Although the prevalence of leukemia is increasing, the agents responsible for this increase are not definitely known. While ionizing radiation (IR) was classified as a group one carcinogen by the IARC, the IR-induced cancers, including leukemia, are indistinguishable from those that are caused by other factors, so the risk estimation relies on epidemiological data. Several epidemiological studies on atomic bomb survivors and persons undergoing IR exposure during medical investigations or radiotherapy showed an association between radiation and leukemia. IR is also known to induce chromosomal translocations. Specific chromosomal translocations resulting in preleukemic fusion genes (PFGs) are generally accepted to be the first hit in the onset of many leukemias. Several studies indicated that incidence of PFGs in healthy newborns is up to 100-times higher than childhood leukemia with the same chromosomal aberrations. Because of this fact, it has been suggested that PFGs are not able to induce leukemia alone, but secondary mutations are necessary. PFGs also have to occur in specific cell populations of hematopoetic stem cells with higher leukemogenic potential. In this review, we describe the connection between IR, PFGs, and cancer, focusing on recurrent PFGs where an association with IR has been established. The incnovo AML . In totanovo AML . Howevernovo AML . It has novo AML ,102,103.Klymenko et al.  studied PML-RARA PFG is the most critical event involved in the pathogenesis of acute promyelocytic leukemia. This derives from a cytogenetic translocation t leading to the rearrangement of PML and RARA genes [The RA genes . PML-RARRA genes .p > 0.05). Thus, the data indicate that the PML and RARA genes are not particularly susceptible to the clastogenic effect of IR.Quina et al.  studied + HSPCs in bone marrow.Using the same approach as described by Holmberg , we haveUnder the assumptions of the random distribution of IR-induced DNA strand breaks through the PFG break point cluster regions and total genome, the proportion of BCR-ABL1, RUNX1-RUNX1T1, and PML-RARA in the total yield of all translocations was estimated (A).3 × 3 × 103)/(6.4 × 109)2 = 5.8 × 10−11A = 4 × (200 × 10−11 × 0.057 = 3.3 × 10−12B = 5.8 × 10−12/(20 × 106 × 1400 × 10−2) = 1.18 × 10−4C = 3.3 × 10For BCR-ABL1:For RUNX1-RUNX1T13 × 15 × 103)/(6.4 × 109)2 = 3.6 × 10−11A = 4 × (25 × 10−11 × 0.057 = 2.05 × 10−12B = 3.6 × 10−12/(20 × 106 × 1400 × 10−2) = 7.3 × 10−5C = 2.05 × 10For PML-RARA3 × 15 × 103)/(6.4 × 109)2 = 4.1 × 10−12A = 4 × (2.8 × 10−12 × 0.057 = 2.3 × 10−13B = 4.1 × 10−13/(20 × 106 × 1400 × 10−2) = 8.2 × 10−6C = 2.3 × 10Along with size of the break point cluster regions for ABL1 (200 Kbp) and BCR (3 Kbp), we took into account the break point cluster regions for the RUNX1 (25 Kbp), RUNX1T1 (15 Kbp), PML (2.8 Kbp), and RARA (15 Kbp) genes and the size of the whole human genome (6.4 Gbp). Factor 4 accounts for the fact that the cell has a diploid number of chromosomes.−12, 2.05 × 10−12, and 2.3 × 10−13, respectively (B).A linear dose response was observed between ionizing radiation doses and chromosomal translocations in lymphocytes, with 0.057 translocations yielded per whole genome cell equivalent per Gy . Consequ3) of the 70 kg human body [+) is approx. 20 × 106 mL−1 [+ HSPCs, which are supposed to be the cells of origin for leukemia, constitute approx. 1% (1 × 10−2) [+ HSPCs, respectively, was calculated to be 1.18 × 10−4, 7.3 × 10−5, and 8.2 × 10−6 per Gray and individual (C). Of note, the C value estimated by us for BCR-ABL1 was slightly lower in comparison to Holmberg’s data (C = 0.007) due to the updated numbers, as described above. For further estimations, we will solely consider BCR-ABL1 because almost all (95%) CML patients carry the BCR-ABL fusion gene. Taking into account the data for CML cases among the survivors of the atomic bombs at Hiroshima and Nagasaki [−4 estimated by us for BCR-ABL1 in CD34+ HSPC per individual and Gy, we calculate the number of individuals (D) among the atomic bomb survivors carrying a stem cell with radiation-induced t translocation resulting in BCR-ABL1:The human bone marrow represents approx. 1.4 kg  ,115,116.Nagasaki , and traNagasaki , a totalHowever, this calculation did not take into account the fact that genetic mutations are influenced by the sequence context, structure, and genomic features, what might actually lead to a ~100-fold difference in germline mutation rates across human DNA . It was Of note, the reported number of CML cases in atomic bomb survivors in Hiroshima and Nagasaki were 18 , thus inThe first radiation accident study aiming to estimate the effect of radiation on humans came from the 1960s. The presence of chromosome abnormalities was found in the peripheral lymphocytes of fishermen exposed to radiation ,124. To The study by Ito et al.  observedSeveral studies clearly showed that the frequency of PFGs in healthy newborns significantly exceeds (≥100 times) the incidence of the corresponding leukemia subtypes ,129,130.+ HSPCs [−5), which is close to the sensitivity threshold of the RT qPCR [−6 to 10−7 and 10−4 to 10−5, respectively. In most studies  could induce PFGs. However, the data are fragmentary and not always consistent. From all available studies, only one showed linear dose response induction for BCR-ABL1 in the dose range of 10 to 100 Gy. In addition to the absorbed dose, significant dependence on cell type was reported, likely suggesting the importance of the spatial nuclear organization of chromosome territories. A number of radiological studies suggest that leukemia might occur even after low doses of ≤0.1 Gy. Induction of PFGs by low doses was poorly investigated and further efforts are required to study low dose effects on PFG induction. The data collected in this review partially support the Nakamura hypothesis, suggesting that radiation-induced leukemia arises only in a small number of individuals which already harbor clonally expanded preleukemic cells. However, we assume that leukemia induced via ionizing radiation occurs regardless of whether first hit (PFG) or secondary hit  or both are induced via IR, as far as assuming that both hits have to occur. Of note, our calculations, which are based on the Holmberg approach, indicated that not all radiation-induced BCR-ABL1 are able to result in overt leukemia. The same proportion of BCR-ABL1-positive individuals in the general population and in irradiated persons (about 1%) will develop overt leukemia, indicating the similar mechanism of leukemogenesis.


In this investigation, we examined the impact of CO2 on three variants of O3-δ cathodes and employed the distribution of relaxation times method to distinguish distinct electrochemical processes based on impedance spectra analysis. We meticulously analyzed and discussed the corrosion resistance of these O3-δ cathodes under high CO2 concentrations, relying on the experimental data. Electrochemical impedance spectroscopy results revealed that La0.6Sr0.4Co0.2Fe0.8O3-δ (LSCF−6428), La0.4Sr0.6Co0.2Fe0.8O3-δ (LSCF−4628), and La0.4Sr0.6Co0.2Fe0.7Nb0.1O3-δ (LSCFN−46271) cathodes exhibited persistent degradation when exposed to CO2 at temperatures of 650 °C or 800 °C during the durability-testing period. An increase in electrode polarization resistance was observed upon CO2 introduction to the electrode, but electrode performance recovered upon returning to a pure air environment. Furthermore, X-ray diffraction and scanning electron microscopy analyses confirmed that CO2 did not cause permanent damage to the O3-δ cathodes. These findings indicate that the O3-δ cathodes exhibit excellent resistance to CO2-induced corrosion.Solid Oxide Fuel Cell (SOFC) cathodes operating in ambient atmospheric conditions inevitably encounter CO Solid oxide fuel cells (SOFCs) stand as the most efficient devices devised to date for the direct conversion of chemical fuels into electrical power ,2,3. Nev2, which are challenging to eliminate and contribute to cathode corrosion and degradation [2 on the performance and durability of SOFC cathodes becomes imperative.In both research and practical applications, ambient air is typically directed straight to the cathodes. This air carries contaminants, such as COradation . Consequ2 readily engages in reactions with alkaline-earth elements present in cathode materials, leading to the formation of carbonates [0.5Sr0.5Co0.8Fe0.2O3-δ (BSCF) cathode, recognized for its superior performance, demonstrates limited resilience to CO2 due to the presence of Ba and Sr as A-site alkaline-earth elements [0.4Fe0.4Nb0.2O3-δ, Ba0.5Sr0.5Co0.8Fe0.1Nb0.1O3-δ, and Ba0.9Co0.7Fe0.2Nb0.1O3-δ, substantially enhances corrosion resistance against CO2 [2 on La-based cathodes, such as La0.8Sr0.2MnO3-δ, La0.6Sr0.4CoO3-δ, and La0.6Sr0.4FeO3-δ, have also been systematically conducted [2. It is worth noting that the CO2 corrosion mechanism varies among different cathodes due to the diversity in cathode chemical composition and crystal structure [COrbonates ,10. Conselements ,12. Yi, inst CO2 ,14,15. Ionducted ,17. Notatructure .3-δ cathodes have garnered considerable attention due to their exceptional catalytic activity, mixed electronic–ionic conductivity, and compatibility with other cell components, both chemically and mechanically. Within the diverse spectrum of O3-δ cathode compositions, La0.6Sr0.4Co0.2Fe0.8O3-δ (LSCF−6428) stands out as a favored choice among IT-SOFC developers [2 on surface segregation and phase formation in LSCF−6428 was investigated [2 accelerated the kinetics of Sr surface segregation, primarily due to the heightened thermodynamic driving force for SrCO3 formation. Nevertheless, the crystal structure and performance of both La0.6Sr0.4FeO3-δ and La0.6Sr0.4Fe0.8Nb0.2O3-δ materials remained largely unaffected under a CO2 atmosphere, demonstrating their robust CO2 tolerance [Ovelopers ,20,21. Tstigated ,23. The olerance ,24.0.4Sr0.6Co0.2Fe0.8O3-δ and La0.4Sr0.6Co0.2Fe0.7Nb0.1O3-δ cathodes contain a higher proportion of Sr element. Therefore, it becomes imperative to investigate CO2 corrosion on these two cathode variants and compare them with the state-of-the-art LSCF−6428 cathode.Previous research has demonstrated that to further enhance the performance of LSCF−6428, the partial substitution of La by Sr at the A-site and the addition of a small amount of Nb to the B-site can improve its electrical conductivity and chemical stability ,26,27,282-induced corrosion affecting La0.6Sr0.4Co0.2Fe0.8O3-δ, La0.4Sr0.6Co0.2Fe0.8O3-δ, and La0.4Sr0.6Co0.2Fe0.7Nb0.1O3-δ cathodes.In this study, we delve into the mechanisms of CO2-95% N2, 20% O2-80% N2, 50% O2-50% N2, and 100% O2. From the Nyquist plots, it can be observed that the polarization impedance of the symmetric electrodes increases with decreasing oxygen partial pressure. The same conclusion can be drawn from the modulus curves of impedance in the Bode plots. Additionally, the Bode plots illustrate changes in the phase angle within specific frequency domains, showing that significant variations occur primarily below 400 Hz.To determine the frequency range associated with various electrochemical processes of oxygen electrode reactions, impedance spectra Nyquist plots and Bode plots were obtained for LSCF−6428, LSCF−4628, and LSCFN−46271 symmetric electrodes at 800 °C see . These e2 concentration dropped below 5%, with its appearance closely tied to oxygen concentration. Typically, the frequency range of 0–10 Hz is attributed to gas diffusion within and around the cathodes. The area beneath the P2 peaks displayed variability with oxygen partial pressure, and previous studies indicate its association with chemical reactions [2, featuring a frequency range of approximately 10–400 Hz. At higher frequencies, the DRT curves exhibited overlap, indicating that this region remained unaffected by fluctuations in O2 concentration. Consequently, P3 pertains to the electrode–electrolyte interface process and corresponds to charge transfer reactions at the triple phase boundary, encompassing a frequency range of 400–10,000 Hz. Notably, in As illustrated in eactions ,30,31. P2 concentrations. 