Abstract
Gasdermin D (GSDMD) is the executor of pyroptosis, which is important for host defence against pathogen infection. Following activation, caspase-mediated cleavage of GSDMD releases an amino-terminal fragment (GSDMD-NT), which oligomerizes and forms pores in the plasma membrane, leading to cell death and release of proinflammatory cytokines. The spatial and temporal regulation of this process in cells remains unclear. Here we identify GSDMD as a substrate for reversible S-palmitoylation on C192 during pyroptosis. The palmitoyl acyltransferase DHHC7 palmitoylates GSDMD to direct its cleavage by caspases. Subsequently, palmitoylation of GSDMD-NT promotes its translocation to the plasma membrane, where APT2 depalmitoylates GSDMD-NT to unmask the C192 residue and promote GSDMD-NT oligomerization. Perturbation of either palmitoylation or depalmitoylation suppresses pyroptosis, leading to increased survival of mice with lipopolysaccharide-induced lethal septic shock and increased sensitivity to bacterial infection. Our findings reveal a model through which a palmitoylation–depalmitoylation relay spatiotemporally controls GSDMD activation during pyroptosis.
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Data availability
The RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus under the accession code GSE253306. Sequencing reads were mapped to the reference genome mm10 (GRCm38: GCA_000001635.8, GCF_000001635.26) from Gencode with hisat2 using the default parameter. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.
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Acknowledgements
We thank F. Shao (NIBS) for the immortalized Gsdmd−/− BMDMs, W. Wei (HMS) for the DHHC constructs, and the staff members of the Animal Facility at the National Facility for Protein Science in Shanghai (NFPS). The work of D.X. was supported in part by grants from the STI2030-Major Projects (grant number 2022ZD0213200), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant number XDB39030600), the National Natural Science Foundation of China (grant numbers 32350022, 32070737 and 92049303), the Shanghai Science and Technology Development Funds (grant numbers 20JC1411600, 20QA1411500 and 22JC1410400), the CAS Youth Interdisciplinary Team (grant number JCTD-2022-10), the Shanghai Key Laboratory of Aging Studies (grant number 19DZ2260400) and the Shanghai Municipal Science and Technology Major Project (grant number 2019SHZDZX02). The work of Z.C. was supported in part by grants from the Young Scientists Fund of the National Natural Science Foundation of China (grant number 32101213). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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This project was conceived, designed and directed by D.X. N.Z. designed and conducted majority of the experiments. J.Z. designed and performed the animal experiments. Y.Y. conducted the key experiments on GSDMD processing. M.M. and Z.C. performed the TIRF microscopy assay. S.H. assisted with the experiments on GSDMD membrane translocation. H.F. assisted with the experiments on GSDMD oligomerization. L.T. and H.S. performed the chemical synthesis of 16C-BYA. The paper was written by D.X.
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Extended data
Extended Data Fig. 1 GSDMD is a substrate of S-palmitoylation.
