Less commonly, the short-range configuration Ca(Al2□)Al6(BO3)3[Si5T3+O18]V(OH)3W(OH) could occur in Al-rich tourmalines with a Si deficiency, where T3+  = B, Al. Therefore, tourmalines enriched in cations with charge 2 +  contain only insignificant Y-site vacancies. Aluminum-rich tourmalines with ≥ 7.0 apfu Altotal that usually contain ≥ 0.2 apfu Li may have significant vacancies at the Y site. However, no more than 12% vacancies (0.36 pfu) at the Y site can be observed in such samples. If no chemical data for Li is available it is proposed to calculate the Li content in such colourless or coloured tourmalines  for Y = 2.8 apfu or for Y + Z + T = 14.8 apfu, because this calculation should give more accurate results than calculating the Li content as the difference to 3.0 apfu at the Y site. For Fe2+-rich and Mg-bearing tourmalines from the schorl-dravite series with MgO > 1.0 wt%  the structural formula can still be calculated for Y + Z + T = 15 apfu, because such tourmalines do not appear to contain significant Y-site vacancies. It can further be concluded that the Z site could be only ≤ 1% vacant and therefore such vacancies would be insignificant even in Al-rich tourmaline.Tourmaline has two different [6]-coordinated sites, the Y site and the Z site. Vacancies were reported from both sites. Based on high-quality chemical and single-crystal structural data it usually needs increasing proportions of short-range order configurations Na(Al Based on published high-quality data, the present work considers whether, why and when vacancies occur in the [6]-coordinated sites and in which tourmalines they would be expected. Any effects of such possible vacancies are also discussed.The tourmaline crystal structure contains two different [6]-coordinated sites. Since more than 50 years, the site with the larger (distorted) octahedra is named the Y site and the site with the smaller (distorted) octahedra is named the Z site. Articles from the 1970ies and 1980ies, in which small amounts of Y-site vacancies, up to 0.16 per formula unit (pfu), have been cited, were summarized in Table 5 of Foit . Many st2O3, Li2O, and H2O analysis) and by single crystal structure, show up to 0.21 pfu Y-site vacancies , 0.08 pfu Z-site vacancies (for 6 Z sites) were reported, based on chemical and structural data . This means that the oxy-component at the W site usually increases, when such a tourmaline contains increasing amounts of  and/or increasing X-site vacancies. Hence, the following substitutions can theoretically occur in Al-rich tourmalines to produce Y-site vacancies:2+  + WO =  > YAl3+  + Y□ + W2YXCa2+  + 3Y2+  + WO =  > XNa1+  + 2YAl3+  + Y□ + WX□ + Y2+  + WO =  > XNa1+  + Y□ + W2+  + T3+  + WO =  > Y□ + TSi4+ + WYX□ + YAl3+  + T3+  + WO =  > XNa1+  + Y□ + TSi4+  + WYLi + WO =  > Y□ + WXNa1+  + 3Y2+  + TSi4+  + WO =  > XCa2+  + 2YAl3+  + Y□ + T3+  + W(OH)XCa2+  + 2YLi1+  + YAl3+  + WF =  > XNa1+  + 2YAl3+ + Y□ + W(OH)2+: Fe2+, Mn2+, Mg2+Y3+: Al3+, B3+TUsually, the best way to detect Y-site vacancies is when a tourmaline is fully characterised, including chemical analyses of the light elements B, Li, and H. If the tourmaline formula is normalized to 15 atoms per formula unit (apfu) for the sum of the occupants at the Y + Z + T sites, or for Y + Z + T + B = 18 apfu, it is clear that possible Y-site occupancies cannot be found. Tourmalines, which contain significant amounts of Mg, usually contain almost no Li and almost no tetrahedrally coordinated B. For such tourmalines, it is therefore not necessary to analyse B in most cases, since they usually contain exactly 3 apfu B. Since essentially Al-rich tourmaline samples with significant Y-site vacancies have been reported in the literature, only such samples were used for plotting correlations. Only the best-characterised tourmalines were used, most samples of which were analysed both chemically (including the light elements) and by single crystal X-ray diffraction. Some of the very Al-rich samples contain significantly less Si than 6 apfu. Hence, two different correlations were plotted, one with ~ 6 apfu Si Fig. . Since t2+, Mn2+ and Mg2+) cations, respectively. If