Here we show that mean clinopyroxene δ18O values decrease concomitantly with crustal thickness and that lavas from Agung possess mantle-like He-Sr-Nd-Pb isotope ratios and clinopyroxene mean equilibrium melt δ18O values of 5.7 ‰ (±0.2 1 SD) indistinguishable from the δ18O range for Mid Ocean Ridge Basalt (MORB). The oxygen isotope composition of the mantle underlying the East Sunda Arc is therefore largely unaffected by subduction-driven metasomatism and may thus represent a sediment-poor arc end-member.Magma plumbing systems underlying subduction zone volcanoes extend from the mantle through the overlying crust and facilitate protracted fractional crystallisation, assimilation, and mixing, which frequently obscures a clear view of mantle source compositions. In order to see through this crustal noise, we present intracrystal Secondary Ion Mass Spectrometry (SIMS) δ Subduction zone volcanoes are underlain by extensive magma plumbing systems, which can obscure original mantle source signals. Here, the authors show that intra-crystal oxygen isotope analysis of clinopyroxenes from the Sunda arc (Indonesia) reveal the δ18 O value of the sub-arc mantle. A prime example of this phenomenon is the Sunda arc in Indonesia . These processes can obscure the primary mantle sources of arc volcanism21 and lead to considerable uncertainty surrounding the compositions of primary arc melts that are produced in the mantle wedge due to subduction.Magma storage beneath subduction zone (arc) volcanoes typically comprises a series of interconnected magma bodies that extend vertically from the mantle to the surface, spanning the entire crustal column  are frequently employed to study magma genesis and evolution at arcs (e.g.32). Initially, our knowledge of the δ18O values of subduction zone lavas relied on analysis of pulverised whole-rocks or mineral separates of ca. 3.5 mm3 sample volume (~10 mg) per analysis (e.g.25), but drawbacks include that whole-rocks are prone to groundmass contamination and/or alteration, and their δ18O values represent weighted averages of their mineral and groundmass constituents16  by Laser Fluorination (LF) largely circumvented these problems and saw a significant improvement in our ability to gain insight into the oxygen isotope compositions of arc systems (e.g.32). Limitations to this approach, however, include that mineral grains often contain inclusions or are partially altered, possessing contrasting δ18O values to the host mineral, which may bias the δ18O value of the total analysed material . The most recent generation of multicollector, large-radius SIMS instruments offer a typical spatial resolution of <10 µm and sample at <1 µm depth , which permits a high degree of spatial control and analysis of unadulterated, inclusion-free domains within individual crystals33 , due to issues relating to the paucity of well-characterised reference materials, which only recently became available for augitic clinopyroxene12. Consequently, there now exists an opportunity to see through the arc and constrain primitive, sub-arc mantle oxygen isotope compositions by approaching this problem in a large-scale, systematic fashion, which we have undertaken here by utilising recently erupted, well-characterised materials from the Sunda arc in Indonesia.How, then, should we accurately determine mineral δs33 Fig. . However20). Most of the recent volcanic products of the Sunda arc are typical subduction zone basaltic-andesites (ca. 50 to 57 wt.% SiO2) and contain relatively scant olivine (cf.38). Clinopyroxene, on the other hand, is a ubiquitous mineral phase in Sunda arc lavas with the potential to provide valuable insights into source compositions and subsequent magmatic evolution owing to its wide crystallisation pressure and its refractory nature with respect to oxygen isotope diffusional exchange22. In this contribution, we present 298 SIMS intracrystal δ18O values from clinopyroxene phenocrysts from four volcanoes forming a partial transect of ca. 550 km length of the Sunda arc in Indonesia, comprising Merapi in Central Java, Kelut in East Java, and Batur and Agung on Bali  and sedimentary accretionary trench infill is virtually absent in this region44. The regional tectonics therefore increase the likelihood of obtaining a robust estimate of primitive sub-arc mantle δ18O values by examining erupted products from Batur and Agung on Bali. By the same token, we can test for crustal overprinting of primitive δ18O values by utilising the erupted products from Merapi and Kelut, where this effect is hypothesised to be stronger due to the greater crustal thickness and the presence of the Kendeng sedimentary basin in the upper crust under these volcanoes, which can promote differentiation and crustal contamination of primary arc melts45.The Sunda arc is a ca. 5600 km long, mixed oceanic-continental arc system generated by subduction of the Indo-Australian plate beneath the Eurasian plate. From Central- to East Java to the islands east of Bali , subduction occurs beneath progressively thinning crust, ranging from ca. 30 km underneath Merapi in Central Java to ≤20 km under Batur and Agung on Bali and further east21. Thermobarometric studies have shown that clinopyroxene in both Merapi’s 2006 lava and Kelut’s 2007 dome-forming lava formed in the mid- to upper- arc crust13, while clinopyroxene in the 1974 lava at Batur and the 1963 lava at Agung formed at the crust-mantle boundary and within the upper mantle beneath Bali14  and radiogenic isotope data (host lava Sr–Nd–Pb isotopes) were obtained at the University of Cape Town, South Africa  versus 5.3 ‰  for the Bali sector , Kelut = 5.6 ‰ , Batur = 5.3 ‰ , Agung = 5.2 ‰   mirrors the overall decreasing depth of the Moho from Merapi towards Bali. Furthermore, the difference between the mean δ18O value for clinopyroxene from Merapi (westernmost studied volcano) and Agung (easternmost studied volcano) is 0.6 ‰, which is significant considering the uncertainties on the SIMS data (see “Methods”). Our SIMS data thus allow us to capture a previously unobserved arc-wide trend in clinopyroxene δ18O values.We grouped the volcanoes studied here into two crustal sectors with Merapi and Kelut assigned to the “Kendeng” sector, due to their spatial association with the Kendeng sedimentary basin in Central to East Java, and Batur and Agung to the “Bali” sector, due to their location on the thinner crust beneath Bali Island Fig. . The ovetor Fig. . All mea26) Fig. . This tr206Pb/204Pb, 207Pb/204Pb, and 208Pb/204Pb ratios of the four volcanoes studied here, at 18.445 , 15.591 , and 38.526 , respectively . We therefore argue that clinopyroxene crystals from Bali, and particularly Agung, largely escaped the effects of crustal overprinting and represent robust archives of primitive Sunda Arc oxygen isotope ratios. The mean clinopyroxene SIMS δ18O values from both Batur and Agung are, in fact, indistinguishable within analytical uncertainty, at 5.3 and 5.2 ‰. These combined isotopic characteristics point to preservation of mantle isotopic signatures in ferromagnesian minerals from Batur and Agung, consistent with clinopyroxene crystallisation at the crust-mantle boundary beneath Bali.Focussing first on the eastern part of the studied arc segment, the Agung lava sample utilised in our SIMS study possesses the most primitive 18O values, we calculated equilibrium melt δ18O values for i) grain averaged clinopyroxene analysed by SIMS  and 5.7 ‰ , respectively. Since Agung shows the most primitive radiogenic isotope compositions of all our samples, as well as a tighter range of δ18O values than Batur, we argue that Agung represents the most robust estimate of the primary δ18O value in the region. This argument is supported by the fact that the calculated melt δ18O values for Agung show strong overlap with the range for MORB, which has most recently been assigned a mean value of 5.5 ‰ 48 .To gain an insight into the corresponding magmatic δ18O values of 4.5 and 4.8 ‰. When combined with two existing olivine LF analyses in the literature, the mean δ18O value for Batur olivine equates to 4.7 ‰ . Olivine from mantle xenoliths yield a predominant δ18O value of 5.2 to 5.3 ‰ (refs. 51), which for ultramafic melt equilibrium at 1300°C would suggest melt δ18O values of 5.5 to 5.6 ‰. Thus, olivine from Batur possess mineral δ18O values lower than olivine equilibrated with ultramafic melt at high temperature. However, this difference is perhaps expected since the δ18O values of olivine in our samples are controlled by mineral-melt fractionation in a crystallising basaltic-andesitic magma. Indeed, the δ18O values for Batur olivine translate to a mean melt value of 5.8 ‰ , identical to the mean melt value obtained from the SIMS clinopyroxene data. These results lead us to suggest that the bulk of Batur olivine and clinopyroxene crystallised under equilibrium conditions at the crust-mantle boundary and overall reflect the composition of primitive mantle-derived melts in the region.We also present two new LF analyses of olivine grains from Batur, with δ18O values along the studied arc segment, we performed a mixing model in δ18O-87Sr/86Sr space utilising average MORB48 and Indian Ocean sediment (“sed A” in ref. 52) . The high δ18O values recorded by some fraction of clinopyroxene at all of the studied volcanoes  must therefore arise from crustal contamination, as discussed below. In summary, an important implication of our results is that the mantle source beneath Bali is seemingly not significantly enriched in 18O from crustal oxygen via subduction metasomatism, consistent with earlier estimates of as little as 1 or 2% crustal oxygen in the source of most oceanic arc lavas28.In order to constrain the influence of source contamination (subducted sediment input) on δ52) Fig. . The mod18O values recorded by clinopyroxene crystals from Merapi and Kelut, which contrast with the lower values for Batur and Agung discussed above. We hypothesise that these overall higher mean δ18O values are a function of the composition and thickness of the underlying arc crust, which is continental to transitional under Central Java (ca. 30 km thickness), and thins eastward to become oceanic in character beneath Bali (ca. 20 km thickness)41, thus facilitating the presence of mafic plumbing systems in the mid- to upper arc crust in Central and Eastern Java21. The mean clinopyroxene δ18O values obtained for Kelut and Merapi calculate to melt δ18O values of 6.2 and 6.5 ‰, respectively, which are significantly higher than each of the calculated melt values for the Bali sector (5.8 ‰ on average), Agung volcano (5.7 ‰ on average), and unmodified, MORB-type upper mantle melts (5.5 ‰ on average48). The elevated δ18O melt values from Central and East Java might reflect the addition of a slab-derived fluid possessing exotic oxygen isotope compositions to the magma source region (cf. ref. 32). However, the relatively more evolved compositions of Kelut and Merapi lavas compared to the Balinese lavas  to basaltic andesite (SiO2 of 55 wt.%) under various crystallisation conditions will increase the melt δ18O value by <0.2 ‰  for a bulk lava SiO2 content of 56 wt.