a, Chemical structure of palmitic acid and its alkyne-tagged analogue palmitic acid alkyne (Alk-14). b, Chemical structure of 2-BP and its alkyne-tagged analogue adec-15-ynoic acid (16C-BYA). c, HEK293T cells were transfected with the indicated constructs expressing Flag-tagged DHHC7-WT, catalytically inactive DHHC7-C160S (DHHS7) mutant, full-length GSDMD or GSDMD-NT for 6 hours, then incubated with 50 μM 2-BP or 16C-BYA overnight. Cell lysates were immunoprecipitated using anti-Flag beads, and the immunoprecipitate was subjected to Cu(I)-assisted crosslinking to Biotin-azide. The immunoprecipitated samples were eluted with 6 M urea and subjected to streptavidin pulldown (PD), followed by western blot using anti-Flag antibody. d, Schematic of the immunoprecipitation and acyl-biotin exchange (IP–ABE) assay followed by streptavidin pulldown to purify and detect protein S-palmitoylation. Cells were lysed, and GSDMD was then purified using anti-Flag (for Flag-GSDMD) or anti-GSDMD (for endogenous GSDMD) antibodies. Purified GSDMD is then treated on beads with N-ethylmaliemide (NEM) to irreversibly block free thiol groups along unmodified cysteines followed by treatment with hydroxylamine (HAM), resulting in specific cleavage of thioester bonds at S-palmitoylated cysteines and unmasking of the thiol group. Next, GSDMD was treated with a thiol-reactive biotin molecule, biotin-HPDP, resulting in specific biotinylation of the palmitoylated cysteine. Finally, the biotinylated GSDMD was eluted and removed from the antibodies and beads followed by streptavidin pulldown and palmitoylated GSDMD is detected by Western blotting with anti-GSDMD antibodies. e, BMDMs were challenged with S. Typhimurium (MOI 40) in the presence or absence of 2-BP (50 μM) for 1 hours. The amount of S. Typhimurium taken up by cells was quantified by counting colony-forming units (CFUs). Data are presented as mean ± s.d. of n = 3 independent wells of one representative experiment. Unpaired two-tailed Student’s t-test. f, HEK293T cells were transfected with Flag-tagged GSDMD in the presence or absence of 2-BP (50 μM) for 24 hours. Palm-GSDMD with or without HAM treatment was detected using IP–ABE assay. g, Immunoblotting analysis of GSDMD in each reconstituted BMDMs cell line. The reconstitution levels of WT and Cys-to-Ala mutants of GSDMD were comparable to endogenous GSDMD levels.
Extended Data Fig. 2 Inhibition of palmitoylation suppresses pyroptosis.
a–d, Primary (a and b) or immortalized (c and d) BMDMs were primed with LPS (100 ng/ml) for 3 hours followed by 2-BP (50 μM) treatment for 0.5 hours before stimulation with Nig (10 μM) (a and b) or ATP (2 mM) (c and d) for indicated times (a, c and d) or 2 hours (b). Cell death was measured by SytoxGreen positivity assay (a and c). The resulting supernatants were quantified for LDH release (b and d). e–i, BMDMs were pre-treated with 5z7 (200 nM) with or without 2-BP (50 μM) and Nec-1s (10 μM) for 0.5 hours followed by LPS (40 ng/ml) treatment for indicated times (e), or treated with 10 µM Val-boroPro (VBP) with or without 2-BP (50 μM) for 6 hours (f) or indicated times (h), or 100 ng/ml LFn-Rod plus 1 µg/ml protective antigen with or without 2-BP (50 μM) for 1.5 hours (g) or indicated times (i). Cell death was measured by SytoxGreen positivity assay (e, h and i). Palm-GSDMD was detected using IP–ABE assay (f and g). j, HT-29 cells stably expressing acMLKL were stimulated with DMSO or AP20187 (20 nM) in the presence or absence of 2-BP (50 μM) for indicated times. Cell death was measured by SytoxGreen positivity assay. The Schematic representation of the oligomerizable MLKL (acMLKL) were shown at upper left, and western blotting analysis of lysates from HT-29 cells stimulated with AP20187 with or without 2-BP for 2 hours were shown at lower right. endo., endogenous. Data are presented as mean ± s.d. of n = 4 (c), 6 (a, b and d), 5 (e, h and i) or 8 (j) independent wells of one representative experiment. Unpaired two-tailed Student’s t-test (b). Two-way ANOVA post hoc Bonferroni’s tests (d).
Extended Data Fig. 3 Inhibition of palmitoylation suppresses GSDMD processing.
a, Primary BMDMs were primed with LPS (100 ng/ml) for 3 hours followed by treatment with 2-BP (50 μM) for 0.5 hours before stimulation with Nig (10 μM) for 2 hours. Western blot analyses were performed with anti-GSDMD antibodies. b, BMDMs were primed with LPS (100 ng/ml) for 3 hours followed by treatment with 2-BP (50 μM) for 0.5 hours before stimulation with ATP (2 mM) for the indicated times. Western blot analyses were performed with anti-GSDMD antibodies. c, BMDMs were pre-treated with 5z7 (200 nM) with or without 2-BP (50 μM) and Nec-1s (10 μM) for 0.5 hours followed by treatment with LPS (40 ng/ml) for the indicated times. Western blot analyses were performed with anti-GSDMD antibodies. d, Primary BMDMs were primed with LPS (100 ng/ml) for 3 hours followed by treatment with 2-BP (50 μM) for 0.5 hours before stimulation with Nig (10 μM) for the indicated times. Western blot analyses were performed with anti-NLRP3 antibodies. e, Primary BMDMs were treated with LFn-Rod (100 ng/ml) plus protective antigen (1 µg/ml) with or without 2-BP (50 μM) for 1.5 hours. Volcano plots of RNA-seq showing fold-change and P-value for the comparison of BMDMs treated with 2-BP versus Veh (n = 3) with cutoffs set at fold change (FC) > 2 or < 0.5, and P-value < 0.05 were shown. Representative genes associated with pyroptosis are highlighted. Unpaired two-tailed Student’s t-test.