such tourmalines exhibit low X-site vacancies, they will only have small amounts of [4]Al and/or [4]B. Such compositions will come close to a W-site occupancy of OH + F = 1. Regardless of whether tourmaline has a Si deficiency or not, it usually needs increasing proportions or short-range order configurations of Na(Al2□)Al6(BO3)3[Si6O18]V(OH)3 with OH or F at the W site in order to produce Y-site vacancies. A similar short-range order configuration (with OH at the W site) for Al- and Mn2+-rich tourmaline was proposed by Ertl et al. (Y(Al2□)ZAl6(BO3)3[Si6O18]V(OH)3WF, 16% fluor-tsilaisite and 11% fluor-schorl  increase at the tetrahedral site Al6(BO3)3[Si5T3+O18]V(OH)3W(OH), where T3+  = B, Al. This would be the only proposed substitution with a short-range order configuration, where the T site is not completely filled with Si4+. However, a calculation using bond-valence theory shows that this arrangement also appears to be stable.It is likely that these proposed substitutions depend not only on the chemistry of the whole rock, but also on the pressure and temperature conditions during the crystallization of tourmaline. Therefore, it would be helpful to have a larger number of tourmaline analyses of each locality. In general, complete published data of Al-rich tourmaline from a locality, including light elements and structural data, are not very common. Therefore, one is limited to check the proposed substitutions. There is, however, one locality from which such tourmalines have been extensively investigated. Therefore, the correlation between Al content at the Y site and Y-site vacancies was checked by using the Al-rich tourmalines of the Koralpe, Austria Fig. . A stron2+ and Mn2+ are extremely small in all tourmalines examined, it seems that the substitution (8) is mainly responsible for the Y-site vacancies. This is the only proposed substitution in which the W site is not occupied by oxygen but by fluorine. Interestingly, in substitution (8), an increasing Ca content does not lead to an increasing content of tetrahedrally coordinated B and Al, as in substitution (7). It can only be assumed that the tourmalines from Anjanabonoina, Madagascar crystallized at different temperatures and pressures than the tourmalines from the Koralpe, Austria. Anyway, such vacancies are unlikely to result from incorrect Li2O secondary ion mass spectrometry (SIMS) values, since the Li content is relatively low in many samples examined. The Al-richest sample from the study Ertl et al. (2O content would be much too high (> 30%) compared to the measured Li2O content for these samples. Furthermore, Al cannot fill these vacancies since Al has significantly more electrons than Li and therefore the calculated electron occupancy would be much too high compared to the observed electron occupancy at the Y site.In tourmalines of the fluor-liddicoatite – elbaite series from Anjanabonoina, Madagascar, investigated by Ertl et al. , the vacl et al. , which c3+, Mn3+, Cr3+ and V3+. Apart from a sample with a relatively high content of Fe3+, described by Filip et al.  at the Y site appear to occur. It can be concluded that the calculation of the Li content by completely filling the Y site with Li when no chemical analysis is available is problematic for such Al-rich tourmalines from lithium pegmatites. The average Y-site vacancies in completely characterised Al-rich and Li-bearing (colourless – coloured) tourmalines  is 0.185(7) 1.0, particularly in the tourmaline with a X-site vacancy > 0.3 pfu. Based on the trend seen in Fig. 2+-rich and Mg-rich tourmalines have shown that samples with MgO > 1.0 wt% typically contain < 0.10 pfu Y-site vacancies  the structural formula can be calculated for Y + Z + T = 15 apfu, or for Y + Z + T + B = 18 apfu, because such tourmalines contain no significant Y-site vacancies.Chemical data of Novák et al.  showed tIt can be assumed that possible vacancies at the Z site could be ≤ 0.05 apfu (for 6 Z sites), which corresponds to ≤ 1% vacancies, and thus would be insignificant. The highest chance of detecting Z-site vacancies in tourmalines is in samples with a very large amount of [6]-coordinated cations with a 3 + charge, e.g., in Al-rich tourmalines. Further investigations will be necessary to obtain more high-quality data to be able to definitively prove Z-site vacancies. These conclusions are consistent with the theoretical approach of Bosi  who, usi