% at Merapi.We now consider the relatively high mean δ2 ‰ Fig. . Fractio18O values grew was contaminated in the mid- to upper crust by crustal material enriched in 18O, such as carbonates, calc-silicates and/or siliciclastic sedimentary rocks derived from sedimentary basin fill and altered volcanic debris, which are all present to variable degrees under Central and East Java53. Moreover, recently erupted lavas at Kelut and Merapi contain a variety of crustal xenoliths, including some of siliciclastic and carbonate sedimentary origin, which provide first-hand evidence for magma–crust interaction in the region (e.g. refs. 53). The Sr isotope ratios in lavas from Central and East Java are consistent with this model, as the Merapi and Kelut lavas utilised in this study have mean 87Sr/86Sr ratios of 0.705716 ± 0.000010 and 0.704244 ± 0.000010, respectively, which extend beyond the likely 87Sr/86Sr ratios of both uncontaminated arc volcanics54 and lavas from Batur and Agung. Indeed, the Sr isotope composition of the Merapi 2006 lava samples overlap regional crustal values and are similar to data reported in previous studies that argue for extensive magma–crust interaction at Merapi55. Furthermore, helium isotopes in Merapi fumarole gases range from 5.4 to 5.7 RA (ref. 47) and are distinct from the mantle-like RA values reported for minerals from Batur and Agung , suggesting that Merapi magma interacted with crustal material with characteristically low 3He/4He ratios . By employing a combined Rayleigh fractionation and binary mixing model and utilising a pristine mantle-type starting composition, the elevated melt δ18O values at Merapi and Kelut can be replicated by invoking ca. 10 % fractional crystallisation accompanied by a maximum of 20 wt.% crustal assimilation. This result is similar to mixing models in δ18O–87Sr/86Sr space, where we can see that some of the clinopyroxene crystals at Batur and Kelut record less than 10 % mixing between a source contaminated parent melt and crustal lithologies, whereas some clinopyroxene grains at Merapi record slightly higher degrees of mixing, again with a maximum of 20 %  and low δ18O material  cannot therefore be excluded for Bali sector volcanoes, but this does not seem to be a major process at Agung, which shows a relatively tight range of clinopyroxene δ18O values.We also note here that a small number of clinopyroxene grains from Batur record both relatively high and low δues Fig. . Minor d31 or laser fluorination (LF) of single or several crystals37. The SIMS method differs in that it analyses a sample volume many orders of magnitude smaller than these methods and offers a high degree of spatial control during analysis. Here we compare our new SIMS data to the available LF data for the Sunda arc, disregarding data obtained on relatively large volume, pulverised bulk mineral separates analysed by CF in order to facilitate as direct a comparison as possible12  of powdered mineral separates37 fit into the step-wise decrease in mean pyroxene δ18O values from west to east, giving a sequence of: Merapi = 5.8 ‰, Kelut = 5.6 ‰, Ijen = 5.5 ‰, Batur = 5.3 ‰, and Agung = 5.2 ‰ , similar to Merapi. Two-pyroxene geothermobarometry has shown that Gede pyroxene crystallised at 13–24 km depth within the arc crust30, which is similar to pyroxene crystallisation depths of 9–21 km depth calculated for the Merapi 2006 eruption12, thus offering a possible explanation for their similar δ18O values. Pyroxene grains from Salak volcano, on the other hand, record a relatively low mean δ18O value of 5.3 ‰ (ref. 36), more akin to the δ18O values observed on Bali. We cannot fully assess whether Salak pyroxenes truly record mantle-like δ18O values, as only three analyses are available. However, it is possible that either i) a relatively unmodified δ18O value was sampled at Salak36 or ii) Salak magma assimilated 18O-depleted lower arc crust whose δ18O values were modified by high temperature alteration52. Assimilation of low δ18O hydrothermally altered crust has been suggested based on a small number of low δ18O values in plagioclase at Merapi35, however, in the case of Merapi, the dominant crustal influence appears to come from 18O-enriched crustal sedimentary rocks31 30, one value for Ijen in East Java (5.0 ‰)37, and four values for Batur on Bali . Although the arc-wide olivine dataset is comparatively small, a decrease in mean δ18O olivine values from West Java to Bali can be observed, paralleling the trend in the pyroxene data , offering a unique window into both lower crustal assimilation (Batur) and primitive arc magma compositions (Agung). The clinopyroxene data from Agung are a particularly robust reflection of mantle-derived melts supplying the arc, because their δ18O values are correlated with independent isotopic markers of mantle derivation, including relatively low 87Sr/86Sr, 206Pb/204Pb, 207Pb/204Pb, 208Pb/204Pb, and high 3He/4He ratios. The results of this study lead us to conclude that Sunda arc primitive melts are not significantly metasomatised or enriched in 18O from the subducting slab and they possess a mean δ18O value of 5.7 ‰ (±0.2 1sd), in agreement with the δ18O range for MORB-type upper mantle-derived melts. The intersection of trans-crustal mafic plumbing systems and a relatively thick arc crust in parts of the Sunda arc  exerts a major control in terms of modifying primary clinopyroxene δ18O values and causing elevated oxygen isotope ratios. Intracrystal oxygen isotope analysis of clinopyroxene thus offers an effective tool that, when applied systematically on a regional scale, can disentangle mantle from crustal oxygen compositions in arc systems.A first-order conclusion of this study is that the δ61. Finally, the sample mounts were coated with carbon for Electron Probe Micro Analysis (EPMA), after which the carbon coat was removed from the sample mounts by gently polishing the surface with a 1 µm diamond solution. The sample mounts were then cleaned with pure ethanol and coated with a 20 nm gold layer prior to SIMS analysis. All sample mounts were imaged by Scanning Electron Microscopy (SEM) before analysis to create sample maps to aid the placement of SIMS analysis spots on inclusion-free crystal domains. The sample mounts were also inspected after SIMS analysis using optical microscopy and SEM to verify that the analysis pits did not intersect foreign material.Crystal aliquots of clinopyroxene and olivine were separated from crushed lava samples under a binocular microscope and cleaned in pure ethanol in preparation for oxygen isotope analysis by laser fluorination (LF) and Secondary Ion Mass Spectrometry (SIMS). One split of crystals was analysed by LF (see below) and, in the case of clinopyroxene, another split was mounted on double-sided tape under an optical microscope, adjacent to a fragment of the reference material NRM-AG-1. The mounts were then cast in epoxy resin and polished using an automated polishing machine employing progressively fining diamond suspensions . Care was taken to ensure that crystals were placed within a distance of >5 mm from the edge of the mount, and that the mount was polished flat with minimal relief in order to avoid analytical artefacts associated with sample geometry and topography14. The run conditions were 15 kV accelerating voltage and 10 nA probe current with 10 s on peak and 5 s on lower and upper background, with a beam diameter of 2 μm for pyroxene analysis. The following standards were used for calibration: wollastonite for Ca and Si, pyrophanite (MnTiO3) for Mn and Ti, magnesium oxide for Mg, orthoclase for K, albite for Na, aluminium oxide for Al, fayalite for Fe, nickel oxide for Ni, and chromium oxide for Cr. Analytical precision was measured on Smithsonian Institute mineral standards, including USNM 111312 (olivine), USNM 122142 (Cr-augite), USNM 137041 (anorthite), USNM 115900 (Ca-plagioclase), and USNM 133868 (anorthoclase). Uncertainties on the standards are as follows: SiO2, Al2O3, MgO and CaO ≤ 1.5% s.d., FeO ≤ 2.2% s.d., Na2O in plagioclase and clinopyroxene ≤ 4.5% s.d., and minor elements ≤ 10% s.d.Mineral chemical data were acquired using the field-emission gun source JEOL JXA-8530F Hyperprobe (FEG-EPMA) at Uppsala University, Sweden following the protocols employed by ref. 18O/16O)sample/(18O/16O)V-SMOW-1]*1000. Full analytical details of the laser fluorination method employed at UCT are given in ref. 62. Measured values of the UCT internal standard MON GT  were used to normalise the raw data and correct for drift in the reference gas. The δ18O value of MON GT was established by cross-calibration with the UWG-2 garnet standard of ref. 63 and San Carlos olivine. The long-term average difference in δ18O values of duplicates of MON GT is 0.12 ‰ (n = 341), which corresponds to a 2σ value of 0.16 ‰.Laser fluorination of mineral grains was carried out in the Department of Geological Sciences, University of Cape Town (UCT), South Africa, using mineral fragments weighing ca. 2 mg for each independent run, with the number of grains in each analysis varying from one to three. The results are reported in standard δ-notation relative to V-SMOW (Vienna Standard Mean Ocean Water), where δ = [ of data acquisition using two Faraday detectors in the multicollector system operating at a common mass resolution of ca. 2500. The secondary magnet field was regulated at high precision using a Metrolab NMR teslameter. To monitor external reproducibility during the analytical sessions, a sample-bracketing procedure was employed, whereby every block of four or five sample analyses was bracketed before and after by two analyses of augitic reference material NRM-AG-1 12. In all analytical sessions, the data were corrected for within-session ratio drift and instrumental mass fractionation (IMF) using NRM-AG-1, as described in detail in ref. 12. All oxygen isotope ratios are reported in standard δ-notation.The SIMS instrumentation and methods employed in this study are based on refs. 16O intensity was 1.8 ×109 cps. Throughout the Kelut analytical session, external precision was 0.14 ‰ (1 sd) on NRM-AG-1 and internal precision was on average 0.08 ‰ . The average 16O intensity during the Kelut session was 2.8 ×109 cps. For the Agung 2020 analytical session, external precision was 0.10 ‰ (1 sd) on NRM-AG-1 and internal precision was on average ±0.07 ‰ . The average 16O intensity was 2.7 ×109 cps. Note that the 2020 analytical session utilised the recently installed low noise high-gain (1012Ω) Faraday amplifier at NordSIMS, which improves the signal/noise level for 18O. Reproducibility and precision during the Merapi analytical session is reported in ref. 12.Throughout the Batur and Agung 2017 analytical session, external precision was 0.27‰ (1 sd) on NRM-AG-1 and internal precision was on average 0.12 ‰ . The average 65 and those spots were filtered purely as a precaution, even though we did not observe strong evidence for their isotope ratios being biased.All mounts were analysed by optical microscopy after SIMS analysis to verify that analysis spots were not placed on fractures or foreign phases. No rejections were made on this basis, however, a total of 8 spot analyses were filtered from the Batur dataset due to their relatively high DTFA (field aperture) values, which were in the range -60 to -80. These high DTFA values might indicate a degree of beam misalignment in which isotope fractionation can occur at the entrance slit crossover18O value of each grain before plotting reduced over-representation of outlying individual spot values and that the overall mean δ18O values per volcano remain almost unchanged, while the interquartile ranges per group change very little. In cases where only one analysis point was available for an individual grain, this value was assigned to the mineral grain in question for use in the mineral grain averaged plots. A detailed explanation of our data handling and a comparison between SIMS and LF datasets is provided in Supplementary Figure 2. We also emphasise that we observed no statistically significant relationship between clinopyroxene mean δ18O values and clinopyroxene mean major element composition . Instrumental mass fractionation (IMF) during SIMS δ18O analysis of augitic clinopyroxene due to matrix effects associated with small variations in mineral Ca, Mg, or Fe contents thus appears to be negligible (cf. ref. 66).In the main text, we display and discuss mineral grain averaged SIMS datasets. Note that averaging the δ67) and are summarised here. After sample dissolution, a Thermo iCap quadrupole inductively coupled plasma-mass spectrometer (ICP-MS) was employed for trace element analysis, following procedures similar to those described in ref. 68, with routine internal two-sigma analytical errors for individual analyses < 3% RSD. For isotope ratios, elemental separation chemistry followed routines after ref. 69. All isotope ratio analyses were conducted using a Nu Instruments NuPlasma, with the addition of a DSN-1000 desolvating nebuliser during Nd and Pb analyses. 