Wear‐induced aseptic loosening has been accepted as one of the main reasons for failure of total hip arthroplasty. Ceramic wear debris is generated following prosthesis implantation and plays an important part in the upregulation of inflammatory factors in total hip arthroplasty. The present study investigates the influence of ceramic debris on osteoblasts and inflammatory factors.Ceramic debris was prepared by mechanical grinding of an aluminum femoral head and added to cultures of MC3T3‐E subclone 14 cells at different concentrations . Cell proliferation was evaluated using a Cell Counting Kit (CCK‐8), and cell differentiation was assessed by mRNA expression of alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In addition, cell bio‐mineralization was evaluated through alizarin red S staining, and release of tumor necrosis factor alpha (TNF‐α), interleukin‐1 beta (IL‐1β), and interleukin‐6 (IL‐6) was measured through enzyme‐linked immunosorbent assays (ELISA). Furthermore, mRNA expression of Smad1, Smad4, and Smad5 and protein expression of phosphorylated Smad1, Smad4, and Smad5 were measured by reverse transcriptase polymerase chain reaction (RT‐PCR) and western blotting.P < 0.05, and the values decreased gradually with the increase of ceramic debris concentration), but the results of the CCK‐8 assay showed that cell proliferation was not significantly affected . In addition, the results of ELISA, RT‐PCR, and western blotting demonstrated that ceramic debris significantly promoted the release of inflammatory factors, including TNF‐α, IL‐β, and IL‐6 , and also greatly reduced the mRNA expression of Smad1, Smad4, and Smad5  as well as protein expression of phosphorylated Smad1, Smad4, and Smad5.The ceramic debris had irregular shapes and sizes, and analysis of the size distribution using a particle size analyzer indicated that approximately 90% of the ceramic debris was smaller than 3.2 μm (2.0 ± 0.4 μm), which is considered clinically relevant. The results for mRNA expression of ALP, OCN, and OPN and alizarin red S staining indicated that cell differentiation and bio‐mineralization were significantly inhibited by the presence of ceramic debris at all tested concentrations (Ceramic debris may affect differentiation and bio‐mineralization of MC3T3‐E subclone 14 cells through the bone morphogenetic protein/Smad signaling pathway. Total hip arthroplasty (THA) is an effective surgical procedure for treating hip disorders such as osteoarthritis, rheumatoid arthritis, and avascular necrosis, dramatically improving the quality of life of patients as a result of immediate relief from pain in the hip joint. The first tentative methods of restoration of function for patients with an arthritic hip were developed by researchers through many novel ideas, but a significant contribution to the routine success achieved today is obviously the concept of low‐friction arthroplasty proposed by John Charnleyin vivo service of the hip prosthesis in the human bodyAt present, the biotribo‐acoustics issue has been reported in the literature as a major concern with regard to the ceramic‐on‐ceramic articulation for THAin vitro tests to investigate the influence of ceramic debris on the osteoblast behaviors of MC3T3‐E subclone 14 cells in THA. First, we prepared ceramic debris using an aluminum femoral head by mechanical grinding. The morphology, size distribution, and phase composition features of the ceramic debris were sequentially evaluated to confirm its feasibility for the following tests. Subsequently, the mouse osteoblast‐like MC3T3‐E subclone 14 cells were co‐cultured with different concentrations of ceramic debris  in the incubation at an atmosphere of 37°C and 5% CO2. The cell proliferation behavior was evaluated by Cell Counting Kit  at 1, 3, and 5 days, with the optical density being measured at an excitation wavelength of 450 nm. The cell differentiation was characterized by mRNA expression of alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN) at 3 days. The cell bio‐mineralization was evaluated by alizarin red S staining at 5 days. In addition, the release of typical tumor necrosis factors such as alpha (TNF‐α), interleukin‐1 beta (IL‐1β), and interleukin‐6 (IL‐6) was measured using enzyme‐linked immunosorbent assays (ELISA) at 3 days. Furthermore, the mRNA expression levels of Smad1, Smad4, and Smad5 and protein expression levels of phosphorylated Smad1, Smad4, and Smad5 were measured by reverse transcriptase polymerase chain reaction (RT‐PCR) and western blotting at 3 days. Thes abovementioned in vitro tests were carried out to investigate the potential signaling pathway of the ceramic debris affecting the osteoblast behaviors of MC3T3‐E subclone 14 cells. Finally, the data were processed for statistical analysis of significant difference.In the present study, we performed a series of in vitro tests, it is anticipated that the influence of ceramic debris on the osteoblast behaviors of MC3T3‐E subclone 14 cells can be determined, which has great value for both research and clinical relevance: (i) the effect of ceramic debris concentration on the osteoblast behavior; (ii) the potential signaling pathway for the regulation; and (iii) the importance of reducing generation of wear debris for ceramic‐on‐ceramic THA.Based on the completion of these Ceramic debris was prepared by mechanical grinding of an aluminum femoral head  using a QM‐3SP2 ball mill , and then filtered by a 400‐mesh screen mesh to remove debris of larger sizes. The size distribution and morphology of the ceramic debris obtained were characterized using an HELOS/OASIS dynamic light scattering (DLS) particle size analyzer  and an S‐3400N scanning electron microscope . In addition, phase composition analysis of the ceramic debris was performed using a D8 Advance X‐ray diffractometer  with a scanning angle of 2θ from 10° to 70°.2 incubator  with an atmosphere of 100% humidity at 37°C and 5% CO2 and replaced every 3 days.Mouse osteoblast‐like MC3T3‐E subclone 14 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences  and cultured in α‐minimal essential medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. The cultured cells were placed in a CO3 cells per 200 μL culture medium in each well. Subsequently, the ceramic debris  was added to the culture medium at final concentrations of 0 (as control), 5, 10, and 15 μg/mL. The culture plates were gently transferred to the CO2 incubator for incubation at 37°C in 5% CO2.At 80% confluency, the MC3T3‐E subclone 14 cells were digested by 0.25% trypsin‐1 mmol/L ethylene diamine tetraacetic acid and seeded in 96‐well culture plates at a concentration of 5 × 10Cell proliferation was determined using the Cell Counting Kit (CCK‐8) method. After culturing for 1, 3, and 5 days, 10 μL CCK‐8 solution was added to each well of a 96‐well culture plate, followed by further incubation for 1 h. The optical density of the solution was measured using an MK3 enzyme labeling instrument  at an excitation wavelength of 450 nm. In addition, the MC3T3‐E subclone 14 cells cultured with different concentrations of ceramic debris  in the culture medium for 3 days were collected, and the total RNA was extracted using the Trizol Reagent Kit . Primers for ALP, OCN, and OPN were designed  in the culture medium for 5 days.2 incubator with four different concentrations of ceramic debris  in the culture medium for 3 days, and the concentrations of inflammatory factors in the culture medium were measured based on the manufacturer's instructions for the ELISA kits.The release of inflammatory factors from the MC3T3‐E subclone 14 cells, including TNF‐α, IL‐1β, and IL‐6, was evaluated using commercially available ELISA kits . The cells were cultured in the CO2 incubator for 3 days, the total RNA was extracted using Trizol Reagent Kit. Primers to Smads (sma and mad homologs) including Smad1, Smad4, and Smad5 were designed . One‐way analysis of variance was used to evaluate the differences between the groups, and a Images of the original aluminum femoral head and the prepared ceramic debris along with their morphology and composition characterizations are shown in Fig. P > 0.05), suggesting that the ceramic debris has no significant influence on the cell proliferation capability. The mRNA expression of ALP, OCN, and OPN of MC3T3‐E subclone 14 cells after 3 days in culture is presented in Fig. P < 0.05). Therefore, it is considered that the ceramic debris impairs the differentiation capability of the MC3T3‐E subclone 14 cells, and this impairment is more distinctive with a higher concentration of the ceramic debris. Bio‐mineralization of the MC3T3‐E subclone 14 cells cultured with different concentrations of ceramic debris  after 5 days is shown in Fig. The comparison of the cell proliferation kinetics of the MC3T3‐E subclone 14 cells cultured with different concentrations of ceramic debris  at 1, 3, and 5 days is shown in Fig. P < 0.05). These results suggest that ceramic debris promotes the release of the inflammatory factors, and this effect is more obvious when the concentration of debris is higher.The release of TNF‐α, IL‐1β, and IL‐6 by MC3T3‐E subclone 14 cells after 3 days in culture with different concentrations of ceramic debris  is shown in Fig. P < 0.05). This inhibition is more distinct with the increase of the ceramic debris concentration. In addition, the results in Figs The mRNA expression data for Smad1, Smad4, and Smad5 and protein expression data for p‐Smad1, Smad4, and Smad5 after 3 days in culture of the MC3T3‐E subclone 14 cells exposed to different concentrations of ceramic debris  are presented in Table Reducing the generation of wear debris in THA is of critical importance to further improve the longevity of this surgical procedureet al. in 2012 indicated that synovial fluid microanalysis can be a useful surrogate in predicting ceramic failure, particularly when a strong presence of ceramic debris is observedRecently, ceramic debris has attracted more attention, and the study performed by Stea et al., in which alumina debris was observed to induce osteolytic cytokine production (TNF‐α) by human mononuclear phagocytesIt has been shown in previous studies that cell proliferation can be inhibited by the presence of wear debris, and the influence of ceramic debris is relatively small in comparison with that of metallic debrisin vitro, and an in vivo investigation in an animal model of osteolysis has not been performed as yet. However, it should be noted that osteolysis is a multifactorial problem, and the results of in vivo studies in animals cannot be used or interpreted as directly related to the condition in humans. Therefore, in the present study, we focused on investigating the behavior of MC3T3‐E subclone 14 cells (e.g. proliferation and differentiation) by adding ceramic debris to the culture medium. Further studies will be performed to validate these results when a more suitable animal model representing osteolysis is available. In addition, in future studies, we will focus on methods to promote the proliferation and differentiation of osteoblasts in the presence of ceramic debris: for example, the introduction of osteogenic protein. Finally, because debris of varying sizes may affect osteoblast behaviors differently, we will investigate potential differences in the effects of debris of varying sizes in future research.One major limitation of the present study is that all observations were made using cultured cells In the present study, ceramic debris was prepared by mechanical grinding, and its influence on the osteoblast behaviors of MC3T3‐E subclone 14 cells was investigated. The presence of ceramic debris inhibited cell differentiation, cell bio‐mineralization, mRNA expression of Smad1, Smad4 and Smad5, and protein expression of p‐Smad1, Smad4 and Smad5. It promoted the release of inflammatory factors and had no significant influence on cell proliferation. The mechanism underlying these effects of ceramic debris may be related to the impairment of the BMP/Smad signaling pathway.


