One-third of the Antarctic continental shelf is covered by ice shelves, floating extensions of the Antarctic Ice Sheet. Marine life beneath and bordering ice shelves is rarely investigated, yet likely to be highly impacted by climate change. As ice shelves retreat, marine environments transition into new open-water spaces, with potential for primary production and consequent food-fall to the seafloor. How Antarctic seabed assemblages (benthos) develop in such emerging spaces is influenced by neighboring and oceanographically-connected communities; thus, closing knowledge-gaps of benthic biodiversity near ice shelves underpins understanding of future ecosystem change. This study examined seafloor assemblages, and environmental differences, in a region that has experienced ice-shelf retreat, in a polynya adjacent to a marine margin at the forefront of climate change: the ice-shelf front. The study area, located in the Weddell Sea, is seldom accessible, and lies within a proposed international marine protected area. The study found a physically- and biologically-diverse seabed, complexity in potential environmental influences, and evidence of increasing megafaunal densities with increasing distance from an ice-shelf front. This research provides insights into seafloor habitats and inhabitants close to an evolving marine margin, and establishes ecological baselines from which biological responses to climate change can be evaluated to inform marine management.2 of the Antarctic continental shelf and are sensitive indicators of climate change. With ice-shelf retreat, aphotic marine environments transform into new open-water spaces of photo-induced primary production and associated organic matter export to the benthos. Predicting how Antarctic seafloor assemblages may develop following ice-shelf loss requires knowledge of assemblages bordering the ice-shelf margins, which are relatively undocumented. This study investigated seafloor assemblages, by taxa and functional groups, in a coastal polynya adjacent to the Larsen C Ice Shelf front, western Weddell Sea. The study area is rarely accessed, at the frontline of climate change, and located within a CCAMLR-proposed international marine protected area. Four sites, ~1 to 16 km from the ice-shelf front, were explored for megabenthic assemblages, and potential environmental drivers of assemblage structures were assessed. Faunal density increased with distance from the ice shelf, with epifaunal deposit-feeders a surrogate for overall density trends. Faunal richness did not exhibit a significant pattern with distance from the ice shelf and was most variable at sites closest to the ice-shelf front. Faunal assemblages significantly differed in composition among sites, and those nearest to the ice shelf were the most dissimilar; however, ice-shelf proximity did not emerge as a significant driver of assemblage structure. Overall, the study found a biologically-diverse and complex seafloor environment close to an ice-shelf front and provides ecological baselines for monitoring benthic ecosystem responses to environmental change, supporting marine management.Ice shelves cover ~1.6 million km Previously long-constant Southern Ocean ecosystems are in transition, with climate change considered the greatest influencer ,2. DespiThe majority (~95%) of Antarctica’s known marine biodiversity is seafloor-dwelling, i.e., benthic . Benthos2) and deep (mean depth > 450 m) continental shelf is overlain by a unique environment: ice shelves [Approximately one-third of Antarctica’s extensive . 2) can be viewed as an “open-water” system, typically with regimes of seasonal sea-ice cover (extended winter sea-surface freezes and summer melts) and primary productivity (phytoplankton blooms) [The remaining two-thirds of Antarctica’s continental shelf (~3 million km blooms) ; however blooms) . Althoug blooms) ,41, far  blooms) , as well blooms) , while i blooms) , and nea blooms)  or rapid blooms) .Ice-related seafloor disturbance is a major driver of Antarctic shelf community patterns, and the severity and impact of such disturbances can differ over spatial scales, and temporally. For example, while iceberg scouring or ploughing of the seabed can be catastrophic to benthos at local scales, it can also, over time, promote higher regional diversity as mosaics of different faunal successional stages emerge across the seafloor ,48,49,50Following ice-related disturbance, (re)colonization of the Antarctic continental shelf can involve some predictable taxa and assemblage dynamics and, depth-depending, take from just several to hundreds of years for mature communities to (re)establish ,48,57. FOver the last 50 years, considerable ice-shelf loss has occurred in Antarctica, which is predicted to continue in a warming world. Indeed, a recent study by Gilbert and Kittel  has suggAs a center of ice-shelf loss, and with the regional benthos well-studied by Antarctic standards , vast ice shelves, perennially-heavy sea-ice cover ,39,72, aSA Agulhas II (IMO: 9577135), as part of the Weddell Sea Expedition 2019 data collection program [2 for 128 days per annum (pa), with phytoplankton blooms for 69 days pa [The study area is situated on the inner continental shelf of the western Weddell Sea adjacent to the northeast sector of the Larsen C Ice Shelf (maximum width ~200 km and thickness of ice front ~200 m) ,74, betw program . At the  days pa . Benthic days pa ,77,78. L days pa ), in parTo discern how sites differed environmentally, and to help identify environmental variables potentially responsible for structuring megabenthic assemblages, various environmental datasets  were used.Ice-shelf cover and ice-shelf front proximity. The distance of each site from the nearest ice-shelf front, at the time of sampling, was calculated using Vector Analysis Tools in QGIS  data, spanning 1979 to 2019 and derived from Nimbus-7 SMMR and DMSP SSM/I-SSMIS passive microwave observations [2 and were processed in the same manner as described by Christie et al. [rvations , were obe et al. . Using tPrimary productivity. Mean daily net primary production  data, spanning 1997 to 2018, were generated following the methods of Arrigo et al. [o et al. ,89. FromOceanography. Oceanographical data were acquired by deployment of a CTD (conductivity-temperature-depth) system (Sea-Bird Scientific SBE 911plus), which included auxiliary sensors  and a rosette of 24 12-L Niskin bottles enabling collection of water samples  were obSubstratum and phytodetritus. Both substratum type (hardness) and level of phytodetritus cover were determined during seafloor imagery analysis   (piloted by Eclipse Group Inc. and supported by Deep Ocean Search Ltd. and Ocean In-finity Inc.) equipped with a high-definition (HD) 1080i/29.97 fps color video camera ; the camera was forward-facing and angled obliquely towards the seabed. The ROV was fitted with two red parallel scaling lasers , 10 cm apart, and six lights . During dives, the three-dimensional position  of the ROV was provided by a high-precision acoustic positioning system . Videos were time-stamped, enabling the pairing of observations with positional data. Mean transect length was 847 m and depth within transects varied by <15 m.www.handbrake.fr, accessed on 15 June 2022), an open-source video transcoder. Frames were extracted from re-encoded videos every 20 s to avoid spatial overlap between successive frames. Frames not viable for analysis  were discarded, the mean discard rate being ~25%. A 250 m sample was taken from each transect for analysis . Examples of seafloor imagery from the study are provided in Raw transect videos  were de-interlaced in HandBrake 1.3.3  [2 per frame). To provide further ecological context to the megabenthic diversity observed, and also to potentially identify ecological proxies to facilitate future monitoring, a functional group .As per Jones et al. , video fne 2022) . AnalysiStatistical analysis comprised calculations of univariate indices, including the total number of morphotaxa (richness), as well as the application of multivariate statistical techniques in PRIMER v7  with PERPrior to multivariate analysis, and adopting established procedures  . The PCAThe statistical processes were also conducted for the functional group-based classification.Across and within the study sites, the seafloor varied considerably in terms of rugosity (roughness) and dominant substratum type. Expanses of poorly-sorted coarse-grained (pebbles to boulders) substratum were particularly prominent at site LCP2, closest to Jason Peninsula. Such material appeared similar to that observed northward along the Larsen A Ice Shelf front ; indeed,Recent iceberg-scouring, such as that observed by Gutt and Piepenburg  and GuttPhytodetritus cover of the seafloor, indicative of potential “food banks” ,111, wasCircular/elliptic seabed depressions (~10 to 25 cm in diameter) were observed at each study site. These depressions appeared individually and in clusters, with more and larger clusters observed at the shallower sites, LCP2 and LCP4.Calyptogena-like shells and weedy worm-tubes , and thPerknaster sp. and brisingids, respectively, were observed in higher numbers at LCP2, the latter consistently on large boulders . Instances of commensalism were evident throughout much of the study area, including, for example, crinoids and ophiuroids on top of corals or large sponges [Across the study sites, biota, and associated functional groups, were, at times, highly variable in terms of abundance, diversity and composition—indicating a degree of faunal patchiness. Some areas could be depauperate, with