87Sr/86Sr data were referenced to a value of 0.710255 for SRM 987, with all ratios corrected for interference on mass 87 using the measured 85Rb signal and the natural 87Rb/85Rb ratio. Instrumental mass fractionation was corrected for using the exponential law and a value of 0.1194 for 86Sr/88Sr. 143Nd/144Nd data were referenced to a value of 0.512155 for JNdi-1, with all ratios corrected for Sm and Ce interferences using measured 147Sm and 140Ce signals and the natural isotope ratios. Instrumental mass fractionation was corrected for using the exponential law and a value of 0.7219 for 146Nd/144Nd. All Pb isotope data were referenced to values for SRM 981 of 208Pb/204Pb, 207Pb/204Pb, 206Pb/204Pb using normalising values of 36.7219, 15.4963, 16.9405. Instrumental mass fractionation was corrected for using the exponential law and a 205Tl/203Tl value of 2.3889 for SRM 997 added to all standards and samples (Pb:Tl of ±10:1). The andesitic reference material GSJ-JA-2 was analysed as an unknown alongside the samples in this study and gave the following ratios: 87Sr/86Sr = 0.706406 ± 0.000013; 143Nd/144Nd = 0.512543 ± 0.000012; 208Pb/204Pb = 38.6703 ± 0.0029, 207Pb/204Pb = 15.6054 ± 0.0010, 206Pb/204Pb = 18.4008 ± 0.0009. These results are in good agreement with accepted values from the GeoReM database . Total procedural blanks in the radiogenic isotope facility at the University of Cape Town are < 250 pg Sr; < 100 pg Nd and < 200 pg Pb and are insignificant relative to the concentrations in Indonesian lavas. Thus, no blank corrections were applied.Most of the radiogenic isotope data reported here were obtained in 2020 by Multicollector Inductively Coupled Plasma Mass Spectrometry (MC-ICPMS) at the Department of Geological Sciences, University of Cape Town (UCT), South Africa, including Sr–Nd–Pb isotope ratios for two samples of the Agung 1963 lava, one sample of the Batur 1974 lava, one sample of the Kelut 2007 dome-forming lava, and two samples of the Merapi 2006 lava. All analysed materials consisted of pulverised aliquots of fresh, bulk lava samples that were split from the same lava samples utilised for SIMS analysis of clinopyroxene phenocrysts. The sample preparation and analytical methods employed have previously been described in detail . Procedural blank values at the VU are typically ≤ 100 pg, which is negligible when compared to the 500 ng Sr loaded per sample and therefore no blank corrections were made. Neodymium separation was carried out as a two-column procedure, utilising conventional TRU Spec and LN Spec cation exchange chromatography69. Neodymium isotopes were analysed using a Finnigan Neptune MC-ICPMS, employing the method in ref. 72. During the course of this study, repeat analysis of the CIGO internal standard gave a mean 143Nd/144Nd ratio of 0.511349 ± 0.000010  in agreement with previous TIMS measurements of the CIGO standard of 0.511342 ± 0.000007 73. Repeated analyses (n = 2) of BHVO-2 gave a mean 143Nd/144Nd ratio of 0.512962 ± 0.000007 (2SE). Nd blanks were less than 10 ppb which is negligible compared to the 200–400 ppb (Nd) measuring solutions used and hence no blank corrections were applied.In addition to the samples analysed at UCT, one sample of each of the Kelut 2007 dome-forming lava and the Merapi 2006 lava were analysed in 2011 for trace element concentrations by ICP-MS, Sr isotope ratios by Thermal Ionisation Mass Spectrometry (TIMS), and Nd isotope ratios by MC-ICPMS at the Vrije Universiteit Amsterdam (VUA), Netherlands. These data formed part of the MSc thesis of M. van HeldenSupplementary Information


This study also explored how soil available nutrients are affected by different forest types. Lutou forest research station, located in Hunan Province, central China, was used as the study area. Results demonstrated that the topsoil layer had higher TC and TN content in the mixed forest stand, resulting in a better quality of organic materials in the topsoil layer in the mixed forest than NBF and CPF. In general, soil TC, TN, and δ15N varied significantly in different soil depths and forest types. However, the forest type did not exhibit any significant effect on δ13C. Overall, soil δ13C was significantly enriched in CPF, and δ15N values were enriched in mixed forest. Foliar C content varied significantly among forest types, whereas foliar N content was not significantly different. No big differences were observed for foliar δ15N and δ13C across forest types. However, foliar δ13C and δ15N were positively related to soil δ13C and δ15N, respectively. Foliar N, soil and foliar C:N ratio, soil moisture content (SMC), and forest type were observed as the major influential factors affecting isotopic natural abundance, whereas soil pH was not significantly correlated. In addition, forest type change and soil depth increment had a significant effect on soil nutrient availability. In general, soil nutrient availability was higher in mixed forest. Our findings implied that forest type and soil depth alter TC, TN, and soil δ15N, whereas δ13C was only driven by soil depth. Moreover, plantations led to a decline in soil available nutrient content compared with NBF and mixed forest stands.Natural isotopic abundance in soil and foliar can provide integrated information related to the long-term alterations of carbon (C) and nitrogen (N) cycles in forest ecosystems. We evaluated total carbon (TC), total nitrogen (TN), and isotopic natural abundance of C (δ SpeciN cycles ,7,8. OveN cycles . Despiteprofiles . Therefo13C and δ15N, whereas a higher decomposition rate enhances δ13C and δ15N [15N [Land is an indispensable natural resource for ecosystem functioning. In general, soil organic carbon usually changes with human activities and land-use change. Litter inputs usually lower the soil δand δ15N ,11. Howeand δ15N ,12. In a15N [15N ,15,16. T15N [15N ,18.13C and 15N natural abundance and their relation with influential factors is scarce regarding the different sub-tropical forest types of eastern Asia. Soil nutrient availability is a crucial indicator of soil fertility [15N, δ13C, TC, and TN content in leaves and soil, and variability in soil fertility is expected to provide insights into the species’ resource utilization efficiency in an ecosystem and the consequences of different silvicultural methods for these biogeochemical cycles.Plantations are usually fast-growing species with shorter rotation cycles compared to broadleaved natural forests and mixed-species forests. Ever-increasing forest product demand has led to an upsurge in plantation forests worldwide . Since tertility . Soil feertility ,29,30. Tertility ,32,33. T13C and 15N natural abundance in vertical depth soil profiles in different forest types; and (ii) to analyze the effect of forest type change (different forest types) on soil nutrient availability. Soil and foliar stable isotopic ratios are influenced by atmospheric N2 inputs and processes that regulate N cycling and soil organic matter (SOM) transformation. Moreover, different forest management regimes influence the C and N isotopic abundance, for example, clear-cutting practices in plantations increase soil N availability, which can enrich δ15N. Therefore, we hypothesize that: (a) TC and TN content and δ13C and δ15N in soil and foliar would significantly vary in different forest types and at different soil depths; moreover, (b) forest structure and composition would also considerably influence the soil nutrient availability.The objectives of this study were: (i) to characterize the variability of soil and foliar total C (TC), total N (TN) content, and The study was conducted at the Lutou forest ecosystem observation and research Station, Pingjiang County, Yueyang, Hunan Province, China. The geographic coordinates of the research station are 113°51′52″~113°58′24″ E, 28°31′17″~28°38′00″ N . The totNatural broadleaved forest (NBF), coniferous plantation forest (CPF), and mixed forest stands were used in the study. NBF and mixed forest stands were adjacent to each other, and the CPF stand was at a 300–400 m (0.19–0.25 mile) distance from the first two forest stands. All three forest stands were present in the same research station. There were no water channels present on the ground in any forest type and watering was natural because of the abundant rainfall. Litterfall biomass and understory vegetation were apparently more in the mixed forest stand. We selected those trees where shrub presence around the canopy projection area of trees was significantly less than the usual shrub presence in the stands. Therefore, the direct influence of the shrub layer would be minimal. Still, these are field conditions; hence indirect effects might be possible. The characteristics of the study area and forest types are shown in Castanopsis eyeri (Fagaceae) is one of them [C. eyeri is a vital part of forest ecosystems and provides services such as water conservation, biodiversity protection, biomass maintenance, and local climate regulation [C. eyeri (NBF) stand in the research station is the largest and most complete community of Castonpsis, and therefore has crucial protection, scientific research, and landscape values. The forest is neat, and the forest canopy undulating. At the Lutou forest farm, it is mainly distributed on mountain slopes. The average slope gradient is 20–25°. The shrub layer includes species such as Rhododendron simsii, Ilex formosana, Daphniphyllum oldhami, Toxicodendron vernicifluum, Cyclobalanopsis glauca, Eurya tetragonoclada, Symplocos sumuntia, Rhododendron simiarum, Lindera aggregate, Eurya muricat, Dendropanax dentiger, and Myrica rubra.The mid-subtropical zone of China is covered with natural forests dominated by broadleaf evergreen species, and  of them . C. eyergulation . The C. Cunninghamia lanceolata) is a typical evergreen coniferous timber tree species. The pure natural forest of C. lanceolata is rare to find, and mainly presents as an artificial forest plantation. The CPF stand at Lutou forest station are present on both flat soil and mountain slopes. The average slope gradient is 18–23°. Besides timber production, climate regulation, and soil and water conservation, it also provides biomass energy [Litsea cubeba, Eurya mauricata, Rhus chinensis, Sapium discolor, Rhododendron mariesii, Diplospora dubia, Ilex chinensis, and Rubus lambertianus.Chinese fir  NBF, (2) CPF, and (3) mixed forest stands is mentioned below.Sampling was carried out in the first week of October 2020. Soil samples were collected from nine selected locations (three locations from each forest type). Three 20 × 20 m plots were established in each forest stand. The soil samples were obtained with a steel soil auger (3.5 cm diameter) up to a depth of 0–10, 10–20, and 20–40 cm soil. After collection, soil samples were appropriately cleaned, and roots/stones were sorted out. Three composite samples per forest type per soil depth were prepared for the analysis. A sufficient number of mature and healthy foliage (5–8 per tree) of each forest type (three plots in each stand) was collected from four to six different individuals (at least 5 m apart from each other) using a long-handled pruner and pooled three composite samples for each forest type. Leaves were placed in a clean, perforated plastic bag . We trie13C and δ15N, C and N content, SOC, and soil nutrient availability analysis [Field moist soils were air-dried at room temperature and sieved through a 2 mm mesh size to remove stones, roots, and plant residues. The foliage samples were gently rinsed with distilled water and oven-dried for 72 h at 65 °C. Later, soil and foliar samples were finely ground with a ball mill  and used to measure δanalysis ,15.13C, δ15N, TC, and TN content were measured using an isotope ratio mass spectrometer (IRMS) , connected to a CN elemental analyzer . C and N isotopic abundances were calculated as δ13C and δ15N (‰) using the following formula:sample is the stable isotopic ratio in the samples and Rstandard is the ratio in the standard. The Vienna Pee Dee Belemnite (VPDB) was used as a standard for δ13C and atmospheric N2 was used as the standard for δ15N. The precision of isotopic composition was checked using internal standards  and positive and negative values were observed. Positive delta values indicate that there is a greater percentage of isotope presence relative to the standard whereas negative values indicate a lesser percentage of isotope presence relative to the standard. In general, the analytical precision for δ13C and δ15N was better than 0.2‰ [δhan 0.2‰ ,15.Available nitrogen (AN) was determined by the Kjeldahl method . Availab13C, δ15N and, foliar TC and TN content among forest types. A regression analysis was conducted to analyze the foliar and soil isotopic abundance relationship. The Pearson correlation test was performed to observe the relationship between isotopic dynamics and influential factors. Means that exhibited significant differences were compared using post-hoc Tukey’s HSD significance test. All statistical analyses were performed using the SPSS Statistical Package .Two-way analysis of variance (ANOVA) at a 5% probability level was performed to determine the effects of forest types and different soil depth profiles on the natural abundance of C and N isotopes, soil TC and TN content, and soil nutrient availability. One-way ANOVA at 5% probability level was performed to determine the differences between foliar δp < 0.05) and soil depths (p < 0.001)  . Soil TNto 14.06 . Soil TNto 14.06 .p < 0.05), whereas foliar N content and foliar C:N ratio were not significantly different   .15N varied significantly among forest types (p > 0.001) and soil depths (p = 0.048). However, forest type (p = 0.078) and interaction of forest type × soil depth (p = 0.329) had no significant effect on soil δ13C, whereas soil δ13C varied significantly with soil depth (p = 0.028) (15N-soil depth (r = 0.606) and soil δ13C-soil depth (r = 0.470). Overall, soil δ15N values ranged from −1.61‰ to 5.19‰, and soil δ13C values ranged from −28.23% to −25.00% (Two-way ANOVA depicted that soil δ= 0.028) . A posit −25.00% .13C (p = 0.03) and foliar δ15N (p = 0.047). Foliar δ15N values ranged from 6.39‰ to 8.90‰, and foliar δ13C values ranged from −25.4‰ to −29.7‰. Foliar δ15N was enriched in the NBF stand, and δ13C was less negative in the mixed forest stand  and soil and foliar δ15N  across forest types and soil depths  (−1 and 109 mg·kg−1) was observed to be higher in the mixed forest; in 10–20 cm, it was higher in the NBF (155 mg·kg−1). AN content decreased as the soil depth increased in the mixed forest and NBF. AP content ranged from 29 mg·kg−1 to 82 mg·kg−1. In the 0–10 cm soil layer, the highest AP content was observed in CPF (71 mg·kg−1), while in the remaining two layers (10–20 and 20–40 cm), the maximum content was measured in the mixed forest. Among all forest types and soil depths, AK ranged from 12 g·kg−1 to 147 g·kg−1. In the topmost soil layer, it was higher in the mixed forest stand. In both the 10–20 cm and 20–40 cm layers, it was higher in NBF. Generally, soil nutrient content decreased as the soil depth increased across all forest types  . In the st types .Acer psedoplatanus, Fraxinus excelsior, and Tilia platyphyllos significantly affect the C:N ratio and it was related mainly to litter quality. Lovett et al. [Among all forest types, soil TN content decreased as the soil depth increased. In the topsoil layer, both TC and TN contents were enriched in the mixed forest. It indicates that litter input was higher in the mixed forest due to mixed species composition and more understory vegetation, which increased soil organic matter (SOM) more than both NBF and CPF stands. Since soil organic carbon changes with the vertical soil depth profiles, soil depth is widely used as a SOM decomposition rate indicator ,45,46. It et al.  also revt et al. ,51.15N and 13C was observed in CPF. Enriched soil δ15N and δ13C in CFP could be due to more intensive forest management practices than that in the NBF and mixed forest stands because the plantations are artificially managed forests with added activities of humans and livestock. Pardo et al. [13C and δ15N in plantation forests because of higher soil C and N loss such as enhanced nitrification followed by nitrate loss, which could have a direct impact on isotopic abundance. Rehman et al. [In this study, a higher abundance of soil o et al.  also repn et al.  reportedn et al. ,51.15N, whereas soil δ13C was only impacted by soil depth; moreover, both the soil δ13C and δ15N were enriched in the topmost soil layer. Higher isotopic values in the upper soil layer can be due to higher SOC. Ellert and Janzen [13C in the topsoil, whereas lesser C content in the deeper layers determines the lesser δ13C values. Ngaba et al. [15N values increased under N-rich conditions; in our study, higher foliar and soil δ15N were observed in CPF. On average, foliar and soil δ15N patterns in sub-tropical forests are higher than other forest types because of the gaseous N losses related to microbial activities. Our study showed foliar C and N positively correlated with the soil isotopic values; this indicates that foliar isotopic values of different species are consistent concerning soil values.Forest type and soil depth significantly impacted soil δd Janzen  reporteda et al.  also rep13C and δ15N in soil and foliage, we observed that SMC, foliar TN, and foliar C:N ratio was usually significantly related to soil foliar 13C and 15N natural abundance, either positively or negatively. These factors can affect biogeochemical processes such as N mineralization, nitrification/denitrification, soil respiration, root dynamics, thus altering the rate of δ15N [13C of the plant and soil. Philips et al. [13C depends on moisture conditions, either caused by low atmospheric humidity, less precipitation, or low soil moisture. Moreover, drought can also potentially influence the C fluxes and soil δ13C [13C. While analyzing the 15N abundance between the north and south slopes of forests, Chen et al. [When we studied the correlation of potentially influential factors concerning δ of δ15N ,56. Moiss et al.  stated toil δ13C . C isotooil δ13C . Ngaba eoil δ13C  also staIn our study, a significant difference was observed in variability in macronutrient availability among all forest types. Generally, AN and AK were higher in mixed forest stand and AP in CPF. Plantation forests mostly have fast-growing species with successive short rotations; hence, the fast growth demands more extensive nutrient availability, which causes an imbalance in soils under plantations compared to mixed-species forest. Kooch et al.  reported13C, δ15N, TC, and TN content of soil and foliage and soil nutrient availability is affected by different forest types. Forest type and soil depth significantly affected the soil δ15N, while forest type effect on soil δ13C was not significant. Significant changes in soil δ15N among different forest types indicated that 15N signatures might dominate the processes of N cycling in different forest types. Across forest types and soil depths, the observed δ13C and δ15N variations were attributed to SMC, soil TC, soil TN, and soil C:N ratio. Variations in foliar δ13C were accredited to differences in soil BD and foliar C:N ratio, whereas variation in foliar δ15N was accredited to SMC, foliar TN, and TC. Our study also highlights that, on average, available forms of soil nutrients were less in monoculture Chinese fir stands (CPF) than NBF and mixed forests. This difference was possibly caused by higher litter biomass accumulation from broadleaved and mixed-species litter compared to CPF. Thus, the presence of broadleaved species within a monoculture stand along with other management practices such as the introduction of multi-layering and multi-aged plantations, avoiding clear-cutting practice, and burying the cutting leftover rather than burning could improve soil health, sustainable management, and production, which would enhance both economic and environmental aspects to the forest industry. Overall, our results highlight the importance of forest types and composition when studying biogeochemical cycling and its response in different terrestrial ecosystems. Moreover, this study will also help to further understand the role of C and N cycles concerning soil productivity.This study explored how δ


Resistance to Fusarium head blight (FHB) of spelt wheat was investigated in field trials carried out at three European locations between 2016 and 2018. Resistance was assessed after artificial inoculation by visual scoring of symptoms and the determination of the contamination of grains and glumes with the mycotoxin deoxynivalenol (DON). It was found that typical spelt traits such as tall plant height, lax spikes, and tough glumes play a role as passive resistance factors. Across all test environments, modern spelt varieties with a significantly reduced plant height showed a significantly higher susceptibility to FHB and a higher contamination of the grains with DON compared to old landraces/varieties and plant genetic resources. Similarly, the lowest mycotoxin levels in grains were found only in old landraces and varieties, while the highest DON concentration was observed mainly in modern varieties. The results obtained can be used for the selection of suitable parental material for breeding spelt with improved FHB resistance. Fusarium species (Fusarium head blight (FHB) is a devastating fungal disease of wheat provoked by various  species . Since t species . In the  species . An inte species . Resista species . Resista species , whereas species . A few s species , wild em species , cultiva species , Georgia species , spelt  Thell.] is a hulled hexaploid wheat with a long cultivation tradition in southwest Germany, Switzerland, Austrian Alpine valleys, and a few other remote areas of Europe , Agroscope , Crop Research Institute , Institute of Agrobotany , IPK , LSA Hohenheim , and NordGen . The diversity panel contains landraces, old and obsolete varieties, as well as modern varieties and breeding lines, which were classified into three groups in order to investigate the effect of breeding: landraces and old varieties from (i) Switzerland and Germany; (ii) modern varieties (post 1970) from Switzerland, Germany, and Belgium; and (iii) plant genetic resources and genebank accessions of uncertain breeding status (PGR). The Czech variety “Rubiota” (released 1981), Austrian “Ebners Rotkorn” (released 1999), and Hungarian “Öko 10” (released 2000) were treated as landraces/old varieties, as they were selected from landrace accessions.A spelt diversity panel including 80 genotypes of winter spelt was established within the FP7 project HealthyMinorCerealsField experiments with artificial inoculation of the HMC spelt diversity panel were conducted in Austria, Czechia, and Switzerland from 2016 to 2018, resulting in nine test environments. Artificial inoculation was performed at full flowering of the spikes (BBCH 65) by spraying a spore suspension. FHB symptoms were scored several times after inoculation by visual evaluation of the percentage of diseased spikelets of whole plots. Plant height was measured from the ground to the top of the spike.F. culmorum isolate IFA-91015 at a concentration of 2.5 × 104 conidia per ml in tap water at a rate of 100 ml m–2. Inoculations were repeated four times in 2 day intervals in order to inoculate each plot at least once at maximum flowering. After each inoculation, the crop stand was mist irrigated for 21 h with a total of 4–5 mm water. FHB severity scoring (% diseased spikelets) started on day 10 after