Mycobacterium tuberculosis complex (MTBC). In this study, we correlated previously published classifications on a collection of more than 10,000 genomes and proposed a new, comprehensive nomenclature that unifies the existing ones. In total, we identified 169 lineages and sublineages of M. tuberculosis/M. africanum and 9 animal-adapted species. For the purpose of organizing these genotypes in a more streamlined manner, we stratified them into five hierarchical levels. To represent the classification and compare it with the reference, we compiled a confirmatory data set of 670 high-quality isolates, which includes all genotypes and species of MTBC, and this confirmatory data set can serve as a basis for further studies. We proposed a set of 213 robust barcoding single-nucleotide polymorphisms and a suitable workflow for reliable differentiation of genotypes and species within the complex. This work integrates the results of all the major systematized studies to date to provide an understanding of the global diversity of the MTBC population structure. The results of this work may ultimately help to reliably determine the pathogen genotype and associate it with traits that reflect its prevalence, virulence, vaccination, and treatment efficiency, as well as to reliably find natural features revealed during its spread.The development of whole-genome sequencing technologies is gradually leading to a more detailed description of the population structure of the Mycobacterium tuberculosis complex (MTBC), a number of ambiguous phylogenetic classifications have emerged, which often overlap with one another. In the present study, we have combined all major studies on MTBC classification and inferred a unified, most complete to date classification and accompanying SNP barcodes.Through years of research into the  The cause of tuberculosis, the MTBC, remains a pressing issue for both public health and scientists around the world. To date, nine human-adapted lineages and nine animal-adapted species have been identified within the MTBC ,2. SeverA landmark study on phylogenetic polymorphisms was conducted by Coll et al. in 2014 , which whttps://www.ncbi.nlm.nih.gov/sra). To construct the most complete phylogenetic structure of the MTBC, a subset of 670 isolates was selected to represent five isolates per each genotype and animal-adapted species . A system of SNP barcodes, a workflow for reliable MTBC genotyping, and a companion web app were created at the final stage  was used, including all phylogenetic units: lineage 1 (n = 125), lineage 2 (n = 180), lineage 3 (n = 55), lineage 4 (n = 190), lineage 5 (n = 35), lineage 6 (n = 45), lineage 7 (n = 5), lineage 8 (n = 1), lineage 9 (n = 5), M. microti (n = 5), M. pinnipedii (n = 5), M. orygis (n = 5), M. bovis (n = 5), M. caprae (n = 5), M. suricattae (n = 1), M. mungi (n = 1), Dassie bacillus (n = 1), and Chimpanzee bacillus (n = 1)  . From th (n = 1)  1,2). F . From t. F . Fro (n = 1) . The mai (n = 1) .Lineage 2 classification is the most revised one. In 2021, Thawornwattana et al.  presenteM. tuberculosis/M. africanum genotypes and 5 animal-adapted species . Two SNPs were chosen for each genotype at the first and second levels for better reliability and false-positive exclusion. We verified these 213 barcodes using the confirmatory data set of 670 isolates. All genotypes were correctly called using the proposed barcodes  using Streamlit (https://streamlit.io/) to improve the representation of our study results. TB-gen provides a graphical interface to explore our findings. The application includes a curated set of barcoding SNPs and a reference data set with detailed information about isolates. Additionally, phylogenies that visualize the relationships between these isolates are available within the application. Users can also barcode MTBC lineage from the variant call format (VCF) file via the “Genotype lineage” page.We have developed a web application called TB-gen (https://github.com/dbespiatykh/tblg), which facilitates the classification of MTBC lineages from a VCF file. The tool employs a curated panel of reference barcoding SNPs, identified in this study, for precise lineage classification.Additionally, we have developed a Python-based command-line tool TbLG (Taken together, we have introduced the most extensive and flexible level-based MTBC classification scheme, comprising 169 genotypes and 5 animal-adapted species. From this analysis, we identified 213 robust barcoding SNPs and created a workflow for reliable MTBC genotyping. Furthermore, we report the confirmatory data set of 670 MTBC isolates available in public databases, which can serve as a basis for further phylogenetic studies. Overall, our findings might help to more reliably associate the genotype of isolates with phylogeography and such traits of individual genotypes as the incidence of drug resistance, transmission, virulence, vaccination efficiency, and disease severity.


In mouse aortas lacking functional endothelium, BRD4 knockout did not alter the inhibition of contractile responses by (+)-JQ1. In primary cultured SMCs, (+)-JQ1 inhibited Ca2+ influx. In aortas with intact endothelium, (+)-JQ1 inhibition of contractile responses was reversed by NOS inhibition (L-NAME) or guanylyl cyclase inhibition (ODQ) and by blocking the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. In cultured human umbilical vein endothelial cells (HUVECs), (+)-JQ1 rapidly activated AKT and eNOS, which was reversed by PI3K or ATK inhibition. Intraperitoneal injection of (+)-JQ1 reduced mouse systolic blood pressure, an effect blocked by co-treatment with L-NAME. Interestingly, (+)-JQ1 inhibition of aortic contractility and its activation of eNOS and AKT were mimicked by the (−)-JQ1 enantiomer, which is structurally incapable of inhibiting BET bromodomains. In summary, our data suggest that (+)-JQ1 directly inhibits smooth muscle contractility and indirectly activates the PI3K/AKT/eNOS cascade in endothelial cells; however, these effects appear unrelated to BET inhibition. We conclude that (+)-JQ1 exhibits an off-target effect on vascular contractility.(+)-JQ1, a specific chemical inhibitor of bromodomain and extraterminal (BET) family protein 4 (BRD4), has been reported to inhibit smooth muscle cell (SMC) proliferation and mouse neointima formation via BRD4 regulation and modulate endothelial nitric oxide synthase (eNOS) activity. This study aimed to investigate the effects of (+)-JQ1 on smooth muscle contractility and the underlying mechanisms. Using wire myography, we discovered that (+)-JQ1 inhibited contractile responses in mouse aortas with or without functional endothelium, reducing myosin light chain 20 (LC20) phosphorylation and relying on extracellular Ca The family of bromodomain (BRD) and extra-terminal domain (BET) proteins comprises four members—BRD2, BRD3, BRD4, and bromodomain testis-specific protein (BRDT)—and plays a critical role in the development and pathogenesis of cardiovascular diseases (CVDs), including heart failure, atherosclerosis, pulmonary arterial hypertension, and renal ischemia-reperfusion injury ,2,3,4,5.More recently, Dutzmann et al. reported that local application of (+)-JQ1 significantly attenuated neointimal lesion of the femoral artery in C57BL/6 mice in response to wire-induced injury . In sponIn the present study, we demonstrate that (+)-JQ1 administered through intraperitoneal injection ameliorates mouse carotid arterial neointima formation induced by complete carotid ligation. Importantly, (+)-JQ1 inhibits contractile responses of mouse aortas through activating eNOS in endothelial cells. Interestingly, (+)-JQ1 also directly inhibits smooth muscle contraction without functional endothelium. It is critical to note that (−)-JQ1 has the same effects on smooth muscle contractility, and knockout (KO) of BRD4 in mouse SMCs does not affect (+)-JQ1’s inhibitory effect. In conclusion, (+)-JQ1 exhibits BET protein-independent inhibition of smooth muscle contractility through both endothelium-dependent and -independent mechanisms.Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), 1H- [#C2687 and β-actin #A5441 were purchased from Sigma-Aldrich .Human umbilical vein endothelial cells C2519AS), pooled donor, were purchased from the Lonza Walkersville . Cre recombinase adenovirus  and Null adenovirus  were purchased from Vector Biolabs . Endothelial cell medium (#1001) was purchased from ScienCell  10,000 units/mL penicillin (#15140122), and 10,000 units/mL streptomycin solution (#15140122) were purchased from Thermo Fisher Scientific . (+)-JQ1 and (−)-JQ1 were purchased from Adooq . 9AS, pool2. The medium also included 100 U/mL penicillin and 100 μg/mL of streptomycin and was changed every 48 h.HUVECs were cultured in an endothelial cell medium supplemented with fetal bovine serum (FBS) and endothelial cell growth supplement, and incubated at 37 °C in an atmosphere of 5% CO2 and used for experimentation when they were in passages 4 to 10.Primary culture of SMCs was performed from the aortas of 8-week-old male C57BL/6J mice using an enzyme dispersion method, as we previously described . In brieg for 15 min at 4 °C, and the supernatant was kept for analyses. Total proteins were quantified using the measurement of their concentrations with an iMark™ Microplate Reader . The same amount of protein per sample was separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose filter membranes. Before immunodetection, membranes were blocked within 10% non-fat dry milk and then incubated with primary antibodies (1:1000) overnight at 4 °C. Subsequently, membranes were incubated with a secondary antibody  for 1 h at room temperature after washing with TBST . The abundance of immunolabelled proteins was detected using the enhanced chemiluminescence (ECL) detection system, acquired by ImageQuant LAS-4000 , and quantitated using Gel-Pro Analyzer version 4.0 software . Each experiment was repeated in triplicate.Cultured HUVECs or mouse aortic tissues were washed with cold PBS three times followed by lysis with RIPA buffer  for 30 min on ice. Aortic tissues were lysed in RIPA buffer for 2 h under agitation, following homogenization with mortar and pestle. Samples were centrifuged at 14,000× 2PO4, 1.2 mM MgCl2, 11 mM D-glucose, 25 mM NaHCO3, and 2.5 mM CaCl2, pH 7.4. The thoracic aortas were dissected and cut into rings (2.5 mm in length), mounted in 5-mL myograph chambers  by two metal hooks, and maintained in Krebs solution bubbled with a 95% O2/5% CO2 gas mixture at a resting tension of 0.5 g (4.9 mN). Isometric tension was recorded using MyoDaq/MyoData 2.1 software . After equilibration, each ring was contracted with 1 µM PE, followed by relaxation induced by 1 µM ACh. Vessel segments displaying >50% relaxation to ACh were considered to have intact endothelium. The aortic ring was perfused with a 1 mg/mL solution of sodium deoxycholate in saline for 30 s to remove the vascular endothelium, as described previously [C57BL/6J male mice were purchased from the Jackson Laboratory . All animal studies were approved by the Institutional Animal Care and Use Committees at the University of Calgary and were performed in accordance with the US National Institutes of Health guidelines. Mice were euthanized by decapitation under halothane anesthesia, followed by removal of