little or no perceptible biota, or comparatively dominated by a particular taxon or functional group . In contrast, other areas could host multiple encrusting fauna  on hard substratum (mainly larger rocks and boulders). Giant predatory and suspension-feeding asteroids, e.g.,  sponges .Ice-shelf cover and ice-shelf front proximity. Observations of changing areal extent of Larsen C throughout the satellite era revealed varying degrees of ice-shelf cover over the study sites. Specifically, it was found that LCP1 and LCP2 had intermittently been covered by ice shelf in the observation period (1963–2018), consistent with the natural advance and calving of small icebergs along this particular stretch of the Larsen C front through time [ period 163–2018, ugh time . LCP1 anugh time , and posugh time . LCP3 waSea-ice concentration. Mean monthly SIC (±1 SD), for the 1997–2018 period, was lowest at LCP1 (65.1% (±22.7)) and highest at LCP2-4 (69.6% (±25.7)). The low SIC values observed are indicative of the presence of the polynya.Primary productivity. Mean daily NPP and mean peak NPP, for the 1997–2018 period, were highest at LCP4 and showed a significant increase with increasing distance from the ice-shelf front . For LCP1-4, the mean daily NPP values (±1 SD) were 123.5 (±202.8), 165.9 (±226.4), 199.4 (±245.2), and 243.8 (±309.3) mg C m−2 d−1, respectively, and mean peak NPP values (±1 SD) 264.5 (±329.7), 372.6 (±207.7), 435.4 (± 309.5), and 791.7 (±552.9) mg C m−2 d−1, respectively.Overall, a total of 98 morphotaxa from 23 classes of 10 phyla were observed across the study zone.2), the latter peaking at 32 morphotaxa (p > 0.2), while all other pairwise comparisons indicated slopes to be significantly different.The relationship between richness and area surveyed was examined at each site and compared to determine if megafaunal richness accumulated differently across sites. As expected, faunal richness increased from the area of a single frame to a sample , including “singletons” (morphotaxa that only appeared once), and this mainly drove the increased richness observed at LCP1 and LCP4, with different taxa contributing to the morphotaxa count of each site.2 samples (three per site), the lowest richness was observed in an LCP2 sample and the highest in an LCP4 sample, with 11 and 31 morphotaxa recorded, respectively. An LCP1 sample exhibited the second-highest richness at 27 different morphotaxa. The richness of LCP4 samples was varied, with the value of one sample being almost double of another (16 and 31), whereas the richness of LCP3 samples was noticeably more uniform . Marked variability in richness was found at the sites closest to the ice-shelf front, with richness more than doubling from the poorest to richest sample (11 and 27).When comparing standardized 100 mp = 0.33). However, when LCP1 samples were excluded from the exploration, as they were located within a different water mass .In terms of a relationship with proximity to the ice shelf, richness did not significantly alter with increasing distance from the ice-shelf front , the rep = 0.03), although richness did halve from predominantly soft-sedimented (with incidences of mixed) substratum through to assorted hard sediment-boulder fields.Overall, a weak (inverse) relationship was observed between richness and “hardness” of substratum . To control for water mass differences, this trend was also explored without LCP1, where it became more significant (p < 0.01).With increasing distance from the ice-shelf front, it was observed that density generally increased , ophiuroids  and encrusting lithophilic fauna . Bottlebrush corals and encrusting fauna mainly drove the high variability in density and occasionally high values at LCP1.The general relationship of increased faunal density from the ice-shelf front to farther offshore was principally caused by increases in densities of bottlebrush corals . The lowest density values were observed in the samples categorized as having the “hardest” substratum type. This trend was principally driven by decreased densities across the majority of faunal groups at LCP2 (closest to Jason Peninsula), but particularly in encrusting suspension feeders, and the apparent absence of certain taxa, including bottlebrush corals . However, in contrast, functional group richness did not exhibit a relationship with substratum hardness, including when removing singleton groups .With respect to richness, functional group richness, like morphotaxa richness, did not significantly change with increasing distance from the ice-shelf front, including when removing groups represented by singletons , pioneer sessile suspension feeders (primarily encrusting lithophiles) and hard sessile predator/scavengers  all showed similar patterns to the overall faunal trend A–C; thisp = 0.02, 0.001 and 0.007, respectively). Whereas other functional groups (and component taxa), e.g., climax suspension feeders, flexible strategists , sedentary suspension feeders , and mobile hard predator/scavengers  did not show clear trends with substratum hardness .With regard to substratum type, deposit-feeding crawlers, hard sessile predator/scavengers, and pioneer sessile suspension feeders, like the overall fauna pattern, decreased in density with increased substratum hardness  for the wider faunal patterns observed, e.g., in density, deposit-feeding crawlers appeared to be the strongest surrogacy candidate (Sterechinus antarcticus (as well as other echinoid species), principally when comparing LCP1 with offshore sites, and, in some site comparisons, encrusting lithophilic organisms and Pyura sp.   see .Peniagone vignoni was observed. Notably, LCP1 had higher densities of echinoids (of at least three different morphospecies). At LCP4, an athecate hydroid species occurred in higher densities than at other sites. Compared to LCP3, LCP4 had more bottlebrush corals, the aforementioned hydroid species, and the Pyura sp. In general, LCP3 and LCP4 had higher densities of holothurians (of differing species), particularly those  identified as Pseudostichopus mollis.The differences between LCP2 samples and those of other sites appeared to be driven by absences and much lower densities of various taxa, although LCP2 was the only site in which the holothurian HAC analysis with a SIMPROF test identified four distinct groupings among samples, corresponding to the four individual sites . FurtherWhen HAC (plus SIMPROF) analysis was performed for functional groups, results were similar to that of the morphotaxa in that the samples were again clustered by their respective study site .A range of environmental variables . This subset included mean depth, water physico-chemistry and substratum hardness. The single variable showing the highest Rho correlation (0.77) was substratum type, considered a local-scale variable.The BEST analysis indicated that a subset of three environmental variables most highly correlated with (or “best explained”) the variation in faunal assemblage structure . As such, there appears to be some consistency in which environmental variables emerged as potential key drivers of the biotic variation observed, irrespective of whether the benthic biodiversity was classified by taxonomic levels or functional groups.When comparing BEST analysis results of all faunal grouping methods, i.e., morphotaxa, class and functional groups, the most highly correlated subsets of potential explanatory variables each included water physico-chemistry and substratum hardness . When individual taxa and functional groups were explored, some groups were found to reflect the overall density trend , some contrasted, and some showed no clear pattern. Substratum type appeared to strongly influence megafaunal density, although it did not appear to exert the same impact on richness. Distance from the ice-shelf edge, which, in the confines of the study, correlated with the remotely-sensed primary-production-related variables, did not emerge as a significant environmental driver of assemblage composition. Instead, different environmental variables appeared to be important locally at each study site. Compared to offshore sites, assemblages closest to the ice-shelf front could be expected to be more similar to each other , yet they were actually the most dissimilar. Overall, results suggested that a study zone, where a polynya abuts the ice shelf, can be a complex coastal environment, both physically and biologically.Large-scale alterations in faunal richness have been widely observed across geological time  and spacPotentially having experienced greater historic disturbance , Antarctica’s coastal open-water shelf areas can harbor a great array of habitat types and niches, and hence resident biota, including those associated with different successional stages. As such, it was envisaged that a positive richness gradient would be observed with increasing distance from the ice-shelf front . However, within the study confines, no significant increase (or change) in faunal richness was discerned (including when examining Jason Peninsula sites exclusively). Furthermore, richness values were particularly variable at the sites closest to the ice-shelf front. Similarly, Grange and Smith  found noBiota responses to major environmental gradients can be reflected in faunal density in both space and time. In the polar regions, this can be exemplified by (re)colonization of seafloor on ecological timescales in response to ice scour, glacier retreat and ice-shelf collapse ,132,135,Additionally, the seafloor (hence benthos) at LCP1 may have been more protected from ice-mediated disturbance than