Triticum turgidum) and hexaploid wheat (Triticum aestivum), the spikelet is a short indeterminate branch with two proximal sterile bracts (glumes) followed by a variable number of florets, each including a bract (lemma) with an axillary flower. Varying levels of miR172 and/or its target gene Q (AP2L5) result in gradual transitions of glumes to lemmas, and vice versa. Here, we show that AP2L5 and its related paralog AP2L2 play critical and redundant roles in the specification of axillary floral meristems and lemma identity. AP2L2, also targeted by miR172, displayed similar expression profiles to AP2L5 during spikelet development. Loss‐of‐function mutants in both homeologs of AP2L2 (henceforth ap2l2) developed normal spikelets, but ap2l2 ap2l5 double mutants generated spikelets with multiple empty bracts before transitioning to florets. The coordinated nature of these changes suggest an early role of these genes in floret development. Moreover, the flowers of ap2l2 ap2l5 mutants showed organ defects in paleas and lodicules, including the homeotic conversion of lodicules into carpels. Mutations in the miR172 target site of AP2L2 were associated with reduced plant height, more compact spikes, promotion of lemma‐like characters in glumes and smaller lodicules. Taken together, our results show that the balance in the expression of miR172 and AP2‐like genes is crucial for the correct development of spikelets and florets, and that this balance has been altered during the process of wheat and barley (Hordeum vulgare) domestication. The manipulation of this regulatory module provides an opportunity to modify spikelet architecture and improve grain yield.The spikelet is the basic unit of the grass inflorescence. In tetraploid ( APETALA 2‐like genes AP2L2 and Q (= AP2L5), which are targets of miR172, play essential and redundant roles in floret development. These genes have additional effects on heading time, plant height, spikelet and floret number, and lodicule development. We identified two induced AP2L2 alleles resistant to miR172 that can be used to modify these traits in wheat.A better understanding of wheat spike development is essential to engineer varieties that are more productive. This study shows that wheat  Modifications in cereal inflorescence development facilitated increases in grain number and size, and fine‐tuned factors limiting shattering while improving threshability  and hexaploid wheat (Triticum aestivum), the primary and secondary branches of the inflorescence are absent, resulting in spikelets attached directly to the rachis, forming a derived structure called a spike. The wheat IM generates a determined number of lateral spikelet meristems (SMs) in an alternating distichous pattern along the central rachis before forming a terminal spikelet  transitions from the vegetative to the reproductive phase. In the ancestral grass inflorescence, the panicle, the inflorescence meristem (IM) generates lateral primary and secondary branches, each ending in a spikelet (‘little spike’). In tetraploid (Hordeum vulgare (barley), Oryza sativa (rice), Sorghum sp. and Zea mays (maize), whereas wheat generates an indeterminate number of lateral florets with only the most basal florets surviving to support grains ‐like family of transcription factors (TFs) are good candidates for this function. Combined mutations in two closely related AP2‐like genes from maize, INDETERMINATE SPIKELET 1 (IDS1) and SISTER OF INDETERMINATE SPIKELET 1 (SID1)   or a single mutation within the miR172 binding site of IDS1  resulted in the production of additional florets in the spikelet and a lack of pistil abortion in the tassel  or an extra point mutation in the miR172 binding site of Q showed a similar conversion from glumes to florets in spikelets located in distal positions of the spike , which targets UBIQUITIN promoter (Ubi::miR172) also results in alterations in inflorescence and spikelet development. Rice Ubi::miR172 panicles showed reduced branching and additional glume‐like bracts similar to those observed in snb osids1 double mutants. The Ubi::miR172 plants with the highest miR172 expression levels showed more severe effects than the double mutants, however, suggesting that additional miR172 targets were likely involved in the regulation of these phenotypes  and AP2L7, with numbers corresponding to their chromosome locations. Henceforth, and to avoid confusion, we will use the symbols Q and q to refer specifically to the A‐genome alleles with or without the mutation in the miR172 binding site, respectively, AP2L5 when referring to the overall function of the different homeologs, and ap2l5 when referring to the loss‐of‐function mutants for all homeologs.To prioritize which mutant AP2‐like genes targeted by miR172 from wheat, barley, rice, maize, Brachypodium and Arabidopsis showed that the wheat AP2L5 gene belongs to the IDS1/SID1 cluster , Triticum urartu , Aegilops tauschii (a D‐genome progenitor) and Triticum turgidum ssp. dicoccoides (wild emmer), accession Zavitan , but AP2L1 and AP2L7 expression decreased after the reproductive transition (from W2 to W3.5). Expression of AP2L5 and AP2L2 did not change significantly in the different developmental stages and were expressed at the floret primordia stage (W3.5), when floral meristems and floral organs are specified  on cDNA derived from the vegetative SAM and early stages of spike development identified using the Waddington scale . Sequencing of K2233 ap2l‐A2 transcripts revealed that the splice site mutation causes the use of a nearby intronic GT site, resulting in four additional nucleotides and a frame‐shift mutation . The encoded protein (393 amino acids) lacks the two critical AP2 domains and is most likely not functional. Transcripts from the K3634 ap2l‐B2 allele were not detected in the expression experiments , suggesting that the mutation may affect transcript stability. We backcrossed the individual mutant lines with wild‐type Kronos twice to reduce background mutations, and then intercrossed the ap2l‐A2 and ap2l‐B2 mutants to generate an ap2l2‐null mutant, which for simplicity will be referred to hereafter as ap2l2.Using a sequenced mutant population in the tetraploid wheat variety Kronos , although we did not detect clear developmental defects in floret organs to explain this observation.We next compared the phenotypes of ap2l2 and ap2l5 mutations. The ap2l2 ap2l5 mutant plants (four homozygous mutations) displayed more severe spike phenotypes than the single‐gene mutants . B‐class TaPI1 and TaAP3  and C‐class genes TaAG1 and TaAG2  are both expressed mainly after the glume primordium differentiation stage. Finally, E‐class genes can be divided into two groups based on their expression profiles, an earlier group including TaSEP5 and TaSEP6 that is expressed at the double‐ridge stage  and a later expressing group including TaSEP2, TaSEP3 and TaSEP4, which is upregulated at or after the glume primordium differentiation stage .ap2l2, ap2l5 and ap2l2 ap2l5 mutants. No significant differences in expression among genotypes was detected for the A‐class genes VRN1, FUL2 and FUL3 , but increased expression of AG‐like genes (C‐class).Taken together, the expression results described above show that the young spikes of ap2l2 loss‐of‐function mutations in Kronos, we explored the effect of mutations in the miR172 binding site of AP2L2 in tetraploid Kronos and in hexaploid wheat. First, we identified a tetraploid wheat tilling mutant (K2236) with a point mutation in the miR172 target site of the AP2L‐A2 homeolog (henceforth resistant Ap2l‐A2 or rAp2l‐A2), in exactly the same position as the one that generated the Q allele in AP2L‐A5 . Sequencing of the miR172 target site of the AP2L2 homeologs revealed an SNP at a different position in the miR172 target site of the AP2L‐B2 gene  when compared with the wild type , with the exception of the specific effects on lodicules.Taken together, the results from two independent mutant lines show that mutations in the miR172 site of AP2L2 and AP2L5 genes have overlapping roles in the regulation of homeotic changes between glumes and lemmas, the development of axillary floral meristems, spike compactness and plant height. The simultaneous absence of both genes results in spikelets with multiple sterile bracts that form a few distal florets with no lodicules and with other altered floral organs. In addition to their overlapping functions, AP2L2 affects lodicule size and AP2L5 affects spikelet number, heading time and floret number. We discuss first the traits for which both genes have overlapping effects and then the specific effects of each gene.Wheat AP2L2 resulted in reduced plant height in tetraploid and hexaploid wheat, with a stronger effect in hexaploid wheat. This stronger effect is likely to be associated with the more disruptive effect of the rAp2l‐B2 mutation in hexaploid wheat relative to the rAp2l‐A2 mutation in tetraploid wheat. An induced mutation in the miR172 binding site of AP2L‐A5, which already had the Q mutation, also resulted in a severe reduction in plant height , but the differences were significant only for ap2l5. However, the ap2l2 ap2l5 mutant showed a significant reduction in spikelets per cm relative to the ap2l5 mutant . To understand better the floral phenotypes observed in the AP2‐like mutants, we characterized the transcript levels of several MADS‐box genes known to be involved in the ABCE model, for the determination of floral organ identity , suggesting a limited role of AP2L2 in the regulation of B‐class genes. This result may explain the increase in lodicule size observed in ap2l2  and the decrease in lodicule size in the rAp2l2 plants  and TaAG2 (OsMADS3), have been identified in monocots , are divided into two subfamilies  and TaSEP6 (OsMADS5) are expressed earlier than the other SEPALLATA genes in both rice  is the earliest expressed among the SEP‐like genes and regulates the timing of the transition between branches and spikelet meristems . This result is consistent with the effect of AP2L2 and AP2L5 in the regulation of B‐ and C‐class but not A‐class genes, as described above. The 10‐fold downregulation of TaSEP2 (OsMADS1) in ap2l5 and ap2l5 ap2l2 may contribute to the conversion of lemmas to glumes, as overexpression of the rice ortholog OsMADS1 results in the conversion of rudimentary glumes to lemmas  is also reduced, whereas the fourth whorl is normal in all ap2 mutant alleles  genes in the wheat ap2l2 ap2l5 mutant result in the conversion of lodicules into carpelloid organs.The homeotic conversion of lodicules to carpels observed in the et al., et al., Cly1/Zeo) was mapped to the distal region of