2 content in the air led to an increase in the electrode polarization resistance (Rp).The LSCF−6428, LSCF−4628, and LSCFN−46271 electrodes underwent testing at temperatures of 650 °C and 800 °C under various gas atmospheres, each featuring different CO3-δ cathodes appears to be more responsive to CO2 at the lower temperature of 650 °C than at 800 °C. Specifically, for LSCF−6428 at 650 °C, Rp values of 0.510 Ω cm2 (air), 0.534 Ω cm2 (20% CO2-air), and 0.556 Ω cm2 (50% CO2-air) were recorded. In comparison, LSCF−4628 and LSCFN−46271 exhibited Rp values of 0.390 Ω cm2, 0.414 Ω cm2, and 0.443 Ω cm2 and 0.400 Ω cm2, 0.415 Ω cm2, and 0.430 Ω cm2, respectively, under the same gas atmospheres. Notably, the Rp increased with rising CO2 content in the air. Under identical atmospheric conditions, LSCF−6428 exhibited the highest Rp, while the difference between LSCF−4628 and LSCFN−46271 was relatively minor.From these findings, it becomes evident that the performance alteration of O2, 0.039 Ω cm2, and 0.043 Ω cm2, respectively. Comparative analysis revealed that the Rp values of LSCF−4628 and LSCFN−46271 were lower than those of LSCF−6428.However, at 800 °C, Rp did not exhibit a clear trend under the three atmospheres. For instance, the initial impedances of the LSCF−6428, LSCF−4628, and LSCFN−46271 electrodes were approximately 0.050 Ω cm2 content on EIS at 650 °C, DRT analysis was applied to these results. 2 content, the P2 peaks shifted towards lower frequencies, and their area also expanded. As depicted in 2. Consequently, it can be inferred that CO2 predominantly impacts the oxygen surface exchange reaction [2 and O2 for available oxygen vacancies, leading to a competitive mechanism that affects cathode performance.Given the significant influence of COreaction . The obs3-δ cathodes when exposed to CO2 was further scrutinized at 800 °C under a 50% CO2-air atmosphere for durations of up to 60 h. 2 at 1 h to 0.085 Ω cm2 at 60 h. Similarly, LSCF−4628 and LSCFN−46271 increased from ~0.053 Ω cm2 to 0.084 Ω cm2 and ~0.057 Ω cm2 to 0.073 Ω cm2, respectively. The evolution of Rp values over time is illustrated in 3-δ cathodes, the trends were remarkably similar, with all three Rp values showing a relatively rapid increase during the initial 10 h, followed by a more gradual rise. Notably, LSCF−4628 exhibited the most pronounced degradation rate.The endurance of O2 partially covered the catalytically active sites of O3-δ for the oxygen reduction reaction, thus hindering the diffusion of O2.The DRT analysis of the EIS results is illustrated in 2 effect, a baseline test was conducted. The three O3-δ cathodes were subjected to testing at 800 °C under 100% air for 70 h . The results are presented in 2, the degradation rates of Rp were considerably lower than those observed under the 50% CO2-air mixed gas atmosphere. According to previous studies, the performance degradation of O3-δ cathodes over time might be attributed to Sr migration and segregation on the electrode surfaces at high temperatures [To assess the magnitude of the COeratures .2-rich environment, at the 60 h mark, the gas flow was reverted to atmospheric air for a duration of 10 h to assess the possibility of reversing the increase in Rp upon CO2 removal. EIS measurements demonstrated that the Rp values decreased and closely approximated those of the electrodes that had not been exposed to CO2. In other words, the electrode performance experienced a substantial recovery. This observation suggests that exposure to CO2 likely did not result in any permanent damage to the O3-δ cathodes. For cathodes subjected to a COIn the figure within 3-δ cathodes at 650 °C was also investigated, and the results are presented in 2-air mixture, maintained for a duration of 60 h, before reverting to 100% air conditions. In the case of LSCF−6428, the Rp increased from 0.510 Ω cm2 to 0.611 Ω cm2 within the initial 10 h. Nevertheless, in comparison with EIS conducted at 800 °C, the impedance value did not exhibit significant alterations after 50 h. This observation suggests that LSCF−6428 demonstrated a higher degree of stability at lower temperatures. Upon replenishing the tube furnace with 100% air for 10 h, the EIS readings dropped back to approximately 0.520 Ω cm2. Similar trends were observed for cathodes LSCF−4628 and LSCFN−46271. To better illustrate the changes in impedance values, the impedance variations during the process are presented in 2 did not lead to permanent damage to the electrodes, as the electrode performance could be readily restored upon CO2 removal.The corrosion stability of the O2-induced performance degradation is most likely attributable to the competition between CO2 and O2 molecules vying for active sites on the electrode surface during oxygen exchange reactions [This COeactions ,35.3-δ cathodes, both before and after annealing in a 50% CO2-air environment at 800 °C, are illustrated in 3-δ cathodes retained their singular-phase perovskite structure with a cubic lattice. No evidence of any additional impurity phases was detected, or if present, their content was below the detection limit of the XRD analysis. These findings serve as strong indicators that all three cathodes remained stable when exposed to a CO2-containing atmosphere and did not undergo any reactive interactions with CO2 during the testing period.XRD patterns of the O3-δ cathodes, both prior to and after the CO2 stability testing, is depicted in The morphological evaluation of the O2-air at 800 °C. A comparison of these two sets of images clearly indicates that there was virtually no discernible alteration in electrode porosity or particle size following the 60 h CO2 exposure period. The combined results from XRD and SEM provide compelling evidence that the LSCF−6428, LSCF−4628, and LSCFN−46271 cathodes remained stable when exposed to high-temperature, high-CO2 atmospheres. Importantly, they did not undergo any reactive interactions with CO2 throughout the testing process, and their surfaces remained smooth and devoid of impurities at the conclusion of the test.0.6Sr0.4Co0.2Fe0.8O3-δ (LSCF−6428), La0.4Sr0.6Co0.2Fe0.8O3-δ (LSCF−4628), and La0.4Sr0.6Co0.2Fe0.7Nb0.1O3-δ (LSCFN−46271) cathode powders were synthesized using the solid-phase synthesis method. Details regarding the material selection, preparation procedures, and the formulation of the electrode ink can be found in our prior publication [Lalication . As the 3-δ electrodes were meticulously fabricated. O3-δ powder was admixed with a slurry composed of pine oil alcohol and ethyl cellulose. LSCF−6428, LSCF−4628, and LSCFN−46271 electrode inks were screen-printed on both sides of the GDC substrates, followed by sintering in air at 1100 °C for 2 h. The thickness of O3-δ and GDC was about 30 μm and 150 μm. This process yielded three distinct types of symmetric electrodes: LSCF−6428||GDC||LSCF−6428, LSCF−4628||GDC||LSCF−4628, and LSCFN−46271||GDC||LSCFN−46271. The active area of the electrodes was 0.81 cm2.For the measurement of electrochemical impedance spectroscopy (EIS), symmetric O2 + O2 mixtures at 800 °C, followed by exposure to air containing different levels of CO2 at temperatures ranging from 650 °C to 800 °C. To assess changes resulting from these tests, the phase compositions of the electrodes were examined using a Bruker D8 Advance X-ray diffractometer (XRD). Additionally, the microstructure of the sample surfaces was characterized through the utilization of a ZEISS Merlin scanning electron microscope (SEM).Initially, the symmetric electrodes underwent testing under varying oxygen partial pressures within N−1–106 Hz, and the bias potential was set to 0. The symmetric electrodes were placed inside a controlled-atmosphere furnace, with a collector layer consisting of gold paste and silver wires serving as conductive leads. During the measurement, various gases were introduced to the symmetric electrodes at a flow rate of 6 L/h.The electrochemical impedance spectroscopy (EIS) responses of the symmetric electrodes subjected to various atmospheres were investigated using a Solartron 1260 instrument employing a signal amplitude of 10 mV across a frequency range of 10HF, RMF, and RLF correspond to the high-frequency, mid-frequency, and low-frequency polarization resistances, respectively. The total cell Rp is the sum of these three components. The fitting results of this equivalent circuit in the figure closely match the experimental data [tal data . Using ital data .DRT analysis entails fitting complex impedance data to a relaxation time distribution. The fundamental concept behind this technique is to disentangle the impedance spectrum into a sequence of relaxation processes. The Kramers–Kronig transformation establishes a mathematical integral relationship between the real and imaginary components of impedance. The general mathematical expression for this transformation is expressed as follows:DRT(τ) signifies the distribution of relaxation times as a function of τ, which represents the relaxation time. Z′(ω) denotes the real component of impedance with respect to angular frequency (ω). Z′∞ corresponds to the high-frequency limit of the real impedance. f(τ/ω) is a weighting function employed in the analysis. The identification of characteristic peaks within the DRT spectrum was conducted through a comprehensive analysis. Variations in the DRT peaks, as an integral part of the relaxation time spectrum, were associated with distinct electrochemical processes. Impedance data analysis was carried out using the rapid and versatile data analysis/visualization (Ravdav) software version 0.9.7 .3-δ cathodes when exposed to CO2-enriched air environments. Utilizing DRT analysis, we successfully identified three distinct electrochemical processes along with their corresponding frequency ranges. Under CO2-containing atmospheres at temperatures ranging from 650 °C to 800 °C, EIS measurements revealed a consistent increase in impedance across all three cathodes as CO2 concentrations increased. This phenomenon was attributed to the adsorption of CO2 molecules onto the active oxygen sites present on the electrode surfaces, leading to a reduced oxygen exchange rate and, consequently, an increase in Rp.In this research, we conducted a comprehensive investigation into the performance and stability of three distinct O2 exposure. No secondary phases were detected, demonstrating the resilience of the cathode materials to CO2-induced corrosion or degradation. This suggests that O3-δ cathodes do not undergo chemical reactions with CO2 and maintain their structural integrity during exposure to CO2-enriched atmospheres.Crucially, XRD and SEM analyses confirmed the absence of any adverse reactions or corrosion of the electrodes induced by CO2 from the electrode surface, allowing the active oxygen sites to become available for oxygen exchange reactions once again. In essence, our findings reveal that the performance deterioration observed in O3-δ electrodes when exposed to CO2 is entirely reversible upon CO2 removal, highlighting the excellent corrosion resistance exhibited by these cathodes in the face of CO2 exposure.Furthermore, when the gas supply was reverted to 100% air, the recorded EIS spectra indicated a remarkable recovery in electrode performance. This recovery can be attributed to the removal of COAmong the three different cathode compositions examined in this study, it is noteworthy that LSCF−6428 and LSCFN−46271 displayed superior durability compared to LSCF−4628. Both LSCF−6428 and LSCFN−46271 emerge as promising cathode materials for SOFCs.