the Jason Peninsula sites due to being more consistently ice-shelf-covered, and for a more extended period, at least within the satellite era. Furthermore, when open water, LCP1 may have been afforded further protection from disturbance by Bawden Ice Rise and associated shallow seafloor, known to constrain iceberg-flow into and within the area . NonetheThe site with the lowest faunal densities was LCP2, closest to Jason Peninsula. Such density values may be explained in reference to the aerial/satellite imagery that showed the site had often been proximate to the ice-shelf edge and had undergone ice-shelf advance and retreat (at least twice) in recent-time. Additionally, the site is at a water depth within reach of modern iceberg keels in an area of iceberg transit . Hence, the respective site may have been especially subject to disturbance such as scouring and the deposition of ice-rafted debris  ,143.Although all sites in the study occurred within a polynya, and therefore an area of potentially elevated primary production and related organic matter export to the seabed ,144, megBiological patterns were not only explored by looking at the megafaunal assemblage as a whole, but also through the 12 functional groups represented by the fauna detected . Such anPartitioning faunal density and richness into various taxonomic levels, or by function, permits further insight into how different elements of biodiversity respond to environmental conditions. Finding appropriate surrogacy, or indicators, can also help simplify ecosystem monitoring, accelerate data processing and results-delivery, and thus enhance the timeliness of informing marine management, which is particularly important for rapidly changing environments. Suitable surrogates should reflect the overall biotic pattern of interest while not driving it. In the present study, deposit-feeding crawlers, all of which were holothurians, were found to be the strongest surrogacy candidate for overall megafaunal density . AlthougOrdination techniques, such as nMDS, can help reveal underlying structural differences in biodiversity at various scales, for example, between islands and seamounts , along fOne of the striking biological differences between Jason Peninsula site LCP2 and other sites was that it did not appear to host calcaxonian corals, despite the availability of plentiful hard surfaces, i.e., attachment points. However, the distribution of such biota is also thought to be strongly influenced by water depth, coincident iceberg scouring, and the flow of organic matter , so thesA priori considerations that ice-shelf proximity may be a major influencer of megafaunal assemblage composition were not directly supported by the BEST analysis findings of this study. However, such a factor (as aforementioned) could be linked to other environmental variables such as prevailing substratum type. As found in other polar locations and farther afield e.g., ,153,154),154153,1In the polar seas, sea-ice cover  is a key influencer of seabed biodiversity dynamics, in part through associated impacts on light penetration, primary production, and flux of particulate organic matter to the seafloor ,157. HowThis study explored benthic assemblages of four western Weddell Sea continental shelf sites, all within an ice-shelf-adjacent coastal polynya, and two of which have recently experienced ice-shelf retreat. The research revealed distinct faunal patterns in density (but not in richness), and identified a taxonomic and functional group ) that could potentially be a surrogate for the wider megafauna, hence contributing to simplifying, and potentially expediting, future marine environmental monitoring efforts. Distance from the ice shelf was not identified as a key driver of assemblage composition; however, results evidenced that at the ice-shelf front, habitats and assemblages can be very different across space. The study further showed that different environmental variables were potentially important in structuring benthic assemblages at each study site , emphasizing the complexity of Antarctic continental shelf environments, particularly those at the edge of ice shelves.The current findings provide an indication of how seabed communities may develop along the eastern Antarctic Peninsula as further ice-shelf cover is lost, and narrow biological knowledge gaps of a very rarely accessed and distinctive area within a CCAMLR-proposed MPA.


Notably, Takeda G-protein coupled receptor 5 (TGR5) deficiency partially reverses the inhibitory effect of LCA on DCs in vitro and in vivo. TGR5 activation also downregulates the NF-κB and MAPK pathways by inhibiting glutathione production and inducing oxidative stress in DCs, which leads to apoptosis and autophagy in DCs. In addition, LCA or INT-777 treatment increases the TGR5 expression in monocyte-derived dendritic cells (MD-DCs) of patients with active BD, whereas both LCA and TGR5 agonists inhibit the activation of MD-DCs. These results suggest that LCA and TGR5 agonists might be potential therapeutic drugs for the treatment of autoimmune uveitis.Dendritic cells (DCs) are the major antigen-presenting cells and play an important role in autoimmune uveitis. Emerging evidence suggests that bile acids (BAs) regulate DCs maturation. However, the underlying mechanisms by which BAs regulate the function of DCs still need to be clarified. Here, we demonstrate that lithocholic acid (LCA) inhibits the production of pro-inflammatory cytokines and the expression of surface molecules in bone marrow-derived dendritic cells (BMDCs). LCA attenuates the severity of EAU by modulating the maturation of splenic CD11C Uveitis is an autoimmune inflammatory disease marked by inflammation of intraocular entities Primary bile acids (BAs) are produced in the liver and converted to secondary BAs by intestinal bacteria Takeda G protein-coupled receptor 5 (TGR5) is a membrane receptor activated by BAs, deoxycholic acid (DCA) and lithocholic acid (LCA) are preferential agonists Glutathione (GSH) regulates several metabolic and cell cycle-dependent functions, such as scavenging of exogenous toxins and free radicals, protein synthesis, and maintenance of intracellular redox balance In the present study, we found that BAs regulate the secretion of cytokines in DCs and their subsequent antigen presentation ability via TGR5 signaling. In addition, TGR5 activation promotes apoptosis and autophagy in DCs by inhibiting intracellular GSH production.-/- mice were obtained from Viewsolid Biotech . TGR5+/+ (C57BL/6J) mice were purchased from Jackson Laboratory . EAU was induced in 6- to 8-week-old female mice through subcutaneous injection of 300 µg human IRBP651-670 dissolved in 0.2 ml emulsion 1:1 (vol/vol) supplemented with CFA containing 2.5 mg/mL Mycobacterium tuberculosis strain H37Ra . The mice also received intraperitoneal injections of 0.5 µg Bordetella pertussis toxin  suspended in 0.1 ml PBS. The toxin was injected once on the first day and after 2 days. Then, EAU mice were fed a special LCA diet (0.01%) or normal diet. Clinical manifestations and histopathological changes in the mice were evaluated as previously described TGR5Healthy individuals (n=16), patients with active BD (n=8) and patients with inactive BD (n=8) were enrolled in this study. BD diagnosis was performed according to the international nomenclature committee guidelines TRIzol  was used to extract the total RNA from Bone marrow dendritic cells (BMDCs), retinal tissues, and monocyte-derived dendritic cells (MD-DCs). The PrimeScript RT reagent kit  was used to synthesize cDNA from the RNA. The resultant DNA was amplified by real-time PCR as previously reported 5 BMDCs were stimulated using lipopolysaccharides (LPS)  and bile acids  or INT-747 or INT-777  for 24 h.BMDCs from the mice were extracted and cultured as previously described Mice CD4+ T cells were isolated using mouse CD4 microbeads  and cocultured with BMDCs pretreated with LCA or INT-777 at a ratio of 5:1 (CD4+ T cells: BMDCs) for 5 days.5 MD-DCs were treated with 100 ng/ml LPS and 10 µM LCA or 100 µM INT-777 for 24 h.Human CD14+ monocytes in PBMCs were isolated using Human CD14 microbeads (Miltenyi Biotec). CD14+ monocytes were incubated with 50 ng/mL IL-4  and 100 ng/mL GM-CSF (AcroBiosystems) to induce DC maturation. 7 days later, 5×10To explore the role of TGR5 and FXR in BMDCs, the cells were transfected with TGR5-siRNAs or FXR-siRNAs using Lipofectamine 3000  in accordance with the manufacturer's protocol. An adenovirus encoding human TGR5-siRNA was constructed  and transfected into MD-DCs as previously reported The integrity of the BRB was evaluated using Evans blue  as previously described Anti-mouse IL-17A-PE, CD4-APC and IFN-γ-PE-cy7, anti-mouse IgG isotype, anti-human IFN-γ-PE-cy7, CD4-APC and IL-17A-PE and anti-human IgG isotype were obtained from eBioscience. For intracellular expression of IFN-γ and IL-17, CD4+ T cells were incubated for 1 hours with ionomycin (1 μg/mL), PMA (50 ng/mL) and for another 4 hours with brefeldin A , harvested, washed and fixed before permeabilization.BMDCs or MD-DCs were stained for 30 minutes at 4 °C with anti-mouse CD86-FITC (eBioscience), CD11c-APC (eBioscience), CD80-PE (eBioscience), CD40-FITC , MHCII-PE (eBioscience), anti-human CD86-PE-cy7 (BioLegend), CD40-FITC (BioLegend), HLA-DR-PE-cy5.5 (BioLegend), and CD80-PE (BioLegend).The concentrations of human IFN-γ, IL-1β, IL-17, IL-6 and TNF-α as well as mouse IL-1β, IL-12/p70, IL-6, IL-23, IL-17, IFN-γ and TNF-α in the supernatants of BMDCs, MD-DCs or mouse serum were analyzed using Duoset ELISA kits . The concentration of human IL-12/p70 in the supernatants of MD-DCs was assessed using an ELISA kit (Thermo Fisher Scientific).