The phosphorylatedpeptides were identified by liquid chromatography-high-resolutionmass spectrometry and the site of phosphorylation by tandem mass spectrometry.The results clearly show that the oxidized tyrosine residues are phosphorylated,displaying a specific immonium ion peak in the MS2 spectra.Furthermore, we detected this modification in our reanalysis (MassIVEID: MSV000090106) of published bottom-up phosphoproteomics data. Themodification, where both oxidation and phosphorylation take placeat the same amino acid, has not yet been published in PTM databases.Our data indicate that there can be multiple PTMs that do not excludeeach other at the same modification site.Post-translationalmodifications (PTMs) alter the function andfate of proteins and cells in almost every conceivable way. Proteinmodifications can occur as a result of specific regulating actionsof enzymes, such as tyrosine kinases phosphorylating tyrosine residuesor by nonenzymatic reactions, such as oxidation related to oxidativestress and diseases. While many studies have addressed the multisite,dynamic, and network-like properties of PTMs, only little is knownof the interplay of the same site modifications. In this work, westudied the enzymatic phosphorylation of oxidized tyrosine ( Abnormalities in phosphorylation can have pathogenic effects,as they disrupt the regulatory mechanisms and signaling pathways ofcells.Protein phosphorylation catalyzed by kinasesis a reversible post-translationalmodification (PTM) that has a central role in the regulation of cellfunctions. It is estimated that up to 75% of proteins encoded by thehuman genome can be phosphorylated.6 Tyrosine phosphorylation is especially peculiar, as 90 of the 518protein kinases are tyrosine kinases, but phosphorylated tyrosine(pTyr) residues constitute below 1% of protein phosphorylation intotal, suggesting heavy and wide regulatory activity.8 Disruptions in signaling pathways occur in the pathophysiologicalmechanisms of many diseases, among them several types of cancer.9 Indeed, many tyrosine kinases show oncogenicactivity, which can be caused either by mutation or by overexpression.10 Chronic myelogenous leukemia (CML) is a well-knownexample of a disease in which tyrosine kinases play a role.11 A remarkable step in treating this form of cancerwas the development of different tyrosine kinase inhibitors to suppressthe activity of the overactive enzymes, making CML, previously a lethaldisease, treatable.12Proteins in the human proteome are phosphorylated by518 proteinkinases and dephosphorylated by ∼200 protein phosphatases.13 ROSand RNS can react with a large number of biomolecules such as lipids,carbohydrates, nucleic acids, and amino acid residues of proteins.13 Oxidative modifications of proteins can resultin changes in their physical and chemical properties, including conformation,structure, solubility, susceptibility to proteolysis, and enzyme activity.14 Protein oxidation has been associated with variousbiological consequences, including aging and disorders such as Alzheimer’sand Parkinson’s diseases and amyotrophic lateral sclerosis(ALS).16 Several amino acids can be directly modifiedvia side-chain reactions with ROS, but the most reactive amino acidresidues are sulfur-containing amino acids (cysteine and methionine)and those with aromatic structures .Besides phosphorylation,protein oxidation can have a huge impacton cellular activity. Oxidative stress from endogenous  and exogenous  sources forms reactive oxygen and nitrogenspecies .17 are especially prone to oxidation, resultingin protein-bound l-3,4-dihydroxyphenylalanine (l-DOPA) as well as other products.18 Ruokolainenet al. have demonstrated that tyrosine oxidation is inhibited afterthe initial phosphorylation of the tyrosine residues.19 Gow et al. showed that peroxynitrite-mediated nitrationof tyrosine residues significantly inhibits the