This relationship is useful to identify kinetic effects in sample measurements and recover formation temperature. Here, we use experimental measurements and theory to constrain the equilibrium relationships between heavy isotope substitution in mass 47 and mass 48 CO2 isotopologues, and their relationships to formation temperature.A powerful tool for reconstructing past temperatures is based on the extent of carbonate ions in carbonate minerals with more than one heavy isotope substitution, termed carbonate clumped isotope thermometry. The use of the clumped isotope thermometer assumes that the mineral formed under isotopic equilibrium conditions, however, some carbonate minerals do not form at equilibrium. When carbonate minerals do not form at isotopic equilibrium, they are typically not useful for temperature reconstructions. It is now possible to simultaneously determine the extent of heavy isotope substitution in mass 47 and mass 48 CO The acid digestion of minerals containing carbonate ion groups with m/z 63 yields m/z 47 CO2, which can be measured by isotope ratio mass spectrometry (m/z 48 CO2 isotopologue derived from acid digestion of m/z 64 (12C18O216O) carbonate ion groups could be used for geothermometry  has two 18O substitutions and is therefore in extremely low abundance at 4.1 ppm in air, which is an order of magnitude lower than m/z 47 isotopologues at 45 ppm (m/z 48 CO2 isotopologue (13C18O17O) has an abundance of 16.7 ppb , DH-2–11 (163 ± 5 ka), DH-2–12 (57 ± 5 ka), and DH-2–13 (151 ± 4 ka) (−1) in water with a low calcite saturation index (0.16–0.21)  mammillary calcite samples from core DH-2 for paired Δ ± 4 ka) , that pr16–0.21) . Devils 16–0.21) , 1992.2.3.m/z 44, 45, and 46 are registered through 3 × 108, 3 × 1010, and 1011 Ω resistors, respectively, while detectors for m/z 47 through 49 are registered through 1012 Ω resistors.Standards and samples were analyzed on three mass spectrometers using five configurations , includim/z 47–49 detectors. These ESAs consist of two curved plates with a voltage difference placed directly in front of each of the Faraday collectors. The addition of the ESAs as well as ion lenses following the magnetic sector of the flight tube removes secondary ion and electron signals from the mass detection. This removal results in a drastic reduction in the interfering signals on all masses (m/z 44–49) during operation, producing flatter and more stable baselines, relative to the older MAT 253  before the  MAT 253 . In addi2 (g) was cryogenically purified through traps containing dry ice-cooled ethanol and liquid nitrogen, which remove low vapor pressure gases such as H2O (g). The CO2 passed through elemental silver wool (Sigma Aldrich) to remove sulfur compounds, followed by a −20°C gas chromatograph (GC) that contains Porapak Type-Q™ 50/80 mesh column pack material with He carrier gas. The m/z 44 beam intensity is 16 V. Data are acquired in 9 blocks of 10 cycles, with each consisting of 8 s of integration and 16 s of changeover delay, for a total integration time of 720 s.The Thermo Fisher MAT 253 used an autosampler similar to what is described in m/z 44 ion beam intensity was 24 V before 6/2017 and 18 V thereafter. Nu Perspective-1a analyzed 0.5 mg samples, with sample and working gas volumes depleted in microvolume mode at precisely matched rates, with m/z 44 ranging from 24–9 V during sample acquisition. Microvolume mode allows for a full hour-long measurement per sample. Data were taken in 3 blocks of 20 cycles, including 8 s of changeover delay and 20 s of integration per cycle, for a total integration time of 1,200 s. The sample preparation system was operated by software in Labview that controls the sampler, GC column, cryogenic dewar lifters, and valves. The Labview software is integrated with the Perspective Stable Gas Control software interface that controls the Nu Perspective mass spectrometer.Nu Perspective-EG, Nu Perspective-1, and Nu Perspective-1a used the same mass spectrometer and a similar autosampler setup as the MAT 253. Samples weighing 5 mg were analyzed in bellows on the Nu Perspective-EG and Nu Perspective-1 in 4 blocks of 20 cycles, including 8 s of changeover delay and 20 s of integration per cycle, with a total integration time of 1,600 s. The ™ 50/80 and silver wool. This instrument operates under vacuum pressure and does not use a carrier gas. The sample and working gas volumes were matched precisely during depletion into the mass spectrometer, with m/z 44 ranging from 24–9 V. Sample data were analyzed in 3 blocks of 20 cycles, with each cycle integrating for 20 s, for a total integration time of 1,200 s.Nu Perspective-2 used a Nu Carb Sample Digestion System instead of a common acid bath, where 0.5 mg of carbonate mineral was digested at 70°C in individual glass vials with 105 wt% phosphoric acid. The sample gas was cryogenically purified in liquid nitrogen-cooled tubes called coldfingers before passing through a relatively short GC column packed with Porapak Type-Q2.4.2 gas cylinder and was equilibrated with 5–10 mL of 25°C deionized (DI) water. The heavy isotope enriched 2 was equilibrated with evaporated DI water held at 25°C. Aliquots of the two 25°C gases were re-equilibrated at 1000°C by heating the gases in quartz tubes inside a muffle furnace for >1 hr, and then flash cooling the tubes, to produce gases with near stochastic clumped isotope values.We analyzed equilibrated gas standards on Nu Perspective-EG . We utilocedures . The hea2.5.2 equilibrated at 25 and 1000°C with samples digested at 90°C. The 2 or carbonate standard values originally determined using equilibrated CO2, we want to note again that the only instrument here that used equilibrated CO2 normalization was Nu Perspective-EG, while the others use carbonate standard-based transfer functions for normalization  , meaning(I-CDES) , meaninglization .m/z 44–49) on all instruments before any further data normalization. The background is measured  at the start of an analysis and is subtracted from the measurement. For the nonlinearity slope correction, a slope was determined over a ±10-replicate moving average for the regression lines between 2 gas standards equilibrated at 25 and 1000°C and/or ETH-1 and ETH-2 , was fit through experimentally determined A polynomial,  calcite .All 3.3.The calculated equilibrium T and T relationships (2 = 0.99) and 2 = 0.99).The ionships  are descThe AFFs for the compositionally-dependent fractionation of O isotopes during phosphoric acid digestion of carbonate minerals  are repr4.4.1.m/z 48 isotopologue is an order of magnitude lower in abundance than the m/z 47 isotopologue  and a brachiopod (Magellania venosa) and observed similar enrichments in All sample and standard gression , with thof 4.2°C . Guo  and 19 (r2 = 0.99),T is in Kelvin. We also report the inverse of the relationships for ease of use for samples with unknown precipitation temperature in These offsets are well within the bounds of what we observed when comparing differences between ETH standard ratories . This isurements . Therefond Δ48-T , represeT equation , as were mean P < 0.0001). The Devils Hole mean There are multiple lines of evidence that Devils Hole calcite has achieved quasi-equilibrium oxygen and clumped isotope values . It has 2 degassing from groundwater, which is observed in other speleothems (It is unlikely that the offsets are the result of Devils Hole samples exhibiting kinetic effects from COleothems . Clumped ∼−0.793 . The sam ∼−0.793 . AlthougThe combined average o 34.3°C . This fu5.This study contributes to establishing research  that carT, and T equilibrium relationships with experimental values for standards and samples, and theoretical equilibrium values, and formed regressions using data from this study and previously published work. These regressions are useful for determining if unknown samples precipitated at isotopic equilibrium and can therefore be used in accurate temperature reconstructions, or potentially to recover primary temperatures by using We have further constrained the Additionally, we compared readmeinster-instrumental comparisonscustom R functionsanalysesuser manualSupporting Information Tables S1 - S27Text S1 to S3; Figures S1 to S6


U6 snRNA is a catalytic RNA responsible for pre-mRNA splicing reactions and undergoes various post-transcriptional modifications during its maturation process. The 3'-oligouridylation of U6 snRNA by the terminal uridylyltransferase, TUT1, provides the Lsm-binding site in U6 snRNA for U4/U6 di-snRNP formation and this ensures pre-mRNA splicing. Here, we present the crystal structure of human TUT1 (hTUT1) complexed with U6 snRNA, representing the post-uridylation of U6 snRNA by hTUT1. The N-terminal ZF-RRM and catalytic palm clamp the single-stranded AUA motif between the 5'-short stem and the 3'-telestem of U6 snRNA, and the ZF-RRM specifically recognizes the AUA motif. The ZF and the fingers hold the telestem, and the 3'-end of U6 snRNA is placed in the catalytic pocket of the palm for oligouridylation. The oligouridylation of U6 snRNA depends on the internal four-adenosine tract in the 5'-part of the telestem of U6 snRNA, and hTUT1 adds uridines until the internal adenosine tract can form base-pairs with the 3'-oligouridine tract. Together, the recognition of the specific structure and sequence of U6 snRNA by the multi-domain TUT1 protein and the intrinsic sequence and structure of U6 snRNA ensure the oligouridylation of U6 snRNA. U6 snRNA plays a catalytic role in pre-mRNA splicing. Here the authors report the structure of human terminal uridyltransferase, TUT1, complexed with U6 snRNA revealing the mechanism of specific uridylation of U6 snRNA by TUT1. The U6 snRNP enters the splicing cycle by forming di-U4/U6 snRNPs in a reaction catalyzed by the p110/SART3 and Lsm2-8 ring protein complexes, which together promote the annealing of U6 and U4 snRNAs4. The di-U4/U6 snRNPs are used to form the U4/U6·U5 tri-snRNP, which is then recruited into the pre-spliceosome consisting of the pre-mRNA and the U1 and U2 snRNPs. The U6 snRNA forms an alternative helix with the U2 snRNA, and the splicing reactions proceed with the structural rearrangements of the U6 snRNA. In the splicing reactions, U6 snRNA catalyzes the trans-esterification splicing reactions by forming the active site with the coordinating divalent cations for the catalysis5.In eukaryotes, the pre-mRNA splicing reaction is catalyzed by the spliceosome, a ribonucleoprotein (RNP) complex. The major spliceosome comprises five small RNP complexes—U1, U2, U4, U5, and U6 snRNPs—and a large number of proteins7. The U6 snRNA primary transcript undergoes multiple processing steps, including modifications of the nucleosides, 5'-γ-phosphate methylation, 3'-oligouridylation, and 3'-uridine trimming8. The primary transcript of the human U6 snRNA has four genome-encoded uridines (4 Us: UUUUOH) at its 3′-end. After transcription, the 3′-end is oligouridylated by a terminal uridylyltransferase (TUTase), TUT1 (TENT1)11. Subsequently, the 3'-oligouridylated tail is trimmed by Mpn1 (Usb1), a 3′−5′ exonuclease14. The mature U6 snRNA has five uridines with a 2′,3′-cyclic phosphate (5 U > p: UUUUU>p). These 3′-maturation processes protect U6 snRNA from degradation. The 3'-oligouridylated tail is required for the di-U4/U6 snRNP formation. The Lsm2-8 ring complexes bind the oligouridylated tail of U6 snRNA, and together with p110 facilitate the annealing of the U6 and U4 snRNAs to form the di-U4/U6 snRNP16. The 3′-oligouridylated tail is also required for the U6 snRNA recycling after the splicing reaction17, and thus contributes to efficient pre-mRNA splicing in cells.U6 snRNA is transcribed by RNA polymerase III. The U6 snRNA transcript has 5'-short stem and 3'-telestem-internal stem-loop (ISL) secondary structures, which are separated by a single-stranded region containing the AUA motif11, and is a member of the TENT family20. hTUT1 is a multi-domain enzyme consisting of the N-terminal zinc finger (ZF), the RNA recognition motif (RRM), the central catalytic core domain, and the C-terminal kinase associated-1 (KA-1) domain25. The detailed mechanism for the nucleotide specificity of hTUT1 was clarified by structural and biochemical analyses of the catalytic core domain of hTUT1 complexed with UTP22. However, the detailed mechanisms for the specific recognition of U6 snRNA by the multiple domains of hTUT1 are not fully understood. Furthermore, the regulatory mechanism ensuring the oligouridylation of the 3'-end of U6 snRNA also remains enigmatic.Human TUT1 (hTUT1) specifically oligouridylates U6 snRNA using UTP as a substrateHere, we present the crystal structure of a shortened hTUT1 complexed with a short U6 snRNA, representing the post-uridine addition to the 3'-end of U6 snRNA by hTUT1. Crystallographic and biochemical studies of hTUT1 have now revealed the molecular mechanism underlying the specific oligouridylation of the structured U6 snRNA and the regulation of the 3'-oligouridylation of U6 snRNA by hTUT1.23. A proline-rich region (PRR), which is less conserved among vertebrate TUT1 proteins and dispensable for the oligouridylation of U6 snRNA in vitro22, is present within the palm. The nuclear localization signal (NLS) is inserted within the C-terminal KA-1 domain  is a multi-domain protein consisting of the N-terminal ZF, RRM, catalytic core domains , and the C-terminal KA-1 domain22, as described below. The C-terminal KA-1 domain of hTUT1 is bound around the stem of the ISL and the bulged region between the telestem and the ISL of U6 snRNA22  complexed with an in vitro transcribed full-length U6 snRNA ending with four 3'-uridines (3'− 4 Us) were unsuccessful. Thus, we designed a shorter hTUT1 and a shorter U6 snRNA for the crystallization of the hTUT1-U6 snRNA complex, based on our previous biochemical and structural analyses of hTUT1A22 Fig. . In addiA22 Fig. . While t_ΔC Fig. . For a s_ΔC Fig. .22  as efficiently as the hTUT1_FL and rapidly produces U6 snRNA with six 3'-Us (U103-U108) Fig. . hTUT1_ΔP6522 and contains one hTUT1_ΔC-U6_mini complex in the asymmetric unit cell. The initial phase was determined by the molecular replacement method, using the structures of the catalytic core domains of hTUT1 (PDB ID: 5WU1 and 5WU6)22 as search models. Finally, the structure was refined to an Rwork of 30.2% (Rfree = 33.9%) at 3.7 Å resolution. The scaling and refinement statistics are summarized in Supplementary Table The X-ray diffractive crystal of hTUT1_ΔC complexed with U6_mini was obtained, and its structure was analyzed. The crystal belongs to the space group The structure of the hTUT1_ΔC-U6_mini complex revealed the extensive interactions between U6_mini and multiple domains of hTUT1_ΔC Fig. , 5. The The structure of the catalytic core domain, the palm and fingers, in the hTUT1_ΔC-U6_mini complex is virtually identical to those of the previously determined structures of hTUT1 lacking the ZF and KA-1  and U6_mini-2 were 258 ± 46, 204 ± 41 and 574 ± 78 nM, respectively  did not increase the oligouridylation of the RNA by hTUT1_FL, the removal of the AU in the 5'-single-stranded AUA motif of U6_mini (U6_mini-2) decreased the oligouridylation Fig. . Based oely Fig. . Thus, tely Fig. . The gelely Fig. . The Q5328. Thus, the present structure represents the post-U-addition stage. In the structure of the hTUT1_ΔC-U6_mini complex, the 5'-anchor  in the palm interacts with the bases at the top of the stem region, while the groove loop (amino acid residues 423 − 427) in the fingers interacts with the minor groove of the stem  to produce U6 snRNAs with six 3'-uridines (6 Us: U103-U108). However, hTUT1_FL cannot produce U6 snRNAs with more than seven uridines efficiently Fig. .It was assumed that the four-adenine tract (A27—A30) in the telestem of U6 snRNA could define the number of uridines added to the 3' end of U6 snRNA, regulate the uridine incorporation and ensure the oligouridylation. The oligouridylation proceeds until U103—U106 form a duplex with the four-adenine tract (A27—A30), and U107 and U108 occupy positions −1 and +1 in the catalytic pocket, respectively Fig. . In suppAfter the U108 incorporation, the telestem of U6 snRNA translocates, and U107 and U108 move to the −2 and −1 positions, respectively Fig. . U107 at29, and the motif sequence matches that between the 5'-short stem and 3'-telestem of U6 snRNA  of TUT4/7 to pre-let746. The stable ternary complex facilitates the processive oligouridylation of pre-let7 by the C-terminal catalytic module (CM) of TUT4/7 . Residues L527 and Y592 in URT1 contribute to its preference for purine over pyrimidine at the -2 position (the 3'-priming position corresponds to -1) of single-stranded RNA47. This preference enables URT1 to control the optimal number of uridines and URT1 added two uridines to the 3′ extremity of polyA. L527 and Y592 of AtURT1 corresponds to V361 and G426 of hTUT1, respectively. V361 is located at the 5'-anchor of hTUT1 and stacks with the bases at the top of the stem : TUT1, TUT4 and TUT7. While TUT1 and TUT4/7 belong to the TENT family, their domain compositions and substrate RNAs are different30 Fig. . TUT4 anUT1 Fig.  is distiet7 Fig. . In the 4/7 Fig. . The oli 43 Fig. . In cont 43 Fig. . hTUT1 ctem Fig. . hTUT1 utem Fig. . Recentltem Fig. . G426 istem Fig. . Thus, tIn sum, the intrinsic sequence and structure of U6 snRNA and the recognition of the U6 snRNA structure by the multi-domain hTUT1 protein together regulate and ensure the oligouridylation of U6 snRNA, which is essential for the splicing of pre-mRNAs.The synthetic DNA encoding human TUT1 (hTUT1) and its variants were purchased from Eurofins Genomics (Japan). The nucleotide sequences of the synthetic hTUT1 gene and its variants are shown in Supplementary Table E. coli BL21(DE3)  was transformed by each plasmid, and grown at 37˚C until the A600 reached 1.0. The expression of the hTUT1 protein or its variants was induced by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and continuing the culture at 18 °C for 16 h. The cells were harvested and lysed in buffer, containing 20 mM Tris-HCl, pH 7.0, 500 mM NaCl, 10 mM β-mercaptoethanol, 20 mM imidazole, 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and 5% (v/v) glycerol. The proteins were first purified on a Ni-NTA agarose column , followed by a HiTrap Heparin column . Finally, the proteins were purified on a HiLoad 16/60 Superdex 200 column , in buffer containing 20 mM Tris-HCl, pH 7.0, 300 mM NaCl, and 10 mM β-mercaptoethanol. The purified proteins were concentrated and stored at −80 °C.48, was used as the template. After in vitro transcription, the RNA was phenol-chloroform-extracted and dissolved in buffer containing 10 mM Tris-HCl, pH 7.0, and 20 mM MgCl2. The RNA solution was subjected to 15 cycles of incubations (at 60˚C for 3 min and 25 °C for 3 min) to self-cleave the RNA by the HDV ribozyme. The 3'-cyclic phosphate of RNA after the HDV self-cleavage was removed by T4 kinase , in buffer containing 50 mM MES, pH 5.8, 20 mM MgCl2, and 10 mM DTT. Finally, the RNA was purified by 10% (w/v) polyacrylamide gel electrophoresis under denaturing conditions. The synthetic oligonucleotide sequences of the U6 snRNA gene and its variants used for in vitro transcription are listed in Supplementary Table Synthetic human U6 snRNA and its variants were synthesized by T7 RNA polymerase using the corresponding DNA fragments as templates. To prepare U6 snRNA (or its variants) with the homogeneous 3´-end, the DNA fragment encoding the U6 snRNA gene (or its variants) carrying the T7 promoter and the HDV ribozyme sequence upstream and downstream of the U6 snRNA gene (or its variants) sequence, respectively49. The initial phase was determined by molecular replacement with Phaser50, using the catalytic core structures of hTUT1 (PDB IDs: 5WU1 and 5WU6)22. The structure was refined with phenix.refine51 and manually modified with Coot52. All figures were prepared with PyMOL (http://www.pymol.org).The crystal used for the structural determination was generated by the sitting drop vapor diffusion method at 4˚C. Before crystallization, 25 μM protein and 30 μM RNA were mixed in a solution containing 10 mM Tris-HCl, pH 7.0, 150 mM NaCl, 5 mM β-mercaptoethanol, 10 mM DTT, and 50 mM zinc acetate. A 200 nL portion of the protein-RNA solution was mixed with 100 nL of the reservoir solution, containing 18–20% (w/v) PEG3350, 100 mM Bis-Tris, pH 6.4–6.5, 2% (v/v) Tacsimate, pH 6.0 , and 8% (v/v) acetonitrile. The data set was collected at beamline 17 A at the Photon Factory at KEK, Japan. The crystals were flash-cooled with the reservoir solution supplemented with 30% (v/v) PEG400. The dataset was indexed, integrated, and scaled with XDS2, 10 mM β-mercaptoethanol, 1 mM UTP, and the indicated concentrations of RNA transcript and TUT1 (or its variants), was incubated at 37 °C. At the indicated time points, a 5 μL portion of the reaction mixture was withdrawn, and the reactions were stopped. The RNAs were separated by 10% (w/v) polyacrylamide gel electrophoresis under denaturing conditions and stained with ethidium bromide. The band intensities were quantified with Gel Doc EZ, using the Image Lab software .A reaction mixture (30 or 40 μL), containing 50 mM Tris-HCl, pH 8.5, 100 mM NaCl, 10 mM MgCl2, 10 mM β-mercaptoethanol, and 10% (v/v) glycerol. The mixture was incubated at room temperature for 15 min. The RNAs were separated by 6% (w/v) or 8% (w/v) polyacrylamide gel electrophoresis under native conditions at room temperature, and the gels were stained with ethidium bromide. The shifted band intensities were quantified with Gel Doc EZ, using the Image Lab software .RNAs (100 nM U6_mini and its variants or 50 nM U6 snRNA and its variants) were mixed with various concentrations (0–1000 nM) of hTUT1_ZF-RRM (residues 1-140) or its variants in 10 μL of buffer, containing 50 mM Tris-Cl, pH 8.5, 100 mM NaCl, 10 mM MgClFurther information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Adenomatosis polyposis coli down-regulated 1 (APCDD1)—a conserved single-span transmembrane protein containing a large extracellular domain—negatively regulates WNT signaling and plays important roles in hair follicle