anthesis and was repeated four times in 4 day intervals. Anther retention was recorded in the 2017 trial, as described by In Austria, the field trials were carried out in Tulln . The field experiments were performed similar as described in F. culmorum  on a 1–9 scale, as described . The spelt genotypes were sown manually as spikelets in hill plots with three replications. At the flowering period, bunches of 10 spikes were inoculated with the highly pathogenic isolate B of culmorum  at a conescribed .F. culmorum spores. Spores were kindly provided by Agroscope, Changins. Inoculation started at the beginning of flowering and was repeated three times in 2016 and 2017 and two times in 2018 in a 2 day interval. Scoring of visual symptoms  was started 10 days after the last inoculation and repeated three times in a 7 day interval.In Switzerland, the trial was sown under organic conditions in Feldbach . The genotypes were sown in 50 cm-long double rows with two replications. The inoculum was a conidial suspension of ® FAST DON enzyme immunoassay . Additionally, the DON content of the glumes was also determined in the Czech samples from 2017 and 2018. A representative grain sample was ground by a Yellowline IKA A10 Analytical Grinder Universal Mill  and thoroughly mixed. Afterward, 100 ml of distilled water were added to 5 g ground sample and shaken for 3 min in a horizontal rotary shaker at 200 rpm. After filtration, 50 μl of the filtrate were used for the ELISA test together with standards according to the manufacturer’s instructions. The absorption of the final solution was measured at 450 nm using a SunriseTM spectrophotometer. Data were processed with RIDASOFT®Win.NET software (R-Biopharm AG).Dehulled seed samples from all sites were analyzed for deoxynivalenol (DON) content by the RIDASCREENPi for each individual genotype according to Pi is defined as the distance mean square between a genotype’s response and each environment’s best response averaged over all environments. The Pi values for FHB and DON were further used to calculate a Fusarium rating as devised by FHB scoring values of the last evaluation date from each test site were converted to the 1–9 scale as described by p < 0.0001) for all years in Austria  and Czechia in 2016 (ρ = 0.54–0.57), however, not significant for Switzerland and Czechia in 2016 and 2017, respectively. In the other environments, the correlation between field replications was significant (p < 0.05) but weak (ρ = 0.21–0.34).The genotypic variation in the FHB score across environments is displayed in 2oh = 0.77. Considering the single countries, the highest heritability was observed for Austria (2oh = 0.79), where the genotypic variance component was 1.4 times the variance due to the residual error. Heritabilities for Czechia (2oh = 0.54) and Switzerland (2oh = 0.43) were medium. In these cases, the variance component for the residual error was four to five times higher than the variance explained by the genotypes.Heritability for FHB score was high with 2oh = 0.61 lower than for FHB and did not change if the two before mentioned environments were excluded from analysis. Similarly, to FHB score, the heritability was highest for the Austrian trials (2oh = 0.67) and significantly lower for Switzerland and Czechia . Similarly, to the visual scoring of FHB symptoms, the correlations between the single field replications vary between countries and years. For Austria, the correlations are significant (p < 0.0001) for each year ; for Czechia (ρ = 0.61–0.75) and Switzerland (ρ = 0.58), similar correlations were observed in 2016, whereas the field replications were not correlated in Czechia 2017 (ρ = 0.15–0.27) and weak (ρ = 0.35–0.47) but significant (p < 0.01) in the other environments.For DON contamination of the grains, genetic variation was negligible in Austria 2017 and Switzerland 2016 . HeritabPi = 0.18. Considering a cutoff of 10%, the other genotypes with a low Pi were “Toess 6D” (Pi = 0.24), “Fuggers Babenhauser Zuchtvesen” (Pi = 0.28), “Sofia 1” (Pi = 0.45), “Gugg 4H” (Pi = 0.48), “Roter Schlegeldinkel” (Pi = 0.57), “Riniken Weißkorn” (Pi = 0.71), and “Strickhof” (Pi = 0.75). Hence, all genotypes with low disease severity were from the group of old landraces/varieties and plant genetic resources, respectively. The best modern variety was not ranked before 25 . In contrary, among the 10% most susceptible genotypes with the highest Pi values, six out of eight were modern varieties, i.e., “Alkor” (Pi = 5.31), “Schwabenspelz” (Pi = 5.46), “Titan” (Pi = 5.6), “Badenkrone” (Pi = 5.96), “Zollernspelz” (Pi = 6.02), and “Cosmos” (Pi = 6.09). “Thürig Rotkorn Nr. 4” (Pi = 5.84) and “Waggershauser Hohenheimer Weißer Kolben” (Pi = 6.33) were the only old varieties within the group of highly susceptible spelt varieties , “Farnsburger Rotkorn Nr. 6” (Pi = 10.4), “Gugg 4E” (Pi = 13.8), “Ebners Rotkorn” (Pi = 20.4), “Gugg 9F” (Pi = 27.9), “Toess 6D” (Pi = 32.7), “Roter Schlegeldinkel” (Pi = 35.2), and “Ostro” (Pi = 36.2), whereas the highest DON concentration was observed mainly in modern varieties, i.e., “Badenkrone” , “Titan” , “Cosmos” , “Alkor” , and “Goldir” . In this case, however, also a few old landraces showed high DON contents, i.e., “Zuzgen Nr. 15A” , “Schnotwiler Weißkorn Nr. 35” , and “Thürig Rotkorn Nr. 4”   .P(FHB) = 0.36; P(DON) = 9.5] from which “Rubiota” was selected. On the other hand, “Franckenkorn” showed a similar medium resistance level as the abovementioned varieties but having a significantly reduced plant height. High disease severity and mycotoxin contamination were observed for “Titan” and “Zollernspelz,” two varieties released in the early 2000s. Due to its reduced plant height and therefore increased lodging tolerance, “Zollernspelz” is the most popular variety in Germany and is also widely grown in Austria and Czechia.In The Fusarium rating based on the ranks of the superiority indices for both FHB and DON summarizes the results obviously. Only 16 genotypes were rated as “resistant” , and allp < 0.05). The closest relationship was observed between FHB scores and DON content of grains (r = 0.66) . Considering correlations only within the three test countries, higher correlation coefficients were observed between FHB scores and grain DON content for Austria (r = 0.80) and between FHB scores and plant height for Czechia (r = −0.43)  , whereas = 0.66)  and/or g= −0.43) .r = 0.56 (p < 0.001)  correlated to both FHB scores (r = 0.65) and grain DON content (r = 0.63).In two environments, i.e., CZ17 and CZ18, also the mycotoxin content of the hulls was determined, which was, on average, three to five times higher than in the grain . The cor< 0.001) . In one severity  and ρ = Heritage varieties and “ancient grains” gained attention and market in the last decade. Often, they are promoted without reasonable knowledge about their performance under current agronomic and processing practices . Spelt, r = −0.35 was observed between plant height and FHB severity, which is similar  and thereby changes in the canopy microclimate. Similar conclusions were reached by Rht genes is available. It can only be assumed that, in modern varieties released after 1970, major Rht genes are present as a result of crosses with common wheat and selection for lodging tolerance . Considering the limited presence of the two major dwarfing genes in Central European common wheat germplasm  was significantly lower than the reported r = −0.59 to −0.70 by In common wheat, dwarfing genes and their effect on plant height were shown to be also associated with anther extrusion/retention , which iFusarium species in glumes and grains of naturally infected field samples. On the other hand, Alternaria infection: the contamination with the mycotoxins alternariol and alternariol monomethyl ether was fourfold higher in the hulls than in the grains of spelt.The higher anther extrusion in spelt is surprising in view of the tough, sharply keeled glumes that are firmly enclosing the floret and/or grain. The tough glumes (hulls) may constitute physical barriers to pathogens and thereby protect the developing grain. The protective effect of spelt glumes against colonization by soilborne fungi and therefore increased germination under unfavorable conditions were demonstrated by Fusarium strain, etc.) on especially the mycotoxin accumulation but also FHB severity is well documented .The original contributions presented in the study are included in the article/JC, VW, and HB conceived the field experiments. VW, MB, JP, JK, MT, QN, and JM collected the data. HG and JC analyzed the data and drafted the manuscript. DJ contributed to manuscript editing and organized funding. All authors reviewed and approved the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Using the SERS method to analyze OTC in soil samples, the relative standard deviation is 1.35–4.78%, and the recovery rate is 94.3–104.9%.In this paper, dicyandiamide (Dd) and p-benzaldehyde (Bd) were heated at 180 °C for 3 h to prepare a new type of stable covalent organic framework (COF) DdBd nanosol with high catalysis. It was characterized by molecular spectroscopy and electron microscopy. The study found that DdBd had a strong catalytic effect on the new indicator reaction of polyethylene glycol 600 (PEG600)-chloroauric acid to form gold nanoparticles (AuNPs). AuNPs have strong resonance Rayleigh scattering (RRS) activity, and in the presence of Victoria Blue B (VBB) molecular probes, they also have a strong surface-enhanced Raman scattering (SERS) effect. Combined with a highly selective oxytetracycline (OTC) aptamer (Apt) reaction, new dual-mode scattering SERS/RRS methods were developed to quantitatively analyze ultratrace OTC. The linear range of RRS is 3.00 × 10 Pd) that can catalyze a Au@NiP nanoreaction, combined with aptamer reactions to establish resonance Rayleigh scattering (RRS) methods for the detection of melamine [A covalent organic framework (COF) is a new type of porous conjugated crystalline polymer material. Its framework is mainly composed of C, N, O, and H elements, with strong covalent bond connection, low density, and good chemical stability. In addition, it also has the advantages of large specific surface area, regular pore structure, simple functional design, and easy functionalization; in some cases, it has the characteristics of luminescence and has been applied to analytical chemistry . In analmelamine . TherefoAptamers (Apts) are the main specific oligonucleotides including single-stranded DNA or RNA for varieties of targets obtained through in vitro selection. Under the action of intermolecular forces, for instance, van der Waals forces, hydrogen bonds, hydrophobic, and electrostatic interactions, Apt reactions usually fold into complex and stable three-dimensional structures. Compared to conventional molecular biology cognitive elements, for instance antibodies and enzymes, Apt has considerable merits, including stability in a wider temperature range and pH range, ease of synthesis and modification, lower production costs, and long shelf life . It has 4-fructose reaction to produce AuNPs as bifunctional materials of SERS substrates and indicators. On the basis of this, a new method was developed to determine As3+ by SERS with a linear range of 0.10–0.60 μg/L and a detection limit of 0.06 μg/L. The combination of the two scatterings with Apt controlled nanocatalytic amplification has made great progress, and has the advantages of both. Li et al. coupled a sensitive carbon dot catalytic multiplicative reaction and a high-selectivity Apt reaction to develop a SERS-RRS scattering spectral technology to determine 5–150 nmol/L potassium ions [Surface-enhanced Raman scattering (SERS) is a sensitive molecular spectral technique. Generally, the nanostructured substrates functionalize with Raman reporter molecules, and strong characteristic peaks are generated in the SERS spectrum, thus achieving quantitative detection of the analyte . SERS suium ions .