recovery and Climate Experiment (GRACE) satellite mission (2002–2017) and GRACE Follow On  are used to estimate ice mass variations. While separating ocean mass change from land ice mass change and correcting for vertical land motion requires careful processing, gravity measurements produce high-quality data on ice mass changes, especially for large ice areas like Greenland –15. WithRepeat altimetry and digital elevation model (DEM) differencing allow measurements of volume change, which is then converted to mass change . Laser and radar altimetry data from airborne  and satellite  platforms is useful for small and large ice areas –20. It cThe mass change of an ice body is the difference between the input (mainly snow accumulation through precipitation) and the output . Scientists calculate this mass budget by combining data or modeling of the surface mass balance (SMB)—the difference between accumulation and ablation at the ice surface—with information about ice dynamics, such as iceberg calving.  The result is not a measure of total mass but of mass change over some period  •, 22, 23Changes in length, volume, and mass have been monitored routinely for a small number of individual glaciers, some for more than a century. Those glaciological data  and geodetic observations have been compiled by the World Glacier Monitoring Service (WGMS) for several hundred glaciers, including many in the Arctic. Although labor-intensive and limited in spatial coverage , these measurements provide a high-fidelity record of glacier mass balance changes from the early twentieth century through the present •.Another technique applied to glaciers and ice caps is volume-area scaling . Based oSophisticated dynamical ice sheet models are used to estimate future Greenland mass changes resulting from modified SMB and ice flow •, 29, 30rates. Ice loss fits into two broad categories: loss due to changes in SMB and loss due to iceberg calving, often called dynamic ice loss. SMB changes arise from variations in solar and longwave radiation, air temperature, and the amount and phase of precipitation (rain or snow). Other surface processes, like refreezing of surface melt water in the glacier firn [The warming atmosphere and ocean are the foundational cause for accelerating Arctic ice loss, but it is useful to consider the specific mechanisms driving ice loss. Understanding these mechanisms is particularly important for the challenge of understanding ice loss ier firn • or incrier firn  or changier firn ), also aSolid ice discharge requires a marine connection. The rate of ice discharge is determined by the speed of glacier motion and the advance or retreat of the glacier ice front . Ice front stability can be influenced by ocean temperature and melt at the terminus and by glacier thinning from surface or bottom melt •, 40. ToFor the GrIS and other large marine-connected ice caps and glaciers, the timescales and range of variability for SMB and dynamic discharge are different. SMB has higher year-to-year variability than solid ice discharge because it can respond quickly to atmospheric changes. For example, record GrIS melt years with exceptional mass loss in 2010 and 2012 helped to shift the SMB-to-discharge ratio from roughly 50:50 during 2000–2005 to 68:32 during 2009–2012 . Due to The observational record of widespread Arctic  land ice loss over the last two to three decades is well established, with significant contributions to global sea level rise. The magnitude of ice loss varies across the Arctic. Current GrIS contributions are roughly double those from other Arctic glaciers and ice caps. The ranking of recent (~2003–2009) Arctic mass loss from most to least is GrIS alone (~ 360 Gt/year), Alaska (~ 50 Gt/year), Greenland peripheral glaciers and ice caps (~ 38 Gt/year), North Canadian Arctic (~ 33 Gt/year), South Canadian Arctic (~ 27 Gt/year), Russian High Arctic and Iceland (~ 11 Gt/year each), and Svalbard (~ 5 Gt/year) •, 48, 492 [Ice loss from Greenland accelerated over the beginning of the twenty-first century and will continue to be the largest source of Arctic land ice loss. The ice sheet was likely close to a balanced state during the 1970s and 1980s but transitioned into negative mass balance beginning in the early to mid 1990s ••, 50. I2 , and the2 , 48. The2 ••, thoug2 .As Greenland transitioned to negative mass balance, changes in SMB and solid ice discharge contributed roughly similar amounts to mass loss , 23. RecModes of climate variability, such as the Atlantic Multidecadal Oscillation (AMO), North Atlantic Oscillation (NAO), and atmospheric blocking events, all influence ice loss , 53, 54•Natural climate variability was the primary driver in earlier Greenland ice loss. Records back to the nineteenth century show that there were periods of substantial Greenland glacier retreat and ice loss following the Little Ice Age  and during the 1930s–1940s warm period. However, glaciers at the start of the LIA were longer than today, reaching lower elevations and flatter terrain at their termini , and thuArctic regions outside of Greenland are also losing substantial quantities of ice ••. AlaskThe Canadian Arctic archipelago contains roughly one third of the Earth’s ice outside the AIS and GrIS . GRACE eThe Russian High Arctic contains roughly 20% of Arctic glacier ice outside of Greenland ••, with The Vatnajökull Ice Cap contains ~ 80% of the glacial ice in Iceland, with other small glaciated areas also mostly in the southeast . GRACE o3 of ice, equivalent to 17 ± 2 mm of potential sea level rise [Svalbard has had the smallest mass loss of the regions discussed here. Surface mass balance estimates over 2003–2009 give a loss of 5 ± 2 Gt/year •, and tovel rise .The Arctic will be a major source area for future sea level rise. Greenland is now a larger contributor than the world’s glaciers and ice caps (Antarctica currently trails both), while Arctic glaciers and ice caps (including Greenland’s periphery) are the largest combined source outside of the ice sheets .2 additional radiative forcing in 2100 as compared to pre-industrial values) [Ice loss from the GrIS will continue to 2100 under all of the Intergovernmental Panel on Climate Change (IPCC) Representative Concentration Pathways (RCPs) used for projecting the climate response to varying additional levels of radiative forcing  ••. In fa values) ••, 74. O values) , 46, wit values) •, 45, 46 values) , similarUnconnected and weakly connected glaciers and ice caps in the GrIS periphery, typically assessed separately, are projected to contribute roughly 20 mm to sea-level rise under RCP8.5 . SignifiWhile we have presented estimates of the mean Arctic contributions to sea level rise, local sea level rise can be much higher or lower than the global mean. Local sea level rise is influenced by factors including land subsidence, ocean currents, atmospheric circulation, and the origin of ice loss. The loss of gravitational pull caused by ice mass loss, along with vertical land motion associated with unweighting, means that sea level can fall locally (within ~ 2000 km of the location of ice mass loss) while rising in more distant locations . FortunaSubstantial research, particularly over the last one to two decades, has focused on understanding and projecting Arctic ice loss, leading to the various estimates outlined above. Earlier papers have discussed progress along the way ••, 65••.Evidence has been mounting that glaciers are declining worldwide, with the few exceptions attributed to local differences in climate change response . Recent statistical analysis confirms that local glacier retreat  provides categorical evidence of climate change •, 81. QuThe limited length of observational records continues to pose a challenge for assessing long-term trends, particularly before the satellite era. Data recovery from old photos, application of field techniques to map previous ice extents, and model hindcasts are helping to fill out the record •, 82–84.Technological advances continue to improve direct observations of, for example, ice flow speed , 86, chaBecause surface mass balance will play an increasingly dominant role in Arctic ice loss, surface processes such as darkening have received increased focus. Recent work establishes that Greenland summertime surface albedo decreased significantly (0.02 per decade) during 1996–2012, in contrast to 1981–1996, which showed no significant albedo trend ••. This Perennial firn aquifers, liquid water bodies in the firn, were found in southeast Greenland  on the hCorrelation between substantial glacier retreat and increased subsurface ocean temperatures , 97 sparSubglacial topography is a primary control on the rate and extent of glacier retreat in response to climate forcing. Fortunately, scientific efforts continue to measure subglacial topography and improve interpolation methods in unsampled regions . For GreResearchers continue to improve models for projecting individual glacier changes. For example, several studies have used the Ice Sheet System Model (ISSM) to project changes in Greenland glaciers, including Store Gletscher in western Greenland  and NiogIn translating land ice loss into sea level rise, the mass loss is commonly treated as total regional sums. However, the location of land ice loss  influences the location and magnitude of sea level rise. With recent improvements in projecting localized land ice loss and interpreting that loss into far-field sea level rise, local and regional sea-level