-/- and TGR5+/+ mice treated with LCA was sequenced. The technique was performed using the Illumina NovaSeq 6000 platform. DEGs were identified using DESeq2. P values were adjusted to control for the false discovery rate using Benjamini and Hochberg's method. DEGs with an adjusted P value <0.05 and |log2foldchange|>1 were considered statistically significant.RNA from BMDCs of TGR5http://www.geneontology.org) provides functional classification, including categories of cellular component (CC), molecular function (MF) and biological processes (BP). Enriched pathways were evaluated by the KEGG database (http://www.kegg.jp/kegg/pathway.html). GO terms and KEGG pathway enrichment analyses were performed on DEGs using the DAVID database. FDR<0.05 was considered significant.The GO database . Before LPS stimulation, the cells were pretreated with DCFH-DA for 20 min. The fluorescence DCFH-DA signal was analyzed using flow cytometry. Briefly, frozen sections of eyeball tissue were stained with dihydroethidium  at 4 °C for 30 min before being observed under a fluorescence microscope.Cell extracts of BMDCs and MD-DCs were collected to detect the concentrations of GPx (Cayman), CAT (Cayman) and GSH (Cayman) using the appropriate assay kits according to the manufacturer's instructions. Intracellular GSH levels in the retinal tissues of EAU mice were quantified using monochlorobimane . Frozen sections of eyeballs were stained with MCB for 30 min, rinsed and observed under a fluorescence microscope.The expression of proteins of interest in BMDCs was analyzed using western blotting as previously described The protocols of all studies were approved by the ethics committee of our hospital (2018-049). All participants agreed to participate in this study and gave informed written consent. All experiments involving human participants strictly followed the principles of the Helsinki Declaration. The animal protocols were approved by the Animal Care and Use Committee of our hospital and were conducted in line with the ARVO Use of Animals in Ophthalmic and Vision Research guidelines.Differences between groups were analyzed using one-way ANOVA, whereas Dunn's correction analysis was used for multiple groups. Differences in independent groups were analyzed using unpaired Student's t test and the Mann-Whitney U test. Comparisons between paired groups were performed using the t test and the Wilcoxon test. Continuous data are expressed as the mean ± standard deviation (SD). Statistical significance was set at *p<0.05 or **p<0.01.To study the effects of different BAs on DCs, LPS-primed BMDCs were treated with CDCA, LCA, DCA, taurolithocholic acid (TLCA), ursodeoxycholic acid (UDCA), cholic acid (CA) or glycocholic acid (GCA) Figure A. The reIn vitro studies using TGR5-/- BMDCs found a similar result , glucocorticoid receptor (GR), α5β1, pregnane X receptor (PXR) and constitutive androstane receptor (CAR) t Figure G. These +/+ mice an LCA diet significantly ameliorated the severity and slowed the progression of EAU . We found that LCA diet significantly disrupted the maturation of DCs to CD11+MHCIIhigh subtypes in splenocytes of TGR5+/+ EAU mice (in vivo (data not shown).To further explore the involvement of TGR5 signaling in DCs, we weighed the spleens of EAU mice Figure D and anaEAU mice A-B. ConsEAU mice A-C. LCA +/+ and TGR5-/- EAU mice fed either ND or LCA diet were co-cultured with naïve CD4+ T cells from TGR5+/+ EAU mice at a ratio of 1:5 (CD11c+ DCs: CD4+ T cells). CD11c+ DCs isolated from TGR5+/+ EAU mice fed LCA diet significantly inhibited the differentiation of Th17 and Th1 cells . LCA-pretreated BMDCs significantly inhibited the differentiation of Th1 and Th17 cells and the production of IFN-γ and IL-17  in TGR5+/+ BMDCs compared with TGR5-/- BMDCs, of which 641 DEGs were upregulated and 943 DEGs were downregulated  and glutathione reduced form ethyl ester (GSH-OEt) were used to block or increase GSH expression in TGR5-/- and TGR5+/+ EAU mice fed a diet with or without LCA were examined by fluorescence microscopy. However, LCA had no effect on ROS and GSH levels in the retinal tissues of EAU mice  inhibited the secretion of IL-18 and IL-1β in intestinal epithelial cells +MHCIIhigh DCs.The anti-inflammatory roles of secondary BAs were first described in chronic cholestasis, characterized by under-secretion of TNF-α, IL-6 and IL-1β and by macrophages -/- mice in vitro, which was caused by the low expression of TGR5 in T cells.BAs exert anti-inflammatory effects by activating the expression of BA receptors such as GPCRs as well as nuclear receptors Glutathione, a major component of the antioxidant defense system of living cells, is present in both prokaryotes and eukaryotes. GSH and oxidative stress are thought to play a key role in immunity, including autoimmunity In macrophages, TGR5 inhibits the activation of NF-κB via the cAMP-PKA pathway TGR5 was found decreased in macrophages of active VKH patients and decreased in DCs of active BD patients In summary, BAs inhibit the expression of pro-inflammatory cytokines and surface markers in DCs, with LCA exerting the greatest effect. Although FXR can be activated by LCA, TGR5 is the most potent downstream target of LCA. LCA inhibits DC activation through TGR5 signaling to regulate the development of autoimmune uveitis. This effect of LCA is associated with a decreased intracellular GSH in DCs. Inhibition of GSH attenuates the release of pro-inflammatory cytokines via the NF-κB and MAPK pathways. Additionally, LCA promoted apoptosis and autophagy in DCs by inhibiting GSH. Therefore, LCA and TGR5 agonists are potential therapeutic drugs for the treatment of autoimmune and inflammatory diseases, including autoimmune uveitis.Supplementary figures and tables.Click here for additional data file.


The ratio of the distance between defects >7 mm was high, and more cases required ≥2 devices in the 3D TEE group than those in the ICE group (p < 0.0001). All patients in the 3D TEE group and seven patients (5%) in the ICE group were operated on under general anesthesia (p < 0.0001). The fluoroscopic time was shorter in the ICE group . No difference in the complete closure rate and complications was observed. ICE-guided transcatheter and 3D TEE were feasible, safe, and effective in successful multiple ASD device closures, especially for young children and patients at high risk under general anesthesia.We aimed to determine the feasibility, efficacy, success, and safety of intracardiac echocardiography (ICE) in transcatheter multiple atrial septal defect (ASD) closure. Of 185 patients with multiple ASDs who underwent transcatheter closure, 140 (76%) patients who weighed <30kg with a narrow distance between defects or in whom single device closure was anticipated were guided by ICE and 45 patients were guided by three-dimensional (3D) transesophageal echocardiography (TEE) with or without ICE. Patients in the ICE group were relatively younger and weighed less than those in the 3D TEE group ( Transcatheter atrial septal defect (ASD) closure has become the treatment of choice as a minimally invasive approach that allows for rapid recovery and earlier hospital discharge. This procedure, guided by proper echocardiographic imaging, is becoming the gold standard treatment ,2. PreopTwo-dimensional (2D) echocardiography, specifically transesophageal echocardiography (TEE) with or without ICE, has conventionally been used for transcatheter ASD closure. However, three-dimensional (3D) TEE plays a central role, especially in complex cases with multiple ASDs ,7,8,9,10Overall, 185 patients with multiple ASDs who underwent transcatheter device closure between January 2010 and August 2020 were enrolled in the study. All cases with more than single interatrial communication or multi-fenestrated atrial septal defects were included in the multiple ASD. We excluded patients who were referred to surgery before undergoing the procedure or with contraindications to ASD closure because of severe pulmonary hypertension. The following data were collected from the medical records: demographic data, number of defects, defect size, number of devices, total fluoroscopy time, procedural complications, and clinical outcomes. Before performing the procedure, information on the number of defects and adequacy of the septal rims for the device was obtained from TEE or cardiac computed tomography, and 2D ICE or 3D TEE was performed before the procedure based on the defect characteristics and patient factors, including age, body weight, the risk to general anesthesia, comorbidities, and overall general condition. In particular, we preferred using ICE, predominantly in small children weighing <30 kg regardless of age, or with a closer distance between the multiple defects in the pre-procedure transthoracic echocardiography. This procedural strategy was primarily based on a recommendation regarding multiple ASD device closure, which stated that multiple ASDs with an inter-defect distance of <7 mm can be successfully closed using a single device ,17. SomeAccording to the pre-procedural and intraprocedural assessments, we classified atrial septal anatomy into aneurysmal type, fenestration type, and septal aneurysm with fenestration type and classified whether there are fenestrations. If it did not correspond to aneurysm or fenestration, it was classified as “neither”. The fenestrated ASD was defined as an ASD with small, multiple fenestrations or pinpoint defects. According to Silver et al.  and PearProcedural success