−9–1.0 × 10−4 g/mL OTC. Some of the above methods are not very sensitive, and some of the analysis processes are complicated. As far as we know, there are no reports of a polycondensation reaction of dicyandiamide (Dd) containing C≡N bonds with p-benzaldehyde (Bd) to synthesize the COF of dicyandiamide/p-benzaldehyde (DdBd) with high catalytic activity, and a polyethylene glycol 600 (PEG600)-HAuCl4 nanocatalytic amplification indicator reaction, coupled with a highly selective aptamer, to establish a double scattering assay for OTC. In this study, the specific Apt reaction of OTC was combined with the novel COF-DdBd nanocatalytic amplification to establish a simple and sensitive new SERS/RRS dual-mode method to detect the ultratrace OTC in a soil sample.Oxytetracycline (OTC) is a compound in the tetracycline class of drugs. It is a broad-spectrum antibiotic and is broadly utilized for veterinary purposes in the livestock and poultry industry. Owing to its low absorption efficiency, any intake above 70% is excreted into the environment through excrement. The non-point-source emissions of municipal waste and the farmyard manure application release antibiotics into the surroundings. They enter natural surface water through urban sewage and wild outflows . The conInstruments: A model F-7000 fluorescence spectrophotometer  with the parameters of volt = 350 V, excited slit = emission slit = 5 nm; a DXR smart Raman spectrometer with a laser wavelength of 633 nm, a laser power of 3.0 mW, a slit of 25 µm and an acquisition time of 5 s ; a model TU-1901 dual-beam UV−visible spectrophotometer ; a high-speed freezing centrifuge , an electric thermostatic drying oven ; a Fourier transform infrared (FTIR) absorption spectrometer ; an FEI Tecnai G2 F20 field-emission transmission electron microscope  with stem volt = 200 kV and camera constant 200; an X-ray diffractometer ; Belsorp-max II automatic specific surface area and porosity distributor , sonic cleaner SK3300B ; KQ3200DB CNC ultrasonic cleaner ; SXZ-4–10TC ceramic fiber muffle furnace ; and a model HH-S2 constant temperature water bath kettle  were used.4) solution was prepared by taking 250 μL of 4% chloroauric acid in a 10 mL centrifugal tube and diluting to the mark. An amount of 0.0496 g of OTC solid was put in a tube and diluted to 10 mL with water to obtain a 10.0 mmol/L OTC solution, then gradually diluted to 0.100 nmol/L with water. All reagents used were analytical grade, and the experimental water was secondary pure water.Reagents: Dicyandiamine ; p-benzaldehyde ; dimethyl sulfoxide solution (DMSO) ; ethanol ; and 0.1 nmol/L Apt of OTC with a base sequence of 5′-CGA CGC ACA GTC GCT GGT GCG TAC CTG GTT GCC GTT GTG T-3′  were used. A 1.00 g/L chloroauric acid (HAuCl−3 mol) and 0.101 g of p-benzaldehyde (7.5 × 10−2 mol) were dissolved in 20 mL DMSO at a ratio of 6:1, and were completely dissolved via ultrasound, then the solution was transferred into the reaction kettle and heated for 3 h in the muffle furnace at 180 °C. It was then taken out and a uniform orange-yellow solution was obtained. The prepared bright yellow solution was different from the reference prepared [Bonding the requirements of nanocatalytic analysis and referring to previous research , the preprepared . The molOTC solution, and 0–1000 μL 0.100 nmol/L OTC solution were added to a 5 mL test tube with stopper and mixed evenly. Then, 120 μL 1.00 g/L HAuCl4 solution, 35 μL 10.0 mmol/L HCl solution, and 200 μL 15.0 g/L PEG600 solution were added, and diluted to 1.50 mL. The tube was put in an 80 °C water bath to heat for 45 min, then taken out and cooled with ice water. The RRS intensity was measured by a fluorescence spectrophotometer and the value at 370 nm (I370nm) was recorded. The sample without OTC solution was prepared as the blank (I370nm)0, and ∆I = I370nm − (I370nm)0 was calculated. When 100 μL 10.0 μmol/L VBB solution was added, the SERS intensity at 1613 cm−1 (I1613) was measured; OTC was not added as the blank (I1613)0, and ∆I1613 = I1613 − (I1613)0 was calculated.Amounts of 100 μL 6.75 mmol/L DdBd solution, 100 μL 0.100 nmol/L Apt4-PEG600 to form gold nanoparticles (AuNPs) is very slow; in this gold nanoreaction system the COF could catalyze the reaction to form more AuNPs, and the generated AuNPs have RRS and SERS signals. Using the slope procedure, COF (DdBd) prepared by dicyandiamide-p-benzaldehyde showed the best catalytic effect and was selected for use. Considering the fact that both AptOTC and DdBd have functional groups and π-π overlapping hydrophobic groups, DdBd will be wrapped by AptOTC through hydrogen bonds, hydrophobic interactions, and intermolecular force interactions, causing its catalytic activity to be inhibited. When the OTC target was added, it could specifically bind to the Apt to form a stable complex and release free DdBd, thereby restoring the catalytic activity of DdBd and gradually increasing the content of AuNPs in the system. As a result, due to the increase in AuNPs, the RRS/SERS signal also gradually increased. Within a certain concentration of OTC range, the signal intensity of RRS/SERS is proportional to the concentration of added OTC. On this basis, a sensitive SERS/RRS double-scattering spectroscopy method can be established to determine OTC  images of DdBd and the analysis system were recorded. As shown in sorption D. The Ausorption F,G.4 is extremely slow, and the RRS signal is very weak due to fewer AuNPs. However, the prepared DdBd has a strong catalytic effect on this nanoreaction. The system has two RRS peaks at 370 nm and 520 nm /(IRS/CRS), the Raman spectra of the VBB solution with AuNPs and VBB solution were recorded  and crystal violet (CV) common molecular probes, the EF of R6G and CV in the study of Wang et al. were 6.12 × 107 and 3.02 × 105, respectively [VBB molecular probe in this study was not as good as R6G, there exists fluorescence interference. Compared with the CV, the VBB shows a better Raman enhancement effect.Highly sensitive SERS and simple RRS are a kind of inelastic and elastic scattering, respectively, and both have developed rapidly with nanoparticle preparation technology. The dimode molecular spectra were chosen to study the COF catalytic indicator reaction. The AuNP indicator reaction of PEG600-HAuCld 520 nm A, and thntration B, so we NH2 bond C. The sicompared , and it recorded ; ISERS =ectively . Althoug4 by PEG600 to form gold nanoparticles. Within a certain concentration range, with the concentration of the catalyst DdBd increases, the catalytic capacity increases, and the generated gold nanoparticles increase. At the same time, the RRS and SERS effects of the system increase and show a certain linearity with the concentration of DdBd. Here we still compare the catalytic effects of DdBd by changing the reducing agent  is 1.1 × 10−3 nmol/L. The SERS linear range is 3.00 × 10−3–7.00 × 10−2 nmol/L, the linear equation is ΔI1613 = 4.58 × 104 C + 181, the coefficient is 0.9872, and the detection limit is 9.0 × 10−4 nmol/L. The RRS and SERS working curves are shown in Under the chosen conditions, different concentrations of OTC, and the according RRS, SERS signal values were used to draw the working curves to obtain their analytical features . The lin−2 nmol/L OTC by RRS and SERS was researched. The outcomes of the experiment indicate that within a relative error range not exceeding ±10%, in the RRS spectrum analysis, 1000 times Na+, Co2+, Ca2+, SO32−, PO43−, NO2−, CH3COO−, lysine (Lys), histidine (His), and tryptophan (Trp); 500 times Fe3+, SO42−, HCO3−, HPO42−, CO32−, and alanine (Ala); 100 times Mg2+, P2O74−, glycine (Gly), and threonine (Thr); 50 times K+; 10 times H2PO4− and penicillin sodium; and 5 times tetracycline did not interfere with the RRS measurement of OTC , Lys, and Ala; 500 times Ca2+, HPO42−, P2O74−, and His; 100 times K+, HCO3−, CO32−, and Trp; 50 times Al3+; 10 times Zn2+, SO42−, and Gly; and 5 times ofloxacin and tetracycline did not interfere with OTC SERS determination -PEG reaction, which can amplify the analytical signal. This is a foundation for developing a highly sensitive SERS/RRS method. Secondly, the interaction of COF and Apt should be appropriate. The strong interaction between them leads to the difficulty in Apt desorption, and the assay method cannot be developed. Too weak an interaction caused a high blank. Finally, the specificity of the Apt is high. After reacting with the target molecule, the Apt can be desorbed from the COF surface so as to obtain a highly sensitive and selective method.4 nanoreaction, and the generated AuNPs show intense SERS signals in the presence of VBB molecular probes. Via combining the Apt reaction with the DdBd nanocatalytic amplification reaction, a new SERS quantitative analysis method to detect OTC was developed. It has advantages of high sensitivity, selectivity, accuracy, and low blank value. In addition, RRS can also be used to detect OTC without adding molecular probes. This work can provide new ideas for the design of the Apt-controlling COF catalytic AuNP reaction SERS system, and this system can be used to detect residual antibiotic substances in the environment and eliminate the potential harm of these substances to the natural environment and human body.According to the solvothermal reaction of dicyandiamide and p-benzaldehyde, DdBd nanosols with high catalysis and stability were prepared and characterized via TEM, EDS, infrared spectroscopy, and other techniques in detail. It was found that DdBd has an intense catalytic activity on the new PEG600-HAuCl


Endometrial cancer is one of the leading gynecological cancers diagnosed among women in their menopausal and postmenopausal age. Despite the progress in molecular biology and medicine, no efficient and powerful diagnostic and prognostic marker is dedicated to endometrial carcinogenesis. The canonical TGFβ pathway is a pleiotropic signaling cascade orchestrating a variety of cellular and molecular processes, whose alterations are responsible for carcinogenesis that originates from different tissue types. This review covers the current knowledge concerning the canonical TGFβ pathway (Smad-dependent) induced by prototypical TGFβ isoforms and the involvement of pathway alterations in the development and progression of endometrial neoplastic lesions. Since Smad-dependent signalization governs opposed cellular processes, such as growth arrest, apoptosis, tumor cells growth and differentiation, as well as angiogenesis and metastasis, TGFβ cascade may act both as a tumor suppressor or tumor promoter. However, the final effect of TGFβ signaling on endometrial cancer cells depends on the cancer disease stage. The multifunctional role of the TGFβ pathway indicates the possible utilization of alterations in the TGFβ cascade as a potential target of novel anticancer strategies. Endometrial cancer is one of the most commonly diagnosed gynecological tumors. According to GLOBOCAN 2020 data, endometrial cancer is diagnosed in around 417,367 women worldwide, and is the cause of almost 97,370 deaths each year, with the world morbidity around 8.7/100,000 of the female population. Overall lifestyle risk factors of endometrial cancer determine its higher incidence rate among women in developed countries in contrast to less-developed ones [−6 mutations per Mb). The MSI subgroup is related to deficiencies in a DNA mismatch repair system leading to common mutations of ARID5B, PTEN, PIK3CA, and PIK3R1 genes. The copy-number low subgroup is described also as microsatellite stable and corresponds to more than half of low-grade endometrioid tumors, whereas the copy-number high subgroup reflects serous histopathology [Along with the progress in molecular biology, the above-mentioned classification of endometrial cancer, proposed by Bokhman in 1983, is extended now by molecular findings based on a large-scale, comprehensive genetic analysis of endometrial cancer according to The Cancer Genome Atlas. Molecular