estimates can be tied to the spatial partitioning of ice loss, reducing the range of predictions , 79•.Together, these advances give a clearer view of ongoing Arctic ice losses, improve understanding of the processes that control ice loss rates, and aid translation of ice loss observations into actionable sea level projections for decision makers.While estimates of total land ice mass changes consistently point to accelerating loss ••, land Observations provide the foundation for understanding the physical processes that govern ice mass loss and key boundary conditions and parameterizations for models. Improvements across these four highlight areas are key to improving process understanding and reducing uncertainties.Since even small scale (< 1 km wide) features can alter retreat rates, subglacial topography is a key dataset for projecting ice loss rates. Improved knowledge of other subglacial properties  and ice thickness changes across space and time are also critical for accurately modeling rates of change.Understanding ice sheet and glacier hydrologic processes, including the potential for water storage, will help constrain the rate of freshwater flux to the ocean, which influences ocean properties, circulation, nutrient flux, and ocean ecosystems along with sea levels. Results that help distinguish among contributors to surface darkening will aid in projecting future surface albedo changes and associated variations in surface melt, which are also key to determining potential melt event magnitude and spatial extent.Researchers must continue to work towards appropriate parameterizations for this multi-stage process so that it can be accurately predicted and incorporated into models in a realistic fashion . This inGiven the profound changes already occurring in the Arctic and the dramatic shift expected into the future, efforts must continue to investigate the influence of a changing ocean and atmosphere on land ice. Climate states like the AMO, NAO, and atmospheric blocking events all influence ice loss , 102. WiAs observational records continue to improve, one can envision near-term advances that combine observational records and computational techniques, such as machine learning, to make short-term projections of glacier change for both mountain glaciers and large ice-sheet outlet glaciers. Observational advances will also support improved model projections of short-term and long-term ice mass loss.Future modeling advances can also reduce uncertainties. First, ice sheet models must incorporate a wider range of spatial and temporal scales. Higher-order ice flow models are now capable of simulating the whole GrIS on century to millennial scales •. Some mHigh-resolution models with more realistic physics will enable detailed simulations of small, distinct glacier regions. For example, modeling efforts project a slow, steady retreat for Greenland’s Nioghalvfjerdsfjorden (79 North), in contrast to a rapid multi-decade retreat for Zachariae Isstrøm once it loses its floating ice tongue, with subglacial topography acting as the primary control •. This tSince the start of the century, mass loss from the Greenland Ice Sheet has accelerated. The ice sheet contributed an average of 0.8 ± 0.2 mm/year to sea level rise during 2003–2013 , with higher contributions during high-melt years such as 2012. Smaller Arctic glaciers and ice caps in Alaska, the Canadian Archipelago, the Russian Arctic, Iceland, Svalbard, and the periphery of Greenland are also losing mass, with a combined sea level contribution of about 0.5 mm/year [Research continues to affirm that human-caused climate change is the primary influence on Arctic  ice mass loss. Until ~ 2050, most sea-level projections show little divergence across the range of RCP scenarios . PlannerDecisions on action can also be aided by current progress in translating ice loss into local sea level rise projections. At least two techniques have been recently established to link local sea level changes at far field locations, like the coastal US or southern hemisphere cities, with regional ice loss from Greenland, Arctic North America, and elsewhere , 79•. Su


AB23A inhibited spleen tyrosine kinase (Syk) and the downstream signaling molecules including phospholipase Cγ (PLCγ), serine-threonine protein kinase/inhibitor of nuclear factor kappa-B kinase/nuclear factor kappa-B (Akt/IKK/NF-κB), and mitogen-activated protein kinases/cytosolic phospholipase A2 (MAPK/cPLA2). Furthermore, AB23A blocked mobilization of Ca2+. Similar results were obtained in other mast cell lines Rat basophilic leukemia (RBL)-2H3 cells and a human mast cell line (HMC-1). In addition, AB23A attenuated allergic responses in an acute allergy animal model, passive cutaneous anaphylaxis (PCA). Taken together, this study suggests that AB23A inhibits the activation of mast cells and ameliorates allergic reaction, and may become a lead compound for the treatment of mast cell-mediated allergic diseases.Alisol B 23-acetate (AB23A), a natural triterpenoid, has been reported to exert hepatoprotective and antitumor activities. Aiming to investigate the anti-inflammatory activity, this study examined the effect of AB23A on mast cells and allergic reaction. AB23A inhibited the degranulation of mast cells stimulated by immunoglobulin E/antigen (IgE/Ag), and also decreased the synthesis of leukotriene C Mast cells play an important role in the initiation and propagation of various inflammatory disorders such as asthma, atopic dermatitis, arthritis, etc . Mast ce2, and SLP76, guanosine triphosphate exchangers including Sos and Vav1, and the signaling enzymes phospholipase Cγ1 (PLCγ1) and PLCγ2 [2+ [Interaction of a multivalent antigen with FcεRI induces the activation of the spleen tyrosine kinase (Syk), and phosphorylation of linker for activated T cell (LAT). These proteins then serve as scaffolds for multi-molecular signaling complexes for the binding of cytosolic adapter molecules such as Gads, Grbnd PLCγ2 . ActivatLCγ2 [2+ . ActivatLCγ2 [2+ . These s4, generation of interleukin (IL)-6, and expression of cyclooxygenase (COX)-2 in IgE/Ag-stimulated mouse BMMCs. To investigate the possible mechanism involved, the effect on molecules in the FcεRI signaling pathway including nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPKs), Syk, Src-family kinases Fyn and Lyn, PLCγ, serine-threonine protein kinase C (Akt), cytosolic phospholipase A2 (cPLA2), and cytosolic Ca2+ mobilization was examined. In addition, we used other mast cell Rat basophilic leukemia (RBL-2H3) and human mast cell line (HMC-1) to demonstrate the anti-inflammatory effects of AB23A. Finally, we evaluated the anti-allergic effect of AB23A on AN IgE/Ag-mediated passive cutaneous anaphylaxis (PCA) reaction in vivo.Alisol B 23-acetate (AB23A), a natural triterpenoid from a Chinese medicinal herb, Alismatis Rhizoma, has multiple physiological activities including anticancer, hepatoprotective, and antibacterial activities ,7,8. Howp < 0.001), and the induced-release of histamine was suppressed by treatment with 2, 5, and 10 μM of AB23A significantly (p < 0.001) and in a concentration-dependent manner, as shown in p < 0.001).We initially used a 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assay to determine the cytotoxicity of AB23A, see 3, resulting in the release of Ca2+ from endoplasmic reticulum (ER) [2+ concentration is crucial for mast cell degranulation [2+ mobilization. As shown in 2+ was increased by IgE and Ag stimulation. As expected, the intracellular Ca2+ levels were inhibited by AB23A in IgE/Ag-stimulated BMMCs, especially at concentrations of 5 (p < 0.01) and 10 μM (p < 0.001). Furthermore, the IgE/Ag-induced phosphorylation of PLCγ in BMMCs, whereas the increased phosphorylation was dose-dependently inhibited by AB23A treatment, as shown in FcεRI engagement induces phosphorylation of PLCγ, and production of IPlum (ER) . The incnulation . Thus, w4 after stimulation with IgE/Ag was significantly increased and was about 9.6-times greater over that of the non-treated group. AB23A dose-dependently inhibited LTC4 generation in IgE/Ag-stimulated BMMCs, especially at a concentration of 10 μM (p < 0.001). LTs are derived via the 5-lipoxygenase (5-LO) pathway of arachidonic acid (AA) metabolism [2 and 5-LO reside in the cytoplasm. The increase of intracellular Ca2+ leads to the translocation of 5-LO to the nuclear membrane, where it associates with the scaffold protein 5-lipoxygenase-activating protein (FLAP). Meanwhile, cPLA2 translocates from the cytosol to the nuclear membrane. These make up the core of the LT biosynthetic complex [2, BMMCs were treated with AB23A, and the cytosolic and nuclear phosphorylated cPLA2 were measured. The phosphorylation of cPLA2 in BMMCs was significantly increased by the IgE/Ag challenge. However, the phosphorylation, as well as the translocation of cPLA2, was strongly suppressed by AB23A in activated BMMCs, see LTs as a pro-inflammatory factor can cause increased endothelial permeability, contraction of vascular smooth muscle, and enhanced mucus secretion . As showtabolism . Under r complex . To dete2 precedes the increase of intracellular Ca2+ to fully activate the enzyme and mobilize AA for eicosanoid production, and cPLA2 activation correlates with the activation of p38 and ERK [2, next, we measured the effect of AB23A on the phosphorylation of ERK1/2 and p38 MAPK. The Ag challenge for 15 min increased the phosphorylation of p38 and ERK1/2 in BMMCs, which was down-regulated