development, CNS vascular development, and glial differentiation. We report here the three-dimensional structure of the ECD of APCDD1, revealing an unusual architecture. The APCDD1 ECD consists of two closely apposed β-barrel domains (ABD1 and ABD2). ABD2 contains a large hydrophobic pocket that accommodates a bound lipid. In an in vitro assay, the ECD of APCDD1 bound to WNT7A, which contains a covalently linked palmitoleate. Collectively, the results of this study suggest that APCDD1 serves as a negative feedback regulator of WNT signaling by neutralizing WNT ligands. APCDD1 transcripts are highly up-regulated. We have determined the three-dimensional structure of the extracellular domain of APCDD1, and this structure reveals an unusual architecture consisting of two closely apposed β-barrel domains (ABD1 and ABD2). ABD2, but not ABD1, has a large hydrophobic pocket that accommodates a bound lipid. The APCDD1 ECD can also bind to WNT7A, presumably via its covalently bound palmitoleate, a modification that is common to all WNTs and is essential for signaling. This work suggests that APCDD1 functions as a negative feedback regulator by titrating WNT ligands at the surface of responding cells.Diverse extracellular proteins negatively regulate WNT signaling. One such regulator is adenomatosis polyposis coli down-regulated 1 (APCDD1), a conserved single-span transmembrane protein. In response to WNT signaling in a variety of tissues,  WNT signaling plays a central role in embryonic development and tissue homeostasis. WNT signaling is negatively regulated by diverse extracellular and intracellular pathways . These iApcdd1  is associated with hereditary hypotrichosis simplex, a defect in hair follicle development  lesions  myelination, astrocyte migration, and adipocyte differentiation. In mice, APCDD1 promotes oligodendrocyte precursor cell (OPC) differentiation ex vivo and it enhances regenerative myelination after white matter injury , consist lesions , 24. In  lesions . In cult lesions .At present, the mechanism of APCDD1 action is uncertain, as APCDD1 has been variously reported to associate with LRP5, WNT3A, and β-CATENIN –19. To gSI Appendix, Fig. S1A and Table S1). Attempts to use molecular replacement for structure determination failed because of a lack of discernable homology to known structures and the low quality of predicted structures. Therefore, we produced, purified, and crystallized a fusion protein between engineered maltose-binding protein (eMBP) , collected X-ray diffraction data to 2.3 Å resolution, and used molecular replacement with eMBP to obtain phase information . Although the resulting electron density map provided a good fit to the eMBP half of the fusion protein, the map of APCDD1 was of insufficient quality for model building , we produced this domain as a glycosylated secreted protein using human embryonic kidney (HEK293) cells, crystallized it, and collected X-ray diffraction data to 1.95 Å and 2.15 Å resolution from two crystal forms (n (eMBP)  and APCDbuilding A.SI Appendix, Table S1). Molecular replacement with this structure was then used to solve the structure of APCDD1 in the two crystal forms described above . ABD1 and ABD2 have, respectively, three and two intradomain disulfide bonds, a large area of interfacial contact, and one interdomain disulfide bond (Cys52–Cys466). This structure is observed in all three crystal forms (eight unique APCDD1 structures), with a global rmsd of 0.7 to 1.1 Å among structures . ABD1 and ABD2 present highly similar folds, consisting of two curved antiparallel β-sheets connected by an α-helix and loop domain (AHLD) . For both ABD1 and ABD2, the interior surface of the β-barrel is lined by hydrophobic amino acids. The barrels are open to the solvent on the side adjacent to AHLD, whereas the other side is closed by tight packing between side chains from β1 and β2 for ABD1 and from α3 and β12 for ABD2  that differ in orientation by ~90° (n (AHLD) B and C. for ABD2 A and B.SI Appendix, Fig. S3 A and B). Therefore, we modeled a molecule of stearic acid into the electron density in the ABD2 pocket; the subsequent structural refinement revealed a good fit between stearic acid and the electron density .Intriguingly, we observed an electron density consistent with the acyl chain of a lipid molecule in the hydrophobic pocket of ABD2 in two of the four eMBP-APCDD1 structures .Since APCDD1 inhibits WNT signaling and WNTs have a covalently linked palmitoleate , as calculated with CASTp .Despite the nearly identical backbone configurations of the β-barrel residues in ABD1 and ABD2, the interior volumes of their hydrophobic pockets are dramatically different: ~35 Åth CASTp . To defith CASTp . Two looth CASTp . In ABD1th CASTp . The difth CASTp B–D. In pth CASTp E–H. ThesWith respect to the function of the individual ABD1 and ABD2 domains, our attempts to address this question by producing the individual domains have thus far failed, most likely due to the instability of the individual domains. We note that the two domains have a large area of interdomain contact and an interdomain disulfide bond, which likely stabilizes them.3 to ~400 Å3 . At ~350 Å3, the ABD2 pocket is at the high end of this distribution and is predicted to accommodate hydrophobic ligands in the 230 to 440 Da range . The shallow ABD1 pocket might accommodate a small ligand or part of a larger ligand.Binding to the PAM that is covalently linked to WNT ligands is a recurrent theme among protein that transduce or modulate WNT signals. These proteins include FRIZZLED , NOTUM , 8, WNTLSI Appendix, Fig. S5). Detailed structural comparisons between APCDD1 and members of the lipocalin and P2 families showed marked differences in the shapes of their β-barrels and the orientations of β-sheets, giving an average rmsd of ~2.4 to 3.7Å . Proteins in the lipocalin and P2 families bind a wide variety of hydrophobic molecules via the pocket within their β-barrel domains  and AlphaFold  database domains . Across th CASTp .SI Appendix, Fig. S6). The APCDD1L sequences retain the principal features of the APCDD1 sequence, including the 12 conserved and disulfide-bonded cysteines . Interestingly, APCDD1L amino acid sequences show a several-fold greater rate of evolutionary change compared to APCDD1 sequences, as seen in the dendrogram in SI Appendix, Fig. S7A. Threading the rat APCDD1L sequence into the mouse APCDD1 structure suggests that APCDD1L adopts very nearly the same structure, with an ABD2 pocket that can also accommodate a lipid . At present, the function of APCDD1L is unknown.Nearly all vertebrate genomes code for two APCDD1 homologous sequences: APCDD1 and APCDD1-like (APCDD1L), which exhibit ~50% amino acid identity throughout their length  and Triton X-114], which would be expected to both compete for occupancy of the lipid-binding pocket and stabilize the unbound PAM. Both detergents reduced the WNT7A-APCDD1 binding signal, with 1% DDM showing a greater potency than 0.1% DDM A. In conTo determine whether APCDD1 directly interacts with the extracellular four tandem β-propeller-epidermal growth factor-like domain pairs (PE1-4) of LRP5/6 coreceptors, we produced PE1-4 of LRP5 and LRP6 with a C-terminal biotin tag, captured them on streptavidin-coated wells, and probed the wells with APCDD1-AP and AP-APCDD1 fusion proteins or, as a positive control, with DKK1-AP and AP-DKK1 fusion proteins B. We obsApcdd1 transcripts are 5 to 100 fold more abundant than transcripts coding for WNT receptor (FZD4), coreceptor (LRP5 and LRP6), and coactivator (GPR124 and RECK) proteins . Although the corresponding protein abundances in vivo are not presently known, the relative transcript abundances suggest that in brain vascular endothelial cells APCDD1 may be substantially more abundant than WNT receptors, coreceptors, and coactivators.The lower WNT7A-APCDD1 binding signal compared to the WNT7A-FZD4 CRD binding signal presumably reflects a lower affinity for the former interaction. If this differential binding also applies in vivo, it may be partially offset by the greater abundance of transcripts coding for APCDD1 relative to transcripts coding for WNT receptors and coreceptors. For example, in mouse brain vascular endothelial cells, in which a high level of canonical WNT signaling maintains the blood–brain barrier, In sum, the experiments described here reveal APCDD1 to be a transmembrane lipid-binding protein, and they suggest that APCDD1 exerts its inhibitory effect on WNT signaling by binding to lipidated WNTs C.The present study shows that the ECD of APCDD1 consists of two homologous β-barrel domains (ABD1 and ABD2), linked by one interdomain disulfide bond. Protein sequence searches and protein fold comparisons show that APCDD1 exhibits an unusual architecture. Interestingly, structural analyses show that ABD2, but not ABD1, has a hydrophobic pocket that is larger than the hydrophobic binding pockets in FRIZZLED , NOTUM , 8, and 17SI Appendix, Fig. S4, the hydrophobic PAM-binding pockets in these WNT-interacting proteins exhibit substantial shape and size diversity. In the FRIZZLED CRD monomer and dimer-binding modes, and in WLS and DLP, the PAM-binding pockets are extended and narrow, whereas NOTUM contains a compact and globular hydrophobic PAM-binding pocket. By contrast, the structures of APCDD1 determined here reveal that the ABD2 pocket has a large, open, and globular shape that is predicted to accommodate hydrophobic ligands with molecular masses in the 230 to 440 Da range, including PAM (254 Da). Determining whether the APCDD1–WNT interaction also involves protein–protein interactions will likely require a high-resolution structure of the complex.The PAM that is covalently joined to WNT proteins plays a central role in WNT-CRD binding , 41–44, 41SI Appendix, Fig. S7A), but many invertebrate genomes carry one or more APCDD1/APCDD1L sequences with approximately the same degree of similarity to APCDD1 and APCDD1L. Since WNT signaling arose early in metazoan evolution  marker Sox2 (Apcdd1l morpholino oligonucleotide knockdown and TALEN-mediated germ-line elimination of Apcdd1l in zebrafish show that a) homozygous loss of Apcdd1l is dispensable for viability and fertility, and b) progeny embryos from Apcdd1l homozygous null parents, which lack both maternal and zygotic Apcdd1l function, show expansion of the Spemann organizer region , a phenotype that appears to be of little consequence for subsequent development  signaling  in a PAM-dependent manner to maintain WINGLESS solubility  was carried out over the PDB database using the HHpred server . For the49For phylogenetic analysis, the amino acid sequences of APCDD1 and APCDD1L were aligned using ClustalΩ , and theFor mouse APCDD1 constructs, a series of coding segments of the APCDD1 ECD were cloned into pHLsec-mVenus-12H , 56, 57 Escherichia coli AP and finally an 8xHis tag, generating Nb-AP. All constructs were confirmed by DNA sequencing.For protein–protein interaction experiments, coding segments of the APCDD1 ECD and the human FZ4 CRD  were clo2 in Dulbecco’s modified eagle medium (MilliporeSigma) supplemented