chromosome arm 2H , which reduces mRNA cleavage  in barley accession SV235 relative to cly1.b, in spite of having the same miR172 target sequence . By contrast, wheat lines with the rAp2l‐A2 and rAp2l‐B2 alleles developed florets with smaller lodicules in both tetraploid , and we failed to detect variation in the miR172 binding site of the different ap2l5 mutants showed a significant reduction in spikelet number per spike (SNS), which indicates a premature transition of the inflorescence meristem to a terminal spikelet. This effect was not observed in the ap2l2 mutant, and was not enhanced in ap2l2 ap2l5 relative to ap2l5 . Mutations in the miR172 binding site of AP2L5 resulted in increased SNS, indicating a delay in the transition between IM and the terminal spikelet  resulted in increased tassel branching  into spikelets.The AP2 in Arabidopsis in maintaining the stem cell niche  and the proliferative nature of the shoot meristem  result in significant increases in SNS and number of grains per spike  showed a significant increase in floret number per spikelet  than in the SM .The ap2l5 mutants, which flowered approximately 4 days earlier than the wild‐type controls ; however, Ubi::miR172 plants produce one fewer leaf than the wild type, suggesting that one or more AP2‐like genes also affect the transition of the SAM from the vegetative to the reproductive stage. AP2‐like genes are known repressors of the flowering promoting gene FT in many species, and mutation in several ap2‐like genes or overexpression of miR172 produce early flowering  than AP2L2, which seems to be more restricted to spikelet and floret development.In summary, AP2‐like genes is crucial for the correct development of the grass spikelet, and that this balance has been altered during the domestication of wheat and barley. Both the domesticated allele of wheat gene Q, a major determinant of the free‐threshing and compact spike character, and the barley Cly1/Zeo1 gene, which confers compact spike and cleistogamy, resulted from spontaneous mutations in their miR172 target sites that reduce miR172 cleavage activity. These examples show the potential for the modulation of the activity of the AP2‐like genes to control important agronomic traits. The two rAp2l2 alleles identified in this study provide tools to explore the value of the resulting modifications in plant height and spike compactness in different wheat classes and/or in different environments.The results from this and previous studies show that the balance in the expression of miR172 and AP2L2 and AP2L5 in the development of axillary floral meristems and the differentiation of lemma characteristics in the subtending bract. Finally, our study showed an essential role of these genes in the development of lodicules and on the regulation of B‐, C‐ and late E‐class MADS‐box floral genes.In addition to its potential practical applications, this study provided insights on the critical and redundant roles of Vrn‐A1c allele. Kronos also has the Q allele, which confers the subcompact spike phenotype and the free‐threshing character. TILLING populations of Kronos mutagenized with ethyl methane sulphonate (EMS)  adjusted to 16 h of light (22°C) and 8 h of darkness (18°C). The intensity of the sodium halide lights measured at the height of plant heads was (~260 μm m−2 s−1). The line with the mutation in the miR172 target site of the AP2L‐B2 mutant was obtained in the winter hexaploid variety Wedgetail, which was mutagenized using sodium azide . We followed Protocol A that allows for the purification of total RNA including small RNA molecules. Total RNA was treated with RQ1 RNase‐free DNase . cDNA synthesis was carried out using SuperScript II Reverse Transcriptase . mRNAs were reverse transcribed starting from 1 μg of total RNA and using OligodTv primer. The product from the first‐strand synthesis was diluted 1 in 20, and 5 μl of diluted cDNAs was used in the qRT‐PCR reaction, which was performed using SYBR Green and a 7500 Fast Real‐Time PCR system . The ACTIN gene was used as an endogenous control for mRNAs. Primers are listed in Table S5.RNA samples were extracted using the Spectrum Plant Total RNA Kit , mounted on aluminum stubs, coated with gold (Denton Desk II Sputter Coater) and examined with a ThermoFisher Quattro scanning electron microscope operating at 5 kV. Images were recorded with a slow scan for high definition and saved as TIFF files.Spikes at different developmental stages were dissected, fixed for a minimum of 24 h in FAA , rinsed twice in the same buffer, and dehydrated through a graded ethanol series to absolute ethanol. Samples were critical‐point dried in liquid COAP2‐like genes from Brachypodium, maize, rice and Arabidopsis were obtained from the Phytozome web resource . Sequences from barley were obtained from the International Barley Sequencing Consortium (https://webblast.ipk-gatersleben.de/barley_ibsc/viroblast.php). Protein sequences from wheat were obtained from Wheat Genome RefSeqv 1.1. Evolutionary analysis was conducted in mega x. For analysis, we used a region that included the two AP2 domains.The complete protein sequences of the different http://ucdptf.ucdavis.edu/) using the Japan Tobacco (JT) technology licensed to UC Davis. Immature embryos from Kronos were transformed using Agrobacterium EHA105. The selection of transgenic plants was conducted using hygromycin, and transgene insertion was validated by DNA extraction and PCR.Transgenic wheat plants were generated at the UC Davis Plant Transformation Facility  and the ap2l2, ap2l5 and ap2l2 ap2l5 mutants.Figure S4. Transcript levels of wheat MADS‐box genes involved in floral organ identity during spike development.Figure S5. Mutation in the miR172 binding site of AP2L‐B2 in hexaploid wheat (rAp2l‐B2).Click here for additional data file.Table S1. Locus name for the different wheat AP2‐like genes.Table S2. Reciprocal BLASTN searches for wheat homologs of SNB/SID1.Table S3. Wheat orthologs of MIKC‐type MADS‐box genes involved in the ABCE flowering model.Table S4. Natural variation in miR172 target site of AP2L2.Table S5. Primers used in this study.Click here for additional data file. Click here for additional data file.


The procedure is based on the anodic etching of sintered Nd–Fe–B magnets in a nonaqueous dimethylformamide (DMF)/0.3 mol L−1 FeCl2 bath. Selective recovery of Nd2Fe14B grains was realized within the applied current density <5 mA cm−2 based on the etching priority of phases: metallic Nd > intergranular NdFe4B4 > matrix Nd2Fe14B. The total energy consumption of the proposed recycling route is estimated to be 2.99 kWh kg−1, which is comparable to the state‐of‐the‐art methods. However, the proposed recycling route is currently the only procedure that enables repeated recycling of sintered Nd–Fe–B magnets in a closed‐loop system.Recycling of end‐of‐life Nd–Fe–B magnets is an important strategy for reducing the environmental dangers associated with rare‐earth mining and overcoming the supply risks associated with the rare‐earth elements. In this study, a novel concept for recycling of sintered Nd–Fe–B magnets by directly recovering the matrix Nd Here comes the grain again: Selective extraction of Nd2Fe14B grains from sintered Nd–Fe–B magnets has been achieved electrochemically by the preferential etching of their surrounding rare‐earth element‐rich grain boundaries. Recycling of sintered Nd–Fe–B magnets based on the Nd2Fe14B grain recovery represents a circular economy with low energy footprint. Neodymium–iron–boron (Nd–Fe–B) permanent magnets (PMs) are widely used in many applications, such as hard‐disk drives, wind turbines, acoustic transducers, and electric vehicle drivetrains, owing to their combination of high remanence and high coercivity.2O3) cannot be extracted, resulting in reprocessed sintered magnets, lacking full density and exhibiting poor magnetic properties. Therefore, extra REE hydrides are generally added to compensate for the existing REE oxides.The recycling of Nd–Fe–B PMs can be classified into: i) direct re‐use methods, ii) pyrometallurgical processing, and iii) hydrometallurgical separation and recovery.2Fe14B grains, which is practically oxygen‐free, accounting for 85–87 % of the magnet.2Fe14B grains, leaving REE oxides behind as a starting point would provide a sustainable recycling route for fresh Nd–Fe–B PMs production with high magnetic properties.Sintered Nd–Fe–B PMs consist of REE‐rich grain boundaries, representing about 10–12 % of the magnet, and the Nd2Fe14B matrix grains from sintered Nd–Fe–B magnets based on the etching priority of different phases in the magnets. As a result, the Nd2Fe14B matrix grains and the REE oxides were disconnected from each other after electrochemical etching, which allowed magnetic separation of the matrix Nd2Fe14B grains.Herein, we describe an electrochemical process to recover the Ndx1−Dyx)2Fe14B matrix phase, labeled as “Nd2Fe14B” for simplicity, surrounded by the REE‐rich grain boundary phases, which mostly consists of metallic Nd and a mixture of different Nd‐based oxides.4B4 and a mixture of Nd2O3 and Dy2O3 phases sitting in some of the triple points are also observed. The electrochemical etching preference of different phases in the Nd–Fe–B magnet was then studied by linear sweep voltammetry . The initial sintered Nd–Fe–B magnet exhibited a typical microstructure that consists of the (NdAll the possible anodic reactions at the Nd–Fe–B magnet anode are given by Equations (1)–2+ (reaction 1), which includes also the oxidation of the [FeCl3(DMF)]−[16] complex and might explain the mild current peak at approximately 0.55 V and the peak current (P1) attributed to [FeCl4]2−[16] oxidation at the potential of 0.75 V. When the Nd–Fe–B magnet was used as the working electrode, the current density started to increase at the potential of −0.40 V, shown by the red curve. The peak (P2) of 5 mA cm−2 recorded at −0.02 V was related to the oxidation of metallic Nd in the grain boundaries (reaction 2) owing to its very negative electrode potential .3) at 0.30 V is likely the result of the combined oxidation of the NdFe4B4 phase (reaction 3) with oxidation of Fe2+ (reaction 1). From point C on, the current density regularly increases along CD on the red curve, which is the response of the oxidation of all the Nd‐containing phases together with the Fe2+ oxidation (reactions 1–4). Accordingly, the etching priority of the phases inside the magnet is as follows: metallic Nd (in the grain boundary) > NdFe4B4 > Nd2Fe14B (magnetic phase). This is in good agreement with previous reports.When using Pt as the working electrode (black curve), the current density started