by AB23A, see Phosphorylation of cPLA and ERK . In ordep < 0.001). Further treatment with AB23A markedly reduced IL-6 production in a dose-dependent manner, see IL-6 is a pro-inflammatory cytokine that plays an important role in inflammatory diseases such as rheumatoid arthritis, systemic juvenile arthritis, psoriasis, and ankylosing spondylitis . WithoutCOX-2 is well-known to play a key role in the pathogenesis of inflammatory diseases. This enzyme is upregulated in inflammatory situations and responsible for the production of prostaglandins (PGs) from AA . In ordeThe transcription factor, NF-κB, is a central regulator of inflammation and modulates several biological processes. In normal resting cells, p65/p50 heterodimers are maintained in the cytoplasm as an inactive form by inhibitor of NF-κB (IκB). During activation of NF-κB, NF-κB was liberated from the IκB and rapidly translocated to the nucleus to transcript various inflammatory mediators, such as IL-6 and COX-2 . IκB is 2 and signaling enzymes, PLCγ1 and PLCγ2, were activated [FcεRI requires the recruitment of the Src family tyrosine kinases (Lyn and Fyn) and Syk to control the early receptor-proximal signaling events . Syk is ctivated . BecauseWe also tested the effect of AB23A on other mast cells, RBL-2H3 and HMC-1. The concentrations of 2, 5, and 10 μM of AB23A didn’t significantly affect the cell viabilities in RBL-2H3 and HMC-1 (data not shown). As shown in 2 were inhibited by AB23A. Moreover, AB23A also attenuated the phosphorylation of PLCγ, Akt, p-IKKα/β, and phosphorylation and degradation of IκBα, and NF-κB p65 translocation induced by PMA plus A23187, see Because HMC-1 cells lack expression of FcεRI, researchers use phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore (A23187) to investigate the related mechanism. As shown in Anaphylaxis is known to be mediated by IgE/Ag through cross-linking of FcεRI . Mast ce4, PGD2, IL-6, and TNF-α [2+ mobilization causes degranulation, and phosphorylated PLCγ results in an increase of cytosolic Ca2+ concentration, and phosphorylated Syk can activate PLCγ. Our results showed that AB23A inhibited the mobilization of Ca2+ and the phosphorylation of PLCγ and Syk, see 2+ mobilization.Mast cells start to activate when allergen binds to a specific IgE antibody via the transmembrane protein of a high-affinity receptor (FcεRI) . Upon acnd TNF-α . Lee et nd TNF-α . Howevernd TNF-α . The rel4 and IL-6 production and COX-2 expression, see 2 can be phosphorylated by MAPKs on Ser505 [2 preferentially hydrolyzes AA from the sn-2-position of membrane phospholipids in response to diverse cellular stimuli [2 from cytosol to the nuclear envelope, see 4 generation by suppressing the phosphorylation of p38 and ERK1/2 to prevent the translocation of cPLA2.AB23A was also shown to inhibit LTCn Ser505 , and the stimuli . LTs are stimuli . Here, w2+ concentrations activate degranulation and also activate NF-κB which regulates the transcription of various cytokines [2+ was attenuated with AB23A treatment, see 2+/NF-κB pathway.Transcription factor NF-κB dysregulation is associated with inflammatory diseases such as arthritis, asthma, and bowel disease. Therefore, NF-κB has been shown to be the target of several anti-inflammatory drugs ,28. PI3Kytokines . Our res2+. Syk activation is central to mast cell effector function and allergic response . IgE-med2, PLCγ, Akt, IKKα/β, phosphorylation and degradation of IκBα, and translocation of NF-κB into the nucleus, see To confirm the anti-allergic mechanism of AB23A in mast cells, we used an RBL-2H3 cell, which is a mast cell line originating from rat basophilic leukemia and has been widely used to study IgE-FcεRI interactions. The results demonstrated that AB23A inhibited the activations of ERK, p38 MAPK, PLCγ, Akt, IκBα, and NF-κB, see PCA is well known as a local allergic reaction mediated by antigen-stimulated mast cells in vivo, and the IgE-mediated mast cell activation is evaluated by vascular permeability increase . Our stu2, NF-κB p65, lamin B, Lyn, Fyn, and Syk, as well as Bay 61-3606 reagent were obtained from Santa Cruz Biotechnology, Inc. . The LTC4 enzyme linked immunoassay (EIA) kit, and the antibody for COX-2 were from Cayman Chemical . Histamine enzyme linked immunosorbent assay (ELISA) kit was purchased from Demeditec Diagnostics GmbH . AB23A was obtained from Push Bio-technology Co., Ltd , of which the purity is ≥98%.RPMI1640, fetal bovine serum (FBS), and the enhanced chemiluminescence (ECL) Western blot detection reagent were purchased from Thermo Fisher Scientific Inc. . Mouse anti-dinitrophenyl (DNP) IgE was purchased from Sigma Chemicals . DNP-HSA was from Biosearch Technologies . The antibodies specific for phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-PLCγ, phospho-IκB, IκB, phospho-IKKα/β, β-actin, and the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody were purchased from Cell Signaling Technology, Inc. . The antibodies specific for phospho-cPLA4, histamine and IL-6 were determined using ELISA according to the manufacturer’s protocol. The HMC-1 cells were incubated with AB23A in the presence or absence of calcium ionophore (A23187) and phorbol 12-myristate 13-acetate (PMA) for further analysis.BMMCs were isolated from the bone marrow of Balb/c mice and differentiated as described previously by us . RBL-2H3Cell viability was assessed using an MTT assay. Briefly, BMMCs were seeded onto a 96-well culture plate and incubated with various concentrations of AB23A for 8 h. A total of 20 μL of MTT (5 mg/ml) was added to each well; RBL-2H3 and HMC-1 cells were seeded onto a 96-well culture plate and incubated with various concentrations of AB23A for 24 h, then MTT was added to each well. After 4 h incubation, the formazan was dissolved in DMSO, and optical density (OD) at 490 nm was measured using microplate reader iMark .2+ level was determined with the FluoForte Calcium Assay Kit , as described previously [TM dye-loading solution for 1 h, and then treated with AB23A or Bay 61-3606 for 1 h. After addition of DNP-HSA and incubation for 5 min, the fluorescence was monitored with a multilabel plate reader at Ex = 485 nm/Em 535 nm .The intracellular Caeviously . BrieflyIgE-sensitized BMMCs and RBL-2H3 cells were pretreated with AB23A or Bay 61-3606 for 1 h, followed by incubation with DNP-HSA for 30 min; HMC-1 cells were pretreated with AB23A for 1 h and incubated with PMA plus A23187 for 20 min. Then, the nuclear proteins were prepared by using Nuclear Extraction Kit according to the manufacturer’s protocol .IgE-sensitized BMMCs or RBL-2H3 were pretreated with AB23A or Bay 61-3606 for 1 h and stimulated with DNP-HSA. To assess COX-2 expression, IgE-sensitized BMMCs were pre-incubated with 1 μg/ml aspirin for 2 h to eliminate preexisting COX-1. After washing, the BMMCs were stimulated with DNP-HSA in the presence or absence AB23A for 7 h. For HMC-1, cells were pretreated with AB23A for 1 h stimulated with PMA plus A23187 for 20 min, then collected to prepare the cell lysates for follow-up experiments. Western blot analysis was carried out as we reported previously . Equal aIP was performed as we described previously . Total cw/v) Evans blue. After 1 h, mice were euthanized and the ears were removed. The ear tissue absorbed dye was extracted with 400 μL of formamide at 63 °C overnight and the absorbance was measured at 630 nm using a microplate reader . The ears were fixed with 4% formaldehyde and embedded in paraffin. Five μm sections of the tissues were prepared and stained with toluidine blue to count the number of mast cells and calculate ear thickness. Procedures used in this study were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University .ICR mice  were subcutaneously injected with 80 ng of anti-DNP IgE (Sigma) into each ear, 24 h before the Ag challenge. After IgE-sensitization, the mice were orally administered AB23A or Dexa. One hour later, the mice were intravenously challenged with 60 µg of DNP-HSA in 200 μL of PBS containing 1% . Differences were considered statistically significant when 2, COX-2, and Ca2+ release, see In conclusion, the present study demonstrated that AB23A has anti-allergic effects in IgE/Ag-activated BMMCs and RBL-2H3 and PMA/A23187-activated HMC-1 cells. The mechanisms responsible for the anti-allergic effects may involve Syk-mediated down-stream signals including PLCγ, ERK, and p38 MAPK, NF-κB, cPLA