the thoracic aortas, which were immersed at 37 °C in Krebs solution containing the following: 114 mM NaCl, 4.7 mM KCl, 0.8 mM KH2+ influx, extracellular and intracellular calcium were eluted by Ca2+-free Krebs solution with 1 mmol/L EGTA. The aortic rings were incubated with 10 μmol/L (+)-JQ1 for 20 min, and then 50 mM KCl or 1 μmol/L PE was added to the organ bath and co-incubated for 10 min. Subsequently, a final concentration of 2.5 mmol/L CaCl2 solution was added to the water bath. The KCl or PE in the bath caused a Ca2+ influx into the SMCs and vasoconstriction. The vascular tone was recorded to reflect the extracellular calcium influx levels.To investigate the inhibitory effect of (+)-JQ1 on extracellular CaCalcium signaling experiments were performed as previously described . BrieflySMMHC-CreERT2 in the Y chromosome  to generate male Myh11Cre/+: flox/floxBRD4 mice. Given that the Cre gene is inserted in the Y chromosome, only male mice express the Cre protein, and Cre-adenovirus induces KO of the BRD4 gene only in male mice. Mouse aortic rings with denuded endothelium from male Myh11Cre/+: flox/floxBRD4 mice were cultured with Ad-Null-GFP or Ad-Cre-GFP for 3 days to specifically knock out BRD4 in SMCs. Then, these tissues were used for myography. Before myography, vascular perfusion with a solution of sodium deoxycholate was used to remove the vascular endothelium.The BRD4 flox line was generated by Dr. Hill’s lab at University of Texas Southwestern [Before the non-invasive blood pressure test, 12 male mice were randomly divided into 3 groups: DMSO, (+)-JQ1 (10 mg/kg/day), (+)-JQ1 (10 mg/kg/day) plus L-NAME (25 mg/kg/day) , via int2 chamber and subsequently perfused. Carotid arteries were then isolated, and arterial tissues were fixed in 4% paraformaldehyde at 4 °C overnight before being embedded in paraffin. Serial 5 μm-thick paraffin sections were prepared, covering a region 2 mm proximal to the ligation site, and were subsequently stained with hematoxylin and eosin (HE).Male C57BL/6J mice, approximately 18 weeks old, were randomly assigned to either a vehicle control group or a (+)-JQ1 treatment group . The mouse left carotid artery (LCA) was completely dissected and ligated proximal to the bifurcation of the internal and external carotid arteries, as previously described . Male mit-test (Student’s t-test). For each statistical test, a p-value of < 0.05 was considered significant.Data are representative of at least three independent experiments unless otherwise stated. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed with IBM SPSS Statistics version 25.0  using one-way analysis of variance (ANOVA), or an unpaired two-tailed Previous studies have shown that (+)-JQ1 inhibits SMC proliferation both in vivo and in vitro ,3. In thWe then determined the mechanism underlying the acute inhibitory effects of (+)-JQ1 on contractile responses induced by KCl and PE. We first examined the involvement of eNOS activation and NO release, and NO-sensitive GC on smooth muscle. Our results showed that co-treatment with L-NAME, a well-known inhibitor of NOS , or ODQ,Pretreatment with indomethacin, an inhibitor for cyclooxygenase, did not affect (+)-JQ1-induced inhibition in any preparation . In addiIn sum, our data from pharmacological studies suggest that (+)-JQ1 induces acute inhibition of contractile response of mouse aortas through activating endothelial eNOS with a functional endothelium, and a direct effect on smooth muscle when endothelium is denuded.2+. Our results showed that the inhibition of contractile responses in (+)-JQ1 in both preparations with -JQ1 significantly inhibited PE-induced contraction in the presence of 1 μM Ca2+, but not 0.1 μM, in the preparations with functional endothelium -JQ1 inhibitory effects on extracellular Caons with A and witons with B functioothelium A. As a clium -JQ1. As shown in 2+ influx in SMCs. To verify this possibility, we performed Ca2+ signal assays in primary cultured mouse aortic SMCs. Interestingly, a high concentration of PE (50 μM) induced an increase in intracellular Ca2+, which was reduced by pre-incubation with 50 μM (+)-JQ1 but not by α receptor antagonist, prazosin at 5 μM , the proteinase-activated receptor 1 (PAR1) agonist, which is known to induce intracellular Ca2+ release [2+ in primary cultured mouse aortic SMCs. Thereafter, we examined the effects of (+)-JQ1 on Ca2+ induced by calcium ionophore A23187 (CI), which is known to induce Ca2+ influx across cell membranes [2+-free buffer , a well-established Ca2+ channel blocker [2+ influx in aortic SMCs.Our myography data suggested a direct inhibitory effect of (+)-JQ1 on Ca at 5 μM . Thus, w release ,18. As sembranes . Note the buffer , the addr stores . In the ores [2+ , howeverlar Ca2+ . Importaed by CI C,D and Ced by CI . Notably blocker  -JQ1 has been widely used to characterize the role of BRD4 in various biological functions. The rapid effects  we observed do not support the hypothesis that (+)-JQ1 inhibits BRD4 and exerts its effects through an epigenetic mechanism. To examine the role of BRD4 in (+)-JQ1-induced inhibition of mouse aortic contraction, we measured the effects of (+)-JQ1 on the contractility of mouse aortas with BRD4 knockout. As described in Methods, the aortas from knockout A. Our fuknockout B–D. Our In subsequent studies, we focused on how (+)-JQ1 activates eNOS in endothelium cells (ECs). It is well known that activation of the PI3K/AKT pathway results in eNOS activation in ECs. To determine this possibility, we examined whether inhibition of PI3K or AKT reversed (+)-JQ1-induced inhibition of contractility. As shown in To further establish this mechanism, we took advantage of cultured HUVECs and examined the phosphorylation levels of AKT and eNOS in response to (+)-JQ1 treatment. Our results showed that (+)-JQ1 at 10 μM time-dependently increased the phosphorylation of ATK at the Ser473 site B,C and eGiven that the evidence suggests a role for BRD4 in the activation of NF-κB and autophagy , we examDue to the rapid metabolism of (+)-JQ1, its half-life is only about 1 h in CD1 mice that are genetically engineered to express CD1 proteins . Recentl(+)-JQ1 binds to the bromodomain of BET proteins and prevents them from binding to specific regions of chromatin, thereby disrupting the normal regulation of gene expression. To date, many studies demonstrated that (−)-JQ1, an enantiomer of (+)-JQ1, does not have the same pharmacological effects on BET proteins as (+)-JQ1 ,23. ThusTo further verify the inhibitory effect of (+)-JQ1 on vascular smooth muscle contractility in vivo, we measured the blood pressure of mice treated with DMSO, (+)-JQ1 or (+)-JQ1 plus L-NAME for up to 14 days using the tail-cuff method as described in Methods. Our results showed that (+)-JQ1 significantly reduced systolic blood pressure A but notPrevious studies demonstrated that local application of (+)-JQ1 reduces neointima formation in rat carotid and mouse femoral arteries induced by mechanical injury ,3. Our f2+ influx in primary cultured SMCs. In aortas with an intact endothelium, (+)-JQ1’s inhibition of contractile responses was reversed by NOS or GC inhibition, and by PI3K/AKT pathway inhibition. Activation of eNOS by (+)-JQ1 was further validated in cultured HUVECs). Additionally, intraperitoneal injection of (+)-JQ1 also reduced mouse systolic blood pressure and neointima lesions in mouse carotid arteries induced by complete carotid ligation. However, (+)-JQ1’s effects on smooth muscle contractility were mimicked by (−)-JQ1 and were not modulated by BRD4 deficiency in SMCs, suggesting that (+)-JQ1 has an off-target, BET protein-independent effect. These findings are novel and significant given that (+)-JQ1 has been used in clinical trials and has been implicated in the inhibition of SMC proliferation and neointima lesion formation through BRD4, as indicated in previous studies.Our study revealed that (+)-JQ1 can inhibit the contractile responses of mouse aortas, reduce LC20, and block Ca2+. Inhibiting PI3K, AKT, eNOS, or GC activation reversed (+)-JQ1’s inhibition of aortic contraction, suggesting the involvement of eNOS activation. This possibility was confirmed with cultured HUVECs, in which treatment with (+)-JQ1 rapidly increased phosphorylation of AKT and eNOS. LY-29004 and AKTi IV, which inhibit PI3K and AKT, respectively, blocked this effect. These findings support our conclusion that (+)-JQ1 stimulates eNOS activity, which was further confirmed by our in vivo study, in which intraperitoneal injection of (+)-JQ1 reduced mouse systolic blood pressure that was blocked by co-treatment with L-NAME. Our findings are consistent with previous observations that (+)-JQ1 stimulates eNOS in SHR, but opposite to the finding that (+)-JQ1 inhibits eNOS in HUVECs [First, we have demonstrated that (+)-JQ1 inhibits the contractile responses of mouse aortas with functional endothelium through the downregulation of LC20 phosphorylation and dependence on extracellular Can HUVECs ,9. The an HUVECs ,25. 2+, but was not affected by any inhibitors for PI3K, AKT, eNOS, or GC. In our calcium signaling assay with primary cultured mouse aortic SMCs, our results showed that pretreatment with (+)-JQ1 significantly suppressed the influx of extracellular Ca2+, which provides some insight into the mechanism underlying the direct inhibition of (+)-JQ1 on smooth muscle contraction. However, the exact mechanism remains unclear and requires further investigation.Another interesting discovery in our study was that (+)-JQ1 directly inhibits contraction in mouse aortas without endothelium, indicating a direct inhibitory effect on smooth muscle contraction. Importantly, this inhibition by (+)-JQ1 was also through the downregulation of LC20 phosphorylation and dependence on extracellular CaImportantly, our data suggest that the effects of (+)-JQ1 on endothelial cells and smooth muscle are off-target effects. First, (−)-JQ1, a compound structurally incapable of BET inhibition, mimics the effects of (+)-JQ1 on contractile responses and activation of AKT and eNOS in HUVECs, indicating that the effects of (+)-JQ1 were independent of BET inhibition. Our data from mouse aortas with BRD4 KO support the conclusion that (+)-JQ1-induced direct inhibition of mouse aortic smooth muscle contractility is independent of smooth muscle BRD4. Indeed, based on the required time of 10–20 min for (+)-JQ1’s inhibitory effect, it is conceivable that its effects are independent of gene transcription or protein translation, which was confirmed by our studies using ActD and CHX, inhibitors for gene transcription and protein translation, respectively. In addition, the inhibitory effect of (+)-JQ1 on mouse aortic contractility may be independent of its metabolism. It is known that (+)-JQ1 metabolism occurs in human and mouse liver microsomes, and CYP3A4 has been identified as the main contributor to the production of (+)-JQ1 metabolites in vitro . TherefoNevertheless, our conclusion has been supported by several published studies. In prostate cancer, for example, (+)-JQ1, but not (−)-JQ1, significantly inhibited cancer growth but promoted invasion and metastasis through its direct interaction and inhibition of forkhead box protein A1 (FOXA1), an invasion suppressor in prostate cancer, independent of any BET proteins . To our In summary, our results demonstrate that (+)-JQ1 inhibits smooth muscle contractility of mouse aortas through both endothelium-dependent and -independent mechanisms. In aortic preparations with intact endothelium, (+)-JQ1 stimulates the PI3K/AKT pathway and subsequently activates eNOS to exert its inhibitory effect on smooth muscle contractility. (−)-JQ1 mimics (+)-JQ1’s effects on eNOS activation in vitro and directly inhibits smooth muscle contractility. Therefore, we conclude that (+)-JQ1 has off-target effects on vascular smooth muscle contractility, which may have therapeutic implications. Since (+)-JQ1, as an inhibitor of the BET protein family, has been widely used to treat various diseases ,28,29,302+ influx in SMCs. (−)-JQ1 mimics (+)-JQ1’s effects on eNOS activation in vitro and directly inhibits smooth muscle contractility ((+)-JQ1 inhibits smooth muscle contractility in mouse aortas through a dual mechanism involving activation of eNOS and inhibition of Caactility . BRD4 de