to increase at approximately 0.15 V along the BC line owing to the onset of the oxidation of Fe−2) on the anode, whereas applying potentials of higher than 0.40 V (corresponding to >9.9 mA cm−2) would lead to the nonselective etching of all the phases present, for example, the metallic Nd phase, the NdFe4B4, and Nd2Fe14B .Based on the etching priority, selective etching of the metallic Nd from the grain boundary could be realized by applying a potential of <−0.02 V  were formed after preferential etching of the metallic Nd in the grain boundaries, indicating that some etching of the Nd2Fe14B grain edges also occurred. The edges of the Nd2Fe14B grains were further etched perpendicular to the polished surface with a prolonged etching time of 8 min . The second layer of the Nd2Fe14B grains (labeled with white arrows) was much less damaged with the grain boundary completely etched. This indicates that timely removal of the Nd2Fe14B grains from the magnet body can reduce the further etching the Nd2Fe14B grains.To reduce the etching of the Nd−2) for 360 min to recover the Nd2Fe14B grains. The magnetically collected particles shown in Figure 2Fe14B grains can be extracted through selective etching. X‐ray diffraction  confirms that these magnetic particles maintain the Nd2Fe14B crystal structure, which can be re‐used for making new PMs. For the 1.61 g of the sintered Nd–Fe–B magnet treated at 10 mA (2 mA cm−2) for 40 h, 1.08 g of Nd2Fe14B grains were obtained. Accordingly, 67.2 % of the Nd–Fe–B magnet was recovered in the form of Nd2Fe14B grains and the energy consumption per kilogram of the obtained Nd2Fe14B grains was calculated to be 0.58 kWh. Around 20 % of the Nd2Fe14B grains was etched and dissolved into the electrolyte .2Fe14B grains according to the etching mechanism  and ii) untimely removal of the Nd2Fe14B grains from the magnet anode after the complete etching of the surrounding grain boundaries. However, the recovery of the Nd2Fe14B grains can be further improved by using an ultrasonic bath during the electrochemical etching process to remove the Nd2Fe14B grains from the magnet anode in time. The nonmagnetic particles collected by filtering the electrolyte after electrolytic etching are presented in Figure 2O3 and Dy2O3 phases, whereas the elongated ribbed particles consist of Nd2O3, Dy2O3, Nd, and NdB4 phases, as confirmed by the energy‐dispersive X‐ray spectroscopy (EDS) and XRD analysis .The sintered Nd–Fe–B magnet was electrochemically etched with an applied current of 10 mA (current density of 2 mA cm2+ was consumed (deposited) on the cathode, while Fe2+ and REE ions (REE3+), for example, Nd3+ were generated from the partly etched magnet anode, the concentration of Fe2+, as a whole, decreased in the electrolyte with increasing etching time. In contrast, the concentrations of REE3+ in the electrolyte increased linearly with the increasing etching time . Therefore, the whole electrolysis process, including the magnet etching on the anode and the Fe deposition on the cathode, ends up with the Nd2Fe14B grains, REE‐containing electrolyte and REE‐based particles, and pure Fe metal as the final products with only the consumption of FeCl2 and electricity.In parallel, pure Fe metal was deposited on the cathode with the current efficiency of 99.6 % . As Fe2Fe14B grains are used as raw materials for making new magnets. The REE‐containing electrolyte and REE‐based particles can be further treated by the conventional hydrometallurgical process for a high purity of >99 % REE recovery2Fe14B grains to make new Nd–Fe–B magnets. DMF can be recovered by distillation and re‐used in a closed‐loop with minimized safety risk and environmental impact. Based on that, the overall REE mass balance from the initial magnet is 100 % preserved, which forms a circular economy. The total energy consumption of the magnet‐manufacturing process using the proposed electrochemical recycling route is estimated to be 2.99 kWh kg−1, which is directly comparable to the re‐use methods  should be addressed to the authors.SupplementaryClick here for additional data file.


A highly stable and reproducible radiosynthesis is a prerequisite for clinical studies applying [11C]metoclopramide or other 11C-labelled radiotracers, therefore all potential pitfalls must be identified and monitored to allow a stable process.[n = 94 in a time range of approximately 2 years) of [11C]metoclopramide synthesized on two commercially available synthesizers yielded 3.9 ± 2.0 GBq of product with a molar activity of 132 ± 164 GBq/μmol and an overall success rate of 93%. During all successful productions, the product quality was in accordance with the recommendations of the European Pharmacopoeia. The most common pitfalls that were identified for the radiosynthesis included poor turnover into [11C]CH3OTf, decomposition of the solvent or insufficient semi-preparative HPLC performance.Long-term production (11C]metoclopramide for human use.The study provides long-term insight in the improved, robust and stable preparation of [ Consequently, [11C] metoclopramide may possess a better sensitivity to measure moderate changes in P-gp function at the blood-brain barrier than previously described radiotracers  as well as the reference compound metoclopramide (4-amino-5-chloro-N-[2-(diethylamino)ethyl]-2-methoxy-benzamide) were purchased from ABX . The Ni catalyst  was purchased from Shimadzu . SepPak® C18 plus cartridges for solid-phase extraction were purchased from Waters . Low-protein binding Millex GS® 0.22 μm sterile filters were purchased from Millipore® . The silver triflate column was prepared according to the recommendation of the module producer General Electric Medical System, Uppsala, Sweden. Silver triflate and carbopack adsorbent C, 80–100 mesh was obtained by Sigma Aldrich and used as received.All chemicals were obtained from commercial sources and used as received. The precursor 11C]CO2 was produced in a GE PETtrace 860 cyclotron  via the 14N11C nuclear reaction by irradiation of a gas target  filled with N2 + 1% O2 . Typical beam currents were 60–65 μA and the irradiation was stopped as soon as the desired activity level was reached . The synthesis of [11C]metoclopramide starting from [11C]CO2 .1CCO2 wasThe flow charts of the TRACERlab™ FX C Pro and FX2 C are depicted in Fig. O-desmethyl-metoclopramide (3.5 μmol) was dissolved in 400 μL butanone and transferred to the reaction vessel. Then 7 μL of 3 M aqueous NaOH solution  was added to deprotonate the phenol.The reaction vials were filled with 1 mL water , 5 mL sodium chloride 9% (V4), 1.5 mL ethanol (V5) and 10 mL water . The bulb was filled with 80 mL of water (aqua ad injectabilia), a C18 Plus Sep-Pak (equilibrated with 10 mL ethanol and 20 mL water) was placed on the Sep-Pak position and 6 mL of phosphate buffered saline  were placed in the product collection vial. For patient application, a product vial with sterile filter and sterile air ventilation was prepared in a laminar air flow cell and connected to the product tubing. Prior to use, the reaction vessel had been stored at 100 °C for at least 4 h. Immediately before start of the radiosynthesis, 1 mg of The synthesis sequence started with a 15–20 min pre-run including heating of the carbon dioxide trap to 400 °C and subsequent cooling to < 50 °C. During the heating process, the trap was continuously flushed with helium (50 mL/min). When 400 °C was reached, 3 min of hydrogen pre-equilibration of the carbon dioxide trap took place. Afterwards the carbon dioxide trap was cooled to < 50 °C and the methane trap was cooled to − 75 °C ending the pre-run with the comment ‘ready to receive activity’.11C]CO2 to the carbon dioxide trap and confirmation of the end of delivery. The gas phase transformation of [11C]CO2 via the formation of [11C]CH4 produced approximately 50 GBq of [11C]CH3I and has been described in detail elsewhere  from Merck  was eluted with freshly prepared 20 mM aqueous NaH2PO4 / acetonitrile (80/20) at a flow rate of 5 mL/min with UV absorption measured at 254 nm , insufficient separation or no product formation at all besides other technical issues. Thirty-eight radiosyntheses were performed so far on the GE TRACERlab™ FX2 C, of which six syntheses were performed for validation of the new module (excluded data) and four synthesis failed. Out of these productions , 81 radiosynthesis were released for clinical application, the remaining six productions were released but for other applications. Details on optimization radiosyntheses performed, which were not released for human application but are included in the overall analysis are shown in Table 11C]metoclopramide produced on TRACERlab™ FX C Pro or GE TRACERlab™ FX2 C was 95 and 88%, respectively. The productions were performed between August 2017 and September 2019. The change of the synthesis module was implemented in December 2018.In total, 70 syntheses were performed on the TRACERlab™ FX C Pro, of which the first eight syntheses were performed for establishment and evaluation of the automated radiosynthesis and analytical and preparative HPLC establishment. These syntheses were excluded from statistical analysis as the final activity yield was not always measured. Three syntheses failed, caused by insufficient transfer of the activity into the reactor  column eluted at a flow rate of 1 mL/min with a gradient program .The retention times were checked prior every radiosynthesis by injection of the respective reference compounds. Both, the semi-preparative as well as the analytical HPLC systems were tested for the retention times of the reactive intermediates methyl iodide, methyl triflate and methanol (from the reaction of methyl triflate with water) as well as [11C]CO2 in GBq and is expressed in % if not stated otherwise , in accordance to previously published data  at early retention times between 3 and 6 min, followed by a minor unknown side product at 8 min and the desired product fraction at approximately 12 min. In contrast to a previous study  or 23 ± 11% .Starting from 116 ± 17 GBq of [The molar activity of the final formulated product was 132 ± 164 GBq/μmol (range: 5–659 GBq/μmol). The molar activity was calculated according to the recently published nomenclature and harmonization guidelines  The quality control parameters are listed in Table 11C]metoclopramide at the end of synthesis was slightly higher for the TRACERlab™ FX C Pro than for the TRACERlab™ FX2 C .In general, the productions with the TRACERlab™ FX C Pro afforded significantly lower molar activities (62 ± 80 GBq/μmol) than the newly installed TRACERlab™ FX2 C (299 ± 184 GBq/μmol). This considerable difference in molar