was defined as a stable, adequately positioned device in the absence of trauma to the surrounding structures. The medical report in the pointed data registry demonstrating residual defects, major and minor complications, including cardiac tamponade, erosion, device embolization, thromboembolism, and complete atrioventricular block, and the need for re-intervention were included in the follow-up outcomes. The study endpoint was the evaluation of the difference in the procedural success and complications between the 2D ICE and 3D TEE (±ICE) groups.We defined “feasibility” and “safety” of using ICE as the cases where closure was successfully complete without any significant residual shunt with no minor and major complications.Echocardiographic imaging was performed to assess the defect diameter, intra-defect distance, superior, inferior, and anterior rims of the defect, total antral septal length, the proximity of the defect to surrounding structures, and final device positioning. Our institution performed ASD closure with TEE guidance under general anesthesia until mid-2011. Since 2011, ICE has been used as the standard echocardiographic imaging during transcatheter closure of ASDs and patent foramen ovales in the pediatric and adult populations at our institution. TEE was rarely performed, except for the cardiac catheterization laboratory when 3D TEE was required. Procedures in pediatric patients guided by ICE were performed under local anesthesia with mild sedation. Patients with a minor distance between two significant defects on pre-procedural TEE and those in whom one device was expected to close another defect underwent transcatheter closure with 2D ICE. Otherwise, 3D echocardiography was used.Furthermore, ICE was used when a patient’s body weighed <30 kg. We followed the ICE -guided ASD closure methodology previously described by Earing et al. . ICE wasThe ASDs in our patients were closed using the Amplatzer septal occluder , Cocoon Septal occluder , Occlutech Figulla Flex II device , and Gore Cardioform Septal Occluder . When more than one device was required, the operator ensured that all devices were positioned at adequate distances from important structures, such as the vena cava entrances and coronary sinus, because the devices may interfere with blood flow and increase the risk of thrombosis. TEE was performed immediately after each procedure to confirm the position and stability of the device(s) and residual shunt. All patients were evaluated using transesophageal echocardiography, electrocardiogram, and chest radiograph on the day of and on the day after the procedure. Patients were followed-up after 1 week; at 3, 6, and 12 months; and annually thereafter until the first documented event or time of final follow-up, whichever came first.t-test was performed for continuous variables. A p-value of ≤0.05 was considered statistically significant.All analyses were performed using SPSS version 25.0 . The results were reported as numbers (percentage), mean ± standard deviation, and range. Comparisons of the baseline data, procedure-related characteristics, and outcomes between the 2D echocardiography and 3D TEE ± ICE groups were performed. Fisher’s exact test was performed for categorical variables, whereas the independent This study was approved by the Yonsei University College of Medicine Institutional Review Board and Research Ethics Committee of Severance Hospital . The requirement for individual consent was waived because of the retrospective study design.p < 0.0001). The mean bodyweight of the 2D ICE group was 28.7 kg , which was less than that of the 3D TEE group (p < 0.0001). The 2D ICE and 3D TEE (±ICE) groups most commonly had two defects; however, the proportion of patients with more than three defects was significantly higher in the 3D TEE group (p = 0.02).In total, 185 patients with multiple ASDs underwent transcatheter device closure at our institution during the study period. p < 0.0001), and 17 (40%), 22 (49%), and 5 (11%) patients required one, two, and three devices, respectively. The fluoroscopy time was significantly shorter in the 2D ICE group (p = 0.005); however, this may be because a majority of the patients in the 2D ICE group only required one device or had simple defects. All patients in the 3D TEE group were operated under general anesthesia.The procedural data of both groups are demonstrated in Among the 185 patients who underwent ASD closure, 181 (98%) cases were successful . Three cIn all children weighing less than 10 kg, device closure was performed safely and successfully with ICE. The youngest child was 9 months old and the smallest weighed 5 kg; 79% of the children had an inter-defect distance of less than 7 mm. All of these patients weighing less, including those with an inter-defect distance of larger than 7 mm, were treated using one device for ASD closure. For this reason, either a residual shunt or an isolated another defect was observed immediately after the procedure. However, at the latest follow up, 75% of patients had complete closure. These results are similar to those of all other patients . Mobitz Transcatheter ASD closure under echocardiographic guidance is the gold standard treatment for most cases of ASDs, including complex cases with insufficient rims or multiple ASDs ,21. In tNotably, 3D TEE is an excellent procedure for patients with ASDs of complex anatomies, such as multiple defects, because it provides a high spatial resolution of all defects, implanted devices, and any residual shunting ,26,27,28On the contrary, the major limitation of 3D TEE is that it cannot visualize the entire defect in a single window, principally when the posteroinferior rim of the defect is blocked by the inferior vena cava, limiting the visualization while passing the wire and catheter during or after device implantation . FurtherCompared with 3D transesophageal echocardiography, ICE provides relatively clearer images of the posterior and inferior walls of the atrial septum ,20,34,35n = 129, 92%) needed only one device because a narrow enough inter-defect distance device disc can close multiple defects and/or other isolated defects were not significant enough to require additional device closure  vs. 43.9 ± 18.8 (10 months–76 years)) and small body weight  vs. 60.9 ± 11.0 (44.4–94.9 kg)). Children under 10 kg treated with ICE showed similar results compared with all children. In particular, it suggests the feasibility and safety of ICE in multiple ASDs in that even children under 10 kg treated with ICE showed similar outcomes compared with all other patients.Because the shape of the defects can be elliptical or complex ovoid, and asymmetric shape, the more complex the defect, the greater the difference in size in 2D ICE and 3D TEE. A study comparing ASD dimensions using 3D TEE vs. 2D TEE and ICE showed that 2D TEE and ICE are more likely to provide a smaller long-axis dimension and larger short-axis dimension when compared with RT3D TEE . The difSince each echocardiographic modality has advantages and disadvantages in multiple ASD device closure, the stability and efficiency of the procedure can be improved by selecting each modality in consideration of the characteristics of the defect and the patient’s condition; 3D TEE is particularly useful for ASDs with complex geometries because it provides the complex morphology and spatial relationship between multiple defects. However, ICE can be considered the first choice in a case requiring a single device, one large defect with one or more small defects and an inter-defect distance less than 7 mm, or one large defect with a distance of less than 7 mm from the farthest point of another defect. If the distance between one large defect and another is greater than 7 mm but these multiple defects are not of significant size, ICE could be considered in patients with low body weight and those at risk of general anesthesia.Our study has some limitations. First, due to its retrospective design, the patients were not evenly distributed between the groups. Therefore, it is difficult to directly compare ICE and 3D TEE in this study. More patients with multiple ASDs are required, and long-term complications are needed to be observed to determine the transcatheter closure experience. Second, the 3D TEE with and without ICE groups could not be compared because of the small number of patients. However, our study was meaningful, in that, it demonstrated that ICE was a reliable method for closing multiple ASDs, particularly in children and in those with simple multiple ASDs where multiple devices are not required. According to our institution’s standard practice strategy, we were able to maximize the advantages of ICE and to provide procedural guidance for multiple ASD closure through tailored decisions for individual patients.In conclusion, transcatheter multiple ASD device closure using 2D ICE or 3D TEE was successfully performed without fatal complications. Both modalities can provide good images for successful transcatheter closure of multiple ASDs. Although the benefits of 3D TEE are obvious, 3D TEE may not be necessary in all cases with multiple defects. 2D ICE can be safely and efficiently performed even in multiple ASD device closure as an alternative to 3D TEE. ICE can be considered first in relatively simple ASD cases where the distance between defects is short or two or more devices are not expected to be needed, especially for patients who weigh less and those at risk of general anesthesia. There is a need for improvement in the cost aspect of ICE, and the design and function of echocardiographic tools need to provide more successful and efficient results for these interventions in the future. Furthermore, there are existing technologies that can implement 3D images using ICE. However, there are not many clinical trials yet, and there is a problem with the cost of the catheter as well. Overcoming these limitations, improving the imaging capabilities, and mastering the manipulation techniques could lead to good echocardiographic modality in cardiac interventions in patients with complex defects. As a new technology, attempts to apply EchoNavigator , a new t