We present a dataset of reconstructed three-dimensional (3D) englacial stratigraphic horizons in northern Greenland. The data cover four different regions representing key ice-dynamic settings in Greenland: (i) the onset of Petermann Glacier, (ii) a region upstream of the 79° North Glacier , near the northern Greenland ice divide, (iii) the onset of the Northeast Greenland Ice Stream (NEGIS) and (iv) a 700 km wide region extending across the central ice divide over the entire northern part of central Greenland. In this paper, we promote the advantages of a 3D perspective of deformed englacial stratigraphy and explain how 3D horizons provide an improved basis for interpreting and reconstructing the ice-dynamic history. The 3D horizons are provided in various formats to allow a wide range of applications and reproducibility of results. The resulting trend in mass loss of the GrIS contributes to sea-level rise of approximately 0.8 mm/a3. Approximately half of the mass loss is attributed to the ice-dynamical contribution from the acceleration of GrIS’ marine-terminating glaciers5. However, in contrast to the ice-mass loss driven by melting, the projected contributions to sea-level rise due to ice dynamics are associated with high uncertainties6. Over the last decade, critical regions in the GrIS experienced extensive speedup and thinning due to frontal ice retreat, affecting the inland ice-flow dynamics7. To better predict future dynamic mass losses, studying past ice-dynamical processes can provide important insight and constraints on forward ice-flow modelling.Over the last decades, the Arctic, and thus, the Greenland ice sheet (GrIS), has warmed more intensively than other regions11. The transmitted electromagnetic waves from the RES system get reflected at interfaces of dielectric contrasts12 within the ice column at so-called internal reflection horizons (IRHs). In the shallow and middle section of the ice column, IRHs primarily represent paleo surfaces that are caused by the former deposition of volcanic material13 at the surface and subsequently buried by subsequent snow accumulation, and hence, represent a horizon of a consistent time of deposition14. These IRHs provide a detailed insight into the englacial stratigraphy18.The GrIS has been extensively mapped with radio-echo sounding (RES) surveys to quantify the thickness of the ice and to map the englacial stratigraphy19 (which we will refer to as 3D horizons). This way, many of the processes occurring in the past, such as changes in the flow and melting of ice sheets, are preserved in the ice and can be decoded25.Viewing the englacial stratigraphy of IRHs at high spatial resolution and in three dimensions has spatial advantages, potentially allowing better process understanding within and at the boundaries of the ice sheet, such as deformation, accumulation, melting and freezing. Three-dimensional views of IRHs within the ice sheets are obtained by tracing IRHs along RES profiles and then interpolating surfaces of equal age between different profiles, allowing the reconstruction of the 3D character of IRHs19. Furthermore, 3D horizons revealed the past activity of paleo-ice streams in currently slow-flowing regions in northern central Greenland21. In addition to the publication of the data in various file formats, we explain in detail how and on what basis the 3D horizons were generated. The datasets are archived and available in a Pangaea Publication Series: 10.1594/PANGAEA.95499126.This paper describes the publication of 3D horizons in the Greenland ice sheet. The horizons reflect the deformation history in different ice-dynamic regimes. A previous study has shown that 3D horizons can provide a holistic overview of the spatial variations in the character of the ice, such as deciphering folding processes due to the mechanical anisotropy of ice at the onset of ice streamsThe data presented in this study originate from four different regions in northern Greenland Fig.  that rep27. The ice shelf of the PG is confined by a fjord and represents the longest floating tongue in Greenland28. Recent calving events in 2010 and 201229 have substantially shortened the ice shelf, which caused an average ice-flow acceleration of ∼10% between 2012 and 201730. Further concerns have been raised that subsequent future ice shelf loss might lead to speedup of the grounded part of PG, leading to irreversible grounding line retreat and accelerated mass loss33.The first data set presented here covers the onset of the Petermann Glacier (PG). PG is located in northwest Greenland and represents one of Greenland’s largest outlet glaciers, draining about 4% of the GrIS34. The basal conditions at the onset of the PG show a complex thermal transition at the base near the onset of fast ice flow35. Moreover, PG’s fast flow onset region is associated with folded and discontinuous IRHs buried deep in the ice sheet19, 38. The folding of the stratigraphy at PG’s onset has been attributed to various processes: Several studies point to basal conditions in the form of moving patches of subglacial slip39 and basal freeze-on36. By contrast, Bons et al.19 used the complete and extensive grid of NASA’s Operation IceBridge (OIB) RES profiles11 to construct 3D horizons, thus overcoming the limitations of using the interpretation on individual RES profiles only, as done in the previous two studies39. For the first time, this revealed the entire 3D geometry and orientation of the englacial folds . The resulting thinning and flow acceleration has the potential to propagate upstream45. Acceleration rates in the order of a few cma−1 at EastGRIP (East Greenland Ice-core Project)46 are first indications that the whole of NEGIS may not be in equilibrium.The third data set covers the upstream part of the Northeast Greenland Ice Stream (NEGIS). Its onset region is close to the central divide, more than 500 km inland from its outlets Fig. . In the 48. However, the geometry of the ice stream, the position of its shear margins, and the origin of NEGIS itself are still not fully understood21,54. Progress in this field is expected from analyzing the EastGRIP ice core56, GPS measurements57, an extensive grid of RES profiles flown over NEGIS59, ground-based phase sensitive RES (pRES) measurements61, and modelling64.Apart from its exceptional length in the Greenlandic context, NEGIS is also unique regarding its distinct and continuous shear margins along its entire length. The position of these shear margins appears not to be constrained by the underlying bed topography65 and numerous plume-like folds, which were attributed to basal freeze-on processes38.The fourth data set covers a ∼700 km wide strip extending across the central ice divide over the entire northern part of central Greenland Fig. . In the 19, appear very different depending on the orientation of the RES profile 67 with AWI’s polar research aircrafts68. The radar specifications for the RES data used in each of the four survey regions are shown in Table The basis for generating the data products published here is airborne RES data. The RES data used to create the 3D horizons presented here were acquired with multi-channel coherent depths sounders (MCoRDS) flown by the Center for Remote Sensing and Integrated Systems (CReSIS) and NASA’s Operation IceBridge69 and consisted of four main steps: (1) GPS synchronization, (2) pulse compression of the chirped waveform in the vertical range to improve the range signal quality and the signal-to-noise ratio (SNR), (3) Along-track Synthetic Aperture Radar (SAR) processing (frequency-wavenumber migration), and (4) cross-track (array) processing to achieve a coherent combination of the return signals of the antenna array to increase SNR and reduce surface clutter66. For detailed acquisition and processing settings of AWI’s 2018 Greenland Polar6 campaign, see Franke et al.58. For acquisition and processing details of NASA’s OIB campaigns, we refer to the CReSIS Open Polar Server70 and the CReSIS RDS (radar depth sounder) documentation71.The RES data processing for all MCoRDS systems was performed with the CReSIS Toolboxair = 1 and for ice with εice = 3.1572. To attribute the absolute elevation to the RES data, we linked the ice surface elevation from the Greenland Ice Mapping Project (GIMP)73 with the location of the ice surface reflection in the RES data. Thus, all elevations are in height above the WGS84 ellipsoid73. We defined suitable IRHs as those that were easily recognizable in all radargrams and had a drill-core constrained age relevant to the respective research questions.We used the SAR-processed RES product as data basis for IRH tracing for the subsequent construction of the 3D horizons. We converted the RES data from the two-way travel time (TWT) domain into the elevation domain. For the conversion from TWT to elevation, we used a two-dimensional dielectric constant (ε) model for air with ε74. The radargrams in the SEGY format were imported into the 3D structural modelling software Move. A slightly modified workflow was used for the Petermann data set. The elevation-converted radargram plots (jpeg files) from CReSIS75 for the respective RES profiles in this region were cropped and provided with a corresponding coordinate system. Similarly to the SEGY format, these radargram plots with coordinates were imported into the Move 3D canvas. Compared to converting the RES data into the SEGY format, this method does not provide the full resolution of the radar data and also has slight inaccuracies with the correct representation of the IRHs in 3D space. For example, small turns in the flight trajectory will be displayed as straight in the radargram plots. However, it is sufficiently accurate for mapping the geometries of IRHs in the frame of the research question for the study by Bons et al.19.For the NEGIS, FINEGIS and NC Greenland data sets, elevation-converted RES data were exported to the SEGY format with the ObsPy Python frameworkMove, they were inspected for the predominant deformation patterns. For the subsequent construction of the 3D horizons, the RES segments and thus the orientation of the IRHs to be traced must be oriented at a high angle to the fold structures where possible . All IRHs were traced manually in Move without an auto picker.After the RES sections for a given