activity can presumably be explained by the long usage of the TRACERlab™ FX C Pro leading to carbon deposits. A detailed analysis of potential sources of carbon load in synthesizers has been published by our group recently . The identified problems are depicted in Fig. 11C]CH3I production, for which in some cases no conversion of [11C]CH4 into [11]CH3I was observed. A higher failure rate was due to insufficient transformation of [11C]CH3I into the more reactive intermediate [11C]CH3OTf. In these cases activity was either trapped on the silver triflate column or the activity was successfully transferred to the reactor, but no reaction took place due to insufficient amounts of [11C]CH3OTf. During the semi-preparative HPLC purification step two possible failure scenarios occurred: firstly, there was a poor separation between the side product and the [11C]metoclopramide fraction leading to an impure product and subsequently inhibiting product release. Secondly, there was no product formation at all, which may be attributed to insufficient conversion of [11C]CH3I into [11C]CH3OTf or resulting from traces of decomposed butanone. A test synthesis using [11C]CH3I as reactive synthon proved the necessity of [11C]CH3OTf for the preparation of [11C]metoclopramide  gas-phase [11C]CH3I production units are not trapping the formed [11C]CH4 on a cooled methane trap  instead of helium prior to the [11C]CH3OTf production was more prone to failure than the [11C]CH3I production and was the main source of synthesis failure. With the set-up of the TRACERlab™ FX2 C module, two different unwanted scenarios were observed. In the first scenario, the [11C]CH3I was either almost completely trapped on the silver triflate column or insufficiently transferred from the silver triflate column into the reactor. In the second scenario, a very good transfer of the radioactivity into the reactor was observed, but there was no conversion of [11C]CH3I into [11C]CH3OTf. The trapping of the [11C]CH3I on the silver triflate column primarily occurred in our facility, when the silver triflate column was exposed to humidity. However, the main source of failure was the insufficient transformation of [11C]CH3I into [11C]CH3OTf. Consequently, the silver triflate column must be checked and replaced regularly in order to guarantee a stable process. The exchange of the silver triflate column was performed every 15th – 20th synthesis for the GE TRACERlab™ FX C Pro. The successful transformation of [11C]CH3I into [11C]CH3OTf was indirectly detected by analytical HPLC analysis of the crude reaction mixture. Although [11C]CH3I reacts with water of the mobile phase to [11C]CH3OH, a certain amount of non-reacted [11C]CH3I was detectable  a detectable [11C]CH3I peak indicated an insufficient conversion to [11C]CH3OTf, which considerably lowers the radiochemical yield; (ii) if only the [11C]CH3OH peak was present, the reaction of [11C]CH3I to [11C]CH3OTf took place, so that other factors, like insufficiently inert atmosphere, among others, were responsible for low product conversion.Overall, the [see Fig. . [11C]CH11C] metoclopramide it was necessary to prepare the mobile phase freshly at least once per week. Otherwise the tailing of the [11C]metoclopramide peak enhanced  or at all (1H-NMR) in butanone, which was stored over molecular sieves for more than 6 months. Still, the olfactory differences between the bottle of butanone stored over molecular sieves and a bottle of butanone without molecular sieves suggests that butanone underwent molecular sieve-catalyzed reactions. This small amount of formed aldol product might still be enough to interfere with the radiolabeling via [11C]CH3OTf. Therefore, a regular exchange of butanone at least every 3 months is recommended.A reduced or no formation of the product was either due to poor [age Fig. . Acetoneage Fig. . However11C]Metoclopramide was reliably synthesized on both, a GE TRACERlab™ FX C Pro and GE TRACERlab™ FX2 C synthesis module, for more than 100 synthesis in good yields and high molar activities. The quality of the final product was in accordance with the recommendations of the European Pharmacopoeia for other 11C-radiotracers. The main technical problems were identified, and individual solutions were developed in order to keep synthesis failures at a minimum.[


Presence of these interactions is evidenced in the solid state using X‐ray diffraction, with Rh−F contacts of 2.36–2.45 Å, and in solution using NMR spectroscopy, through hindered C−CF A–D particularly notable  ancillary ligand for the systematic study of agostic interactions,3→M bonding interactions.The coordination chemistry of the transition elements is extensive, but notable for the paucity of well‐defined complexes featuring explicit C−F→M bonding interactions.e Figure .4, 5 Com3 group, we focused our efforts on probing the intramolecular coordination chemistry of this commonly used appendage and identified PPh2ArF as a prospective ditopic ligand (dtbpm)Cl] (dtbpm=bis(di‐tert‐butylphosphino)methane) is an established source of the {Rh(biph)Cl} fragment in solution2ArF in CH2Cl2 at RT proceeded, as anticipated, with substitution of the small bite‐angle diphosphine alongside precipitation of chloro‐bridged dinuclear complex 1 . There are very few crystallographically characterised transition‐metal precedents for coordination of the CF3 appendage and, to the best of our knowledge,D ° and Rh⋅⋅⋅F>5 Å] demonstrating that this phosphine ligand is sufficiently conformationally flexible as to not enforce the chelation observed in 1–4.The solid‐state structures of 2Cl2 solution at 298 K, coordination of PPh2ArF in 1–5 was confirmed by 31P NMR spectroscopy with the associated resonances exhibiting large 103Rh coupling (1JRhP=124–170 Hz). Further coupling to magnetically equivalent 19F nuclei (2JPF≈5 Hz) is evident from the 31P{1H} NMR spectra of 1–4, but absent in that of 5, consistent with the presence of weak and time‐averaged CF3→Rh interactions in solution. At ambient temperature, fast rotation of the CF3 groups on the NMR time scale and coupling to both 31P and 103Rh, with 1JRhF≈2JPF are also apparent from the 19F{1H} NMR spectra of 1–4 .3→Rh interaction in solution inferred from these data is fully in line with expectation and further vindicated through pronounced structural dynamics of asymmetric 1–3 evident by 1H NMR spectroscopy at 298 K (400 MHz), that results in higher than expected time‐averaged symmetry of the biph ancillary ligand and invokes dissociation of the CF3 group. Equivalent exchange processes are presumably occurring in 4, although the spectroscopic signatures are asymptomatic due to the inherently higher symmetry of this complex.In CD 1JRhP=4–170 Hz.2–5 in CD2Cl2 was possible by variable‐temperature NMR spectroscopy , and inducing the onset of decoalescence of the CF3 resonances . Although a full line shape analysis of the latter was not possible, as the slow exchange regime was not reached, the enthalpies of activation for hindered C−CF3 bond rotation could be estimated from the temperature dependence of the line width −F bonds and our future work will be focused on testing this hypothesis experimentally.Through the isolation and structural characterisation of RhThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


To the Editor: We read with interest the recently published article in Diabetologia by Tang et al on the risk of cancer in patients with type 2 diabetes treated with sodium–glucose cotransporter 2 (SGLT2) inhibitors [hibitors . We agrehibitors . DocumenTo elucidate the potential association between a substance and carcinogenesis, length of exposure needs to be considered. We undertook an additional analysis of patients with at least 6 months’ drug exposure. Bladder cancer with an onset after 6 months’ cumulative exposure to study drug was reported in 4/2187 patients (0.2%) in the placebo group, 3/2216 patients (0.1%) in the empagliflozin 10 mg group, and 7/2190 patients (0.3%) in the empagliflozin 25 mg group (Table With the number of events being so small, a few additional cases can make a difference to the conclusions drawn. Based on the totality of the data, no imbalance in bladder cancer cases between empagliflozin and placebo were observed in the EMPA-REG OUTCOME trial.


After publication of this article , the folThe FUT8 panel in Fig 2B appears similar to the ST6Gal1 panel in Fig 6A when horizontally flipped;○0 μg/ml panel of Fig 4B  and 16h ○5 μg/ml panel of Fig 4B  and 20 μ○0 hr panel of Fig 4C  and 8h pThere are similarities between cell staining patterns in the following panels of Fig 4B and 4C in  and the Several stained cells in Fig 4B and 4C appear to have highly similar morphologies and staining patterns;Areas within Fig 4B (1 and 10 μg/ml panels) of ;An area within the 0 μg/ml panel of Fig 4B  appears There are clusters of stained cells that appear to be duplicated within and across panels of Fig 5B and 6B in . Also, wPLOS ONE Editors retract this article due to concerns about the integrity, validity, and reliability of the published results.We raised these concerns to the authors, who apologized and provided supporting image files as well as replication data for the results in question. However, the image data provided did not resolve the published image concerns. Therefore, the All authors agreed with the retraction.PLOS ONE article [Note: Permissions were not obtained to use the images described above in the second, fourth, and fifth bullet points, in Fig 4B and 4C of this  article , and so  article –4 for in


After publication of this article , concernThe authors note that the original blot underlying the SRB1 panel of Figure 5A is no longer available, and they are unable to clarify the seeming discontinuity between lanes 1 and 2 of this figure. Confirmatory data from replication experiments are provided in Regarding similarities between the β-actin panels in Figs The original raw data underlying other figures in this article  are no lThe updated version of S1 FileThe Experiment I blots included for (PPTX)Click here for additional data file.S2 FileIn Exp II° data for Figure 5B, the membrane was cut to enable probing with two antibodies and the SRB1 western blot resulted in high molecular weight non-specific signals. The lower band in this SRB1 blot is the correct size for the SRB1 protein.(PPTX)Click here for additional data file.S3 FileNote that the SRB1 blot on the final slide (labelled “5° exp. 12-01-2010”) includes a lower set of bands not present in other replicates of this experiment. The authors were unable to clarify the identity of these bands.(PPT)Click here for additional data file.