Extended Data Fig. 4 Palmitoylation promotes GSDMD-NT-induced pyroptosis.
a–d, HEK293T cells were transfected with the indicated constructs in the presence or absence of 2-BP (50 μM) for 24 hours. Cell death was measured by SytoxGreen positivity assay (a and c). The resulting supernatants were quantified for LDH release (b and d). Data are presented as mean ± s.d. of n = 5 (a and b), 4 (c), or 6 (d) independent wells of one representative experiment. Unpaired two-tailed Student’s t-test (a and b). One-way ANOVA post hoc Dunnett’s tests (c and d). e, f, HEK293T cells were transfected with the indicated constructs in the presence or absence of 2-BP (50 μM) for 24 hours. Cell lysates were then collected and subjected to Western blot analyses with the indicated antibodies. g, h, HEK293T cells were transfected with the indicated constructs in the presence or absence of 2-BP (50 μM) for 24 hours. Cell death was measured by SytoxGreen positivity assay (g). The resulting supernatants were quantified for LDH release (h). Data are presented as mean ± s.d. of n = 3 independent wells of one representative experiment (g and h). Unpaired two-tailed Student’s t-test (g and h). i, HEK293T cells were transfected with the indicated constructs in the presence or absence of 2-BP (50 μM) for 24 hours. Cell lysates were then collected and subjected to Western blot analyses with anti-GSDMD antibodies. j, k, HEK293T cells were transfected with the indicated constructs in the presence or absence of 2-BP (50 μM) for 24 hours. Cell death was measured by SytoxGreen positivity assay (j). The resulting supernatants were quantified for LDH release (k). Data are presented as mean ± s.d. of n = 3 independent wells of one representative experiment (j and k). One-way ANOVA post hoc Bonferroni’s tests (j and k).
Extended Data Fig. 5 Palmitoylation promotes plasma membrane translocation of GSDMD-NT.
a, Gsdmd-deficient BMDMs reconstituted with Dox-inducible GSDMD-NT were incubated with the indicated concentrations of Dox in the presence or absence of 2-BP (50 μM) for 12 hours. Cell lysates were then collected and subjected to Western blot analyses with anti-GSDMD antibodies. b, c, HEK293T cells were transfected with GSDMD-NT (b) or GSDMD and caspase-1 (c) in the presence or absence of 2-BP (50 μM) for 24 hours. The distribution of GSDMD-NT in subcellular fractions was determined by Western blot analyses with anti-GSDMD antibodies. d, Gsdmd-deficient BMDMs reconstituted with Dox-inducible GFP–GSDMD-NT were incubated with or without Dox (2 μg/ml) for indicated times. Cell death was measured by SytoxGreen positivity assay. Data are presented as mean ± s.d. of n = 4 independent wells of one representative experiment. e, Flow cytometry gating strategy. Representative schema describes the gating to identify specific GFP+ BMDMs. Dox-inducible GFP–GSDMD-NT-reconstituted BMDMs were stimulated with DOX (2 µg/ml) for 12 hours to produce GFP fluorescence. Single cell suspensions were first gated using forward scatter/side scatter (FSC vs SSC) to exclude debris (Gate 1), then gated using Green-B to identify GFP+ cells (Gate 2). f, g, Gsdmd-deficient BMDMs reconstituted with Dox-inducible GFP–GSDMD-NT (WT or C192A) were incubated with Dox (2 μg/ml) in the presence or absence of 2-BP (50 μM) for 12 hours, then flow cytometry analysis of GFP activity was performed. h, i, Gsdmd-deficient BMDMs reconstituted with Dox-inducible GFP–GSDMD-NT (WT or C192A) were incubated with or without Dox (2 μg/ml) in the presence or absence of 2-BP (50 μM) for 12 hours. Fluorescent images of GFP–GSDMD-NT (green) translocation from the cytoplasm to the basal plasma membrane were determined by TIRF microscopy. Images shown are representative of three independent experiments.