Infectious bronchitis virus (IBV) infection in chickens can lead to an economically important disease, namely, infectious bronchitis (IB). New IBV variants are continuously emerging, which complicates vaccination-based IB control. In this study, five IBVs were isolated from clinical samples submitted to a diagnostic laboratory in Ontario, Canada, and subjected to detailed molecular characterization. Analysis of the spike (S)1 gene showed that these five IBVs were highly related to the Delmarva (DMV/1639) strain (~97.0% nucleotide sequence similarity) that was firstly isolated from an IB outbreak in the Delmarva peninsula, United States of America (USA), in 2011. However, the complete genomic sequence analysis showed a 93.5–93.7% similarity with the Connecticut (Conn) vaccine strain, suggesting that Conn-like viruses contributed to the evolution of the five Canadian IBV/DMV isolates. A SimPlot analysis of the complete genomic sequence showed evidence of recombination for at least three different IBV strains, including a Conn vaccine-like strain, a 4/91 vaccine-like strain, and one strain that is yet-unidentified. The unidentified strain may have contributed the genomic regions of the S, 3, and membrane (M) genes of the five Canadian IBV/DMV isolates. The study outcomes add to the existing knowledge about involvement of recombination in IBV evolution. Gammacoronavirus of the family Coronaviridae [Infectious bronchitis (IB), an acute and highly contagious disease, is characterized by respiratory, renal, or reproductive diseases in chickens of all ages . Here, taviridae . The sizaviridae . Ribosomaviridae ,4. The raviridae .The S protein undergoes post-translational cleavage, resulting in the S1 and S2 subunits. The glycoprotein S1 carries the receptor binding site and plays a major role in tissue tropism and the induction of protective immunity . ConsequIn addition to accumulation of mutations, recombination events are also common during IBV replication, which result in the emergence of major IBV variants. It is possible that the recombination events occur at multiple sites of the IBV genome, leading to rapid viral evolution ,10,11,12Despite the routine vaccination of Canadian poultry flocks against IB, repeated outbreaks occur ,16. ViruAlthough the classification of IBV variants based on partial or full S1 gene sequences is feasible, in depth molecular analysis of IBV variants requires the examination of the complete genome of the IBV. Previously, two Mass IBV variants isolated from western Canada were characterized using whole genome sequencing . In the Specific pathogen free (SPF) fertile chicken eggs were used for IBV isolation. The use of embryonated eggs for virus isolation has been approved by the Health Science Animal Care Committee (HSACC) of the University of Calgary, Calgary, Canada. Five diagnostic samples, which were characterized by partial S1 gene sequencing to be related to DMV/1639 strain, were received from the Animal Health Laboratory (AHL), University of Guelph, Canada, for virus isolation. The samples were collected from different farms located in Ontario, Canada, and the relevant history for each sample is summarized in ® reagent , according to the manufacturer’s protocol. A Nanodrop 1000 spectrophotometer  at a 260 nm wavelength was used to determine the concentration of the extracted RNA, and the quality was determined using the Nanodrop spectrophotometer, based on the 260/280 absorbance ratio. Approximately 1000 ng of extracted RNA was used for cDNA synthesis using random primers  according to the manufacturer’s instructions.Total RNA was extracted from 250 µL of the harvested allantoic fluid using the Trizol LSIn order to confirm the propagation of the virus in allantoic fluid, a previously described real-time PCR assay  was usedhttps://www.ncbi.nlm.nih.gov/genbank/). The complete S1 gene sequences were aligned using Clustal Omega [® v10.2.6 (https://www.geneious.com/).The complete sequences of the S1 gene of our five IBV isolates were phylogenetically analyzed against 86 reference sequences , includial Omega , and theal Omega , with 10https://www.ncbi.nlm.nih.gov/orffinder/). The complete genome sequences of our five IBV isolates were submitted to Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI) database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify the IBV strains with the highest sequence identities. For further analysis, 364 complete genome sequences were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/), and CD-HIT-EST [® v10.2.6.The ORF predictions of our five IBV isolates were carried out using the ORF-finder program  using MAFFT. Sequence segments that were different from those of the Conn vaccine strain were submitted to BLAST to identify the IBV sequences with the highest percentages of identity. On the basis of the BLAST search results, the complete genomic sequences of our five IBV isolates were again aligned with those of the Conn vaccine strain and 4/91 vaccine strain (KF377577) using MAFFT.Similar analysis included isolate GA9977/2019 (MK878536), which is a DMV/1639 IBV strain that was isolated from a broiler chicken farm in Georgia, USA, in 2019 . The com® v10.2.6.To identify the presence of recombination events, a multiple sequence alignment was performed using the complete genome sequences of our five IBV isolates and the Conn and 4/91 vaccine strains. This sequence alignment was introduced into Simplot . The nucThe complete genomic sequences of our five IBV isolates, namely, IBV/Ck/Can/17-035614, IBV/Ck/Can/17-036989, IBV/Ck/Can/18-048192T, IBV/Ck/Can/18-048430, and IBV/Ck/Can/18-049707 were deposited into GenBank .The S1 gene sequences of our five IBV isolates were clustered within the lineage GI-17 . This liThe S1 gene sequences from our five IBV isolates showed a high degree of nucleotide identity (99.4–99.9%). They were 96.4–96.7% identical to the MDL_DMV1639 (KX529720) strain, and, conversely, they were distinctly separate from the Mass and Conn strains, with only 77.2–77.3% nucleotide similarity in this case. Nucleotide and amino acid pairwise comparisons between the S1 genes of our five IBV isolates and other strains affecting poultry industry in Canada are listed in The complete genome sequences of IBV isolates IBV/Ck/Can/17-035614, IBV/Ck/Can/17-036989, IBV/Ck/Can/18-048192T, IBV/Ck/Can/18-048430, and IBV/Ck/Can/18-049707 were 27,710, 27,708, 27,638, 27,684, and 27,708 nucleotides in length, respectively. Thirteen ORFs (5′ -1a-1ab-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-3′) were predicted. A deletion of thirteen nucleotides was detected in the reading frame of the 6b gene of isolate IBV/Ck/Can/18-048192T, compared to the other four IBV isolates . The delOur five IBV isolates were nearly identical among themselves, with an overall nucleotide sequence identity of 99.4–100%. A phylogenetic analysis using 146 complete genomes of IBV strains downloaded from GenBank was conducted to investigate their relationships with the sequences of our five IBV isolates. The sequences of our five IBV isolates showed close relationships with the Conn vaccine (93.5–93.7%), Cal56b (93.5–93.7%), and IBS037A/2014 (93.9–94.1%) strains. The nucleotide-based MAFFT alignment of our five IBV isolates with the Conn and 4/91 vaccine strains provided six fragments that showed alternating similarity with the two reference strains . One fraIsolate GA9977/2019 showed 94.7% nucleotide sequence identity with our five IBV isolates, and a 95.5% identity with the Conn vaccine strain. Multiple sequence alignment data, constructed with MAFFT, showed high similarity between isolate GA9977/2019 and the Conn vaccine strain, except for S gene region .The Conn vaccine and 4/91 vaccine sequences were used as putative parents, and each sequence of our five IBV isolates was queried in SimPlot. The result of SimPlot confirmed the result of the MAFFT alignment, where six fragments were changing identity with the Conn vaccine and 4/91 vaccine strains, except for the genomic region between 20,120–25,100 nucleotides, which showed low identity with either of these strains. The SimPlot results for our five IBV isolates were identical, and only representative analysis of IBV/Ck/Can/18-049707 is shown a.Five crossover points were found: The first recombination breakpoint (at approximately 1911 nucleotide position) was located in the nsp2 gene, the second in the nsp3 gene (at approximately the 3735 nucleotide position), the third in the nsp12 gene , the fourth at the 3′ end of 1ab gene , and the last breakpoint was located at the 3′ end of the M gene . In addition, data from the nucleotide similarities and phylogenetic trees constructed using the corresponding gene fragments also confirmed the results b,c.Infectious bronchitis is one of the most economically important viral diseases that affects poultry worldwide, including in Canada. Like most RNA viruses, the causative agent, the IBV, is genetically diverse, due to a high mutation rate and several recombination events . The aimThe level of egg passage of the five IBVs used in this study was low (≤4 passages), which decreases the likelihood of genetic changes that could have been introduced as a result of propagation in embryonated eggs. In a study that examined a highly variable region within the IBV genome, namely, the S1 gene, even 10 passages in embryonated eggs resulted in no changes in five viruses. Additionally, two viruses had only 2 nucleotide changes and one virus had only 1 nucleotide change . The genThe S1 gene is the most variable region within the genome and encodes virus-neutralizing and serotype-specific epitopes. Therefore, it is used often for phylogenetic analysis . ClassifIn general, recombination events that contribute to the genetic diversity of coronaviruses is believed to result from a template-switching copy-choice mechanism during RNA replication . The ConSince co-infection with two different viruses in a given flock is a prerequisite for recombination between IBV strains , it is cIn conclusion, although it is practical for most research studies to genotype the isolated IBV strains using the S1 gene, the complete genome sequencing approach gives more information about the evolutionary origin of such strains. This study is the first one that has characterized the complete genome of the DMV/1639-like isolates that have been circulating in Canadian poultry in recent years. Our findings have implications for the control of IB in Canadian poultry flocks and add to the existing knowledge on the molecular epidemiology of IBV infection.