region were imported into Move software. A complete 3D horizon is thus composed of several small single 3D horizons 77. The closest distance between the OIB Profile to the NEEM ice core is ~170 m, and for the IRH tracing towards the 3D horizons, we used the following OIB RES profiles: 20110506_02_[001–008], 20170413_01_[039–043], 2013042601_[051–055], and 20170413_01_[048–055]75.For the age attribution of the 3D horizons, we traced the corresponding IRHs to one of the nearby deep ice core sites Fig.  and assi78). Nonetheless, as the primary purpose of constructing the 3D horizons is the visualization of the geometry of the stratigraphic IRHs, this is, in most cases, not a critical issue.Using these ice-core stratigraphies, we dated the 3D horizons, which span approximately the last 7–60 ka. We traced three horizons over FINEGIS, with ages between 60 and 45.5 ka. Over Petermann and NC Greenland, we traced two horizons with ages of 12 and 37.5 ka, and 13.8 and 37.5 ka, respectively. Finally, one horizon was traced over NEGIS, with an estimated age of 7.3 ka. Further details are provided in Table 26 and represent 3D horizon geometries constructed using IRHs on the basis of RES data. The data sets represent the geometry of a respective IRH in four regions of the GrIS. For each region, there may be several data sets that represent different time horizons of deposition on the ice surface and are thus located at different depths within the ice sheet . A summary of the key data for the different 3D horizons of the four regions is shown in Table The data related to this publication is available at PANGAEA (10.1594/PANGAEA.954991)The 3D horizons are provided in three data formats Table : (i) Poi73.Move) as single points and provided for the 3D horizons as well as for the traced IRHs. The x and y columns represent the coordinates in the EPSG:3413 coordinate system, and the z value is the elevation in meters. Distance between the points ranges from 30 to 40 meters.We provide high-resolution point clouds of xyz data in a tabular ASCII file format for all 3D horizons. The point clouds are a representation of the 3D geometry (triangulated meshes in 79 (version 2.2.5) Cubic Spline Approximation module80. The module approximates irregular 2D data in specified points using a continuous bivariate cubic spline. Here, we use 3 to 20 points locally involved in the spline calculation with five points per cell and a relative tolerance multiple in fitting spline coefficients of 140. We restrict the DEM generation to all 3D horizons that do not include overturned fold limbs (such as those in the FINEGIS data set). Although this is a considerable shortcoming when considering the complete topological analysis of those structures, the advantage of this file format is that it is a standard raster format for many GIS applications (such as QGIS and ArcGIS) and other software.Based on the high-resolution point cloud data, we generated digital elevation models (DEMs) in the GeoTIFF (.tif) and NetCDF (.nc) format. DEMs were interpolated to a cell size of 50 m with the SAGA GISGoCAD  is a common file format for geological modelling software. The 3D horizon meshes are stored in the triangulated surfaces (TSURF) format (.ts), which represents the shape as well as the meta-data of the respective 3D horizon. The traced IRHs are stored in the Polyline (PLINE) format (.pl), equivalent to the TSURF format, but used for 2D lines. GoCAD objects represent the 3D geometry in the highest possible resolution and have the advantage that they can be easily imported into many (mostly) commercial geology software packages with a 3D canvas and combined with other data containing geographic information, such as GeoTIFFs, shapefiles or SEGY sections Fig. .Fig. 5ExThe following describes the different data formats and discusses their potential applications, advantages, and disadvantages. The coordinate system for data products is the cartesian EPSG:3413 (WGS 84/NSIDC Sea Ice Polar Stereographic North). All elevations are in height relative to the WGS84 ellipsoidThe file names for the different datasets are organized in the following systematic to specify the region, horizon age, geometry and file type: “[Region]_[Horizon]_[Version]_[age]_[subset]_[geometry].[filetype]”, e.g., Petermann_P1_V001_12_0_ka_mesh.dat for the P1 12.0 ka horizon mesh point cloud in the ASCII format. An overview and explanation for the field names are shown in Table 26. Each 3D horizon is stored as a data set in Publication Series: FINEGIS F1, FINEGIS F2, FINEGIS F3, NEGIS N1, Petermann P1, Petermann P2, Northern Central Greenland NG1, Northern Central Greenland NG2  the range resolution of the RES system of ∼4.31 m71; (2) uncertainties in the determination of the surface reflection in the RES data (estimated uncertainty up to ∼5 m); (3) an error due to inaccuracies by the user who traces the isochrones in the radargrams ; (4) an error in the respective age scale (<1 m); (5) spatial variations in the density-depth function, either manifesting in a slightly different permittivity  or a different firn correction (estimated to ∼5 m). Overall, we estimated the uncertainty in the age assignment to the horizons to be approximately ± 25 m relative to the depth of the respective ice-core time/depth scale. This means that the overall age error increases with depth (Table 78.Because we converted our time-domain RES data into the elevation-domain with a constant dielectric permittivity of ε19 consists of two 3D horizons (P1 and P2), which cover an area of approximately 156 × 95 km. Horizon P1 is ∼12.0 ± 0.9 ka old and approximately represents the transition from the Last Glacial period to the Holocene period and is located on average 1 km below the ice surface. The horizon is mainly characterized by open cylindrical folds with the fold axis oriented parallel to ice flow19  and show an increase in the degree of deformation (larger fold amplitudes) from the upper to the lower horizons , which are located close to the ice divide  cylindrical folds are observed outside the ice stream. These folds form a fan-like pattern extending out from NEGIS , which covers an area of ∼250 × 90 km and is centred on the EastGRIP drill site (Fig. GIS Fig. . Close t65, which is imprinted in the 3D horizons that mimic this bed depression. In the centre of the covered area, near the main ice divide, we find upright cylindrical folds that trend to the north and northwest (Fig. The data set in northern central Greenland covers the largest region in this data collection and consists of two horizons. The 3D horizons cover an area of approximately 690 × 55 km, reaching almost from the eastern to the western ice-sheet margins Fig. . The shaest Fig. .21. Future studies can use this three-dimensional information to calibrate and refine ice-sheet models82,83 assess ice mass loss or gain and better predict the response of both ice-sheets to a warming climate. 3D horizons also provide a detailed view of the age structure within an ice sheet and provide, thus, valuable information on ice-sheet structure and dynamics. Furthermore, 3D horizons hold the potential to reveal information about past accumulation84. Altogether, this knowledge is crucial for understanding ice-sheet stability and their influence on ice-sheet dynamics .The utilization of a 3D representation of IRHs in Greenland and Antarctica can contribute significantly to future studies and expand our understanding of the present-day and past behaviour of these environments. The features visible in the single sections are not necessarily linked directly to bed topography and surface ice flow velocity, data which is often used to plan the grid of a radar survey. The added value of a 3D horizon in comparison to the analysis of single IRHs is that it immediately reveals the geometry of structures independently of the grid layout. By examining the depth, continuity and geometry of these 3D horizons we can infer information on e.g., past and present ice flow patterns, basal conditions and properties and internal deformation processes


Q in complex I of the electron transport chain is conventionally assayed during reverse electron transport (RET) from ubiquinol to NAD. However, S1QELs (specific suppressors of superoxide/hydrogen peroxide production by site IQ) have potent effects in cells and in vivo during presumed forward electron transport (FET). Therefore, we tested whether site IQ generates S1QEL-sensitive superoxide/hydrogen peroxide during FET (site IQf), or alternatively, whether RET and associated S1QEL-sensitive superoxide/hydrogen peroxide production (site IQr) occurs in cells under normal conditions. We introduce an assay to determine if electron flow through complex I is thermodynamically forward or reverse: on blocking electron flow through complex I, the endogenous matrix NAD pool will become more reduced if flow before the challenge was forward, but more oxidised if flow was reverse. Using this assay we show in the model system of isolated rat skeletal muscle mitochondria that superoxide/hydrogen peroxide production by site IQ can be equally great whether RET or FET is running. We show that sites IQr and IQf are equally sensitive to S1QELs, and to rotenone and piericidin A, inhibitors that block the Q-site of complex I. We exclude the possibility that some sub-fraction of the mitochondrial population running site IQr during FET is responsible for S1QEL-sensitive superoxide/hydrogen peroxide production by site IQ. Finally, we show that superoxide/hydrogen peroxide production by site IQ in cells occurs during FET, and is S1QEL-sensitive.Superoxide/hydrogen peroxide production by site I It is conventionally measured during forward electron transport (FET) in the thermodynamically-favoured direction from NAD-linked substrates such as glutamate plus malate (G + M) to ubiquinone (Q) in the presence of Q-site inhibitors of complex I such as rotenone or piericidin A to keep the flavin very reduced. Site IQ is the site of superoxide/hydrogen peroxide production in complex I that is dependent on the redox states of the flavin and particularly of the quinone and on the components of the protonmotive force. It is conventionally measured (as site IQr) during reverse electron transport (RET) in the thermodynamically unfavoured direction (i.e. requiring energy input from the protonmotive force) from the Q pool into complex I driven by succinate oxidation, which generates the required conditions of a strongly reduced QH2/Q ratio together with a high protonmotive force and a high transmembrane pH gradient. Site IIIQo is the site of superoxide production at the outer quinone-binding site of respiratory complex III. It is conventionally measured in the presence of sufficient succinate to reduce the Q pool only partially, and of the Qi-site inhibitor antimycin A to inhibit onward electron flow. The characterisation of these sites enabled the discovery of site-specific small molecules that suppress superoxide/hydrogen peroxide production without affecting normal electron transport: suppressors of site IQ electron leak (S1QELs) [Qo electron leak (S3QELs) [There is extensive evidence that mitochondria generate superoxide and/or hydrogen peroxide at substantial rates in intact cells and  disease . Studies disease ,3. Three(S1QELs) ,5, and s(S3QELs) .Q