Extended Data Fig. 6 DHHC7 mediates GSDMD palmitoylation and promotes pyroptosis.
a, HEK293T cells were transfected with Flag-GSDMD and HA-tagged DHHCs for 12 hours. Palm-GSDMD was detected using IP–ABE assay. b, mRNA expression of the indicated DHHCs in BMDMs transduced with shRNA constructs targeting the indicated DHHCs or non-targeting control (NC). c, Primary Dhhc7+/+ and Dhhc7-/- BMDMs were incubated with Alk-14 (100 μM) overnight then stimulated with LPS (100 ng/ml) for 3 hours. The GSDMD immunoprecipitate was subjected to Cu(I)-assisted crosslinking to biotin-azide, then eluted with 6 M urea and subjected to streptavidin pulldown followed by Western blot analyses. d–i, BMDMs expressing indicated shDhhc constructs were stimulated with LPS (100 ng/ml) for 3 hours followed by Nig (10 μM) (d, e and h) or ATP (2 mM) (f, g and i) treatment for indicated times (d and f) or 2 hours (e and g-i). Cell death was measured by SytoxGreen positivity assay (d and f). The resulting supernatants were quantified for LDH release (e and g). Cell lysates were collected and subjected to Western blot analyses (h and i). j, k, Primary Dhhc7+/+ and Dhhc7-/- BMDMs were pre-treated with 5z7 (200 nM) for 0.5 hours followed by treatment with LPS (40 ng/ml) (j), or treated with LFn-Rod (100 ng/ml) plus protective antigen (1 µg/ml) (k) for indicated times. Cell death was measured by SytoxGreen positivity assay. l, m, Primary Dhhc7+/+ and Dhhc7-/- BMDMs were primed with LPS (100 ng/ml) for 3 hours followed by treatment with Nig (10 μM) for 1 hour (l) or stimulated with LFn-Rod (100 ng/ml) plus protective antigen (1 µg/ml) for 1.5 hours (m). Volcano plots of RNA-seq showing fold-change and P-value for the comparison of Dhhc7-/- versus Dhhc7+/+ BMDMs (n = 3) with cutoffs set at fold change (FC) > 2 or < 0.5, and P-value < 0.05 were shown. Representative genes associated with pyroptosis are highlighted. Data are represented as mean ± s.d. of n = 3 (b), 4 (d-g and k) or 5 (j) independent wells of one representative experiment. Unpaired two-tailed Student’s t-test (e, g, l and m).
Extended Data Fig. 7 DHHC7-mediated palmitoylation promotes GSDMD-NT membrane translocation.