Suture zones are abundant on Antarctic ice shelves and widely observed to impede fracture propagation, greatly enhancing ice-shelf stability. Using seismic and radar observations on the Larsen C Ice Shelf of the Antarctic Peninsula, we confirm that such zones are highly heterogeneous, consisting of multiple meteoric and marine ice bodies of diverse provenance fused together. Here we demonstrate that fracture detainment is predominantly controlled by enhanced seawater content in suture zones, rather than by enhanced temperature as previously thought. We show that interstitial seawater can reduce fracture-driving stress by orders of magnitude, promoting both viscous relaxation and the development of micro cracks, the incidence of which scales inversely with stress intensity. We show how simple analysis of viscous buckles in ice-penetrating radar data can quantify the seawater content of suture zones and their modification of the ice-shelf’s stress regime. By limiting fracture, enhancing stability and restraining continental ice discharge into the ocean, suture zones act as vital regulators of Antarctic mass balance. Suture zones are abundant on Antarctic ice shelves and widely observed to impede fracture propagation. Here we show that fracture detainment is principally controlled by the zones’ enhanced seawater contents, reducing fracture-driving stresses by orders of magnitude and therefore greatly enhancing stability. Around one quarter of the ice-shelf area has been lost on the Antarctic Peninsula in the past half century, and some shelves, including Larsen A and B, have largely disintegrated5. The final demise of the Larsen B Ice Shelf in 2002 triggered a twofold to fourfold acceleration of its former tributary glaciers that persists to the present day8, demonstrating the fundamental role that ice-shelf buttressing plays in regulating sea level rise. Current predictions of ice-shelf stability and future loss of grounded Antarctic ice are subject to considerable uncertainty, however, because the processes driving and resisting ice-shelf retreat are not well understood.Half of the Antarctic coastline is fringed by ice shelves Fig.  that are10  and thence embedded between meteoric ice units derived from feeding glaciers in the neighbouring Solberg and Trail Inlets Figs.  and 3, i22 than the relatively cold meteoric ice and firn derived from the ice-sheet interior or accumulated on the shelf (~−5 to −15 °C on the Larsen C Ice Shelf)23. Suture zones are therefore expected to be warmer, softer, more deformable and more heterogeneous in provenance than the cold meteoric ice units that enclose them31. Although actual in situ measurements of these anomalous characteristics of suture zones are very sparse, they are hypothesised to cause stress relief ahead of incipient fractures, thereby detaining them and resulting in the observed, less fractured suture-zone surfaces34. The JP suture zone, for example, has detained multiple rift sequences that originate at Table Nunatak, some 100 km downstream of JP 31 and associated ice softening and enhanced deformation are the root causes of fracture detainment.Here we address the current paucity of in situ observations by presenting new integrated field data of the JP suture zone. These data allow us to evaluate the widely held hypothesis that warmer temperature35. Of the 140 m thickness, around 40–45 m are in situ firn29 and approximately the lower 100 m are in situ meteoric ice. The provenance of these in situ firn and in situ meteoric ice layers is therefore distinct from the underlying glacier-derived blocks.We conducted ground-penetrating radar (GPR) and multi-component seismic reflection surveys to the north of Table Nunatak in December 2008/09, one centrally on the JP suture zone (JP-Seis) and one ~5 km to the south of it on the meteoric ice flow unit derived from Solberg Inlet (SI-Seis) Fig.  and alon13, type B events are typically discontinuous and characterised by overlapping discrete hyperbolae, and coherent energy returns from below them are normally absent  flowline modeleis Fig. . The thi11 but underestimates the depth of this interface by ~50 ± 20 m at JP-Seis  bounds and the Voigt–Reuss–Hill (VRH) average (see ‘Methods’) suggest maximum bulk and shear moduli in meteoric ice of 8.57 ± 0.36 and 3.19 ± 0.12 GPa, respectively  from the meteoric ice in the glacier-derived block to the basal marine ice cannot be explained by a change in ice density or temperature alone. Because seismic velocities are inversely proportional to the square root of ice density, this velocity reduction would require an implausible increase in ice density (to ~1200 kg m−3). Because seismic velocities are also only weakly dependent on ice temperature39, a change of >40 °C is required to change P-wave velocity by 100 m s−1—a temperature change also cannot explain the measured decrease in bulk P-wave velocity change of ~475 m s−1. Because P-wave velocities are much lower in seawater (~1500 m s−1) than in ice , the proposed presence of liquid seawater in the pore space of basal marine ice offers the most plausible physical means of explaining the large observed P-wave velocity reduction41.The ~13% reduction in bulk P-wave velocities .Although our seismic measurements provide unique observational evidence of meteoric and basal marine ice properties, including the latter’s pore seawater contents, they have two main limitations. First, they impose short-term transient strains and thus sample the elastic properties of ice, while, in reality, ice is considered to deform by secondary creep in response to long-term strain. The inferred elastic properties can therefore only serve as an approximation of the actual physical properties that control the deformation of ice. Second, our seismic experiments sampled a glacier-derived block calved from the JP but not the mélange that fuses these blocks together Fig. . To compUndulating reflections are common in our GPR data, but they are particularly pronounced at depths of ~40–140 m in the JP suture zone Fig. . This deh ≈ 100 m thick with characteristic wavelengths of up to λ ≈ 1000 m  and λ (500 ≤ λ ≤ 1000 m) and using an appropriate rate factor range for this meteoric ice , the combined rate factor of the combined softer units of firn, mélange and basal marine ice has a 90% confidence interval of ~1 × 10−24 to ~5 × 10−22 s−1 Pa−3  range of ~−5 °C to +17 °C  is ~2–12%  and the combined softer (As) units is therefore ~6–100 so that, for a given stress, the combined softer units will deform between one and two orders of magnitude more readily than the harder in situ meteoric ice. Conversely, the ratio in the flow law parameter This apparent paradox can readily be reconciled if we allow for the presence of seawater in the pore space of the basal marine ice and mélange. The seawater fraction equivalent to the combined rate factor range of the combined softer units of firn, mélange and basal marine ice  only contributes ~40 m (~20%) to the total thickness of the combined softer units, these inferences particularly apply to mélange (~110 m or ~50%) and basal marine ice (~70 m or ~30%) Figs.  and 9d a11, a distance over which, according to our flowline model , underlying granular ice (formed by surface-derived snow or firn being invaded by seawater). In both the Amery and the King Baudouin ice shelves, therefore, thick layers of marine ice that had accumulated over extended periods of time exhibit either internal gradation or discrete layering. Two types of seismic evidence support the hypothesis that basal marine ice beneath the Larsen C Ice Shelf may be similarly layered. First, our own  and units of mélange (ISU-4) and basal marine ice (ISU-5) will then further modulate the propagation of the rift tip in space and time. For example, in a cross-section through the suture zone at JP-Seis the combined mélange and basal marine ice units are wider near the ice-shelf base than higher up in the ice column Fig. . A full-The fact that suture-zone heterogeneity may play a major role in ice-shelf stability is daunting from the perspective of predictive ice-shelf modelling, because this requires detailed knowledge of englacial structures and physical properties. Alleviating this concern, we have (i) shown that simple analysis of viscous buckles apparent in cross-flow radargrams of suture zones can quantify their modulation of long-term strain rates; (ii) deciphered the signatures of glacier-derived blocks (ISU-3), mélange (ISU-4) as well as basal marine ice (ISU-5) in both along- and cross-flow radargrams; and (iii) demonstrated that simple 1-D models can adequately capture the along-flow evolution suture-zone structures, if constrained by geophysical observations. These developments can provide high-quality constraints for ice-shelf models.Nonetheless, a range of processes controlling the spatial and temporal evolution of suture zones, their seawater content and temperature and their ability to detain rifts and thus stabilise Antarctic ice shelves must be better understood for their effects to be fully implemented in ice-shelf models. These include for example the provenance, evolution and physical properties of the mélange (ISU-4), the processes that govern basal marine ice (ISU-5) accretion and its along-flow compaction and, most significantly, the processes that control the diffusion of fracture-driving stresses that may eventually allow a rift to break through a suture zone.12. We