The methods developed so far use either chemical conversion or isotope equilibration to determine the δ17O value of CO2. In addition, δ13C measurements require correction for the interference from 12C17O16O on 13C16O16O since it is not possible to resolve the two isotopologues.Determination of δ17O, δ18O and δ13C values of CO2 from the fragment ions that are formed upon electron ionization in the ion source of the Thermo Scientific 253 Ultra high‐resolution isotope ratio mass spectrometer (hereafter 253 Ultra). The new technique is compared with the CO2‐O2 exchange method and the 17O‐correction algorithm for δ17O and δ13C values, respectively.We present a technique to determine the δ13C and δ18O values are slightly larger for fragment ion measurements than for molecular ion measurements. The δ17O and Δ17O values of CO2 can be measured on the 17O+ fragment with an internal error that is a factor 1–2 above the counting statistics limit. The ultimate precision depends on the signal intensity and on the total time that the 17O+ beam is monitored; a precision of 14 ppm (parts per million) (standard error of the mean) was achieved in 20 hours at the University of Göttingen. The Δ17O measurements with the O‐fragment method agree with the CO2‐O2 exchange method over a range of Δ17O values of −0.3 to +0.7‰.The scale contractions for δ2 can be used as an alternative method to determine the carbon and oxygen isotopic composition of CO2 without chemical processing or corrections for mass interferences.Isotope measurements on atom fragment ions of CO These abundances can be changed by kinetic and equilibrium fractionation processes and other physicochemical effects. Variations in isotopic abundance are reported as deviations of a heavy‐to‐light isotope ratio in a sample relative to a reference material. In the case of oxygen isotopes, the two isotope ratios are 18R = [18O]/[16O] and 17R = [17O]/[16O] and the international standard is Vienna Standard Mean Ocean Water (VSMOW).Oxygen has three stable isotopes,17O is approximately half of the fractionation in 18O . Most isotope fractionation processes depend on mass. For oxygen isotopes, this results in fractionation patterns where the fractionation in Equation .(3)lnδ1i.e.17R17Rref=18R18Rrefλ ranges from 0.5 to 0.53 for such mass‐dependent fractionation processes.3 and related gases have a large oxygen isotope anomaly, expressed as Δ17O and referred to as mass‐independent fractionation. We use the logarithmic definition to calculate Δ17O of CO2  and 12C17O16O (exact mass 44.9940). Resolving these two isotopologues requires a mass resolving power (m/Δm) of ~56,000, far beyond the resolving power of most traditional mass spectrometer systems. Different alternative techniques have been developed to measure the δ17O value of CO2: (1) CO2 fluorination and isotopic measurement of the released O22 into H2O and CH4 followed by H2O fluorination and isotopic measurement of the released O22 and CeO245CO2 value before and after exchange to calculate the δ17O value of CO2; (4) isotope exchange between CO2 and CeO2 followed by isotope analysis of the equilibrated CeO2 by laser fluorination2 with H2O followed by fluorination of H2O and measurement of the isotopic composition of released O22 and O2 over hot platinum and measurement of the isotopic composition of oxygen before and after exchange to calculate the δ17O value of CO2.17O values and other isotope signatures of CO2 from air samples have been developed.Determination of Δ13C value are used to quantify fluxes between atmosphere and hydrosphere and/or ocean12C17O16O and 13C16O16O,13C values require an appropriate correction for 17O‐interference. Different “17O correction” algorithms are in use to correct for the interference of 12C17O16O on the value of δ13C, causing discrepancies between different correction algorithms used. The discrepancies in the δ13C value introduced by different 17O correction algorithms  are explored by Assonov and Brenninkmeijer2 with δ45(CO2) and δ46(CO2) values of −9.2‰ and +2.180‰ vs NBS19‐CO2 between the algorithm by Allison et al17R and λ. The discrepancies introduced by 17O correction algorithms depend on the δ46(CO2) values17O correction for CO2 having the same δ45(CO2) value but a different δ46(CO2) value. By design, most of the 17O correction algorithms do not consider the Δ17O of the CO2 and the ones that do include Δ17O require precise measurement of the δ17O value of CO2. For instance, the algorithm of Allison et al2. Nevertheless, the error introduced to the δ13C value because of the use of different values of λ is different for CO2 with different Δ17O even if the same algorithm is used. It is desirable to use an alternative technique that enables the determination of the δ13C value without a bias introduced due to the 17O correction algorithm for better use of the δ13C values as a tracer to quantify fluxes between atmosphere and hydrosphere and ocean.Very small variations in the δ2 from the C+ and O+ fragment ions, which are produced from CO2 in the ion source of two 253 Ultra  instruments installed at Utrecht University and the University of Göttingen.Recently developed high‐resolution isotope ratio mass spectrometers15N+ in N2O,Isotope measurement of fragment ions is not a new concept. The method has been deployed, for example, to study the intramolecular distribution of 17O, δ18O and δ13C values of CO2 directly on the C+ and O+ fragment ions of CO2 without any chemical manipulation of the CO2 molecule. Notably, this method provides an independent technique to measure Δ17O of CO2 and the results are validated by comparison with the existing CO2‐O2 exchange method and by measuring CO2 with known Δ17O.Here we establish an analytical method to determine the δ22.1The 253 Ultra is the commercial version of a high mass resolution gas source multi‐collector mass spectrometer, which was pioneered with the MAT 253 Ultra prototype in 2012.The ion source of the 253 Ultra is connected to a sample introduction system of four variable volume reservoirs that can be filled with sample or reference gases. The control of the ion source chemistry  is critical for accurate isotope ratio measurements. The differentially pumped ion source can be baked to high temperature and is fitted with a variable ion source conductance (VISC) window to adjust the source pumping conductance and to control the residence time of the sample gas in the ionization volume, which is one critical parameter for ion source chemistry. The source slit can be switched to three different slit sizes for low‐, medium‐ and high‐resolution settings. For the instruments at Utrecht University and the University of Göttingen the slit widths are 250 μm, 16 μm and 5 μm. The intermediate aperture at the entrance of the magnetic sector allows an extra‐high‐resolution mode to be selected to achieve m/Δm >35,000 mass resolving power. It should be noted that higher resolution comes at the cost of lower ion beam intensities.o deflection angle of the electrostatic sector (r = 22.4 cm) and the magnetic sector (r = 23 cm) as shown in Figure The basic setup of the instrument follows a double‐focusing Nier Johnson geometry with a 90In the 253 Ultra the ions are generated at a potential of 10 kV. The ions are accelerated to the source slit of the double‐focusing mass analyzer at a kinetic energy of 5 keV. After passing through the electrostatic analyzer the ions are further accelerated to 10 keV kinetic energy before they pass through the magnetic sector where the ion trajectories are split up according to their mass. Finally, the ions are focused along the focal detector plane of the mass analyzer. The two‐stage acceleration of the ion beam allows a very compact design of the electrostatic sector geometry, which otherwise would have required the radius of the electrostatic sector to be about twice as large as that of the magnetic sector. Due to its compact geometry, the ion optical setup of the 253 Ultra fits onto just one monolithic base plate. The resonance frequency of this rigid mechanical construction is very high and precise, which makes the system robust against low‐frequency vibrations that usually occur in buildings. In order to achieve ultimate stability, the complete mass analyzer and the electronics are housed in a shielded temperature‐stabilized cabinet to be robust against temperature fluctuations in the lab (±2°C).8 Ω and 1013 Ω. The three collector platforms at the high mass end are additionally equipped with compact discrete dynode ion counting detectorsThe variable detector array supports eight moveable detector platforms, which are equipped with Faraday detectors that can be read out with selectable resistors with resistances between 3 × 102.22 as suggested by Verkouteren et al2 gases  is adjusted before each acquisition to 3.2 × 1011 cps (counts per second) with an allowed difference of 1 × 1010 cps between the two bellows that are used for the measurement. Under these conditions the ion source pressure is 2.5 × 10−7 mbar. The reference measurement is performed with 9.9 kV accelerating voltage, filament emission current of 1.8 mA, equilibration time of 60 s, integration time of 67.1 s and with the VISC window closed.We investigated the effect of equilibration time, emission current, source conductance and signal intensity on the ionization of COm/z 44), three experiments with 2.5 × 1011 cps, 1.5 × 1011 cps and 9 × 1010 cps for m/z 44 are performed. Note that measurements to characterize the effect of emission control current and signal intensity are performed with an equilibration time of 30 s, so they cannot be directly compared with the reference measurement with an equilibration time of 60 s. The effect of cross contamination is calculated according to Meijer et al13C and δ18O values) measured at different equilibration times with the value obtained at 90‐s equilibration time. Similarly, the scale contraction due to the emission current is calculated with respect to the results obtained at an emission current of 1 mA. The cross contamination (η) is calculated as:y is 13 (for δ13C) or 18 (for δ18O), the index a indicates the respective δ value under reference conditions (90‐s equilibration time and 1 mA emission current), and index m indicates the δ value at a different equilibration time or different emission current.To study the effect of equilibration time and source conductance, we measure the two gases with equilibration times of 10, 20, 30, 40, 50, 60 and 90 s with the VISC window open and closed. The effect of the emission current is quantified by setting the emission current to 1 mA, 1.5 mA and 1.95 mA. To investigate the effect of signal intensity  for analysis. This research group also provides high‐precision δ17O values and has established a direct link between the oxygen isotope scales of O2 and CO2. The reported results were assigned to our reference gas cylinders, which were also measured extensively on the Thermo Scientific Delta Plus XL™ instrument in our laboratory and on the 253 Ultra. The appropriate scale contraction factors  with the “reference” source settings mentioned above, i.e., emission current of 1.80 mA, accelerating voltage 9.9 kV, VISC window closed. The ion signals are registered in three Faraday collectors  that are read out with resistors of 1 × 1011 Ω, 1 × 1013 Ω and 1 × 1013 Ω for m/z 16, 17 and 18, respectively. The ion signal intensity is adjusted before each acquisition to 9.2 × 108 cps on m/z 16, which corresponds to a source pressure of ~2.5 × 10−7 mbar, with a tolerance of 3 × 106 between the bellows. Reasonable source pressures for fragment ion measurement are determined to fall between 2.0 and 4.5 × 10−7 mbar , corresponding to the linear portion of the source pressure vs signal