Kimura disease (KD) is a rare and benign chronic inflammatory disease of unknown cause. It is characterized by subcutaneous granuloma of soft tissues in the head and neck region, increased eosinophil count, and elevated serum IgE. Currently, no definitive treatments are recommended. A 57-year-old Chinese man was diagnosed with KD after 7 years of slow subcutaneous masses growth. The patient underwent treatment of oral glucocorticoids for 1 year, but the masses recurred as the dosage was tapered down. Subsequent anti-IgE therapy of omalizumab administered subcutaneously at 450 mg/day at a 4-week interval did not show improvement. The size of masses and serum IgE and circulating eosinophils did not decrease significantly after 19 cycles of continuous treatment. Ultimately, switched strategy of dupilumab was applied at an initial dose of 600 mg, followed by 300 mg every 2 weeks for 4 months. This treatment demonstrated dramatical effects with reduced masses in each area and fast dropdown of eosinophil counts, while the high level of serum IgE remained without changes. Recently, different biologics including anti-IgE, anti-IL-5, and anti-IL-4/IL-13 have been applied to treat KD with satisfied results and help to explore the pathogenesis of this rare disease. To our knowledge, this is the first report that demonstrates the effects of two different biologics in the same patient and reveals the impressive clinical efficacy of dupilumab to treat KD independent of IgE. Therefore, further investigation of the underlying mechanism and the development of diagnosis and treatment of KD is valuable. It maniThe etiology and pathogenesis of KD remain unclear. Recent studies suggest that it might be associated with allergic reactions, viruses or parasites interfering with the immune system, and even with arthropod bites . It is a29/L , with decreased hemoglobin level at 113 g/L . Serum immunology tests demonstrated a dramatically high level of serum IgE over 2,000 IU/ml , together with high immunoglobulin M (IgM) at 2.39 g/L . The counting number of reticulocyte erythrocytes, sedimentation rate (ESR), and urinary renal function were normal. Bone marrow biopsy suggested low-degree local hyperplasia than normal and a slight increase in acidophils. Ultrasound examination showed low echo masses in the front and back of the bilateral ear, patchy low echo area in the left parotid gland, and multiple lymph nodes enlargement in and around the bilateral parotid gland  4B. Ultr3In the present report, we described a case of KD with successful treatment of dupilumab after failure of anti-IgE therapy. KD was first described as “eosinophilic proliferative lymphogranuloma” by Kimm and Szeto in China in 1937 along with the detailed pathological features described by KM et al. in Japan in 1948 . The patvia IL-4 and IL-5. These studies provide more targets for the therapy of KD.Deeper knowledge of the immunological pathogenesis and appearance of new biologics greatly help in the treatment of KD. A Th2 cell immune response from the very beginning was suggested due to its impressive characteristics of elevated serum IgE, peripheral blood eosinophilia, and infiltration of eosinophils in tissues. Recent findings of its correlation to IgG4-related diseases also enhance this viewpoint –12. OhtaAnti-IgE therapy has been tried first with some efficacy. In 2015, Nonaka et al. reported the first trial of a study of anti-IgE  treatment to three Japanese patients with KD uncontrolled by surgery and achieved successful results with decreased size of tumorous region and peripheral blood basophil and eosinophils’ counts . Anti-ILIn this case, the patient first tried anti-IgE therapy with omalizumab after relapse of the masses. Different from a previous report by Nonaka, it seems ineffective without any changes in our patient, even if the treatment period lasts to 19 cycles. One possible reason might be the insufficient dosage, since the serum IgE is more than 2,000 IU/ml. Another possibility is that IgE is not the key role of the mechanism. Inspite the elevation of serum IgE level, knowledge on the real role it plays in Kimura’s disease is lacking. Previous reports on the therapy of cyclosporin A and corticosteroidin showed a decrease in eosinophil level and persistent high serum IgE level , 22, 23.On the contrary, dupilumab demonstrated a quick and dramatic efficacy in this patient with shrank mass and decreased eosinophils. Without the decrease in serum IgE level, the size of masses was remarkably smaller. This is in consistence with Nonaka’s finding that the high expression of IL-4 in the lesions and the inhibition of IL-4 by dupilumab could reverse the proliferation. According to the known clinical investigations, Th2 cytokines such as IL-4 and IL-5 are the major factors in the pathogenesis of KD. The eosinophilic aggregation and IgE synthesis are usually through IL-4 and IL-5 . The inhTo our knowledge, this is the first report that demonstrates the effects of two different biologics in the same patient and reveals the impressive clinical efficacy of dupilumab to treat KD independent of IgE. Therefore, it is valuable to further investigate the underlying mechanism and the development of diagnosis and treatment of KD.4In summary, we presented a typical KD patient, which was comprehensively confirmed by combining clinical manifestations, serological testing, and pathological biopsy. Biological therapy was applied after relapse with oral glucocorticoids. As a rare disease, the treatment for KD is still underexplored with very poor experience. Our case actually adds new evidence of anti-IgE and anti-IL-4/IL-13 therapy in KD.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of the second affiliated hospital of Zhejiang university school of medicine . The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.HYW was the guarantor and revised the manuscript. BYY and HXY wrote the manuscript and collected the data. MYJ interpreted the ultrasound pictures and provided professional comments. WY, RD, BHL, YFW, TL and LYG attended the treatment and follow-up of the patients. All authors contributed to the article and approved the submitted version.


The extracellular palm domain serves as a key link to the channel pore. In this study, we used cysteine-scanning mutagenesis to assess the functional effects of Cys-modifying reagents on palm domain β10 strand residues in mouse ENaC. Of the 13 ENaC α subunit mutants with Cys substitutions examined, only mutants at sites in the proximal region of β10 exhibited changes in channel activity in response to methanethiosulfonate reagents. Additionally, Cys substitutions at three proximal sites of β and γ subunit β10 strands also rendered mutant channels methanethiosulfonate-responsive. Moreover, multiple Cys mutants were activated by low concentrations of thiophilic Cd2+. Using the Na+ self-inhibition response to assess ENaC gating behavior, we identified four α, two β, and two γ subunit β10 strand mutations that changed the Na+ self-inhibition response. Our results suggest that the proximal regions of β10 strands in all three subunits are accessible to small aqueous compounds and Cd2+ and have a role in modulating ENaC gating. These results are consistent with a structural model of mouse ENaC that predicts the presence of aqueous tunnels adjacent to the proximal part of β10 and with previously resolved structures of a related family member where palm domain structural transitions were observed with channels in an open or closed state.The epithelial Na Channelility  reagents, respond to Cd2+, and have an altered Na+ self-inhibition response, suggesting a role in modulating ENaC gating.The palm domains within ENaC subunits are composed of a series of antiparallel beta strands and provide the key link between the extracellular and transmembrane regions of the channel , 10. RecB). The β10 sequence is well conserved among α, β, and γ subunits of human, mouse and rat ENaC, and chicken ASIC1 .A structural homology model of mouse ENaC was built using SWISS-MODEL , based oFig. 1Xenopus oocytes. Amiloride (10 μM)-sensitive Na+ currents were monitored by two-electrode voltage clamp, and the response to extracellular MTS reagents (MTSES and MTSET) was assessed. We chose these Cys-reactive reagents as they are similar in size. While MTSES modification adds a negative charge, MTSET adds a positive charge. If a Cys-containing mutant channel responded to either reagent with a significant change in channel current, this Cys was considered as functionally accessible and important. If a Cys mutant channel did not respond to any reagent, this Cys was described as functionally inaccessible. The latter category includes channels with Cys substitutions that were not chemically modified by MTS reagents as well as channels where MTS modifications did not alter activity.Thirteen β10 residues in the mouse ENaC α subunit were individually mutated to Cys. Wildtype and mutant ENaCs were expressed in +, a process referred to as Na+ self-inhibition  [Na+] results in a rapid increase