remains unproven; our current model [2/Q pool. Others [2 into complex I. However, we have shown that S1QELs suppress superoxide/hydrogen peroxide production at much lower concentrations than they inhibit RET, and therefore we reject that hypothesis [The molecular mechanism by which S1QELs suppress superoxide/hydrogen peroxide generation at site Int model  has S1QE. Others  have propothesis .Q is conventionally assayed during RET, i.e. as site IQr [Q needs a strongly reduced QH2/Q ratio [Q is functionally different from site IF [Q runs in isolated rat muscle mitochondria only during RET from QH2 to NAD+ with succinate or glycerol 3-phosphate (G3P) as respiratory substrate (i.e. as IQr). It does not run during FET from NADH to Q with excess G + M as substrate (i.e. as IQf) [+ and QH2/Q pools, high protonmotive force and high ΔpH in the presence of Q-site inhibitors such as rotenone or piericidin A, it can be shown to run in a stalled and presumably unphysiological IQf mode [Superoxide/hydrogen peroxide production by site Isite IQr ,3,11. To/Q ratio ,12,13, h/Q ratio . Despite/Q ratio ,15 there site IF ,13,14,16 as IQf) . HoweverIQf mode .Q runs in cells [Q can also generate pathological amounts of superoxide/hydrogen peroxide in vivo [As determined by the extent of sensitivity of extracellular hydrogen peroxide release to added S1QELs, site Iin cells , where iin cells . These rin cells . As dete in vivo .in vivo, yet under physiologically plausible conditions in vitro they have only been shown to suppress superoxide/hydrogen peroxide production during RET. Does this mean that RET (and superoxide/hydrogen peroxide production by site IQr) is common in cells and in vivo under normal and pathological conditions, or does it mean that site IQ normally runs during forward electron flow as site IQf, yet is still S1QEL-sensitive? Consistent with the first possibility, S1QELs have been shown to protect strongly against ischaemia-reperfusion injury in the Langendorff heart model [Qr during RET upon reoxygenation in the presence of excess accumulated succinate [in vivo, involving proton pumping and production of ATP from each of complex I, complex III and complex IV, is often incorrect, with complex I operating in reverse and only complexes III and IV generating ATP, which is not an attractive hypothesis.These considerations raise an intriguing and important question. S1QELs have potent effects in cells and rt model , in whicuccinate ,28. Otheuccinate . HoweverQ can generate superoxide/hydrogen peroxide in both the reverse and forward reactions in the absence of complex I inhibitors, and whether both modes of IQ activity, IQr and IQf, are sensitive to S1QELs.To address this question, we introduce here a simple assay of the direction of electron flow through complex I in isolated mitochondria and in cells. We use it to determine whether site IQ of the electron transport chain during forward electron flow when electrons are supplied from NADH (i.e. site IQf). The only existing assay of superoxide/hydrogen peroxide production by site IQf [2/Q ratio, and complex I Q-site inhibitors, such as rotenone, piericidin A, or a high concentration of myxothiazol, to prevent electron escape from complex I during the assay. However, these conditions are unsuitable for the assay of site IQf in intact cells and in vivo, where we want to suppress site IQf without preventing normal electron transport from NADH to Q. The Q-site inhibitors may also interact with the binding of S1QELs to complex I [Qf. Indeed, preliminary experiments (not shown) revealed that S1QELs suppress superoxide/hydrogen peroxide production only weakly or not at all in the Lambert and Brand [Qf assay .The initial aim of this study was to determine if S1QELs suppress superoxide/hydrogen peroxide production from site Isite IQf  employs omplex I , making omplex I  unsuitabnd Brand  IQf assaQf that does not require the addition of rotenone, piericidin A, or myxothiazol except to establish that FET is running under the defined condition. It is straightforward to run complex I under conditions of FET  or conditions of RET . The requirement here is to set up conditions with FET or RET but favouring superoxide/hydrogen peroxide production at site IQ, i.e. with high QH2/Q ratio, high protonmotive force and high ΔpH . To determine the direction of electron flow we employ a rotenone challenge. If complex I is running FET then the NAD pool will become more reduced when rotenone addition blocks NADH reoxidation through complex I. Conversely, if complex I is running RET then the NAD pool will become more oxidised when rotenone addition blocks the reduction in NAD by QH2 through complex I. Once we have established that FET (or RET) is occurring under a specific assay set-up, we can omit the rotenone challenge and determine the rate of superoxide/hydrogen peroxide production from site IQf (or IQr) in that assay condition, and its sensitivity to the addition of S1QELs.Therefore, we devised a novel assay of FET and site I M alone ,32), andQr is succinate, to maintain a high QH2/Q ratio and a high protonmotive force and ΔpH. Under these conditions, even though the NAD pool becomes 90–95% reduced by RET [Qf we would need to poise the reduction state of the NAD pool with succinate (to maintain a high QH2/Q ratio and a high protonmotive force and ΔpH) plus a small concentration of G + M (to provide electrons to the NAD pool to drive FET) then observe the NAD pool become even more reduced on rotenone challenge. Preliminary experiments showed that the dynamic range to observe unambiguous further reduction in the NAD pool on rotenone challenge when using succinate as substrate was too limited (i.e. the NAD pool was too strongly reduced by this substrate). Unlike some other mitochondria, such as those from liver, skeletal muscle mitochondria have an adequate activity of the mitochondrial isoform of G3P dehydrogenase (mGPDH) [2/Q ratio and protonmotive force to support site IQf.The conventional substrate to drive RET and site Id by RET ,17,18, w (mGPDH) ,34, so wQf assay and its use to also demonstrate conventional RET and IQr. If we use G3P alone as the reductant to reduce Q strongly and set up a large protonmotive force and ΔpH by electron flow through complex III and complex IV  allows hallenge , then el2 and protonmotive force, driving reverse electron flow from QH2 through complex I and substantial reduction of the matrix NAD pool. The addition of vehicle (ethanol) at 7.5 min had little effect. The addition of uncoupler at 9 min reoxidised the NAD pool to 0% reduced, showing that the initial 5 min relaxation time was sufficient to give essentially full oxidation of the matrix NAD pool. At 10 min rotenone and then excess 5 mM glutamate plus 5 mM malate were added to give 100% reduction in the matrix NAD pool for calibration. 2 was prevented, so matrix NADH slowly oxidised as electrons leaked out to uncharacterised acceptors. The slow oxidation of matrix NADH on rotenone challenge is the demonstration that RET was running immediately before rotenone addition, as expected with G3P as the sole substrate.Qr, GQ and IIF [Qr component of superoxide/hydrogen peroxide production under these conditions of RET as expected [ and IIF . These cexpected . At thisexpected .Q (site IQf) during FET through complex I.F and S3QEL3 to suppress site IIIQo. t = 0 min except for rotenone challenge at t = 4 min . In the same way as in Qr was suppressed by S1QEL2.1 as it was in Qf was suppressed by S1QEL2.1, as it was in Qr and IQf.Having established the principles of the rotenone-challenge assay for RET and FET in low-throughput cuvette-based assays, we transitioned to microplate assays to improve throughput. In-flight additions were less feasible in plate-reader mode, so 12.5 mM G3P and, where required, 50 µM glutamate plus 50 µM malate were added to the initial reaction mixes. To decrease irrelevant hydrogen peroxide production by other sites we also added atpenin A5 to prevent electron backflow into site IIQo) and atpenin A5  and particularly IIF  are significant contributors during RET driven by oxidation of G3P [Q. Brand et al. [Q but did not formally exclude S1QEL effects on respiration on G3P. Effects of atpenin A5 on oxidation of G3P have not been reported. Therefore, we tested whether S1QELs or atpenin A5 affected mitochondrial respiration on G3P. S1QEL1.1, S1QEL2.1 and S1QEL1.719 [n = 2–3 independent experiments each the average of three to four runs). Thus, additions of S1QELs, S3QELs and atpenin A5 do not interfere with the bioenergetics during the oxidation of G3P.To decrease irrelevant hydrogen peroxide production by other sites, we routinely added S3QEL3  and sites GQ and IF (and maybe OF) (during FET) [Q operating during RET as IQr or during FET as IQf were not different, showing that the capacity of site IQ to produce superoxide/hydrogen peroxide was unchanged whether it ran during RET or FET.What contribution does site Iring RET , but Figing FET) . The calQf using the new FET conditions and the IQf assay to see if it behaved like site IQr. Superoxide/hydrogen peroxide production at site IQr is known to be sensitive to the classic Q-site electron transport inhibitors rotenone and piericidin A, which act strongly as inhibitors to prevent RET into the site [Qr activity [+ concentration, dissipates the ΔpH needed to support the catalytic activity of site IQr while increasing mitochondrial membrane potential and maintaining protonmotive force [Q operating in IQf mode retains sensitivity to these reagents.Next, we explored the characteristics of site Ithe site ,18 and athe site . FCCP acactivity ,18. Nigeve force . Figure Qf and IQr shows that the two modes of site IQ have generally very similar properties. The uninhibited rates attained under our standard conditions of high protonmotive force, high ΔpH and high QH2/Q ratio were very similar  by the complex I Q-site inhibitors confirms our previous conclusion [Q is distinct from site IF, which is not inhibited but enhanced in the presence of these inhibitors. Distinct sites IQ and IF remains the simplest and most obvious interpretation of all the data from our and other laboratories; however, it remains formally possible that inhibition of the Q-site by rotenone or piericidin A alters the conformation of the distant flavin site in some particular way, and ‘site IQ' is really the flavin in a different state, or the inverse — that ‘site IF' is really the Q-site with complex conformational constraints.Comparison of superoxide/hydrogen peroxide production from sites I similar –5. They  similar , and to io FCCP,  or decreio FCCP, . Inhibitnclusion  that sitQf, Lambert et al. [Qf could be measured under conditions of high protonmotive force, high pH, and high QH2/Q ratio, but only in the presence of high concentrations of complex I electron transport inhibitors such as piericidin A, rotenone or myxothiazol. At first sight that is the opposite of what we report in Qr, but also from site IQf. This contradiction can possibly be resolved by the observation that at the highest concentrations of piericidin A  of the mitochondria in our preparations continues to run RET and site IQr  when the remaining 60% run FET and respond to rotenone challenge by reducing their NAD pool but produce little superoxide/hydrogen peroxide. In