intensity relationship for m/z 16  is OH+ (mass 17.0027 u). The mass difference between these two ions is only 0.0036 u. With the 253 Ultra, they are sufficiently separated using the medium‐resolution slit to enable measurement of 17O+ on a narrow plateau without interference from OH+. In this study the medium‐resolution slit is chosen since the plateau is sufficiently flat and gives a sufficient signal to allow stable positioning for 17O+ measurement, as shown in Figure 18O+ (mass 18.9984 u) the mass difference to its main interference H2O+ (19.0148 u) is 0.0164 u which results in a broad shoulder even at medium mass resolution. The potential effect of other interferences is discussed below.The 17O+ shoulder. This can be largely circumvented by resetting the mass scale at regular time intervals during the measurement. The present version of the Qtegra software does not allow automatic positioning on a shoulder of multiple overlapping peaks. Therefore, the collector configuration is carefully arranged such that the center of the m/z 16 peak is precisely located at the shoulder of the m/z 18 and m/z 17 peaks where 17O+ and 18O+ can be measured interference‐free. A peak centering is then performed on m/z 16 before each acquisition which is precise enough to relocate the system on the narrow shoulder of the m/z 17 peak. Nevertheless, instabilities in the mass scale are still considered a main contributor to the remaining error above counting statistics, and an automatic positioning routine that scans the 17O+ shoulder directly to reposition the peak might improve the precision.Small shifts in the mass scale regularly lead to a deterioration of measurement precision, when the mass position shifts away from the small 17O+ fragment ion measurements on the 253 Ultra at the University of Göttingen are performed at medium resolution  with 9.85 kV accelerating voltage and 1.85 mA emission current, with the VISC window closed. The integration and equilibration times are 67.1 and 12 s, respectively, which implies that in a measurement cycle both sample and reference are measured for 67.1 out of 158.2 s, i.e., 42.4% of the time. Three Faraday collectors , equipped with 1 × 1010 Ω, 1 × 1013 Ω and 1 × 1012 Ω resistors, are used to detect the ion signals for m/z 16, 17 and 18, respectively. The signal intensity is adjusted per acquisition on m/z 16, with a target intensity of 1.2 × 109 cps (tolerance 0.2%), corresponding to a source pressure of 4.12 × 10−7 mbar.All 16O18O++ ion is very close in mass to 17O+  with the same emission current, acceleration voltage, integration time and equilibration time as used for the 17O+ fragment method, again with the VISC window closed. The ion signals are registered in two Faraday collectors (L4 and Center) that are read out with resistors of 1.0 × 1011 Ω and 1.0 × 1013 Ω for 12C+ and 13C+, respectively. The mass spectra covering the range for 12C+ and 13C+ are shown in Figure 13C+ (mass 13.0034 u) is 12CH+ (mass 13.0078 u), which requires a mass resolving power of 2900. This is well resolved with the medium‐resolution slit of the 253 Ultra (m/Δm >7500).The 2 gases , tint is the integration time in seconds, n is the number of measurement cycles and the factor 2 accounts for the fact that the reference and the sample both introduce the same error to the δ value. Throughout the manuscript the error of a single measurement series is reported as the standard error of the mean. When we quantify errors for more than one measurement (series), we report the standard error times the Student's t‐factor to cover the 95% confidence interval.The reported internal precision of the fragment technique is compared with the expected error (precision) based on counting statistics (EECS), which is calculated as:2.42‐CO2 exchange experimental setup at Utrecht University is shown in Figure 2‐O2 exchange system is the exchange reactor, which is made of quartz, while the other parts are made from borosilicate glass. The general design is similar to the one in Barkan et al,2 and O2 into the reactor.A schematic diagram of the O2 with known (measured) δ18O value was expanded to the glass line and trapped cryogenically using liquid nitrogen (LN2) in the calibrated volume . The amount of CO2 was precisely determined with a pressure sensor . The CO2 sample was then transferred cryogenically to the quartz reactor. The trapping in the quartz reactor occurs at the horizontal tube that is continuously cooled using LN2 provided by a microdosing system . After introduction of the CO2 sample, an approximately equal amount of pure O2 (IMAU‐O2) with known δ17O and δ18O values is admitted to the small volume above the reactor and then expanded into the reactor. The CO2 is then released from the cold tube by stopping the LN2 microdosing system, and the gases are allowed to react for 30 min in the quartz reactor that contains 0.18 g of platinum sponge  at the bottom, which is heated to 750°C with a temperature‐controlled oven . After 30 min, CO2 is extracted cryogenically in a double U trap, while O2 is collected behind this trap on 3 pellets of molecular sieve 13X  at LN2 temperature. The isotopic composition of the exchanged O2 is measured using a dual‐inlet system on the DeltaPlusXL isotope ratio mass spectrometer (Thermo Fisher Scientific) using three Faraday collectors equipped with resistors of 3 × 108 Ω, 3 × 1010 Ω and 3 × 1011 Ω for m/z 32, 33 and 34, respectively. The value of δ17O (CO2) is then calculated from the change in the δ17O(O2) value before  and after  isotope exchange with CO2 based on the following mass balance equation  = 1.0006657 and α18(CO2/O2) = 1.000998, determined by measuring the isotopic composition of CO2 and O2 after isotope exchange was fully established.Approximately 1.7 mL of pure CO2.52.5.12 with known isotopic composition is prepared by combusting a pure graphite rod  (Thermo Fisher Scientific) in isotopically known pure IMAU‐O2 , the reactor is cooled to below 200°C and the collar trap is heated to room temperature (25°C) to release the CO2. The CO2 is collected in a break seal tube at LN2 temperature. After each conversion experiment the graphite rod is re‐conditioned by heating at 900°C for 1 h to avoid contamination from remaining oxygen.At Utrecht University, COO2 Table . The gra2 was produced from combustion with isotopically depleted O2 using a slightly different setup. Instead of using platinum foil and wire as catalyst, the graphite rod was immersed in chloroplatinic acid and dried before being installed in the quartz reactor. Isotopically light oxygen for the reaction was provided by hydrolysis of Antarctic precipitation . After full combustion, the produced CO2 was transferred into a glass vial, which was kept at LN2 temperature.At the University of Göttingen, isotopically light CO2.5.217O‐enriched CO2 is prepared by inducing oxygen isotope exchange between CO2 (G2) and O2 (IMAU‐O2) (via O3 and O(1D))2 is expanded into the 2‐L reactor and O2 is then expanded into the reactor until the pressure reading reaches around 1 bar. The mixture is then allowed to photolyze for 18 h without regulating the temperature. Due to the heat produced by the UV light the temperature outside the reactor reaches 30°C during photolysis, and is much higher at the Suprasil finger, but this is only a preparative experiment where the exact conditions are not critical. After photolysis‐induced isotope exchange, CO2 is separated cryogenically in a glass spiral trap at LN2 temperature and O2 is pumped out. Finally, the CO2 is collected in a sample vial containing nickel foil . O3 that is formed during photolysis is also condensed with CO2 and is decomposed to O2 by heating the sample vial with a heat gun at 500°C for 10 min. Ni foil catalyzes the decomposition of O3 to O2. The CO2 is then trapped again with LN2 and the O2 that has formed from O3 decomposition is pumped out. Finally, the CO2 is passed through a glass U‐trap at dry‐ice temperature (−78°C) to remove remaining traces of water. Heating the O3 and CO2 mixture above 200°C might cause isotope exchange between O3 and CO2,17O‐enriched CO2.17O‐enriched CO2 sample is measured with the 253 Ultra for both molecular ions (m/z of 44 to 46) to determine δ18O and δ13C values, and atom fragments to measure δ17O and δ18O values. By diluting the 17O‐enriched CO2 with pure non‐anomalous CO2 from the reference CO2 tank (G2), two gas mixtures are prepared with target Δ17O values of approximately 0.25‰ and 0.55‰. The two mixtures are finally measured both with the CO2‐O2 exchange method and with the fragment technique.The isotopic composition of the 33.118O and δ13C values in the same way, but the effects are larger for the δ18O values than for the δ13C values. The origin of the qualitatively different behavior for δ18O and δ13C values could not be identified and requires further study.Scale contraction decreases with equilibration time and source pressure , when the variable conductance window is fully opened and when the emission current is decreased. A detailed investigation of these parameters is presented in the supporting information  measured with the 253 Ultra at Utrecht University and the assigned relative difference by the Hebrew University of Jerusalem. Thus, the final values reported below are linked to the isotope scale of the Hebrew University of Jerusalem.By comparing the results of the molecular ion measurements on the 253 Ultra with the values assigned to our reference gases by the Hebrew University of Jerusalem, a scale contraction factor of 0.981 was established and applied for molecular ion measurements. The scale contraction factor is the ratio of the difference between the two CO2 gases both with the traditional CO2+ method and with the fragment method (both O+ and C+ fragments). For the traditional molecular ion measurements, the 17O‐correction procedure from Brand et al+ fragment than for those on the O+ fragment, but more measurements are required to quantify this more thoroughly. Note that each individual measurement series presented in Tables 2+ molecule plus O+ fragment and C+ fragment) takes one full day. For the evaluation of the Δ17O measurements below we use the relative scale contraction of 0.997 determined for the value of δ18O between the traditional CO2+ method and the O‐fragment method  correction algorithms at a level of precision similar to the reported differences between different ion correction schemes.When the appropriate scale correction parameters are applied, the δ2 gases are measured directly, and via a third intermediate gas for δ13C, δ18O and δ17O values. Tables 13C+ and 18O+ fragment ions are both measured with a precision close to the counting statistics limit.Figures 3.217O, δ18O and Δ17O: reproducibilityδ17O for a pure CO2 (G5) sample with six replicates measured using the O‐fragment method at Utrecht University. The δ17O and δ18O values of the CO2 are given in Table 17O values are measured with an individual measurement error (standard error of the mean) ranging from 37 to 82 ppm, while the δ18O values have an individual measurement error of 11 to 25 ppm . The measurement precision for the δ17O values is worse than that expected from counting statistics by a factor of 1.42 to 1.73. As shown in Figure 17O reproducibility is 19 ppm (standard error times Student's t‐factor for 95% confidence). At the University of Göttingen the reproducibility experiment is performed using CO2 produced by combustion of a graphite rod with pure O2 (GU‐O2)  ranging from 21 to 35 ppm while the δ18O values have an individual measurement error of 7 to 17 ppm (SEM). As shown in Figure 17O reproducibility is 16 ppm (standard error times Student's t factor for 95% confidence). The reproducibility for the δ17O and δ18O values is lower in