classification of endometrial cancer includes four subgroups, i.e., DNA polymerase epsilon ultramutated (POLE), microsatellite instability-high (MSI-H), copy-number low, and copy-number high subgroup. Each subgroup is characterized by distinct clinical, pathological, and molecular aberrations. The POLE subgroup displays polymerase epsilon mutations in the exonuclease domain, which results in a remarkably high mutation rate , whose name comes from TGFβ itself and their isoforms serve as prototype molecules. Transforming growth factor β (TGFβ) is a large superfamily of cytokines that control a plethora of molecular and cellular processes. Up until now, the superfamily comprised more than 40 different ligands that included TGFβ itself as well as bone morphogenetic proteins (BMPs), activins, inhibins, nodal growth differentiation factor , growth and differentiation factors (GDFs), left-right determination factor (Lefty), and anti-Müllerian hormone/Müllerian inhibiting substance (AMH/MIS). TGFβ ligands are key regulators during embryonic development, tissue formation and regeneration, and their alteration results in body malformation, cancer development and progression, fibrosis, and autoimmunological diseases ,20,21,22The inclusion criteria of literature selected for this review included the search in PubMed of the National Library of Medicine using the following keywords: “TGFβ1-3” or “TGFβR1” or “TGFβR2” or “Smad1-7” or “betaglycan/TGFβR3” or “endoglin/CD105” and “human endometrium” or “endometrial cancer”. Neither of the selected articles was published in languages other than English nor were retracted from publication. The relevance of the articles was evaluated by analyzing the title and abstract. The potentially relevant articles were full-text evaluated and included in this review.The TGFβ superfamily consists of 33 ligands that possess cytokine activity. A large number of TGFβ ligands determines the plethora of processes engaged in regulation and mediation of cellular homeostasis, including embryonic development, cell differentiation and proliferation, immune response, angiogenesis, motility, and apoptosis, both in a spatiotemporal manner, as well as dependent on cell and tissue type ,23,24,25The TGFβ ligands are synthetized as precursors—pre-proproteins consisting of an app. 29-residues secretion signal peptide (SP), an app. 250-residue latency-associated peptide prodomain (LAP), and an app. 110-residue C-terminal growth factor (GF) domain ,34,35,36For full bioavailability the latent TGFβ needs several mechanisms, among which can be distinguished the proteolysis of LLC, low pH in the extracellular matrix, reactive oxygen species (ROS), as well as thrombospondin-1, retinoic acid or basic fibroblast growth factor (bFGF) dependent activation ,46. The The canonical TGFβ signaling in contrast to other non-canonical TGFβ induced pathways occurs upon ligand binding by specific transmembrane receptors, which possess serine/threonine kinase activity and can be divided according to their structural features into two types, i.e., type I (TGFβRI) and type II (TGFβRII). Both types of TGFβ receptors are transmembrane kinases, with an app. 100-residues ectodomain, which is highly glycosylated and disulfide-rich, a transmembrane region, a short juxtamembrane domain, and cytoplasmic kinase domain with 11 subdomains organized in N-lobe and C-lobe ,54,55. TThe first step in TGFβ canonical pathway is an interaction of the dimeric ligand with a tetrameric receptor complex consisting of two pairs of TGFβRIs and TGFβRIIs. This interaction provides conformational changes of ectodomains of TGFβ receptors and brings intracellular domains to close proximity; thus, the activated dimer of TGFβRIIs due to its constitutive kinase activity phosphorylates GS motifs of the type I receptor dimers ,65,66,67Successfully transduced through the plasma membrane signal activates in turn cascade of transphosphorylation reactions of TGFβ effector proteins . The sigThe R-Smads assemblage to the TGFβRs complex and their activation can be mediated by chaperon proteins, such as SARA (Smad anchor for receptor activation) or ERBIN (Erbb2-interacting protein) ,63,78. TThe expression of Smads-dependent genes occurs through highly conserved MH1 and MH2 (Mad homology 1 and 2) domains separated by a serine-proline rich linker of high variability  Figure . The MH1−7 M), it seems that modulation of TGFβ-target genes expression is shaped by the association of Smads with different transcription factors both activators and repressors, which enhance overall interaction [The DNA recognition by the heterotrimeric complex of R-Smads and Smad4 occurs effectively, but with low affinity by its direct interaction with the major groove of specific DNA sequences described as SBEs (Smad binding elements) ,95. In teraction . The shaeraction . It can eraction ,100.The specific role in TGFβ canonical pathway is played by Smad7 protein, which belongs to the subclass of I-Smad . The maiThe negative regulation via Smad7 protein can also occur via mobilization of E3 ubiquitin ligases, i.e., SMAD ubiquitination regulatory factor 1 and 2 (Smurf1 and Smurf2), WW domain-containing protein 1 (WWP1), and neural precursor cell expressed developmentally downregulated 4-2 (NEDD4-2), which in turn promote ubiquitin-dependent proteasomal and/or lysosomal degradation of TGFβRI ,111,112.To fully understand the TGFβ receptors’ functioning and their engagement in signal propagation, it is necessary to focus on another important issue connected with TGFβRs activity and cellular metabolism, i.e., trafficking, internalization, and recycling . In contWhile the native TGFβRs are delivered to the plasma membrane, they organize in a well-defined distribution pattern dependent on the cell type. In polarized epithelial cells, TGFβRs localize to the basolateral plasma membrane, where they are involved in TGFβ-induced signaling, leaving the apical side insensitive to TGFβ stimulation ,130,131.The clathrin-coated pits are microdomains in the plasma membrane covered at the cytoplasmatic side with clathrin triskelions, and they are engaged in cargo enrichment and endosomal vesicles formation ,139. TheThe caveolar internalization occurs in the presence of flask-shaped membrane invaginations organized in lipid rafts of cholesterol and sphingolipids microdomains and enriched by caveolin-1 protein . The cavThe partitioning of TGFβRs in the cell membrane determines the fate of TGFβ-induced signal or TGFβRs themselves . FurtherIn the mid-1980s, the third type of TGFβ receptor was identified and termed in the literature as the TGFβ receptor type III (TGFβRIII). Other commonly used terms for this type of receptor are accessory/auxiliary receptors or co-receptors. The name of the TGFβ receptor type III originates from the common function shared by its members, not from their structural similarities. These receptors play a vital role in presenting TGFβ ligands to the signaling receptors, enhancing receptor–ligand interactions, and promoting the cooperation between canonical TGFβ receptors. Structurally, TGFβ co-receptors are transmembrane proteins or proteins anchored in a membrane by glycophosphoinositol (GPI). Unlike TGFβRI and TGFβRII, they are deprived of any known intrinsic motifs possessing enzymatic activity. Some co-receptors mediate ligand binding by increasing its affinity, whereas the others provide an interface for structural changes to allow ligand–receptor interaction. Furthermore, co-receptors ectodomain shedding delivers extracellular molecules responsible for TGFβ ligands sequestration in ECM, thus modulating or antagonizing TGFβ responsiveness at the signal initiation step ,54,64.To date, a few molecules have been identified and described as acting as TGFβ co-receptors, including betaglycan (TGFβR3), endoglin (CD105), CD109, and repulsive guidance molecules a-c , in general, well-known and extensively studied TGFβ receptors type III ,156,157.Among the above-mentioned TGFβ co-receptors, only betaglycan, endoglin and CD109 participate in a modulation of the signal induced by three TGFβ isoforms. Although, betaglycan and endoglin are not only structurally related but also of the highest importance for cancer development and progression ,157,158 Endoglin binding of TGFβ homodimers can be predicted based on its interaction with BMP9 or BMP10. According to these findings, endoglin might bind TGFβ homodimers with 2:1 stoichiometry, and its homodimer forms an antibody-like structure, where homodimerized fragments of ZP domains resemble the Fc region, and both OD domains of endoglin molecules serve as Fab region. In such a Y-shaped structure, TGFβ binding occurs symmetrically, with ligand binding sites located within OD domains  Figure B. DespitAs it has been shown, betaglycan can form stable binary complexes with all three TGFβ isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3, though with the preferential complex formation with TGFβ2, which has 200–500-fold weaker affinity for its cognate TGFβRII, as compared to TGFβ1 and TGFβ3 ,171. IntThe mechanistically, TGFβ ligands presentation to the dedicated TGFβRs includes the formation of a binary complex between betaglycan and TGFβ dimers, what potentiates binding of TGFβRIIs. In turn, once bound, TGFβRIIs leads to recruitment of TGFβRIs, with a simultaneous displacement of betaglycan from signaling complex  Figure . In the The TGFβ co-receptors not only act as significant molecular agents responsible for potentiation of the extracellular signal induced by TGFβ dimers but also are able to modulate TGFβ response due to their ectodomain shedding. Both betaglycan and endoglin may undergo proteolytic cleavage, releasing to the ECM their extracellular domains ,178 Fig. PresentThe human endometrium is a hormone-dependent tissue, which in the literature is often described as “highly dynamic”. This feature associates with subsequent phases of proliferation, differentiation, shedding, and regeneration in a single menstrual cycle in total more than 400 times in a woman’s lifetime. From a biological point of view, the role of the endometrium is to provide an immunotolerant site for embryo implantation and its development in nurturing conditions until labor. In the structure of the human endometrium, we can distinguish two separate layers of different morphology and fate. The outermost layer of the endometrium is the functional one, which lines the uterine cavity and presents the transient physiology. Underneath the functional layer and adjacent to the myometrium, is located the permanent basal layer. The single strand of luminal epithelium, the stroma, and the superficial glands (glandular epithelium) build up the functional layer. As the functional layer covers the deeper basal one, the terminal parts of the glands are anchored in the basal layer . During In human endometrium, the expression of all three TGFβ isoforms has been observed by many research groups ,192,193.Furthermore, TGFβ type I and type II receptors are localized in both endometrial compartments, i.e., stromal and epithelial. Interestingly, TGFβRII shows a much higher expression level as compared to TGFβRI ,197. ThiInterestingly, the expression of another important TGFβ co-receptor, an endoglin in human endometrium, is associated with the vascular endothelium, where it is regarded as a specific marker of activated endothelial cells. Its angiogenic potential is pronounced as endoglin positive staining is observed in eutopic and ectopic endometrium of women with diagnosed endometriosis, with even higher intensity in the eutopic than ectopic endometrium. Conversely, endoglin immunoreactivity is not observed in the endothelial cells of microvessels of a normal endometrium . On the In summary, the TGFβ isoforms are abundantly expressed in endometrial compartments together with their dedicated receptors and co-receptors. As shown, their expression is varied and depends on the layer of the endometrium . Moreover, cyclic changes in the distribution of TGFβ signaling components are observed throughout the menstrual cycle. These observations implicate a potential role of the TGFβ pathway in maintaining endometrial homeostasis. The errors in TGFβ pathway counterparts result in the development and progression of different human pathologies related to the uterus. Impaired TGFβ signaling is noted in many disorders, such as infertility, recurrent miscarriages, uterine-placental dysfunction, endometriosis, and endometrial cancer ,205,206.In addition to the identification of well-defined risk factors of endometrial cancer development, and described in the Introduction paragraph, the mechanism of endometrial carcinogenesis remains poorly understood ,14,15,16To elucidate the involvement of TGFβ pathway alterations in endometrial carcinogenesis, several research groups evaluated TGFβ isoforms expression during neoplastic transformation of human endometrium by comparison of how it has been disrupted in the following stages of cancer progression, ranging from normal endometrium, through simple and complex hyperplasia, to endometrial carcinomas ,212,213 According to the literature data, little is known about the expression levels of TGFβ2 and TGFβ3 isoforms during endometrial carcinogenesis. As reported, TGFβ2 and TGFβ3 present increased immunoreactivity in endometrial carcinoma as compared to the normal endometrium, and their expression reflects the levels observed in the case of complex endometrial hyperplasia in both the glandular and stromal constituents of the endometrium . ComparaThe proper TGFβ signaling could not occur without ligand-specific TGFβRs, of which disrupted expression or functioning have been reported in endometrial cancer. The ALK5 (TGFβR1) and TGFβR2 at the message level are profoundly decreased in cancerous tissue in comparison with normal endometrium. Although, at first glance, the published data could lead to misinterpretation. The TGFBR1 and TGFBR2 genes expression levels are strongly dependent on the use in the comparison control group  and/or applied experimental methodology ,220,221 Some scientific reports have documented the involvement of TGFβR2 in endometrial carcinogenesis, which similarly to TGFβR1 presents a lower expression level than observed in normal proliferative endometrium ,221. Fur9 microsatellite region in exon 1, resulting in loss of three alanine residues corresponding to the boundary between the signal sequence and the mature extracellular domain of the protein [Impaired TGFβRs expression in endometrial cancer can be a result of their transcriptional down-regulation, an increased receptor degradation, and/or deregulated trafficking to the cell membrane. An additional mechanism responsible for observed alterations in TGFβRs expression can be addressed to mutational inactivation, occurring in their genes. When comparing to the other tumor types, mutations leading to the TGFβRs loss or inactivation in endometrial cancer are relatively infrequent . There i protein   of intracellular downstream Smad proteins. In contrast to the other cancer types, the knowledge concerning the alterations of Smad expression in endometrial cancer is limited . Based oSmad proteins, as cellular effectors of the TGFβ pathway, to provide downstream signalization, demand activating phosphorylation by TGFβRI receptors located in the cell membrane. Following, the activated heteromeric Smads complexes translocate to the nucleus, where together with other co-activators or co-repressors they modulate TGFβ-responsive genes. Due to that fact, the critical role in Smads protein physiology is their subcellular distribution, respectively cytoplasmic or nuclear localization. The analysis of Smads expression at the protein level in endometrial carcinoma revealed that their intracellular distribution undergoes changes during uterine neoplastic transformation. For instance, Smad4 protein expression in a cytoplasmic fraction is gradually increased in association with tumor aggressiveness and progression, evaluated by tumor grade and myometrial infiltration. In general, during cancer progression, a decreased number of samples is observed that are characterized by the presence of Smads exclusively in a nuclear fraction . On the Deregulation of Smads expression can be attributed to the genetic mechanisms, but they are not well studied in endometrial cancer. According to TCGA-UCEC, mutations in Smads encoding genes are relatively infrequent, unlike other cancer types with identified Smads alterations. Mutations related to Smads’ genes are observed in 11.4% of endometrioid carcinoma cases, among which 5% in SMAD2, 4.6% in SMAD3, 3.5% in SMAD4, and 6.8% in SMAD7  Table 1Table 1. p = 0.0177), GC  and TT  [p = 0.0026; T—OR= 2.38; 95% CI = 1.16–4.85; p = 0.0151).Induced by TGFβ isoforms extracellular stimuli transduction may also be interfered at the level of signal modulation by altered expression of TGFβ co-receptors. According to the clinical studies, this step in TGFβ signaling is impaired since the expression of two TGFβ co-receptors, i.e., betaglycan and endoglin, is changed in endometrial cancer. TGFBR3 gene, which encodes betaglycan, is significantly reduced in cancer tissue, corresponding to its immunoreactivity loss. Well-differentiated tumors present weak or patchy staining with rare immunoreactivity signals localized to the epithelial glands, whereas poor-differentiated adenocarcinomas present devoid of stains in endometrial components. The lack of betaglycan in epithelial compartments of the endometrium is opposed by strong positive immunostaining of tumor vessels, which suggests that it may be a distinct player in tumor vascularization . The eva 0.0317)  . As confirmed using two endometrial cancer cell lines—HEC 1A and RL95 2—the acquisition of metastatic phenotype is observed upon the stimulation with TGFβ1 isoform. TGFβ1-treated cells present a decreased cell-cell contacts and the promotion of migratory structures as lamellipodia, what is accompanied by an increase of mesenchymal marker vimentin. The described impact of TGFβ1 on endometrial cells can be effectively abolished using the specific TGFβ1 inhibitor SB-431542, which restores epithelial architecture and the formation of compact colonies . RecentlAdditionally, the TGFβ pathway may also enhance cell motility and invasiveness—the features of cells undergoing EMT, through another downstream target prostate apoptosis response 4 protein (Par-4). Par-4 was initially described as a tumor suppressor responsible for activating apoptosis induced by extracellular cytotoxic signals . HoweverDuring endometrial cancer progression, in particular initiation of the metastatic process, a vital role is played by the tumor microenvironment serving as a reservoir of cytokines, growth factors, and other factors. Among these molecules, TGFβ1 is released directly or as cargo in exosomes released from cancer cells, and/or immunological cells trigger the transformation of normal fibroblast (NF) to cancer-associated fibroblasts (CAFs) . TGFβ1-iConditioned-culture media of CAFs significantly induces dose-dependent proliferation of primary endometrial cell cultures, as well as endometrial cancer cell lines when compared with untreated control cells or cells treated with conditioned media obtained from NF. The CAFs-associated proliferation of endometrial cancer cells occurs via the SDF-1/CXCR4 axis, which in turn activates PI3K/Akt and MAPK/ERK signaling pathways in a paracrine-dependent manner. Simultaneously, an increased secretion of MMP-2 and MMP-9 in an autocrine-dependent manner was observed . CAFs coConsidering endometrial cancer risk factors, particularly prolonged exposure to estrogens that are not balanced with progesterone (that issue is discussed in the Introduction paragraph), it has been demonstrated that extended progesterone treatment effectively reduces the metastatic ability of endometrial cancer cell lines HEC-1B, RL-952 and Ishikawa . This obThe TGFβ pathway, which belongs to the signaling network in the cell, is one of the most essential cascades governing a vast of cellular processes, both in health and disease. The plethora of molecular events under control, and/or being dependent on the TGFβ signaling finds its reflection during carcinogenesis. On the contrary to the other well-known suppressors and oncogenic factors, the TGFβ pathway plays a pleiotropic role in orchestrating contradictory processes. As it is observed in the case of TGFβ-induced stimuli, TGFβ signaling contributes to the suppressor program in cancerous cells at the early stages of carcinogenesis. During the progression of neoplastic disease, the TGFβ axis supports increased cell growth and proliferation, leading to pronounced aggressiveness and invasion, thus stimulating cancer cells seeding and establishing new metastatic sites .As presented in this review, TGFβ pathway components undergo deregulation in endometrial cancer. Impaired expression is observed at every level of signal transduction, beginning from signal induction by TGFβ isoforms, signal reception by plasma membrane receptors and co-receptors, and up to downstream cytosolic effector Smad proteins. All these alterations are of great potential, as diagnostic and prognostic markers of endometrial neoplastic transformation. However, further investigations need to be performed to understand the uterine cancerous transformation better and/or increase the clinical value of identified molecular mechanisms. Moreover, extensive studies would open a new perspective for establishing novel anti-cancer strategies targeting TGFβ signaling components. This issue is fundamental since to date there is a paucity of reliable molecular biomarkers dedicated to endometrial cancer diagnosis and monitoring the effectiveness of therapy. Routine treatment of this tumor type is surgery, which includes total hysterectomy with removal of fallopian tubes and ovaries. In the early stages of endometrial cancer, the majority of patients have a favorable prognosis. However, some women are at risk of disease relapse, so the crucial question is which patients would benefit from adjuvant treatment, including chemo- and/or radiotherapy . An answAs discussed in this review, the engagement of the TGFβ pathway in endometrial cancer progression shows that TGFβ-induced signaling is a prominent route determining increased invasiveness and metastasis. Invasion and metastasis are considered as the main reasons for treatment failure and poor patient survival. Furthermore, the accumulation of molecular insults underlies the development of drug resistance to hitherto successfully administered drugs. In effect, there is a need for modification or developing new therapeutic strategies, which could provide a better, and personalized approach in treatment adjusted not only to the cancer type but to the particular patient. The final issue is the identification of molecular events responsible for the switch of TGFβ-induced signaling from tumor suppressor to tumor promoter. Unfortunately, so far there is a lack of such evidence during endometrial carcinogenesis. Taken together, the TGFβ pathway is an underestimated signaling cascade in human endometrium, thereby there is a need for more extensive research covering the involvement of TGFβ in uterine neoplastic transformation. To confirm that fact, there has been only one drug ongoing clinical trial of TGFβ antagonist in endometrial cancer, i.e., tasisulam. Tasisulam is a TGF-β/TGF-β type I receptor kinase (ALK5) inhibitor assigned to clinical trials in patients with refractory or malignant solid tumors. It has been tested in two phase I clinical trials. The first one included only one patient with endometrial cancer, whereas the second phase Ib clinical trial involved 13 patients with ovarian, uterine, endometrial, and cervical cancers. However, the exact number of endometrial cancer cases has not been indicated in the latter trial .