this scenario, these 40% of the mitochondria run site IQr and contribute the entire rate of superoxide/hydrogen peroxide production we assign to site IQf, yet contribute insufficient NADH signal on rotenone challenge to dominate the bulk NAD reduction seen on rotenone challenge.Can the conclusions of active superoxide/hydrogen peroxide production by site IQr population', defined as the fraction of the mitochondrial population in the presence of 50 µM G + M that polarised strongly on the addition of G3P (to favour any RET), and also responded to rotenone challenge with NADH oxidation (rather than reduction), showing that this sub-population was running RET (and site IQr), not FET (and site IQf) as inferred from the bulk results.To test this possibility, we measured the heterogeneity of our mitochondrial preparation using fluorescence microscopy of single mitochondria stained with tetramethylrhodamine methyl ester (TMRM), a probe of the mitochondrial membrane potential (ΔψM), combined with imaging of NAD(P)H autofluorescence . Specifiy-axis). Because a precise zero mV calibration point was not feasible, the polarisation supported by G + M alone was measured indirectly by expressing how much less polarised the individual mitochondria were in the presence of G + M than with G3P and succinate also added (x-axis). Thus, a smaller magnitude (difference) on this axis means stronger polarisation by G + M alone. Overall, the magnitude of the response of individual rat skeletal isolated mitochondria to G3P in the presence of G + M varied as a continuum from no response to strong response, with no clearly distinct subpopulations. With an arbitrary definition of categories, n = 4; blue) showed a medium increase in polarisation for each of the consecutive additions of G + M and G3P. A weaker G3P and stronger G + M signal was observed in 19 ± 8% of mitochondria (green); such a population may run mostly FET and make a disproportionate contribution to the rotenone-evoked increase in NAD reduction. Importantly, the potential ‘RET and IQr population', giving a strong G3P response after very weak polarisation by G + M, was rare . A small population  gave little response to G3P. Thus, only ∼5% of the mitochondrial population gave a large increase in ΔψM when provided with G3P in the presence of 50 µM G + M, suggesting they might still be running RET even when the bulk population ran FET.First, we compared the magnitudes of ΔψM provided by G3P and by G + M. Because the size, and therefore the fluorescence signal, of individual mitochondria varies, this first set of experiments, which measured single-mitochondrion TMRM intensities only, was finished by the addition of succinate as a calibration of maximal ΔψM . This apso added , x-axis.+ pool, showing they were running FET under our experimental conditions. Only 4.2 ± 1.1% (n = 4 different runs), mostly the potential ‘RET and IQr population' that were more strongly polarised by the addition of G3P in the presence of G + M, responded to rotenone challenge by a significant oxidation of NADH to NAD+, showing they were indeed running RET and presumably site IQr.Next, we measured the polarisation by G3P when added in the presence of G + M as in Qr and IQf were indistinguishable H fluorescence significantly in response to the rotenone challenge, indicating a fairly large population with close-to-stalled electron transport. It is possible that superoxide/hydrogen peroxide production from site IQ occurs when FET slows or stalls due to high protonmotive force, ΔpH and QH2/Q ratio. In this scenario 30% of complex I is in (near) equilibrium, site IQ is active, and challenge with rotenone causes no significant change in NAD(P)H.Since the rates of superoxide/hydrogen peroxide production by sites Iuishable –6, the 4Q, running FET (65%) or near equilibrium (30%), but not running RET (4%), is the source of superoxide/hydrogen peroxide in mitochondria under our contrived conditions of FET in isolated mitochondria, and that heterogeneity of our mitochondrial preparation does not lead to erroneous identification of site IQf in the bulk preparation driven by site IQr in a smaller subset.We conclude that site IQ running during FET (site IQf) produces superoxide/hydrogen peroxide at high rates and is fully S1QEL-sensitive. Is the same true of mitochondria in intact cells? From theoretical considerations we expect FET to be the default in cells, generating ATP from the oxidation of NADH using all three mitochondrial proton-pumping complexes, I, III and IV [2 to NAD is expected to occur only under unusual or contrived conditions, such as the transient oxidation of excess succinate during ischaemia-reperfusion [Qf rather than site IQr. Nonetheless, cell populations may be heterogeneous, and, in theory, the observed bulk superoxide/hydrogen peroxide production might originate from a sub-population of cells with unusual characteristics. Note that at 1–2 µM S1QELs do not affect cellular bioenergetics [The experiments in I and IV ,40, whererfusion ,28. Thererfusion . Therefoerfusion  is most ergetics .Q in Fa2N-4 immortalised hepatocytes, and the evidence that this occurs during bulk FET in the mitochondria (i.e. site IQf) within the cells. In these cells, based on the maximal suppression achieved by S1QEL2.1, site IQ was responsible for about half of total cellular hydrogen peroxide production (+ or NADPH/NADP+ pools. All cells responded to FCCP with a decrease in autofluorescence (n = 4) responded to rotenone challenge with an increase in autofluorescence  were estimated to respond poorly or not at all to rotenone, while 74 ± 3% responded with a 50 ± 10% mean increase in fluorescence intensity. The population of mitochondria oxidising on rotenone challenge was measured by the excess of measured pixel counts over the left tail of the distribution of the log2-fold values , it may be best to consider that site IQ is a potent generator of superoxide/hydrogen peroxide in mitochondria and in cells regardless of the net direction of electron flow, and that all modes are equally S1QEL-sensitive. The conventional assay of superoxide/hydrogen peroxide production by site IQ as site IQr during RET in isolated mitochondria [Overall,  and IQr  and 5, i and IQr , so we cse cells 6%, it machondria ,11,45 isEh) closely matched by the resulting protonmotive force set up by proton pumping at complex I or the other electron transport chain complexes (4*pmf) [Eh, and causing complex I to run FET until conditions re-equilibrate. Conversely, increased electron flow into the Q pool from complex II or mitochondrial G3P dehydrogenase can further reduce the Q pool, thereby decreasing 2*ΔEh, or a decrease in ATP demand by the cell can raise 4*pmf, with either effect causing complex I to run RET until conditions re-equilibrate. In this paper, we introduce a simple assay to determine if electron flow through mitochondrial complex I is in the thermodynamically favourable forward or thermodynamically unfavourable reverse direction in any particular situation: on blocking electron flow through the Q-site of complex I, the endogenous matrix NAD pool will become more reduced if the flow prior to the challenge was forward, but more oxidised if the flow was reverse. We use this assay to show in the model system of isolated rat skeletal muscle mitochondria that superoxide/hydrogen peroxide production by complex I, with the diagnostic characteristics of site IQ  can be equally great whether RET or FET is occurring. Importantly, the activity of site IQr can now be demonstrated without the need for the addition of high concentrations of complex I inhibitors as originally reported [Q (IQr and IQf) are equally sensitive to specific suppressors of superoxide/hydrogen peroxide production by site IQ, S1QELs. The observation that two quite different S1QEL chemotypes  . For thireported . We then2 series ) are equin vivo [in vitro under physiologically plausible conditions they had only been shown to suppress superoxide/hydrogen peroxide production during RET [in vivo does not mean that RET (and superoxide/hydrogen peroxide production by site IQr) is common in cells and in vivo under normal and pathological conditions. Instead, it means that site IQ can run during forward electron flow as site IQf, yet can still be fully S1QEL-sensitive, and the effects S1QELs in cells and in vivo do not depend on the direction of electron transport, whether it is forward or reverse . The direction of electron flow cannot be inferred from the effects of S1QELs or complex I inhibitors such as rotenone on superoxide/hydrogen peroxide production, as is sometimes done, e.g. [per se  h and ΔpH  are likeS1QEL1.1, S1QEL2.1 , S1QEL1.2 asphyxiation followed by bilateral thoracotomy. Mitochondria were isolated from rat hindlimbs in Chappell–Perry buffer  at 4°C by standard procedures [All animal experiments took place in the Buck Institute for Research on Aging. Scientific ethics and the animal protocol were approved by the Institutional Animal Care and Use Committee of the Buck Institute (License/protocol No. 10241) in accordance with the National Institutes of Health guide for the care and use of Laboratory animals . Rats were euthanised by COocedures .Qr and IQf  as substrate were conducted at 37°C as described  using 0. and IQf  will havMitochondrial respiration was assayed at 37°C using an Agilent Seahorse XFe-24 as described previously , using 1g in KHEB medium at 4°C. The microplate was warmed in the 37°C heated environment chamber of a Nikon Eclipse Ti-PFS fully motorised wide-field fluorescence microscope equipped with a Lambda 821 LED light source with Lambda 10-3 emission filter wheel and Andor iXon Life 888 EMCCD camera, controlled by NIS Elements 5.20. TMRM (100 nM) was added to the wells, and in successive additions substrates were added without changing the TMRM concentration. TMRM was imaged using a Super Fluor 40× NA1.3 oil lens, a 586/20 nm excitation filter on a 560 nm LED, a 593 nm dichroic mirror, and a 641/75 nm emission filter capturing the centre 512 × 512 pixels area of the camera with no binning and with 1.5× optical zoom (0.17 µm/pixel resolution). Multiple  view fields were recorded in one to three wells for 3 time points per substrate condition. In some experiments, TMRM imaging was followed by recording frames of NAD(P)H autofluorescence in the same view fields using a 340 nm LED, 414 nm dichroic mirror and 460/80 nm emission filter. The first autofluorescence frame matched the substrate condition of the last TMRM frame and then the recording of three more frames followed the application of rotenone (4 µM) or FCCP (4 µM) by 1 min delay. Recordings were analysed in Image Analyst MKII 4.2.1  by performing image registration, background subtraction and ROI-based intensity measurement. Background was subtracted first as a blank image and then frame-by-frame using the ‘Mean of pixels below percentile of max projection (dilated mask)' method. Single-mitochondria ROIs were generated by morphological segmentation using a modification of the ‘Mitochondria : cell volume fractionator (basic)' standard pipeline. ΔψM was calculated using the Nernst equation. Because fluorescence was small in the absence of substrates, polarisation by G + M was calculated from TMRM fluorescence intensities (FTMRM) by comparison to succinate