this method due to incomplete combustion of the graphite rod.Figure ure CO2 G sample wure CO2 G sample wure CO2 G sample w17O and δ17O values  after a measurement time of 20 h  yielded a precision of 14 ppm for Δh Figure . As ment17O and δ17O measurements using the O‐fragment method is evaluated by measuring CO2 with known δ17O and δ18O values, prepared from isotopically known O2  in the process of preparing the CO2. The effect on Δ17O is much smaller.The accuracy of Δd Figure . To enab17O, δ18O and Δ17O values of oxygen and of the CO2 produced by combusting graphite shows that determination of the triple isotopic composition of CO2 using the O‐fragment method is not only reproducible but also accurate. Furthermore, the agreement in the triple isotopic composition of oxygen between O2 and CO2 (produced by combustion) suggests that our isotope scales for CO2 and O2 are very compatible.The good agreement between the δ17O is measured with an average standard error of 39 ppm (standard error of the mean) for four measurements  at an intensity for m/z 16 of 1.18 × 109 cps. When measurements are made at lower signal intensity than the linear range for source pressure vs signal intensity relation for m/z 16 (see above), measurement precision decreases. For instance, the precision drops from 39 to 83 ppm . Two of the gases are commercial CO2 gases  which covers and extends the Δ17O range expected for tropospheric CO2 samples, including international carbonate standards.17O is determined by the O‐fragment method with a precision of 36–79 ppm (standard error times Student's t‐factor for 95% confidence). The excellent agreement between the two totally independent methods provides an independent validation of the fragment ion technique.C‐fragmentAfter confirming the accuracy and reproducibly of the O‐fragment method, we measured the δ13C values of the two CO2 gases G1 and SCOTT were measured against G2 with the C‐fragment method and with the traditional measurement on the CO2 molecule . A possible challenge for measuring δ13C values with the fragment method is the interference from the 12CH+ adduct due to ion source chemistry (e.g. in the presence of water). The 12CH+ adduct is only 0.004 u separated from 13C+ as shown in the mass spectra . Therefore, only interferences from doubly ionized oxygen formed in the ion source (16O18O++) and other doubly ionized molecules with higher masses  per 1‰ difference in the δ18O value between sample and working reference gas. Thus, when the working reference gas is close in isotopic composition to the samples that are measured, the correction is negligible.Oxygen isotope measurements on O fragment ions with low‐resolution mass spectrometers are mainly limited by the interference from water and its OH fragment ions. The background level of water in mass spectrometers is always significant, and it also generally varies when switching between bellows in dual‐inlet measurements. With the 253 Ultra, these interferences can be separated from the O17O and δ18O values of CO2 using the fragment method is the possible interference of O fragment ions from other oxygen‐bearing impurities (OBI) such as H2O, O2 or N2O. The sample and the mass spectrometer background should be very clean to avoid any oxygen contribution from other molecules. The effect of an OBI on the values of δ17O, δ18O and Δ17O measurements of CO2 (δIimp) can be estimated using Equation 2), ionization efficiency and the abundance of the impurity relative to the CO2  for the calculation. Table 17O, δ18O and Δ17O values of CO2 measured with the O‐fragment method for different water levels and isotopic composition of the water. For instance, when the isotopic composition of the water impurity relative to the CO2 is δ17O = −20‰ and δ18O = −40‰, the effect on the δ17O and δ18O values of CO2 will be significant for ρ >0.3% and ρ >0.1%, respectively. Since the isotopic composition of the water is assumed (roughly) to be mass dependent, the effect on the Δ17O will be only significant when ρ >1%. When the isotopic composition is strongly mass independent (δ17O = δ18O = −40‰ relative to CO2), the effect on Δ17O will be significant for ρ >0.3% . Higher precisions can be achieved by (i) increasing signal intensity; (ii) increasing observation/integration time of the 17O+ fragment ions  carbonate standard (NBS‐18) measured by Passey et al13C measurements due to the use of different algorithms for 17O‐correction.The fragment technique is simple and unlike other techniques does not require any additional chemical conversion or exchange steps to measure the δ17O+ and 18O+ in other oxygen‐containing compounds, for example, CO or N2O. Current isotope techniques of these gases rely in many cases on an assumed relation of mass‐dependent fractionation between δ17O and δ18O values and (e.g. in the case of the CO) chemical conversion into CO2.+ fragment can overcome these limitations and provide quantification of Δ17O. Similar to the case of CO2 presented here, the 13C+ content of CH4 and CO can be measured directly on the C+ fragment of these gases, without chemical conversion steps that are known to cause artifacts in traditional isotope techniques.Isotope measurements of atomic ion fragments may have many applications for other molecules. A straightforward extension of the application presented here is the mass‐interference‐free measurement of 15N+ content of N2O is presently determined by measurement of the parent N2O molecule and the NO fragment, which allow the average δ15N value and the 15N content at the central nitrogen position to be quantified, and the δ15N value of the terminal N atom is derived by mass balance, which induces large errors.15N+ content of the terminal N atom could be derived from the N+ fragment, which originates primarily from the terminal N atom in N2O. Similar to the case of O atoms shown here, this requires a very good vacuum system to avoid contamination from the main atmospheric gas N2.The position‐specific 2. Examining these effects further may help to understand the chemistry and surface effects in the ion source of isotope ratio mass spectrometers by studying different fragments. In addition, analysis of fragment ions facilitates measuring the isotopic composition of two different chemical compounds versus each other . This can on the one hand provide information on ion source effects associated with fragmentation, but on the other it may also help to directly compare isotope scales between different compounds.In addition to these environmental applications, the analysis of atomic fragment ions of different compounds may be a useful tool to study fractionation processes in the ion source of an isotope ratio mass spectrometer. As discussed earlier, the scale contractions for isotopic measurements are different for the fragment ions and molecular ions of COFigure S1m/z 16 . The linear range (shaded area) ends at a source pressure of 4.5x10−7 mbar .Relationship between source pressure and signal intensity at Figure S2 Schematic diagram of the O2‐CO2 exchange experimental setup. The quartz reactor has an outer diameter of 21 mm. PID stands for proportional‐integral‐derivative temperature controller.2 to CO2 by combusting a graphite rod. The reactor is made out of quartz with 21 mm outer diameter.Figure S3 Schematic diagram of the setup used for conversion of O2 with a positive Δ17O by photolysing a mixture of O2 and CO with UV light. BR: Borosilicate reactor (2 L), PRL: pen ray Hg vapor lamp, CT: CO2 trap (liquid nitrogen temperature), SV: sample vial, WT: water trap (operated at dry ice temperature), PS: pressure sensor, HVP: high vacuum pump, FVP: fore vacuum pump.Figure S4 Schematic diagram of the setup used for preparing COFigure S5 δ13C and δ18O values of SCOTT measured against G2. A and B) Effect of equilibration time with VISC window closed. C and D) Effect of equilibration time with VISC window fully opened; E and F) Effect of filament emission current on the relative difference between the two gases in δ13C and δ18O values. G and H) Effect of amount of gas in the ion source quantified by the signal intensity in cps for m/z 44. The emission current experiments are performed at 60‐seconds equilibration time and the sensitivity to the amount of gas was determined with 30‐seconds equilibration time. The VISC window was kept closed.Figure S6 Effect of the equilibration time on the δ13C (A) and δ18O (B) differences between measurement with open VISC window (VISCo) and closed VISC window (VISCc).Figure S7 Drop in signal intensity for at m/z 44 (corresponding to the source pressure) when the dual inlet valve was closed for different initial source pressures. A) decrease of the main ion signal of CO2 as a function of time. B) fraction of CO2 remaining as a function of time for the main ion signal of CO2. The emission current and accelerating voltage were 1.95 mA and 9.9 kV, respectively.Figure S8 Comparison of δ17O differences between three different CO2 gases with the fragment technique using the 253 Ultra at Utrecht University The red arrows indicate that the respective measured δ values are combined for comparison with the directly measured third δ value.Figure S9 Comparison of δ18O differences between three different CO2 gases using either the molecular CO2 ion technique (left) or the atom fragment technique (right) with the 253 Ultra at Utrecht University The red arrows indicate that the respective measured δ values are combined for comparison with the directly measured third δ value.Table S1 Effect of equilibration time on the absolute isotopic difference of two gases measured on the 253 Ultra with VISC window open and closed. The errors given are standard errors of the mean.Table S2 Effect of emission control current and source pressure on the absolute isotopic difference of two gases measured on the 253 Ultra (G1 and SCOTT). The errors given are standard errors of the mean. The integration time of individual measurements is 61.7 seconds The effects of source pressure (intensity of m/z 44) are determined at an equilibration time of 30 seconds and an emission current of 1.8 mA.Table S3 Isotopic composition of CO2 produced by combustion with isotopically light O2 at the University of Göttingen. Each CO2 sample is analyzed four times at different intensity to investigate the effect of signal intensity on the precision of Δ17O measurement based on the relationship between source pressure and signal intensity at m/z 16.Table S4: Comparison of the results obtained with the O2‐CO2 exchange method and O‐fragment technique. δ17O, δ18O and Δ17O values are in per mill (‰) with respect to VSMOW. The error of the mean is the standard error multiplied by the student t‐factor for the 95% two‐sided confidence interval while for the individual measurements it is the standard error. Γ is the ratio between measured precision and the precision calculated according to counting statistics, n is number of cycles, i stand for initial (before exchange) and f stands for final (after exchange). For δ18O values the measurement error is similar to the error calculated based on counting statistics.Table S5 List of potentially interfering ions with masses close to 17O+ and 18O+, and the required resolution to avoid the interference. The interfering ions are ordered based on the resolving power requirement.Table S6 Simulated effect of a H2O impurity (contamination level quantified by γ = [H2O]/[CO2] as source of oxygen during Δ17O measurement using the O‐fragment method for an intensity for m/z 16 of 4.05x109 cps. The signal of O atom fragments relative to molecular ions is 10% for CO2 and 1% for H2O5,6. For these conceptual calculations we assumed the same ionization efficiency for H2O and CO2.Click here for additional data file.