with 2 mM L-Glutamine , 0.1 mM nonessential amino acids (Gibco) and 10% [v/v] Fetal Bovine Serum . The FBS concentration was lowered to 2% [v/v] after transfection with the DNA using polyethylenimine  as described previously (Flask® Cell Culture Vessels (Corning) and cultured in the presence of 5 μM of the class I α-mannosidase inhibitor, kifunensine (HEK293T (ATCC CRL-11268) cells were maintained in a humidified 37 °C incubator with 5% COeviously . For cryunensine  and 4 mMunensine  after trE. coli expression and purification of Nb-AP, the plasmid DNA was transformed into E. coli BL21 Star™ (DE3) cells (ThermoFisher) and induced with 0.2 mM isopropyl β-thiogalactopyranoside in Luria broth containing 100 μg/mL ampicillin (MilliporeSigma) at room temperature (~25°C) overnight. The cell pellets were harvested by centrifugation and resuspended in B-PER bacterial protein extract reagent (ThermoFisher) supplemented with 50 mM HEPES, pH 7.5, 0.3 M NaCl, 30 mM imidazole, 1 mM MgCl2, 500 U benzonase (MilliporeSigma), 0.2 mg/mL lysozyme, and cOmplete Protease Inhibitor Cocktail (MilliporeSigma). The cell lysate was clarified by centrifugation, and the supernatant was filtered using a 0.45-μm Steritop filter (MilliporeSigma). Proteins were purified by IMAC using Ni Sepharose 6 Fast Flow resin . The eluted sample was dialyzed against 10 mM HEPES, pH 7.5, 0.15 M NaCl.For Flavobacterium meningosepticum endoglycosidase-F1 (Endo-F1) prepared as described previously  for the B-factors for all atoms. The second round of MR using PHASER  using PHASER  using thg PHASER  was condg PHASER  and subsg PHASER , 74 for g PHASER  and refig PHASER  and PHENg PHASER  with trag PHASER . The subg PHASER  and PHENg PHASER  with TLSg PHASER  was usedStructure-based multiple sequence alignment was performed using Clustal Omega  and ESPrTo identify the bound ligand in APCDD1 structures, HPLC coupled with mass spectrometry (MS) was used. Briefly, purified APCDD1 protein solution or conditioned media without APCDD1  was mixed with the extraction solution (methyl tert-butyl ether/methanol/water in a ratio of 10:3:2.5) and the phases were separated. The organic phase was dried in a SpeedVac concentrator. The dried pellet was reconstituted in a solution of n-butanol and methanol in a 1:1 ratio. The sample was then analyzed by HPLC/MS on an Ultimate 3000 UPLC (using an Accucore C30 column) and Q-Exactive Plus Orbitrap. Ligand identification was performed by comparison of the mass spectrum of the analyte with the library database at Cayman Chemical.For the AP fusion proteins , HEK293T cells were grown in six-well plates and transfected with plasmids and PEI as described previously , 57. ConIMAC-concentrated complexes (100 μL each) were immobilized on 96-well streptavidin-coated plates (Thermo Fisher Scientific) at 4 °C overnight. For the detergent addition assays, 1% DDM (Anatrace), 0.1% DDM (Anatrace), or 2.25% Triton X-114 (MilliporeSigma) were included in all steps from the 96-well plate immobilization to the washes. The wells were then washed three times with wash buffer  supplemented or not with the indicated detergents and incubated with a 10-fold dilution of bovine serum albumin (BSA) blocker buffer (Thermo Fisher Scientific 37525) in wash buffer for 1 h at 25 °C. The wells were washed with wash buffer with the indicated detergents and incubated with conditioned media containing AP probes  or recombinant Nb-AP proteins at 4 °C overnight. The wells were subsequently washed three times with wash buffer with the indicated detergents and incubated with BluePhos phosphatase substrate solution (Kirkegaard and Perry Laboratories 50-88-00) to visualize the bound AP probes. The binding assays were performed twice.Biotinylated baits were immobilized on 96-well streptavidin-coated plates (Thermo Fisher Scientific) at 4 °C overnight. The wells were then washed three times with wash buffer  and incubated with a 10-fold dilution of BSA blocker buffer (Thermo Fisher Scientific 37525) in wash buffer for 1 h at 25 °C. The wells were washed with wash buffer and incubated with conditioned media containing AP probes  or recombinant Nb-AP proteins at 4 °C overnight. The wells were subsequently washed three times with wash buffer and incubated with BluePhos phosphatase substrate solution (Kirkegaard and Perry Laboratories 50-88-00) to visualize the bound AP probes.Appendix 01 (PDF)Click here for additional data file.


About 18 nm, spherical, and highly agglomerated hematite (αFe2O3) NPs were obtained. Sorghum seeds were primed with 5, 10, and 15 mg/L αFe2O3 NPs, and, after seven days of germination, the seedlings were transferred into potting soil, cultivated for fourteen days, and were subsequently water deprived (WD) for a further seven days. A reduction in plant height (78%), fresh  and dry  weights, and chlorophyll (chl) content , chla (135%), and chlb (1827%)) was observed in WD plants, and this correlated with low nutrients  and alteration in the anatomic structure (epidermis and vascular bundle tissues). Oxidative damage was observed as deep blue (O2●−) and brown (H2O2) spots on the leaves of WD plants, in addition to a 25% and 40% increase in oxidative stress markers (H2O2 and MDA) and osmolytes , respectively. Seed priming with 10 mg/L αFe2O3 NPs improved plant height (70%), FW (56%), DW (34%), total Chl (104%), chla (160%) and chlb (1936%), anatomic structure, and nutrient distribution. Priming with 10 mg/L αFe2O3 NPs also protected sorghum plants from drought-induced oxidative damage by reducing ROS formation and osmolytes accumulation and prevented biomolecule degradation. The study concludes that green synthesized hematite NPs positively influenced sorghum growth and prevented oxidative damage of biomolecules by improving nutrient uptake and osmoregulation under drought stress.Drought is a major abiotic stress that confronts plant growth and productivity, thus compromising food security. Plants use physiological and biochemical mechanisms to cope with drought stress, but at the expense of growth. Green-synthesized nanoparticles (NPs) have gained great attention in agriculture due to their environmental friendliness and affordability while serving as potential biofertilizers. This study investigates the role of hematite (αFe Sorghum bicolor (L.) Moench is a grain-producing cereal crop, ranked fifth in the world and second in Africa after maize. Sorghum grain is mainly used as a source of food for humans in Africa and Asia, whereas sorghum stalks and leaves are used for animal feed and energy production in countries such as Australia, Brazil, and the United States of America [ America . Sorghum America ,3, prolo America .2●−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH), among others. ROS, when overproduced, are toxic to plant cells since they lead to the damaging of lipid membranes and biomolecules [Drought is a major abiotic stress that affects plant growth and development, but its effects depend on the severity and duration of the stress and the plant species. Drought limits the absorption of water and nutrients in plants and affects plants’ metabolism, stomatal conductance, and photosynthetic rates, and these subsequently decrease growth and negatively impact crop yield ,6,7. Watolecules . Droughtolecules . Howeverolecules . Generalolecules ,12 Organolecules . Conventolecules .2) [3O4) [Triticum aestivum [Lallemantia iberica [Moringa peregrina [Nanotechnology employs smaller particles at the nanoscale level, having at least a dimension between one and a hundred nanometers . The use2) , iron ox2) [3O4) , and zin2) [3O4) , have at2) [3O4)  and the 2) [3O4) ,25,26,27aestivum ,29, Lall iberica , and Moreregrina  to drougLagenaria Siceraria [Buddleja lindleyana extract [Caesalpinia coriaria (Jacq.) [A few studies have reported on the synthesis of iron nanoparticles ,33 and ticeraria , Buddlej extract , and Cae (Jacq.) , amongst2O3) NPs synthesized from rooibos tea extracts to improve the growth of Sorghum bicolor plants under drought stress. The αFe2O3 NPs were synthesized and characterized using sophisticated techniques and applied at different concentrations of 5, 10, and 15 mg/L to prime sorghum seeds. Several growth parameters including shoot height, biomass (FW and DW), and physiological traits such as the photosynthetic pigments, anatomical structure, and macronutrient content were assayed. The levels of oxidative stress markers and osmoregulation were also assayed to understand how drought affected sorghum growth at a biochemical level.Seed priming is one of the most effective methods to alleviate oxidative stress ; this is2O3 nanoparticles (NPs) were successfully synthesized from rooibos tea extracts, and this was observed by a color change to black, which was attributed to a combination of FeCl3 (yellow) and rooibos extracts (reddish brown). The extract and the αFe2O3 NPs indicated UV-vis absorption peaks at 283 nm and 294 nm, respectively (2O3 NPs (red line) revealed peaks at 3417 cm−1 and 3404 cm−1 that are attributed to the O-H stretch and a hydroxyl group. The CH2 stretch is attributed to peaks at 3241 cm−1 and 3254 cm−1, and C=O is attributed to peaks at 1626 cm−1 and 1620 cm−1, where N-O is attributed to peaks at 1519 cm−1 and 1577 cm−1, C-N is attributed to peaks at 1407 cm−1 and 1425 cm−1, and the C-O bond is attributed to peaks at 1130 cm−1 and 111 cm−1. The formation of αFe2O3 NPs was further confirmed by an FTIR absorption peak at 590 cm−1 [The αFeectively A. Fourieectively B. The sp590 cm−1 .2O4 NPs, were determined based on the XRD patterns (2O4) NPs. The XRD pattern was compared with the available phase identification of the Joint Committee on Powder Diffraction Standards (JCPDS card no. 00-019-0629). The peak positions at 23.70° (012), 32.70° (104), 35.10° (110), 40.40° (113), 49.05° (024), 53.60° (116), 57.1° (018), 62.0° (214), 63.70° (300), 71.65° (1010), 77.729° (220), and 77.729° (306) are indexed with the hematite phase of iron oxide NPs. Using the Scherrer formula, as shown in Equation (1), the average particle size of the prepared NPs was calculated to be 18 nm. The lattice plane was confirmed by SAED, which corroborated findings obtained from the SAED lattice patterns (2O3) NPs.The crystallinity, phase, and diameter of the αFepatterns C. Resultpatterns D, indica2O3 NPs were spherical in shape and were highly agglomerated as shown on HRTEM images (2O3) NPs to be between 3 and 31 nm (2O3 NPs with weight compositions of 79.94% (0.21% Sigma) and 18.6% (0.2% Sigma), respectively , fresh weight (35%), and dry weight (36%) in water-deprived (WD) plants in comparison to the control, referred as the well-watered (WW) plants . Interesectively . Fresh wectively .2O3 NPs improved chlorophyll content, with total chl increasing by 68% (5 mg/L αFe2O3 NPs), 104% (10 mg/L αFe2O3 NPs), and 55% (15 mg/L αFe2O3 NPs). Priming also increased chla by 102% (5 mg/L αFe2O3 NPs), 160% (10 mg/L αFe2O3 NPs), and 101% (15 mg/L αFe2O3 NPs), whereas chlb increased by 1162% (5 mg/L αFe2O3 NPs), 1936% (10 mg/L αFe2O3 NPs), and 1553% (15 mg/L αFe2O3 NPs) in WD plants as compared to the unprimed