a, Gsdmd-deficient BMDMs reconstituted with Dox-inducible GFP–GSDMD-NT were transfected with 50 nM siRNA targeting Dhhc7 for 48 hours and then incubated with Dox (2 μg/ml) for 12 hours. The subcellular localization of GFP–GSDMD-NT was analysed by confocal microscopy. Images shown are representative of four independent experiments. b, Gsdmd-deficient BMDMs reconstituted with Dox-inducible GFP–GSDMD-NT were transfected with 50 nM siRNA targeting Dhhc7 or Apt2 for 48 hours and then incubated with Dox (2 μg/ml) for 12 hours. Fluorescent images of GFP–GSDMD-NT translocation from the cytoplasm to the basal plasma membrane were determined by TIRF microscopy. Images shown are representative of four independent experiments. c, Gsdmd-deficient BMDMs reconstituted with Dox-inducible GFP–GSDMD-NT were transfected with 50 nM siRNA targeting Dhhc7 for 48 hours and then incubated with Dox (2 μg/ml) for 12 hours, then flow cytometry analysis of GFP activity was performed. d, Relative abundances of palmitoyl-CoA in BMDMs with or without LPS (100 ng/ml) stimulation for 3 hours were measured. Data are presented as mean ± s.e.m of n = 6 biologically independent samples in each group. Unpaired two-tailed Student’s t-test. e, Fold change in autopalmitoylation of the indicated DHHCs in response to LPS in RAW264 cells. The palmitoylation of PATs was determined by mass spectrometry using metabolic labelling of RAW264 cells with 17-ODYA, an analogue of palmitic acid functionalized with an alkyne group, followed by detection and enrichment of labelled proteins using biotin-azide/streptavidin. f, g, Dhhc7-deficient BMDMs reconstituted with Flag-tagged DHHC7 or catalytically inactive DHHC7-C160S (DHHS7) mutant were stimulated with 10 µM Val-boroPro (VBP) for 6 hours (f) or 100 ng/ml LFn-Rod plus 1 µg/ml protective antigen for 1.5 hours (g). Autopalmitoylation levels of DHHC7 (Palm-DHHC7) with HAM treatment were detected using IP–ABE assay.
Extended Data Fig. 8 APT2 mediates depalmitoylation of GSDMD and promotes pyroptosis.
a, b, HEK293T cells were transfected with HA tagged-ATP1 or APT2 and Flag tagged-GSDMD or GSDMD-NT for 24 hours. APT1/2 binding was revealed by immunoprecipitation and immunoblotting. c–e, WT (d and c) or Dox-inducible GSDMD-NT-reconstituted (e) BMDMs were transfected with 50 nM siRNA targeting Apt2 for 48 hours followed by LPS (100 ng/ml) treatment for 3 hours (c), or treated with LPS (100 ng/ml) for 3 hours followed by ML349 (50 μM) treatment for 1 hour (d), or incubated with Dox (2 μg/ml) in the presence of ML349 (50 μM) for 12 hours (e). Palmitoylation of GSDMD (c and d) or GSDMD-NT (e) was detected using IP–ABE assay. f–i, BMDMs were transfected with 50 nM siRNA targeting Apt2 for 48 hours and stimulated with LPS (100 ng/ml) for 3 hours followed by Nig (10 μM) (f and g) or ATP (2 mM) (h and i) treatment for indicated times (f and h) or 2 hours (g and i). Cell death was measured by SytoxGreen positivity assay (f and h). The resulting supernatants were quantified for LDH release (g and i). j, k, Primary Apt2+/+ and Apt2-/- BMDMs were pre-treated with 5z7 (200 nM) for 0.5 hours followed by treatment with LPS (40 ng/ml) (j), or treated with LFn-Rod (100 ng/ml) plus protective antigen (1 µg/ml) (k) for the indicated times. Cell death was measured by SytoxGreen positivity assay. l, BMDMs were transfected with 50 nM siRNA targeting the indicated APTs for 48 hours and then stimulated with LPS (100 ng/ml) for 3 hours followed by treatment with Nig (10 μM) for indicated times. Cell death measured by SytoxGreen positivity assay was shown on the left. mRNA expression of the indicated APTs was shown on the right. Data are presented as mean ± s.d. of n = 4 (f and h), 6 (g, i and l, left), 8 (j), 5 (k) or 3 (l, right) independent wells of one representative experiment. Unpaired two-tailed Student’s t-test (g and i).
Extended Data Fig. 9 APT2-mediated depalmitoylation promotes GSDMD-NT oligomerization.