as 61 mV × log(FTMRM_G + M/FTMRM_succinate), which expresses how much less polarisation was provided by G + M than succinate, in millivolts. Polarisation by G3P was calculated by comparison to G + M as 61 mV × log(FTMRM_G3P/FTMRM_G + M), which expresses the millivolts hyperpolarisation when G3P is added in the presence of G + M. Changes in NAD(P)H autofluorescence (ΔNADH) were expressed as the observed fluorescence intensity change normalised to the population mean fluorescence before rotenone (or FCCP) addition. This mean calculation was required because some mitochondria showed very little autofluorescence. This was then converted to log2-fold changes to provide symmetrical distributions. Scatter plots were generated and analysed in Mathematica 13 (Wolfram Research).Mitochondria were immobilised in coverglass-bottomed 96-well microplates (Greiner Sensoplate) by centrifugation for 15 min at 1000 2O2 production) such that on the day of the experiment monolayer density reached 80–90% confluence . Before experiment, the growth medium was replaced with KRB assay buffer  by washing with this medium twice and then leaving 4 ml total in the 35 mm culture dish or 50 µl in 96-well plates.Fa2N-4 immortalised hepatocytes were cultured in collagen-coated dishes according to the purveyor's instructions using their proprietary media  without cyP450 induction. Coating was performed with collagen IV (15 µg/ml in PBS) overnight. Cells were plated 2–3 days in advance of experiment into 35 mm dishes (for NAD(P)H autofluorescence) and 96-well microplates (for HHydrogen peroxide production rates were measured using Amplex UltraRed with a Pherastar FS (BMG) fluorescence microplate reader. Fa2N-4 cells were plated in 96-well microplates in a pattern that utilises 48 wells of the entire collagen-coated plate in a scheme that avoids cells being cultured at edge wells to prevent ‘edge effects', while leaving the other 48 wells to be used as matched background wells for each condition. Before the assay, cultures were washed with KRB assay medium twice and transferred to a 37°C air incubator for ∼10 min. Immediately before recording, KRB was completely removed from the cell assay plate, which was washed once and the medium was replaced with 50 μl per well of pre-warmed RB  pre-mixed with test compounds or vehicle for each well. Fluorescence recording was immediately started at Ex 540 nm/Em 590 nm for 25 of 80 s-cycles using 3 mm orbital well averaging in the bottom readout, kinetic mode at 37°C. Cycles 5–18 were taken for regression analysis. Background rates measured in cell-free, but otherwise identical wells were subtracted from corresponding cell–well rates. The rate of hydrogen peroxide release was expressed as % vehicle. No SOD was added to the RB as only hydrogen peroxide, but not superoxide, is freely diffusible through the cell membrane. Adding SOD impaired the signal-to-noise ratio of the recording (data not shown).x,y-position, and four other positions were imaged for analysis in each dish. Recordings were analysed in Image Analyst by rigid image registration, background subtraction and ROI-based intensity measurement. Unbiased single-cell ROIs were generated in using Cellpose 2.0 [Subcellular resolution micrographs of NAD(P)H autofluorescence were recorded using an upright Zeiss LSM 7MP multiphoton microscope, equipped with a Chameleon Vision II mode-locked laser set to 710 nm (1–1.5% of the 745 mW output power was used) and W Plan-Apochromat 20×/1.0 DIC M27 75 mm dipping water immersion objective lens and detection of emission between 420 and 480 nm. 1024 × 1024 pixels images (0.2 µm/pixel) were recorded at maximal scan speed and 8× frame averaging. Time courses of two frames, one before and one after the addition of rotenone (2 µM), FCCP (4 µM) or vehicle (DMSO 1 : 2000) were recorded using the Zeiss Multi Time Series macro. The cell monolayer was auto-focused at one 2 of the ratios was taken for all pixels. Image histograms were saved to Excel and analysed in Mathematica using a maximum log-likelihood method for fitting the binned log2-fold intensity values with normal distribution(s). Mock treatments were fitted well with a single, symmetric distribution. We attribute this change of pixel intensities between the consecutive frames of the time course to motility artefacts and noise. The distribution of log2-fold pixel intensity changes for rotenone-treated cells was wider, and therefore we coarsely modelled it as sum of two normal distributions . The fraction of mitochondria with reverse electron flow was estimated as the excess pixel count observed at significantly decreased NAD(P)H autofluorescence intensities (below 2σ of the mock) that could not be attributed to effects of motility and noise based on the fit above. This analysis was repeated for individual single cells .The NAD(P)H autofluorescence multiphoton time courses described above were de-noised by Wiener filtering, cropped based on the Cellpose ROIs resulting in 256 × 256 pixels images of single cells, and surrounding structures were masked in Image Analyst MKII. Then, to compensate for the motility of mitochondria during the time courses, B-spline-based deformable image registration was performed in Elastix 5.0.1 . Image rn = 3 independent mitochondrial preparations. Results are either representative traces, or means ± SEM , as indicated.Mitochondrial experiments were repeated using at least n) with multiple technical replicates (e.g. view fields or wells) within each experiment, which were pooled for analysis.Experiments using cultured cells were replicated from at least three different passages (indicated by


Immunoglobulin E (IgE) blood tests are used to detect sensitizations and potential allergies. Recent studies suggest that specific IgE sensitization patterns due to molecular interactions affect an individual's risk of developing allergic symptoms.The aim of this study was to reveal specific IgE sensitization patterns and investigate their clinical implications in Hymenoptera venom allergy.In this cross-sectional study, 257 hunters or fishers with self-filled surveys on previous Hymenoptera stings were analyzed. Blood samples were taken to determine Hymenoptera IgE sensitization levels. Using dimensionality reduction and clustering, specific IgE for 10 Hymenoptera venom allergens were evaluated for clinical relevance.Three clusters were unmasked using novel dimensionality reduction and clustering methods solely based on specific IgE levels to Hymenoptera venom allergens. These clusters show different characteristics regarding previous systemic reactions to Hymenoptera stings.Our study was able to unmask non-linear sensitization patterns for specific IgE tests in Hymenoptera venom allergy. We were able to derive risk clusters for anaphylactic reactions following hymenoptera stings and pinpoint relevant allergens  for clustering. To address this, molecular or component-resolved diagnosis (CRD)New evolving mathematical models, such as UMAP (Uniform Manifold Approximation and Projection),On the other hand, HDBSCAN In this study, we applied current scalable, state-of-the-art mathematical methods to allergology by combining a dimension reduction method with a clustering algorithm to assess a) if Hymenoptera specific IgE sensitization patterns are unmasking visually in the lower dimensional space, b) the implications for laboratory measurements and clinical application of these methods, and c) if these methods can help to optimize the allocation of resources and suggest a more cost-efficient use of laboratory tests.,in vitro testing. The ImmunoCAP™ system measured total and specific IgE levels, along with serum tryptase.In a cross-sectional study conducted in the Greater Munich Area, Southern Germany, during winter 2016 and January 2017, 257 adult individuals were recruited and analyzed from annual winter meetings of three hunting associations and an international hunting and fishing exhibition.Our primary independent variables were the subject age, total IgE (kU/l), tryptase (μg/l), and each subject's individual subset of measured specific IgE levels (kUA/l) to 10 different Hymenoptera allergens  using ImmunoCAP™ Allergy Testing Solutions and Phadia™ Laboratory System by Thermo Fisher Scientific.Dimension reduction was solely performed using specific IgE values. Uniform Manifold Approximation and Projection (UMAP)Manhattan as a distance metric for higher-dimensional.A high-performance implementationData were analyzed using Python 3.8.8 with statistical libraries pandas 1.3.0, SciPy 1.5.2, statsmodels 0.12.0, umap 0.5.1 and hdbscan 0.8.26. In addition to descriptive parameters, we report all results as mean with 95% confidence intervals. Specific IgE graphs were log-transformed for better comparability. Logistic regression was performed for binary outcome variables using Broyden-Fletcher-Goldfarb-Shanno algorithm (BFGS) as an optimizer, and odds ratios with 95% confidence intervals and p-values are shown.UMAP dimension reduction followed by HDBSCAN with the help of the cluster hierarchy dendrogram revealed 3 principal clusters derived from specific IgE sensitizations to Hymenoptera venom allergens in the hunter/fisher group , A-C. SuThe age and tryptase levels of the subjects in the respective 3 clusters did not differ. The total IgE was significantly lower in cluster 0 (46.2 ± 15.8) compared to cluster 2 , whereas both clusters were indistinguishable from cluster 1 . Regarding previous stings, cluster 1 (85.5% ± 8.5) had significantly more subjects with more than 5 stings than cluster 0  and cluster 2 , while the latter 2 clusters did not show any significant difference (p = 0.74).The analysis of whole venom extracts for honey bee (i 1) and wasp (i 3) revealed a different focus for every cluster. While specific IgE for honey bee (i 1) was only different in clusters 0 and 1 , wasp (i 3) was significantly different between clusters 0 and 2  as well as between clusters 1 and 2 .Specific IgE characteristics of Hymenoptera recombinants were analyzed across 3 distinct clusters. The variations in specific IgE levels between clusters for the tested recombinants are summarized in the table below . SpecifiIn summary, cluster 0 was characterized by peaks in specific IgE for honey bee (i 1), the wasp recombinant rVes v 1, and low levels of CCD MUXF3. Cluster 1 showed the highest average specific IgE to all tested allergens, with a peak in specific IgE for honey bee (i 1). Cluster 2 primarily consisted of high peaks in specific IgE for whole wasp venom (i 3) followed by the wasp recombinants rVes v 1 and rVes v 5 , A.In the previous section, we elaborated on the implications of cluster affiliations on Hymenoptera specific IgE sensitization patterns. When cluster association on previous systemic reactions following Hymenoptera stings was evaluated, we found an increasing risk from cluster 0, (23.1%) to cluster 2 (38.5%), and to cluster 1 . A medical history of more than 5 previous Hymenoptera stings had non-significant implications on previous systemic reactions to Hymenoptera stings . Higher specific IgE levels in subjects who had systemic reactions following Hymenoptera stings were seen for rApi m 10  and rApi m 1 , while the whole honey bee venom (i 1) extract showed an opposite trend . All other Hymenoptera specific IgE