plants  layers, of sorghum plants primed with or without αFeng HRSEM . Under nng HRSEM A, whereang HRSEM B. Iron ong HRSEM C–E. This, 119% (10 mg/L αFe2O3 NPs), and 4.8% (15 mg/L α Fe2O3 NPs), whereas a 100% increase in Si content was observed for all αFe2O3 NP concentrations. Furthermore, P content increased by 230%, 284%, and 11% for seeds primed with 5 mg/L, 10 mg/L, and 15 mg/L αFe2O3 NPs, respectively, in WD plants. Similarly, K content increased by 52% (5 mg/L αFe2O3 NPs), 218% (10 mg/L αFe2O3 NPs), and 19% (15 mg/L αFe2O3 NPs).The element distributions of sorghum plants primed with or without αFem shoots . Water d2O3 NPs revealed smooth epidermis layers with improved surface area and less deformation , seen by the clear dark blue spots observed on sorghum leaves  plants. On the other hand, priming with αFe2O3 NPs prevented O2●− production, as shown by less dark blue spots on the sorghum leaves as compared to unprimed WD plants. In fact, priming with 10 mg/L αFe2O3 NPs was highly effective, showing less to no spots, indicating the protective benefits of αFe2O3 NPs against oxidative damage. Compared to WW plants (control), WD (drought-stressed) sorghum plants showed clear injury due to the presence of DAB-stained H2O2 spots on the surface of the analyzed leaves , prevented the over-accumulation of H2O2 in the leaves, as evident by less intense brown spots, with 5 and 10 mg/L αFe2O3 NPs showing lesser spots, resembling the leaves of WW plants. However, 15 mg/L αFe2O3 NPs was less effective in both NBT- and DAB-stained leaves. Histochemical staining was performed to determine ROS production as a sign of drought-induced oxidative stress, which led to the damage of lipid membranes and biomolecules A. The NBm leaves A. From td leaves B. Howeve2O2 by 25% and MDA content by 26% as compared to well-watered plants , 35% (10 mg/L αFe2O3 NPs), and 25% (15 mg/L αFe2O3 NPs).Drought stress significantly increased Hd plants C,D. This−1 was attributed to the O-H group, confirming the presence of phenolic compounds. The transmittance percentages showed a vibration at 3226 cm−1, attributed to C-H stretching of the alkanes, confirming the presence of aliphatic compounds. The peaks at 2045 cm−1 and 1633 cm−1 can be attributed to C=C and C=O stretching vibration, indicating the presence of alkanes and alkenes and confirming the presence of carbohydrates. The peak at 1567 cm−1 can be attributed to C-F stretching vibration for the alkyl halide group. Proteins were confirmed by the presence of amines, seen by peaks at 1390 cm−1 and 1121 cm−1 that are attributed to N-O stretching, confirming the presence of pectin and aliphatic amines, whereas peaks at 1082 cm−1 and 617 cm−1 are attributed to N-H stretching vibration, confirming the presence of proteins due to primary and secondary amines. Shifts and major differences between the FTIR spectrum of WW (black line) plants and WD (red line) plants were observed more visibly at the 3226, 1633, 1390, and 1121 cm−1 peaks, suggesting the alterations of the structure of proteins and carbohydrates. These changes in the WD spectrum were restored for plants grown from primed seeds with 5 mg/L (blue) and 10 mg/L (green) αFe2O3 NPs, as their spectra resembled that of WW plants. However, the spectrum of plants under 15 mg/L αFe2O3 NPs (magenta) priming resembled that of WD non-primed plants, suggesting that this concentration was a bit high. FTIR spectroscopy was used to investigate the effect of drought stress on the damage of biomolecules, including phenolics, carbohydrates, proteins, and lipids . Several2O3 NP priming on the osmoregulation of sorghum plants, osmolytes including proline and soluble sugars were analyzed , plant height (PH) (r = 0.80), chlt (r = 0.98), chla (r = 0.90), and chlb (r = 0.80). The same pattern was observed between DW and PH (r = 0.98), chlt (r = 0.95), chla (r = 0.90), and chlb (r = 0.85). A strong positive relationship was recorded between PH and chlt (r = 0.75), chla (r = 0.75), and chlb (r = 0.70). Elements were strongly correlated to growth parameters; for instance, Mg, P, and k were strongly correlated to FW, DW, and PH with r > 0.75 and highly correlated to Chlt, Chla, and Chlb with r > 0.5. Si was moderately correlated to FW, DW, PH, Chlt, Chla, and Chlb. Elements were highly negatively correlated to oxidative markers (H2O2 and MDA) and osmolytes . Growth parameters  were strongly negatively correlated with MDA, ROS, proline, and soluble sugar. MDA was strongly positively associated with H2O2 (ROS) and proline (r = 0.8), whereas proline was strongly positively associated with MDA (r = 1) and ROS (r = 0.98). ROS was only strongly positively correlated with proline (r = 0.98) and positively with soluble sugar (r = 0.75). The relationship between the different traits was analyzed using Pearson’s correlation . The clo2O3 NPs were synthesized from Aspalathus linearis (rooibos) extract as a capping agent, and ferric chloride was used as a precursor [2O3 NPs was confirmed by a shift in the absorption peak at ~283 nm (corresponding to the rooibos extract) to ~294 nm, which indicated the formation of αFe2O3 NPs  for metal NPs. This absorption peak was attributed to the presence of alpha hematite (αFe2O3) NPs, as previously reported [The physiochemical properties originating from the rooibos extracts that were involved in the synthesis of αFetroscopy . Based oynthesis . The –OHynthesis ,55. Thisreported ,56.2O3 NPs, with a size of 18 nm (Moringa oleifera extracts [2O3) NPs. Transmission electron micrographs revealed spherical, granular, and agglomerated αFeof 18 nm . Analysiextracts , are in Zea mays are cultivated for consumption [2O3) NPs on the growth of sorghum bicolor under drought stress. In agriculture, NPs are commonly exogenously applied during irrigation, or mixed with soil [2O3 NPs (5 mg/L to 15 mg/L NPs) concentration has the ability to improve sorghum growth under drought stress. Drought is by far the most common and dominant abiotic factor threatening food security, predominately in arid and semi-arid atmospheres , where tsumption ,61. Thisith soil ,26, wherith soil ,62,63. Hith soil . Althougith soil , this de2O3 NPs  prevented drought-induced growth reduction, with 10 mg/L αFe2O3 NPs demonstrating the highest growth improvement in sorghum plants. This explains how αFe2O3 NPs improved water absorption, which led to efficient nutrition uptake. Similar results were observed in barley [Mentha piperita L. [2O3. Drought stress significantly reduced the growth of sorghum plants, and this was reflected in reduced plant height, FW, and DW . This ren barley , cotton n barley , and Menerita L.  treated 2O3 NPs alleviates drought-induced growth inhibition (2O3 NPs considerably increased the chlorophyll content of water-deprived (WD) plants, with the highest increase observed for 10 mg/L αFe2O3 NPs. This observation can be attributed to improved water and nutrient absorption that resulted in adequate physiological accomplishments by sorghum [2O3 NPs contributed in the restoration of sorghum’s metabolism and health.Chlorophyll content was assayed to further understand how priming with αFehibition  in sorghhibition , suggest sorghum ; thus, α2O3 NPs on the growth of Sorghum bicolor under drought stress was also evaluated based on the anatomic structure  layers). Drought stress caused shrinkage of the epidermis, xylem, and phloem layers . A higher percentage of Mg, Si, P, and K was also observed, and this was attributed to αFe2O3 NPs facilitating nutrient distribution under drought stress. Furthermore, αFe2O3 NPs improved the epidermis surface layer, facilitating the opening of the metaxylem and phloem. Hence, it is suggested in this study that αFe2O3 NPs mitigated the effects of drought by improving micronutrient uptake [On the contrary, priming seeds with Fet uptake ,86.2O3 NPs in improving the sorghum growth under drought stress, ROS production, integrity of biomolecules, and osmolyte content were investigated. In assessing the level of oxidative damage caused by drought and the ability of αFe2O3 NPs to prevent oxidative stress, ROS accumulation and MDA content were quantified (2O2 and O2●−), and generation of MDA, which led to oxidative stress and hence impaired growth of the plants [2O3 NPs improved the antioxidant capacity of sorghum plants and is responsible for lower oxidative damage as reported previously in Zea mays [2O3 NPs prior to water deprivation. The decrease in oxidative damage, as seen by reduced ROS, might due to the fact that hematite has the ability to regulate the expression of genes responsible for drought tolerance [To understand the biochemical effects of αFeantified . Droughte plants ,88,89. TZea mays . This stolerance , which uolerance .−1, corresponding to carbohydrates, while peaks at 1390 cm−1 and 1121 cm−1 corresponded to proteins. Several reports have indicated the deleterious effects of drought-induced oxidative damage on biomolecules [2O3-NP-primed seeds showed an improved FTIR spectra, which resembled that captured from WW (control) samples. The major shifts observed in spectral peaks were reversed when seeds were primed with 5 mg/L (green) and 10 mg/L (blue) αFe2O3 NPs prior to water deprivation. [2O3 NPs reduced oxidative damage and prevented the degradation of biomolecules [The oxidative damage due to drought stress was further examined by determining any alterations on the integrity or structure of biomolecules such as DNA, proteins, and carbohydrates ,94 usingolecules ,95,96. Sivation.  This indolecules . 2O2 and O2●− and subsequently boosted the plant’s ability to deal with oxidative damage [2O3 NPs decreased both proline and soluble sugars in WD plants as compared to the controls (WD plants without priming). Similar observations were reported in iron-coated-with-chitosan NPs in peppermint [2O3 NPs facilitated the activation of proline and soluble sugars, but these may have also played a role in stabilizing cellular membranes, maintaining turgor, and working as osmoprotectants. This was further confirmed by the impressive maintenance of the membrane structure of plants as viewed under SEM micrographs (The osmolytes (proline and soluble sugars) are efficient osmoprotectants that mitigate stress, and their responses under drought stress have been well documented ,98,99. Ie damage . On the ppermint , foliar ppermint , and in ppermint . Solubleppermint  showed arographs . Pearsonrographs  among th2O3 NPs on sorghum growth by alleviation of oxidative damage. There was a strong positive correlation between all studied traits, including elements, growth attributes , oxidative markers (ROS and MDA), osmolytes (proline and soluble sugars), and chlorophyll contents. A negative correlation was observed between growth attributes  and biochemical traits . The positive correlations between oxidative stress markers and osmolytes may be explained by the reduction of oxidative damage due to the role played by αFe2O3 NPs in facilitating the prevention of ROS and MDA formation under drought stress, as this could be due to enhancement of efficient water and nutrients use subsequently leading to alleviation of drought stress [This study demonstrated the positive effects of αFet stress ,105. MosAspalathus linearis (Rooibos Lager) leaves purchased at Spar supermarket . About 5 g of tea material was boiled in 250 mL of autoclaved distilled water (ddH2O) at 80 °C for two hours. The extracts were then filtered using Whatman filter paper No. 1 and used immediately or kept at 4 °C until further processing [Green extracts were prepared from ocessing . 