in inward Na+ current, which reaches a peak (Ipeak) that is followed by a fall in current, reflecting a reduction in channel open probability due to Na+ self-inhibition , and the ratio of Iss to Ipeak reflects the magnitude of the Na+ self-inhibition response. Based on previous studies showing that extracellular cues often regulate ENaC gating by altering Na+ self-inhibition response . In contrast, three mutants responded to MTSES with significant increases in whole-cell current. Amiloride-sensitive Na+ currents measured in the presence of MTSES (IMTSES) were normalized to currents measured prior to the addition of MTSES (I). We observed significant increases in currents in oocytes expressing αT509Cβγ and αN510Cβγ (3A), while a very modest but statistically significant increase in IMTSES/I was observed with αK512Cβγ (A). In contrast, MTSES significantly inhibited αS514Cβγ (C and 3A). For other Cys mutants, responses to MTSES were similar to wildtype ENaC (A).All mutant ENaCs expressed sufficient amiloride-sensitive currents to permit examination of their response to externally applied MTS reagents. Similar effects of the MTS reagents were noted when examined before or after assessing the Narvations , 26 . For other mutants, responses to MTSET were similar to wildtype ENaC (B). The effects on currents in oocytes expressing ENaC mutants were sustained after the reagent was washed out, consistent with irreversible channel modification.Four of the 13 α subunit β10 mutants  exhibited significant reductions in amiloride-sensitive Naof MTSET  and 3B. ype ENaC B. The ef+ self-inhibition response of wildtype and mutant channels that showed significant changes in current in response to MTS reagents, by measuring the Iss to Ipeak ratio following the switch from a 1 mM to 110 mM Na+ bath (see A). The Na+ self-inhibition response was determined prior to and following MTSES or MTSET application. The percent change in the Na+ self-inhibition response (ΔSI) following MTS treatment, relative to the Na+ self-inhibition response prior to MTS, was determined using the formula described in the + self-inhibition response after MTSES or MTSET treatment, while negative ΔSI reflects a reduced Na+ self-inhibition response after MTSES or MTSET treatment. Wildtype ENaCs showed a similar and modest increase in Na+ self-inhibition after MTSES and MTSET treatment (A–D). This increase is likely not due to the MTS reagent, as we have observed that repetitive tests of Na+ self-inhibition in the same oocyte often led to a modest increase in the Na+ self-inhibition response. To ascertain this was the case, we examined Na+ self-inhibition responses in oocytes expressing wildtype ENaCs twice with the same time interval as the MTS application shown in A and D . The representative recording and summary data are shown in E and F, demonstrating a similar level of increase in the Na+ self-inhibition response of wildtype channels as observed after MTS application (A–D). This increase in Na+ self-inhibition may be related to a time-dependent decrease in ENaC activity that has been observed when oocytes are bathed with a high concentration of extracellular Na+ and clamped at hyperpolarization potential , consistent with an MTSES-dependent increase in channel open probability in the presence of a high extracellular [Na+]. An increase in current could also reflect an increase in unitary channel conductance. To rule out this possibility, we examined the effect of MTSES on αN510C channels that also had a βS518K mutation. Previous studies have shown that βS518K channels have an intrinsic high open probability .The strong activation of αN510Cβγ by MTSES was associated with a significant reduction in the Naresponse B and 3A,bability , 30, 31.lication A.Figure + self-inhibition response of each α subunit β10 Cys mutant and compared it to the wildtype Na+ self-inhibition response. As shown in + self-inhibition response. The data suggest that these residues have a role in Na+ self-inhibition. As the magnitude of Na+ self-inhibition is correlated with open probability . Interestingly, currents in oocytes expressing either of these mutant channels were not significantly altered by either MTSES or MTSET . The MTSES-induced current decrease in αβD448Cγ and αβQ450γ was associated with an enhanced Na+ self-inhibition response (B). Furthermore, the MTSES-induced increase in αβT454Cγ current was accompanied with a decreased Na+ self-inhibition response (B). Finally, the MTSET-induced current decrease in αβT454Cγ was followed by a significantly increased Na+ self-inhibition response (C). These results suggest that several β10 residues of β subunit have a role in Na+ self-inhibition response.Compared to wildtype, αβQ450Cγ and αβM457Cγ showed a significantly reduced Naresponse  and 9A. response B. Furtheresponse B. Finallresponse C. These Among the four γ subunit mutants studied, αβγF465C (homologous to αV508Cβγ), αβγE467C (homologous to αN510Cβγ), and αβγT471C (homologous to αS514Cβγ) were inhibited by MTSES, whereas αβγL474C (homologous to αY517Cβγ) was not affected . MTSET r+ self-inhibition response of αβγF465C (enhanced) and αβγE467C (reduced) differed from wildtype (A), while the Na+ self-inhibition response of the other γ subunit mutants was similar to wildtype. Following the inhibitory effect of MTSES, the Na+ self-inhibition response of αβγF465C and αβγT471C was enhanced compared to the response prior to MTSES. In contrast, the Na+ self-inhibition response of αβγE467C was not altered by MTSES (B). MTSET inhibited αβγE467C and enhanced the mutant’s Na+ self-inhibition response, whereas MTSET activated αβγT471C and reduced the mutant’s Na+ self-inhibition response (C). These data suggest that several γ subunit β10 residues have a role in Na+ self-inhibition.The Nawildtype A, while by MTSES B. MTSET response C. These 2+ (10−7 M to 3 × 10−3 M) on currents in oocytes expressing wildtype channels or channels with a mutant γ subunit (A). The mutants αβγF465C and αβγE467C responded to both low and high concentrations of Cd2+ with significant and large increases in currents in a reversible manner, compared to wildtype. αβγT471C responded to high concentrations of Cd2+ with significant and moderate increases in currents. In contrast, wildtype and αβγL474C channels showed modest increases in current at high [Cd2+], which then fell at a [Cd2+] of 3 × 10−3 M. This modest activation was similar to what we previously observed with wildtype  for ENaC activation by Cd2+  were 11 ± 2, 3 ± 1, and 124 ± 21 for αβγF465C, αβγE467C, and αβγT471C, respectively. We next examined the effects of Cd2+ on αV508Cβγ and αβD448Cγ channels (homologous to γF465C). Both mutants responded to Cd2+ with significant increases in currents (C), with estimated EC50s  of 253 ± 28 (αV508Cβγ) and 8 ± 1 (αβD448Cγ) (D). These results suggest that the binding of Cd2+ to substituted Cys residues at specific sites in the β10 strand of each subunit activates the channel.We found that Cys residues introduced at multiple sites in the proximal parts of the β10 strands in all three subunits were accessible to MTS reagents. These MTS reagents are considered bulky, with estimated dimensions of 5 (short axis) by 10 (long axis) Å . We also subunit A. The muwildtype . The effed in 2+ . The EC5currents C, with eβD448Cγ) D. These A and B) and most of its residues are not exposed to the surface, charged MTS molecules must travel to the introduced sulfhydryl groups through an aqueous tunnel. Inspection of a surface-rendered model of mouse ENaC did not reveal apparent openings to solvents. We used CAVER Analyst 2.0 (D). The most promising aqueous tunnel (blue) with the largest bottleneck radius (1.4 Å) and shortest length (8 Å) opens to the surface at the junction of the α subunit palm, thumb, and β-ball domains (blue tunnel). A similar search starting at αS514 also revealed three tunnels (E). The most prominent tunnel leads from αS514 to the surface at the α  and γ (thumb domain) interface (blue tunnel). Aqueous tunnels were also found to provide access to residues homologous to αN510 in the β  and γ subunits , as well as access to residues homologous to αS514 in the β  and γ subunits . For αV508 and its homologous residues in the β and γ subunits (βD448 and γF465), their locations were too close to the surface to allow for reliable detection of tunnels. Using the same search parameters, no promising tunnel was identified for αY517 and its homologous residues in the β and γ subunits (βM457 and γL474), consistent with the functional insensitivity of their corresponding Cys mutants to MTS reagents. To determine whether substituted Cys affected the predicted tunnels, we repeated tunnel analyses on mutant ENaC models (generated by PyMol) containing the substituted Cys at sites selected as the starting point. These tunnels appear to be somewhat similar to tunnels based on the wildtype ENaC model. Representative tunnel models based on channels with specific Cys substitutions at three sites are shown in Figure 14F: (αC514), 14I (βC454), and 14L (γC471).As the β10 strand is located within a three-fold axis of symmetry , A and Blyst 2.0  and CAVElyst 2.0  to probelyst 2.0 D. The mo tunnels E. The moβ βQ450, G and γ ss γE467, J, as welβ βT454, H and γ ss γT471, K. For αV+ self-inhibition response. At least four of the first eight Cys substituted residues in the proximal aspect of α subunit β10  were found to be functionally accessible to an MTS reagent, based on a large and significant change in current . However, we cannot rule out the possibility that sites in the distal aspect of β10 were chemically modified