levels (wasp (i 3), rApi m 2, 3, 5, rVes v 1, 5 and MUXF3) showed no predictive benefit. In the case of at least moderate systemic reactions to Hymenoptera stings, the only predictor was the cluster association  and wasp (i 3) venom, rApi m 10, and rVes v 1 A–C. As iinvitro tests on allergy clinical manifestations has been a matter of prolonged, scientific debate.,,The role of quantitative specific IgE latent class analysis (LCA) has successfully been conducted in asthmatic patients.k-nearest neighbors method (KNN) for clustering, a downside of LCA though is the number of groups that need to be determined before analysis and that the dependent variables need to be categorical. The methodology around network analysis revealed previously unknown disease entities in the field of psychopathology,More accurate dimensionality reduction methods like UMAPThe risk assessment of more than 5 previous Hymenoptera stings shown in the previous study on the hunter/fisher cohort was canceled out by the higher predictive value of the cluster association in our model. The authors of the previous study were “unable to find a correlation between reaction severity and sensitization to any of the hitherto available recombinant allergens”.To our knowledge, the presented data are the first to apply modern dimension reduction and clustering methods to the field of Hymenoptera venom allergies and show a diagnostic benefit. We believe that complex interactions in IgE-mediated allergies are reflected in different specific IgE sensitization patterns, which cannot be assessed by classic, linear statistics. In line with these findings, other fields in allergology related to IgE-mediated type I allergies, like asthmatic diseases, showed the benefit of a wholesome analysis of more than a single allergen. We present evidence that there is no need for individual cutoff-values for every single specific IgE antibody in Hymenoptera venom allergies, as the raw quantification of specific IgE antibodies can be used in clustering algorithms to subsequently derive meaningful clinical implications.,,,In other fields, the discovery of new recombinant allergens improved diagnosis and risk stratification based on an individual single allergen sensitization. Advances have been shown for recombinant allergens like rPru p 3In line with the previous studies, not all recombinants appear to be of equal value in Hymenoptera venom allergies. While rApi m 10 was a good single-allergen predictor as previously known, all other recombinants  did not provide a clear benefit for clinical interpretation. While Api m10 stands out in bee allergy significance, we still must consider the other recombinants at lower levels, as low-titer sensitizations can also cause severe clinical reactionsTrue dual sensitization to both bee and wasp venom differs significantly from mere cross-reactivity, such as specific IgE against CCDs without elevated anaphylactic reaction risk. Cross-reactivity between venoms often results from shared allergenic components, like CCDs. In contrast, true dual sensitization implies independent sensitizations to each insect's distinct allergens. This differentiation is crucial for diagnosis, management, and therapeutic strategies, including double venom immunotherapies. Certain studies suggest the basophil activation test for differentiating true dual sensitization.The abundance of available specific IgE recombinants in Hymenoptera venom allergies necessitates responsible and economical use. As the previous results suggest, redundancy and clinically irrelevant sensitizations exist when testing the whole Hymenoptera panel. We attempted to replicate the results with a more cost-efficient use of the available recombinants . We were able to retain most clustering characteristics with only 40% of the allergens.Our study has several limitations that must be acknowledged. First, due to the constrained sample size, our findings are primarily based on internal validation, with external testing and predictions not conducted. Second, the specificity of our cohort — comprising largely of hunters and fishers — restricts the general applicability of our results. This group has a heightened exposure to Hymenoptera stings, which might not mirror the broader population's experience. Third, the dendrogram-based clustering, while insightful, is not without challenges; it is not a fully automated method and demands manual parameter calibration by researchers. It does offer the advantage of not requiring pre-set cluster numbers, unlike some other clustering methodologies. Despite these challenges, our study offers valuable perspectives into Hymenoptera venom allergies, aiding risk assessment and suggesting ways to enhance the efficiency of in-vitro specific IgE testing.CCDs, cross-reactive carbohydrate determinants; CRD, component-resolved diagnosis; HDBSCAN, Hierarchical Density-Based Spatial Clustering of Applications with Noise; UMAP, Uniform Manifold Approximation and Projection.Parts of this study were supported by an unrestricted research grant provided by Thermo Fischer Diagnostics GmbH, Freiburg, Germany.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Robert Kaczmarczyk (RK), Alexander Zink (AZ), and Tobias Lasser (TL) were involved in the conceptualization and methodology of the study. RK curated the data and performed the formal analysis. TL, AZ, and RK were responsible for project administration, software development, and supervision. AZ provided resources for the study. Validation was carried out by RK, AZ, and TB. RK created the visualizations for the manuscript. The writing process involved Johannes Ring (JR), Tilo Biedermann (TB), TL, RK, and AZ, with RK, AZ, and TL preparing the original draft and Johannes Ring (JR) and Tilo Biedermann (TB) taking part in the review and editing process. All five authors  have directly accessed and verified the underlying data reported in the manuscript.The study was reviewed and approved by the local ethics committee of the Medical Faculty of the Technical University of Munich under the reference number 604/19 S.All authors approve this manuscript to be submitted to the World Allergy Organization Journal.AZ has received speaker's honoraris from Alk-Abelló and unrestricted research grants from Alk-Abelló and Phadia-Thermo Fisher.TB gave advice to or got an honorarium for talks or research grant from the following companies: AbbVie, Alk-Abelló, Celgene-BMS, Lilly Deutschland GmbH, Mylan, Novartis, Phadia-Thermo Fisher, Sanofi-Genzyme, Regeneron, Viatris.JR gave advice to or got an honorarium for talks or research grant from the following companies: AbbVie, Allergika, Leo, L'oreal, Alk-Abelló and Mylan-Viatris.All other authors have no conflict of interest within the scope of the submitted work.


Arabidopsis thaliana and regulate root development, including primary root growth, lateral root formation and root gravitropism,etc. [A.[AtRGF orAtGLV were identified inA.thaliana, and 15 corresponding genes were reported in the model legumeMedicago truncatulaM.truncatula,MtRGF3 is abundantly expressed in leaves and is inducibly transcribed in roots by inoculation of the symbiontSinorhizbium meliloti containing thenodC gene (required for nodulation factor biosynthesis). Reverse genetic analysis showed that overexpression ofMtRGF3 in the transgenic roots suppresses symbiotic nodulation ofM.truncatula, while the RNAi increases the number of root nodules, suggesting thatMtRGF3 encodes a suppressor peptide of symbiotic nodulation inM.truncatulaM.truncatula seedlings on agar platesM.truncatula is elusive.Root meristem growth factors (RGFs) were first discovered inm,etc. ‒3 . RGFm,etc. ‒3 . Thetc. [A.‒6 . NinM.truncatula seedlings. The transcript level analysis from quantitative reverse transcription-PCR (qRT-PCR) showed that the expression ofMtRGF3 was repressed by 1 μM ACC (MtRGF3 may be associated with plant defense responses. The exopolysaccharide I (succinoglycan) fromS.meliloti was reported to suppress host defense responses and to promote the symbiosis ofM.truncatula-S.melilotiM.truncatula seedlings inoculated with theexoY210 mutant (the EPSI minus mutant) compared with seedlings inoculated with the wild-type Sm1021  bursts act as a defense response in plants. The results showed that 10 μM of MtRGF3p increased the level of superoxide anion in the vascular bundles ofM.truncatula roots compared with the random peptide , was used for the treatment of μM ACC A, sugges Sm1021 B, suggespeptide C. After peptide C. These M.truncatula. Fluorescence microscopy observations indicated that fewer colonies of Sm1021 (35 colonies/cm) were formed on the root surfaces of theM.truncatula seedlings treated with 10 μM MtRGF3p than on those treated with the random peptide  gene may be induced inM.truncatula. From the RNA-seq data, the induction ofMtRbohD (Mtr3g098350) was observed in the roots treated with 1 μM MtRGF3p, but it was attenuated by inoculation with Sm1021  genes, 9 of 13WRKY (transcription factors participate in response to biotic or abiotic stresses) genes, and 36 of 60R (pathogen resistance protein) genes were inducibly expressed in the roots treated with MtRGF3p, while their induction was suppressed by the inoculation with Sm1021 (Supplementary Table S1). These results indicated that a broad defense response or innate immune genes are boosted by MtRGF3p inM.truncatula, so this peptide may be a defense or immunity signal in the plant to control rhizobium infection.Interestingly, among the differentially expressed genes ofM.truncatulaMtNFP,MtLYK3,MtERN1,MtNSP2 andMtNIN was repressed by treatment with 1 μM MtRGF3p compared with the random peptide after inoculation with Sm1021 , a group of lipo-chito-oligosaccharides produced by rhiozobia, are the most important signal during legume symbiotic nodulation. NF is sensed by the LysM receptor kinase MtNFP/MtLYK3 and activates calcium spiking and several transcription factors, such as MtNIN, MtNSP1, MtNSP2 and MtERN1, to reprogram the expressions of downstream genes to regulate rhizobium infection and nodule development. Since MtRGF3p suppresses symbiotic nodulation of Sm1021 B, which  Sm1021 C. These M.truncatula symbiotic nodulation. This peptide activates the plant defense/immune response to restrict rhizobium colonization and infection, while it attenuates NF signaling by repressing key gene expression to reduce the initiation of nodule primordia and infection (MtRGF3 to produce the active peptideMtNFP/MtLYK3, so that NF signal transduction is attenuated. At the same time, the peptide activates the defense/immune response to prevent colony formation ofS.meliloti onM.truncatula roots (A.thaliana[M.truncatula. If the LRR receptor kinase of MtRGF3p is identified fromM.truncatula, the precise signaling mechanism will be resolved in the future. Moreover, whether MtRGF3p is associated with autoregulation of nodulation inM.truncatula may be elucidated. The supernodulation mutant (sunn-1,M.truncatula will be introduced for treatment with the peptide. The possibility that peptide signaling may be linked to ethylene signal transduction also needs to be studied in theskl mutant backgroundIn the present study, we first provide the possible mechanism by which MtRGF3p suppressesfection D. In faca roots A so thatthaliana‒6 , thesunn-1, ofM.tr23032supplementary_data-ll