acquired one trace every 3–4 m at a towing speed of ~12 km h−1, representing a distance-averaged stack of eight individual traces at a sampling interval of 0.8 ns. Each stacked trace was located with a handheld GPS linked to the GPR system, achieving a planimetric precision of approximately ±5 m. Velocity–depth profiles were derived from common-midpoint surveys29 and used for depth conversion of radargrams.The two common offset GPR profiles shown here  and density (ρ)51, surface accumulation rate35 (52 (V), on the assumption that the mass of the Larsen C Ice Shelf is in steady state (∂ρVZ/∂t = 0):To place the section we have imaged with GPR into a broader spatial context, we use our established 1-D flowline model13. All data sets are compiled on the same 1 km × 1 km grid in a polar stereographic projection. We adopt a published formulation20 to calculate the thicknesses of a surface layer of firn and meteoric accumulated on the ice shelf  relative to the upstream edge of that node (Z1):t is the time taken for the ice to advect between the upstream and downstream edge of the node. The vertical strain rate , a central layer of advected glacier ice (FLM-2) and a basal layer of accreted marine ice (FLM-3)  and shear (S-) wave imaging of the interior and base of the ice shelf and also the inversion of refraction travel times for depth profiles of density29. We established the uncertainty in seismic velocity models  and, using the inverted density model, derived models of the elastic moduli of the glacier-derived meteoric ice block and the basal marine ice beneath it  and shear (µn) moduli of the component phases. Equivalent lower bounds, K and µ describe the strongest and weakest blends of two phases. When one phase is a fluid, the upper bound suggests that the blend is matrix supported, while the lower indicates a fluid-supported suspension. Lying between the H-S bounds is the VRH average58; we consider the range of moduli between the VRH and upper H-S bound, thereby assuming that the marine ice is not a fluid-supported suspension of ice crystals. This assumption recognises that the base ice reflection is distinguishable in our seismic data from JP-Seis  of observed buckles  by:Ah and As are the corresponding rate factors in Glen’s flow law and h is the thickness of the harder in situ meteoric ice layer (VBM-2). The harder the VBM-2 layer and the softer the combined layers VBM-1 and VBM-3  versus seawater content (W) in layer softening. First, we use the Arrhenius relationAh), where R is the universal gas constant (8.314 J mol−1 K−1) and Q is equal to 139 × 103 J mol−1 (A0 = 3.615 × 10−13 s−1 Pa−3) and 60 × 103 J mol−1 (A0 = 1.733 × 103 s−1 Pa−3), respectively, for temperatures warmer, or equal to, and colder than −10 °C22. For a plausible temperature range of meteoric ice of −5 to −15 °C, as measured on the Larsen C Ice Shelf in the 2014/15 austral summer23, Ah ranges22 between 1.6 × 10−24 and 2.9 × 10−25 s−1 Pa−3. Second, we measure ranges of in situ meteoric ice thicknesses  of buckles in our GPR data  of the softer layers VBM-1 and VBM-3  range for these softer layers  for them (Fig. 59C ≈ 181.25 is a constant of proportionality57 and A0 = 2.4 × 10−24 s−1 Pa−3 is the average rate factor of mélange and basal marine ice at −2 °C22.By analogy with other geological mediales Fig.  to the vM-3 Fig. , the larM-3 Fig.  from Eq.M-3 Fig. . Third, ers Fig.  and, fouhem Fig.  using598


Escherichia coli entry vectors  was constructed based on a modified pUC18 backbone, which carried newly designed multiple cloning sites, consisting of two facing type IIS enzyme cleavage sites and one blunt-end enzyme cleavage site. These vectors are useful for seamless gene cloning.A series of new  Escherichia coli entry vectors  was constructed based on a modified pUC18 backbone, which carried newly designed multiple cloning sites, consisting of two facing type IIS enzyme cleavage sites and one blunt-end enzyme cleavage site. These vectors are useful for seamless gene cloning.A series of new  Escherichia coli is one of the essential routine works in the field of molecular biology  on a modified pUC18 backbone . We conslacZ′). pUC18 carries two BsmBI sites, one SapI site, and one BsaI site within its backbone. We have removed these sites using site-directed mutagenesis. This will prevent jamming (incorrect) religation in case of the one-pot reaction of GGC  and one blunt-end enzyme cleavage site  . Each den of GGC  5)..lacZ′). E. coli DH5α chemically competent cells (Nippon Gene), following the standard protocol, and colonies were selected on an LB agar plate supplemented with ampicillin (100 μg/ml) and 2% X-Gal . The modified construct (within blue colonies) was confirmed by successful cleavage with SapI or Bsp68I and whole-plasmid sequencing using the BigDye Terminator ver. 3.1 cycle sequencing kit. The sequence data were analyzed using an ABI 3130xl genetic analyzer . The other four pIIS18 vector models have been constructed in the same way as pIIS18-SapI.For pIIS18-SapI construction, pUC18 was doubly digested with PstI and BamHI. The linearized pUC18 was purified and ligated with the SapI DNA linker . The obtpIIS18 series are designed for type IIS restriction enzyme-mediated seamless gene fusion, such as GGC . They alLC459971 to LC459975, respectively. The resource can be obtained from the Addgene depository (https://www.addgene.org/) and the GCMR library of Gifu University (https://www1.gifu-u.ac.jp/~g_cmr/index.html). The raw sequencing reads are available at https://www1.gifu-u.ac.jp/~suzuki/pIIS_plasmids/.The complete sequences of pIIS18-SapI, pIIS18-BsmBI, pIIS18-BsaI, pIIS18-BfuAI-1, and pIIS18-BfuAI-2 have been deposited in the DNA Data Bank of Japan under the accession numbers


Successful behavioral inhibition involves both proactive and reactive inhibition, allowing people to prepare for restraining actions, and cancel their actions if the response becomes inappropriate. In the present study, we utilized the stop-signal paradigm to examine whole-brain contrasts and functional connectivity for proactive and reactive inhibition. The results of our functional magnetic resonance imaging (fMRI) data analysis show that the inferior frontal gyrus (IFG), the supplementary motor area (SMA), the subthalamic nucleus (STN), and the primary motor cortex (M1) were activated by both proactive and reactive inhibition. We then created 70 dynamic causal models (DCMs) representing the alternative hypotheses of modulatory effects from proactive and reactive inhibition in the IFG-SMA-STN-M1 network. Bayesian model selection (BMS) showed that causal connectivity from the IFG to the SMA was modulated by both proactive and reactive inhibition. To further investigate the possible brain circuits involved in behavioral control, including proactive inhibitory processes, we compared 13 DCMs representing the alternative hypotheses of proactive modulation in the dorsolateral prefrontal cortex (DLPFC)-caudate-IFG-SMA neural circuits. BMS revealed that the effective connectivity from the caudate to the IFG is modulated only in the proactive inhibition condition but not in the reactive inhibition. Together, our results demonstrate how fronto-basal ganglia pathways are commonly involved in proactive and reactive inhibitory control, with a “longer” pathway (DLPFC-caudate-IFG-SMA-STN-M1) playing a modulatory role in proactive inhibitory control, and a “shorter” pathway (IFG-SMA-STN-M1) involved in reactive inhibition. These results provide causal evidence for the roles of indirect and hyperdirect pathways in mediating proactive and reactive inhibitory control. The prefrontal cortex (PFC), particularly the inferior frontal gyrus (IFG) and the presupplementary motor area (preSMA)/supplementary motor area (SMA), are critical to reactive inhibition  are also involved in proactive inhibition  and indirect  pathways  into dynamic causal models (DCMs) of proactive and reactive inhibition. Bayesian model selection (BMS) was applied to investigate the effective connections associated with each type of inhibition.We hypothesized that there are overlapping regions in the neural systems involved in proactive and reactive inhibitory processes and that the effective connectivities between the relevant brain regions are modulated differently in those processes. In this study, we used a special neuroimaging contrast to isolate fMRI activity associated with proactive inhibition Twenty healthy right-handed adults , excluding 10 participants with excessive head movement in the MRI scanner defined as translational or rotational displacement greater than 2.5 mm in any direction, performed a stop-signal task during fMRI scanning. All participants were recruited from the University of Tsukuba as paid volunteers, had normal or corrected-to-normal vision and provided written informed consent prior to the experiment. No participant was taking medicine during the experiment. The present study was approved by the Institutional Review Board (IRB) of the National Institute of Advanced Industrial Science and Technology  and all participants gave informed consent prior to participation.In the present study, we divided the “go,” “stop,” and “switch” trials into several substages to isolate proactive and reactive inhibition . Unlike Each run consisted of 40 “go” trials, 10 “stop” trials, and 10 “switch” trials. An equal distribution of the characters “X” and “O” was ensured across trials, in random