by MTS reagents. Our observations are consistent with the notion that distal palm domain provides a structural scaffold, while the proximal palm enables dynamic changes in conformations associated with channel gating  likely gain access to the pore through the fenestrations formed at subunit interfaces near the transmembrane domain  and homologous to αS514C (βT454C and γT471) were functionally accessible to MTS reagents, whereas channels with distal β10 Cys substitutions homologous to αY517C (βM457C and γL474C) were unresponsive to these reagents  were dependent on the charge of the reagent for some mutants . The effects of MTS reagents on other mutants  were not charge dependent. While both reagents inhibited both γF465C and γE467C mutants, the effect of MTSES was much greater than that of MTSET on the γF465C mutant and effect of MTSET was much larger than that of MTSES on the γE467C mutant (C). In contrast, MTSES had a small effect on αβγE467C channel activity (C). Third, at homologous sites within the three ENaC subunits, the effects of both MTSES and MTSET were often subunit dependent. Functional ENaC subunit dependence has been well documented in many studies  were also activated by external Cd2+ (A and B). The most robust activation by Cd2+ was seen with the αβγF465C mutant. Channels with Cys substitutions at homologous sites in the α and β subunits (αV508C and βD448C) were also activated by Cd2+ (C and D). The EC50 of Cd2+ activation varied among the mutants that were studied  likely have a Cd2+-binding site that includes the introduced sulfhydryl group as well as nearby residues that coordinate Cd2+ binding . Coordinated Cd2+ binding is likely not present in ENaC mutants with higher EC50s (γT471C [124 μM] and αV508C [253 μM])  likely perturbs local structures in distinct ways to alter channel activity. For example, the resolved ENaC structure shows a ring of polar residues, including αN510, βQ450, and γE467, which project their side chains to the central vestibule and contact multiple polar side chains from palm domains of the same or neighboring subunit . It is not surprising that modifying an introducing Cys residue with a bulky side chain at one of these sites will either enhance or disrupt interactions that affect channel structure and activity. In addition, Cd2+ coordination by an introduced sulfhydryl group and other native groups may strengthen polar interactions favoring an open state. Cd2+ binding might induce a collapsed central vestibule, favoring an open channel state (described in the later part of the article).The transition metal Cdchannels . We examnal Cd2+ , A and B by Cd2+ , C and D studied . The Cd2ing site . ENaC mu253 μM]) . These oTSET see . While t2+ to reach their targets. Only one tunnel was detected using βQ450 as starting point. The tunnel was similar to the most promising tunnel for αN510. Interestingly, the most promising tunnels (in blue) for αS514, βT454, and γT471 were also similar. No tunnels were detected for αY517, βM457, and γL474 using the same search parameters, consistent with the observations that their corresponding Cys mutants did not respond to MTS reagents. For all sites studied, no tunnels transiting along the three-fold symmetry axis from the top of the extracellular domain were identified.To further explore the aqueous accessibility of the proximal aspect of the β10 strands in the three ENaC subunits, we used CAVER Analyst 2.0  and CAV+ self-inhibition response. Among the 13 α mutants studied, αY517C and αV508C exhibited reduced Na+ self-inhibition and greater amiloride-sensitive currents, compared to wildtype (+ self-inhibition and channel open probability (+ self-inhibition response (+ self-inhibition response (A and 12A), while three mutants  had an enhanced Na+ self-inhibition response (12A). Our results suggest that these eight sites with β10 residues have a role in the mechanism of Na+ self-inhibition, likely influencing allosteric transitions that occur following Na+ binding. These β10 residues are not in the vicinity of the previously defined α subunit Na+-binding site . This hydrophobic seal likely supports the scaffold formed by the distal parts of β10 strands. Our models predict that the αY517C mutation creates a separation between the αY517C, βM457, and γL474 . The βM457C mutation also creates a separation between βM457C and γL474, though it retains close contact with αY517. In contrast, γL474C appears to be less disruptive. The observations are consistent with the suppressed Na+ self-inhibition response observed with αY517C and βM457C, while γL474C did not affect Na+ self-inhibition (A and 12A). Given the proximity of this hydrophobic seal to a hydrophobic patch in the knuckle domain implicated in Na+ self-inhibition response  where introduced mutations alter ENaC gating. Mouse αC506 is homologous to human αC479, where a Cys-to-Arg mutation was found in a pair of siblings with a Liddle syndrome phenotype , E417 (β12), E412 (β11-β12 linker), and E374 (β10) contribute to protonation and Ba2+-binding sites  and native groups is consistent with a reduction of the central vestibule space in association with channel activation. Clearly additional studies are needed to ascertain changes in the structure of ENaC’s central vestibule during gating transitions.Previous studies suggest that ASIC1 palm domain β sheets lining the central vestibule undergo conformational changes in association with channel gating , 20. DurIn summary, our study identified solvent-accessible residues within the proximal parts of the palm domain β10 strands of the three ENaC subunits. Furthermore, several β10 residues were found to have roles in modulating ENaC gating.via T3 RNA polymerase  and purified by RNeasy MinElute Cleanup Kit (QIAGEN). Concentrations of plasmid DNAs and cRNAs were quantified by spectrophotometry.Point mutations in mouse ENaC α, β, and γ subunits were introduced using QuickChange Ⅱ XL site-directed mutagenesis kit (Agilent). Mutations were confirmed by DNA sequencing conducted in the Genomics Research Core of University of Pittsburgh. Linearized DNAs following restriction enzyme digestion were purified by GeneJET PCR Purification Kit (Thermo Fisher Scientific). Wildtype and mutant mouse ENaC cRNAs were generated Xenopus laevis were harvested according to a protocol approved by the University of Pittsburgh’s Institutional Animal Care and Use Committee and treated with type II collagenase (Sigma-Aldrich). cRNA (0.5 or 1 ng) of each mouse ENaC subunit  was injected into stage V or stage VI oocytes, and oocytes were incubated at 18 °C in a modified Barth’s solution: 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 15 mM Hepes, 0.3 mM Ca (NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 10 μg/ml streptomycin sulfate, 100 μg/ml gentamycin sulfate, and 10 μg/ml sodium penicillin, pH 7.4.Oocytes from female Two-electrode voltage clamp studies were performed at room temperature (20–24 °C) 1 to 2 days after injection using Axoclamp 900A Computer-Controlled Microelectrode Amplifier and DigiData 1440A controlled by pClamp 10.4 (Molecular Devices). Oocytes were placed in a chamber with constant flow (∼5 ml/min). Glass pipettes filled with 3 M KCl were inserted into oocytes, and the intracellular potential was clamped at −100 mV.+ self-inhibition response was assessed as previously reported  for 60 s and switched abruptly to a 110 mM Na+ bath solution . Currents were recorded by a two-electrode voltage clamp. After 60 s of NaCl-110 perfusion, 10 μM amiloride was applied to determine the amiloride-sensitive current. Upon the change from NaCl-1 to NaCl-110, currents rapidly increased to a peak (Ipeak) and slowly declined to a steady-state level .The Nareported . Oocytes+ currents prior to and after MTSES or MTSET application were measured as I and IMTSES or IMTSET, respectively.MTSES  powder was freshly dissolved in NaCl-110 at a concentration of 2 mM and used for experiments within 2 h. MTSET  solution was prepared in NaCl-110 at a concentration of 1 mM and used immediately. MTSES and MTSET solutions were perfused for 2 min, when currents were stable. Amiloride-sensitive Na2+ on selected mutant and wildtype channels were examined by monitoring the current changes following 30 s applications of increasing concentrations of CdCl2, in the range of 10-7 to 3 x 10-3 M. A CdCl2 stock (1 M) was prepared by dissolving CdCl2 powder in deionized water and diluted to various concentrations of CdCl2 in the NaCl-110 bath solution prior to use. Oocytes were perfused with NaCl-110 containing 10 μM amiloride after 3 mM CdCl2 or after washout with NaCl-110 for 1 min in order to measure the amiloride-insensitive component of the whole-cell current. The Cd2+ dose response was analyzed using a two-site equation I=(Imax-Imin)(C/(C+ EC50))(IC50/(C+IC50))+Imin, as previously described .The effects of Cdescribed . The I, A structural model of mouse ENaCs was generated with SWISS-MODEL . TemplatXenopus oocytes, all comparisons between mutant and wildtype channels were done in the same batches of oocytes. Significance between two groups was determined by Student’s t test with Welch’s correction and for multiple groups by one-way ANOVA followed by Dunnett’s correction. p＜0.05 was considered statistically significant. All significance comparisons and normality tests (Shapiro–Wilk test) were performed using GraphPad Prism 9.Data are presented as mean ± S.D. Given the inherent batch-to-batch variation in expression levels in All data are contained within the manuscript.The authors declare that they have no conflicts of interest with the contents of this article.