2O3 nanoparticles (NPs) was done following the previous method [3)  was mixed with 250 mL of the green extracts while vigorously stirring at 1000 rpm for 5 h under heat at 80 °C. The reaction mixture was cooled to room temperature, followed by centrifugation at 13,000 rpm for 15 min. The supernatant was discarded while the pellets were resuspended in autoclaved distilled water; this process was repeated three times to remove all the impurities. After washing, the synthesized nanoparticle solution was freeze-dried, and the obtained powder was calcined at 600 °C for 2 h to obtain a more crystallized structure of the NPs. The resulting reddish-brown powder was kept in an airtight container and stored at room temperature until further use.Synthesis of αFes method . Briefly−1. The surface structural morphology of the NPs was examined using High-Resolution Scanning Electron Microscopy , and the elemental composition of the NPs was determined on the SEM using Energy-Dispersive X-ray Spectroscopy (EDX) performed on a Zeiss Auriga detector  [2O3 NPs were determined using an X-ray diffractometer  was used to determine the crystalline size of the synthesized αFe2O3 NPs.To confirm and characterize the synthesis of the iron oxide nanoparticles (NPs), both the green extracts and the synthesized NPs materials were analyzed. Ultraviolet–visible spectroscopy  was used to verify the formation of the NPs using a wider spectral region/window (200 to 800 nm). The range of the UV bands obtained was between 200–500 nm wavelength. The functional groups of the extract and NPs were examined using Fourier-Transform Infrared (FTIR) spectroscopy  Ltd., Midrand, South Africa) at a spectral range between 400 and 4000 cmMA, USA) . High-ReSorghum bicolor (L.) Moench) seeds purchased from Agricol, Brackenfell Cape Town, South Africa were disinfected by firstly soaking them in 70% ethanol while shaking on a motor shaker at 600 rpm for one minute, followed by washing thrice with autoclaved distilled water (ddH2O). Seeds were then soaked in 5% sodium hypochlorite (bleach) while shaking at 600 rpm for one hour. The bleach was rinsed off by washing the seeds three times with autoclaved distilled H2O. Priming of seeds with αFe2O3 NPs was done by soaking 25 seeds in 250 mL of 0 (mock), 5, 10, and 15 mg/L αFe2O3 NPs and leaving them shaking at 200 rpm in the dark overnight on a motor shaker. Seeds were then dried under laminar flow and sown on filter paper placed in a plastic container (28 × 21 and 6 cm height) containing 50 mL of autoclaved ddH2O. Germination was allowed to take place in the dark at 25°C and monitored for 7 days. Germinated seeds were subsequently sown in wooden boxes (sized: 30 × 30 cm and 7 cm height) containing 2.1 double grow all-purpose organic potting soil and vermiculite  and grown in the greenhouse under controlled conditions . Watering was done by sprinkling 300 mL of nutrient solution , Reg. No./Nr. K5293, Act No./Wet Nr. 36 of/van1947)) every second day for seven days for the plants’ establishment, followed by watering every second day for seven days. Thereafter, plants were divided into three groups consisting of the (1) control , (2) drought stressed, plants that are water deprived, (3) plants grown from seeds were primed with NPs , and then water deprived. Plants, which served as the control continued to receive water every second day for 7 days. Sorghum plants were harvested on day seven after water deprivation without re-watering; plants were rinsed thoroughly with dH2O and stored at −80 °C until further use.Sorghum (Plant height was measured using a ruler in the mm range before harvesting. Shoot fresh weights (FW) were weighed using a Mettle Toledo AE50 analytical balance . To determine dry weights (DW), shoots from which the FW was measured were then oven-dried at 70 °C for 72 h, until a constant weight was attained. To analyze the anatomical structure  and element distribution, samples were sent to the Physics Department, University of Western Cape, South Africa and analyzed using High-Resolution Scanning Electron Microscopy (HRSEM) and HRSEM-EDX as previously described ,109. All® Epsilon visible 8 nm bandwidth spectrophotometer . The concentration of chlorophyll content was estimated according to the following formulas listed below:V is the volume of sample extract; W is the weight of the sample; and Chla, Chlb, and total Chl concentration are expressed as mg g−1 FW. Photosynthetic pigments were determined from shoot materials (0.1 g) that were first grinded in liquid nitrogen; the ground materials were incubated in 10 mL of 80% acetone and incubated in the dark for 5 min or until the leaf’s tissues completely lost color. Estimation of chlorophyll (Chl) was performed as reported previously . To dete2●−), hydrogen peroxide (H2O2), and malondialdehyde (MDA) content, a marker of lipid peroxidation, were assayed to determine the extent of oxidative damage due to drought stress. All spectrophotometric measurements in this section were done using a Helios® Epsilon visible 8 nm bandwidth spectrophotometer  unless otherwise stated.Oxidative stress markers including reactive oxygen species (ROS), such as superoxide (O2●−), fresh whole leaves were gently submerged in 0.1% Nitro-blue tetrazolium chloride (NBT) solution and allowed to incubate at room temperature for 12 h in the dark. The formation of H2O2 was detected by immersing fresh leaves in 1 mg/mL 3′,3′-diaminobenzidine (DAB) solution, and the reaction mixture incubated for 12 h in the dark. After incubation, all histochemical stained samples were boiled in 80% ethanol at 90℃ for 15 min to remove chlorophyll before taking pictures. The presence of dark blue (corresponding to O2●−) and dark brown (corresponding to H2O2) stain was captured using a Canon D700 camera (Taiwan/Japan).Histochemical detection of ROS was determined as previously described . For detw/v) acid (TCA), 0.50 mL potassium iodide (1 M), and 0.25 mL potassium phosphate buffer  was used to homogenize about 100 mg of pulverized sorghum plant material. Centrifuging was done in tubes using a vortex for 15 min at 10,000 rpm (4 °C). Samples were centrifuged and then kept at ambient temperature for 20 min while being shielded from light. A 96-well microtiter plate with samples was used to detect the absorbance at 390 nm. H2O2 standard solution was used as the quantification method to obtain the calibration curve.Quantification of hydrogen peroxide content was performed as described previously by ,109. Basw/v). Tubes containing the extract were vortexed to mix well and centrifuged at 13,000 rpm (4 °C) for 10 min. Small holes were punched on the cap of new 2 mL Eppendorf tubes using a syringe needle to avoid tubes bursting due to pressure from the heat and reaction by-products. About 400 µL of the supernatant was transferred into a new 2 mL Eppendorf tube containing 1 mL of 0.5% TBA; the reaction was then boiled at 80 °C in a water bath for 30 min. Tubes were chilled on ice for 5 min and centrifuged for another 5 min at 13 500 rpm (4 °C) to precipitate any remaining TBA. A total amount of 200 µL of the supernatant was transferred into 96-well microtiter plates in triplicates to measure the OD at 532 nm and 600 nm. The content of MDA was calculated using formula 3.The level of lipid peroxidation was determined by measuring malondialdehyde (MDA) content as described previously by . A total−1 cm−1.The absorption coefficient for the calculation of MDA is 156 mmol−1 with 32 scans for a spectrum at 4 cm resolution.The FTIR spectrum of sorghum shoots was analyzed following a method described by Grace et al. (2020) using a PerkinElmer Spectrum 100-FTIR Spectrometer (PerkinElmer (Pty) Ltd., Midrand, South Africa). About 10 mg of dry sorghum shoot tissues were combined with 100 mg of potassium bromide (Kbr), then crushed and ground into a fine powder. The obtained powder was analyzed using a wider window between 400 and 4000 cm® Omega  microtiter plate reader. The proline content was determined from a standard curve using pure proline as a standard.Proline content was measured as previously described . A total® Epsilon visible 8 nm bandwidth spectrophotometer . The total soluble sugar content was estimated by calibrating a standard curve using glucose and the content was expressed as mg g−1 FW.Soluble sugars were determined as previously described , with sl® 21 Statistical Software . Data in the Figures and Tables represent the mean ± standard deviation (n = 3). Statistical significance between control and treated plants was determined by Tukey Test for comparison at a 95% confidence interval. The difference was regarded as significant when p < 0.05. Means that do not share a letter in a column were significantly different. Gally and “my norm” from R software was used for Pearson’s correlation (r) matrix analysis.All experiments were repeated at least three times and data were statistically analyzed by a one-way ANOVA using Minitab2O3 NPs from rooibos tea extracts. The αFe2O3 provide a means of efficiently enhancing the physiological and biochemical traits and growth of sorghum by reducing the oxidative stress. Additionally, the administration of αFe2O3 NPs boosts photosynthesis and water and mineral nutrient use, which strengthens membrane performance such as in xylem and phloem layers. Furthermore, the positive effect of αFe2O3 NPs was more obvious since it prevented the excessive ROS generation brought on by drought stress. The application of 10 mg/L of αFe2O3 NPs, as compared to other concentrations examined in this study, proved to be more effective in reducing drought stress. It is therefore recommended to consider the use of green-synthesized hematite NPs in the search for effective bio-stimulants to improve crop yield under environmental stress. However, there is still a need to further investigate αFe2O3 NPs’ protective effects at the molecular level in cereal crops to better understand αFe2O3 NPs mechanism of action in ameliorating drought stress. Future work will include investigating the molecular response traits in sorghum that might be induced by αFe2O3 NPs, and this will assist in deducing the mechanism of action by αFe2O3 NPs in ameliorating drought stress. It is necessary to develop new environmentally and cost-effective methods that have the potential to alleviate the effects of abiotic stress and hence increase food production. Drought stress is responsible for a significant reduction in agricultural productivity, as shown in this study. Drought stress led to a reduction in sorghum growth parameters and photosynthesis and biomolecule damage. In the current study, we successfully synthesized stable αFe