order. Each participant needs to complete three runs.t-test on mean RTs for “go” and “switch” trials to test if there were significant difference between them. Because the fixed stop-signal delay (SSD) was used in the current procedure, we estimated SSRT with integration method  at the Department of Information Technology and Human Factors, AIST . Each participant’s head was fixed using foam padding to reduce head movement. Single-shot echo-planar imaging (EPI) sequences were used to acquire functional images. EPI parameters were as follows: repetition time (TR) = 2,000 ms; echo time (TE) = 35 ms; flip angle = 90°, 31 ascending slices, thickness = 3.7 mm.The SPM12 software toolboxWe divided the trials into several components . All trip < 0.05 was applied to correct for multiple comparisons.Based on this approach, reactive inhibition was analyzed by comparing the successful “switch” trials to successful “go” trials. We did not apply the classical comparison that used for race model between successful “stop” trials (proactive inhibitory component + reactive inhibitory component) and successful “go” trials (proactive inhibitory component + action component) because the result cannot be explained by isolated reactive inhibitory component. Proactive inhibition was isolated by the conjunction of all successful trials . We used a general linear model for first-level event-related analysis in each participant. Events  were modeled after a duration of 0.5 s from trial onset. A second-level SPM analysis used contrasts from the first level with one-sample tests to investigate the group-level activation. A peak-level false discovery rate (FDR) at We used DCM12  the regions have been reported to be involved in behavioral control in previous research. We constructed 70 DCM models on four regions: the right IFG , the left SMA , the STN , and the M1 .We defined MNI coordinates of the ROIs for DCM analysis that met all of the following criteria: (1) the coordinate of the spherical ROIs should show significant activations in both proactive and reactive contrasts with a cluster-based FDR at The set of 70 DCM models was divided into two groups: a linear and a nonlinear group. Each group was divided into several sub-groups based on the location of the proactive modulatory input  and the connectivity between the IFG and the SMA . For “go, ” “stop, ” and “switch” trials, mean accuracy was 0.890 (SD: 0.117) and 0.853 (SD: 0.165), respectively. The mean SSRT was 454 ms .There were significant differences in mean RTs between “go” trials  and “switch” trials , caudate, SMA, IFG, STN and M1 of both hemisphere . As for We investigated 70 DCMs representing the alternative hypotheses of modulatory effects from proactive and reactive inhibition. BMS with fixed-effect analysis (FFX) provided strong evidence for one model being optimal, above all other models .Two parameters were measured in the BMS to compare the optimal architecture of the models: relative log-evidence and posterior probability. The former corresponds to the balance between accuracy and complexity of the model, while the latter represents the probability that the specific model provides the best explanation for all participants.DCM estimates the observed BOLD responses and models the effects of neuro-vascular processes and spectral noise at different levels. The parameters represent the rate of change in activity in one region that influences the activity in another region, and the effective connectivity was thus expressed in Hz.The average connectivity between two regions represents how rapidly activity (per second) in one region influences the activity in another region  and reactive modulatory inputs (modulation effect = 0.8404 HZ). The results indicate that when people “prepare to cancel” and then “cancel” a planned response successfully, both proactive and reactive modulation influence on the effective connection from the IFG to the SMA. The IFG inhibits the activity in the SMA, and the decreased activity in the SMA influences the subsequent areas p < 0.05 in the second-level SPM analysis; and (2) the regions have been reported to be involved in behavioral control in previous research. We constructed 13 DCM models including four ROIs: the right IFG, the DLPFC , the left SMA, and the caudate . As previous results showed that the modulatory effects were related to the connection from the IFG to the SMA, this modulatory effect should be a direct or indirect effect from other effective connections that end in the IFG.To further investigate the possible brain circuits involved in the implementation of behavioral control including proactive and reactive inhibitory processes, we added extra regions to the DCM models for proactive inhibition. The regions of interests were selected based on the same criteria as the previous DCM models, i.e., (1) these regions should show significant activation in proactive contrasts with a cluster-based FDR at We limited our model for proactive inhibition to the four regions based on the following reasons: (1) the previous studies suggested the top-down control for proactive inhibitory control  the IFG-SMA is the real effective connectivity that is modulated by reactive inhibition; and (2) the real effective connectivity is the other connectivity in prefrontal-STN network. The modulatory effect was transferred to the effective connectivity IFG to SMA and observed in the IFG-SMA-STN-M1 DCM model. Based on the result from whole brain contrast related to reactive inhibition, there are no other activations in frontal areas involved in inhibitory control and prefrontal-STN connections, which means no other effective connectivity responds to reactive inhibition, so the IFG-SMA is the real effective connectivity that modulated by reactive inhibition.The set of 13 DCM models was divided into three groups based on whether the DLPFC receives driving inputs . The location of proactive modulatory inputs varied in the bidirectional connections in the DLPFC-IFG-SMA-caudate network, and locations were chosen based on the requirement that the indirect modulatory effects from other effective connections should end in the IFG. Average self-connections were applied to all nodes. The interconnectivities between nodes are bidirectional . We consF-test of effects of interest. To extract the time series from the ROIs for each participant, we combined the local maximum close to the group peak and extracted the eigenvariate from a 5-mm sphere.We extracted the first eigenvariate of the BOLD time series from two regions of interests, the DLPFC and the caudate. All time series were adjusted for the The results of BMS (FFX) indicated that there was one model that was superior to all other models . In thisIn the present study, we used fMRI data acquired during a stop-signal paradigm task to identify the cortical and subcortical areas involved in proactive and reactive inhibitory processes. To evaluate the modulatory effects of proactive and reactive inhibition on the effective connections between these areas, we first conducted a DCM analysis where 70 DCM models were compared. The results indicate that the increasing activity in effective connectivity from the left SMA to the left STN was modulated by the right IFG, and the decreasing activity in effective connectivity from the right IFG to the left SMA was modulated by both proactive and reactive modulatory effects. We further investigated an alternative hypothesis with 13 additional DCM models in which the causal connection from/to the right DLPFC and the left caudate were considered for proactive inhibition. The results of the additional DCM model comparison show that the increased activity of the effective connection from the left caudate to the right IFG was modulated by proactive modulatory control, which resulted in the inhibitory effects in the connections from the right IFG to the left SMA in the comparison between proactive and reactive inhibitory control.via hyperdirect and indirect pathways. Previous studies suggested that the right IFG and left SMA are critical regions in inhibitory control  are involved in both proactive and reactive inhibition  or ECoG, which have better temporal resolution, need to be considered in future research. Further, we had to exclude participants with excessive head movement, and the reduced number of effective participants might have caused us to miss some regions with significant activations.We show that the effective connection from the IFG to the SMA is associated with reactive inhibition, while the effective connection from the caudate to the IFG is associated with proactive inhibition. The indirect DLPFC-caudate-IFG-SMA-STN-M1 pathway is involved in the implementation of proactive modulation, while the hyperdirect pathway that bypasses the striatum contributes to reactive inhibition. The function of the IFG is more related to attention control, and the caudate more likely acts as a “gate” between proactive and reactive inhibition.The present study was approved by the Institutional Review Board (IRB) of the National Institute of Advanced Industrial Science and Technology  and all participants gave informed consent prior to participation.FZ and SI conceived and designed the experiments, planned and carried out the experiments. FZ performed the data analysis, interpreted the results, and wrote the manuscript with critical feedback from SI.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.