a, b, BMDMs were transfected with 50 nM siRNA targeting Apt2 for 48 hours and then stimulated with LPS (100 ng/ml) for 3 hours followed by Nig (10 μM) (a) or ATP (2 mM) (b) treatment for 2 hours. Cell lysates were subjected to Western blot analyses. c, d, Primary Apt2+/+ and Apt2-/- BMDMs were primed with LPS (100 ng/ml) for 3 hours followed by Nig (10 μM) treatment for 1 hour (c), or stimulated with LFn-Rod (100 ng/ml) plus protective antigen (1 µg/ml) for 1.5 hours (d). Volcano plots of RNA-seq showing fold-change and P-value for the comparison of Apt2-/- versus Apt2+/+ BMDMs (n = 3) with cutoffs set at fold change (FC) > 2 or < 0.5, and P-value < 0.05 were shown. Representative pyroptosis-associated genes are highlighted. e–g, HEK293T cells were transfected with WT or C192A GSDMD-NT (e and f) or GSDMD and caspase-1 (g) in the presence or absence of 2-BP (50 μM) for 24 hours. Cell lysates were subjected to Western blot analyses under non-reducing conditions. h–j, Dox-inducible GSDMD-NT-reconstituted BMDMs (h and i) or WT BMDMs (j) were incubated with Dox (2 μg/ml) in the presence or absence of 2-BP (50 μM) or NAC (15 mM) for 12 hours (h and i) or transfected with 50 nM siRNA targeting Apt2 for 48 hours and then stimulated with LPS (100 ng/ml) for 3 hours followed by Nig (10 μM) treatment for 2 hours (j). Cell lysates were subjected to Western blot analyses under non-reducing conditions. k, BMDMs reconstituted with Dox-inducible GSDMD-NT were incubated with Dox (2 μg/ml) in the presence or absence of ML349 (50 μM) for 12 hours. Cell lysates were subjected to Western blot analyses under non-reducing conditions. l, BMDMs reconstituted with Dox-inducible GFP–GSDMD-NT were transfected with 50 nM siRNA targeting Apt2 for 48 hours and then incubated with Dox (2 μg/ml) for 12 hours. Representative confocal images of GFP–GSDMD-NT are shown. Circles indicate areas for measurement of FRAP. Images shown are representative of five independent experiments. Unpaired two-tailed Student’s t-test (c and d).
Extended Data Fig. 10 Defective palmitoylation or depalmitoylation sensitizes mice to bacterial infection in a GSDMD-dependent manner.
a, Eight-week-old male mice were injected intraperitoneally with LPS (54 mg/kg body weight) for 3 hours followed by intraperitoneal injection of 2-BP (50 mg/kg body weight) or vehicle for additional 3 hours. Volcano plots of RNA-seq showing fold-change and P-value for the comparison of spleens from mice treated with 2-BP versus Veh (n = 3) with cutoffs set at FC > 2 or < 0.5, and P-value < 0.05 were shown. Representative genes associated with pyroptosis are highlighted. b, c, Eight-week-old male mice of the indicated genotypes were injected intraperitoneally with LPS (54 mg/kg body weight) for 6 hours. Volcano plots of RNA-seq showing fold-change and P-value for the comparison of spleens from Dhhc7-/- versus Dhhc7+/+ mice (n = 3) (b) or Apt2-/- versus Apt2+/+ mice (n = 3) (c) with cutoffs set at FC > 2 or < 0.5, and P-value < 0.05 were shown. Representative genes associated with pyroptosis are highlighted. d, Eight-week-old Gsdmd-/- male mice were injected intraperitoneally with 2-BP (50 mg/kg body weight) or vehicle for 2 hours, followed by intraperitoneal infection with 103 CFU of S. Typhimurium. The survival ratio of the mice was monitored at the desired stages. e, f, Eight-week-old male mice of the indicated genotypes were infected intraperitoneally with 103 CFU of S. Typhimurium. The survival ratio of the mice was monitored at the desired stages. g, h, A relay model of palmitoylation and depalmitoylation that regulates GSDMD activation in pyroptosis. In the absence of DHHC7, GSDMD palmitoylation and its processing are decreased, leading to reduction of pyroptosis (g). In the absence of APT2, membrane-associated GSDMD-NT fails to undergo depalmitoylation, which reduces the oligomerization and pore-forming activity of GSDMD-NT (h). n = 6 mice for each group (d–f). Log-rank (Mantel–Cox) test (d–f). Unpaired two-tailed Student’s t-test (a–c).
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Zhang, N., Zhang, J., Yang, Y. et al. A palmitoylation–depalmitoylation relay spatiotemporally controls GSDMD activation in pyroptosis. Nat Cell Biol (2024). https://doi.org/10.1038/s41556-024-01397-9
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DOI: https://doi.org/10.1038/s41556-024-01397-9
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