Preoperative planning of the starting point and safe trajectory for iliosacral screw (SI screw) fixation using CT scans for safe and accurate fluoroscopically controlled percutaneous SI screw placement.Transalar and transforaminal sacral fractures. SI joint disruptions and fracture-dislocations. Non- or minimally displaced spinopelvic dissociation injuries.Transiliac instabilities. Sacral fractures with neurological impairment requiring decompression. Relevant residual displacement after closed reduction attempts. Insufficient fluoroscopic visualization of the anatomical landmarks of the upper sacrum.Preoperative planning of the starting point and the safe screw trajectory using CT scans and two-dimensional multiplanar reformation tools. Fluoroscopically guided identification of the starting point using the lateral view according to preoperative planning. Advancing the guidewire under fluoroscopic control using inlet and outlet views according to the planned trajectory. Predrilling and placement of 6.5 mm cannulated screws.Weightbearing as tolerated using crutches. Immediate CT scan in case of postoperative neurological impairment. Generally no screw removal.Fifty-nine screws were placed in 34 patients using the described technique. There were 2 cases of screw malpositioning . Percutaneous iliosacral screw (SI screw) fixation is a widely used minimally invasive technique for the stabilization of posterior pelvic ring injuries. It is applicable for both high-energy pelvic ring , 15, 19 Intraoperative two-dimensional fluoroscopic guidance of SI screw placement using inlet, outlet and lateral views in the standard technique has its limitations in the presence of sacral dysmorphism. For example, indentations of the anterior cortex of S1 (“notch”) are not visible on lateral and inlet projections endangering the L5 nerve root during SI screw insertion . AccordiPatients sustaining unstable pelvic ring injuries after high-energy trauma are typically transferred to level 1 trauma centers for surgical treatment. Although some of these trauma centers have the facilities for CT-controlled and navigated SI screw fixation, there is still no ubiquitous availability. Moreover, geriatric patients with fragility fractures of the pelvic ring involving the sacral ala are usually treated in peripheral hospitals, which generally have preoperative CT scans and intraoperative fluoroscopy available, while techniques for CT-controlled and navigated SI screw fixation are not provided.The aim of this article is therefore to present a simple preoperative planning technique to determine the starting point and safe screw trajectory for percutaneous SI screw placement using CT scans and commonly available imaging software tools. Furthermore, it is shown how to transfer the preoperative plan into the intraoperative situation in order to allow for safe and accurate fluoroscopically controlled percutaneous SI screw placement.Safe and accurate percutaneous SI screw fixation under two-dimensional fluoroscopic control even in the presence of sacral dysmorphism via the following two steps:First step: preoperative planning of the starting point and the safe trajectory for SI screw fixation using native CT scans and commonly available tools.Second step: Intraoperative fluoroscopically controlled identification of the starting point in the lateral view and advancing the guidewire/screw under fluoroscopic control using inlet and outlet views based on preoperative planning.No expensive planning software requiredNo intraoperative CT or navigation system requiredReduced risk of screw malpositioningSupine or prone positioning feasibleMinimally invasive technique with minor iatrogenic soft tissue damage and bleeding riskRadiation exposureDistinct fluoroscopic identification of the anatomic landmarks is mandatoryTime required for preoperative planningSpatial sense and experience in pelvic surgery required for the intraoperative two-dimensional application of the preoperative planTransalar and transforaminal sacral fractures after high-energy traumaTransalar and transforaminal sacral insufficiency fracturesSI joint disruptionsTransiliac and transsacral SI joint fracture dislocations with small bony fragment and thus amenable to SI screw fixation Non- or minimally displaced spinopelvic dissociation injuries Transiliac instabilitiesSacral fractures with neurological impairment requiring surgical decompressionRelevant residual displacement of the posterior pelvic ring after closed reduction and/or reduction of the anterior pelvic ringInsufficient fluoroscopic visualization of the anatomical landmarks of the upper sacrumGeneral surgical risksResidual risk of screw malpositioning and iatrogenic nerve palsyGenerally no implant removalPreoperative planning is the major step for safe fluoroscopically controlled percutaneous SI screw fixation and is therefore described in detail. Pelvic CT scans with a slice depth of 0.6 mm are recommended for preoperative planning. CT scans with slice depths >0.6 mm are also applicable, but may result in inferior image quality during the reformation process. Any imaging software, which supports two-dimensional multiplanar reformation (MPR), is suitable for the preoperative assessment.In the lateral view, the midsagittal plane is applied Fig. a. The coIn the inlet view Fig. b, the saIn the outlet view Fig. c, the hoThe definition of the corridor type and the assessment of the “notch” is a prerequisite for the determination of the screw trajectory. The corridor type is defined in the inlet view based on the axis of the osseous corridor (white line) in comparison to the coronal axis (blue line).Ascending type Fig. a: The axHorizontal type Fig. b: The axDescending type Fig. c: The axThe corridor type may change within a single sacral body along the craniocaudal extension of the sacrum. Accordingly, the corridor type is assessed in the upper and the lower third of the sacral body as well. Figure A “notch”  are preferable over anterior and posterior transverse trajectories for the following reason. Small intraoperative deviations from preoperative planning may result in posterior screw misplacement for posterior transverse trajectories  with consecutive iatrogenic lesions of sacral nerve roots in the sacral canal. Small intraoperative deviations from preoperative planning may result in anterior screw misplacement for anterior transverse trajectories  with consecutive iatrogenic lesions of lumbar nerve roots. In contrast, central transverse trajectories may compensate for small intraoperative deviations.The assessment of the MC transverse trajectory is the first choice due to its central location. This trajectory is therefore also called “Bullseye trajectory”. In the present case there is an adequate osseous corridor for an MC trajectory. There is also an UC trajectory, while the LC trajectory is in close vicinity to the S1 nerve roots. The transverse screw trajectories with safe osseous corridors (MC and UC) are marked in the grid as depicted. Even in the presence of safe transverse trajectories it is advisable to preoperatively also assess safe oblique trajectories as shown below in order to be able to intraoperatively judge whether small intraoperative deviations from the preoperative plan are acceptable.The term “sacral dysmorphism” is widely used and several radiographic characteristics of dysmorphic sacral anatomy have been defined . NeverthHowever, there are obviously numerous options for oblique trajectories in S1 in the presence of sacral dysmorphism. Safe oblique trajectories typically run from posterior to anterior in the inlet view and from caudal to cranial in the outlet view. It is advisable to assess an oblique trajectory, which runs oblique in a single plane only and transverse in the orthogonal plane, as this facilitates intraoperative two-dimensional fluoroscopic control. In some cases, however, oblique trajectories in both the inlet and outlet view are inevitable. Accordingly, there are three main options for oblique S1 screw trajectories as shown in Fig. In the outlet view, the intersection of the sagittal axis (green line) and the horizontal axis (red line) is moved to an optional starting point in the lower third on the lateral cortex of the ilium. The horizontal axis is then rotated cranially to reach the middle third in the midsagittal plane. Without any further adjustments in the inlet view, both views now show a safe osseous corridor. The screw trajectory is oblique in the outlet view (“outlet-oblique”), but transverse in the inlet view (“inlet-transverse”) and runs from LC to MC.In the inlet view, the intersection of the sagittal axis (green line) and the coronal axis (blue line) is moved to an optional starting point in the posterior third on the lateral cortex of the ilium. The coronal axis is then rotated anteriorly to reach the central third in the midsagittal plane. Without any further adjustments in the outlet view, both views now show a safe osseous corridor. The screw trajectory is oblique in the inlet view (“inlet-oblique”), but transverse in the outlet view (“outlet-transverse”) and runs from MP to MC.This option is required in the presence of sacral dysmorphism and no safe corridor with an oblique trajectory in a single plane. The horizontal axis (red line) is adjusted in the outlet view and the coronal axis (blue line) is adjusted in the inlet view. The screw trajectory is oblique in the inlet view (“inlet-oblique”) and oblique in the outlet view (“outlet-oblique”) and runs from LP to MC.Cannulated large fragment cancellous screws (6.5 or 7.3 mm thread diameter)Guide wire (2.8 mm diameter)Partially threaded screws (16 mm or 32 mm thread) for the application as lag screws Fully threaded screws for the application as position screws General anesthesiaSupine or prone positioningProne positioning is advisable for bilateral SI screw fixationSupine positioning with a pad or pillow supporting the lumbosacral regionFigs. , 6, 7FigWeight-bearing as tolerated using crutchesImmediate CT scan in case of postoperative neurological impairmentPostoperative CT scan for the assessment of screw positionGenerally no implant removalFigs. , 9Fig. 8Osseous corridor types and the presence of a notch were assessed in a series of 1000 CT scans in a recent study using the above described technique . An asceFluoroscopically controlled SI screw fixation using the above-described preoperative planning and insertion technique was performed in 34 patients aged between 22 and 92 years. There were 21 patients with sacral insufficiency fractures and 13 patients with type C injuries of the pelvic ring after high-energy trauma. SI screw fixation was performed bilaterally in all patients with insufficiency fractures and in 4 patients with bilateral posterior pelvic injuries following high-energy trauma, resulting in a total of 59 screws. All surgeries were performed by three pelvic surgeons experienced in fluoroscopically controlled SI screw fixation. Transverse screw trajectories were used for 36 screws and oblique trajectories for 23 screws. There were 2 cases of screw malpositioning. In the first case, fluoroscopic identification of the anatomical landmarks was inadequate and fluoroscopically controlled SI screw fixation should not have been performed. In the second case of a patient with sacral dysmorphism and an ascending type in S1, the preoperative assessment suggested a posterior and/or caudal S1 starting point and an oblique trajectory. Intraoperatively, a different starting point and screw trajectory was chosen resulting in screw malpositioning with iatrogenic L5 nerve palsy (Fig. Some limitations of this study have to be noted. First, the number of patients is relatively small. Second, postoperative CT scans were performed in the case of postoperative neurological deficits only. Accordingly, besides the two described cases with postoperative neurological deficits we were not able to retrospectively assess the rate of screw malpositioning in patients without neurological deficits. Finally, all SI screw fixations were performed by experienced pelvic surgeons. The rate of neurological complications might therefore be higher for surgeons with less experience.