phosphorylation ofa synthetic peptide by a tyrosine kinase.20 It has been suggested that besides direct oxidative damage to biomolecules,cell toxicity by oxidation could be partially induced by the inhibitionof tyrosine phosphorylation and the consequent interference with cellularsignaling.17 Similarly, Zhang et al. reportedthe first proteome-wide survey of endogenous site-specific tyrosineoxidation to DOPA modifications in mouse heart and brain tissues.Many of the oxidation sites were also possible phosphorylation sites,and it was hypothesized that this oxidation would disrupt tyrosinephosphorylation signaling pathways.21The oxidation of phosphorylated amino acid residuesor phosphorylationof oxidized amino acid residues in proteins and peptides has rarelybeen investigated. The residues that are prone to both oxidation andphosphorylation are tyrosine and histidine. In proteins and peptides,tyrosine residues, which take part in signaling processes by phosphorylation,22 So far,the phosphorylation of l-DOPA residues formed by oxidationof tyrosine has not been studied  using an Orbitrapmass spectrometer. Because the phosphorylated l-DOPA is notpublished in the UniMod database,23 thepresence of phosphorylated and unphosphorylated l-DOPA inpublished raw phosphoproteomics data was also searched using variablemodification search and unrestricted modification search.The aim of this work was to evaluate the phosphorylation of  and IR3 1 by a tyl-DOPA). In IR0, all tyrosine residuesare nonoxidized. In IR1 Tyr5 residue, in IR2 Tyr5 and Tyr10 residues,and in IR3, all of the tyrosine residues  areoxidized , and synthetic peptides IR1, IR2,and IR3 were from CASLO ApS . All peptideshave the same sequence TRDIYETDYYRK with a varying number of oxidizedtyrosine residues ,5× kinase buffer A from Thermo Fisher , adenosinetriphosphate (ATP) from Sigma-Aldrich , and IRkinase from Life Technologies . LC-MS grade methanol and acetonitrile were used assolvents. Deionized water was prepared with a Milli-Q water purificationsystem  on site.–1 (0.6 mM) ofindividual peptide solutions of IR0, IR1, IR2, and IR3 and 5 mg mL–1 (9.1 mM) of ATP solution were separately preparedin 1× kinase buffer A. 20 μL of IR kinase  was diluted to 0.1 mg mL–1 (1.4 nM) by adding 58 μL of 1× kinase buffer A. Samplesfor kinase incubation were made according to Table S1. After mixing, incubation was carried out at 37 °Cin a warm-air oven with shaking for 3 h, excluding the samples forthe investigation of the reaction rate .After incubation, the reaction was quenched using 3 μL of 100%formic acid, followed by evaporation to dryness with a vacuum centrifuge(SpeedVac). Dry samples were reconstituted for LC-MS analysis by adding40 μL of 1% methanol and 0.1% formic acid in water (LC-eluentA). The samples were sonicated in an ultrasonic water bath at roomtemperature for 15 min and transferred to autosampler vials.1× kinasebuffer A was prepared by dilution with Milli-Q water from the 5×commercial stock solution. 1 mg mL–1. The gradient startedfrom 0% B and was then increased to 50% B in 8 min, to 95% B withinthe next 2 min, and held at 95% B for 3.5 min. After that, the gradientwas decreased to 0% B within 0.5 min. Subsequently, a cleaning stepfollowed, where the gradient was increased to 95% B in 1 min and afterthat decreased back to 0% in 1 min. The gradient was held at 0% Bfor 4 min, resulting in a total gradient time of 20 min.The LC-MS measurementswere carried out using an Orbitrap Fusion tribrid mass spectrometercoupled to an UltiMate 3000 liquid chromatography setup (both fromThermo Fisher Scientific). Chromatography was performed using a WatersAcquity UPLC C-18 column  at 30 °C. The autosampler temperaturewas 15 °C, and the injection volume was 3 μL. Eluent Aconsisted of 1% methanol and 0.1% formic acid in water in positiveion mode and 3% methanol and 0.1% formic acid in negative ion mode,while eluent B consisted of 0.1% formic acid in methanol in both measurements.The flow rate was 0.29 mL minm/z 200), and the scan range was m/z 150–1500.Automated gain control (AGC) was set to accumulate 1 × 105 ions with a maximum injection time of 100 ms, and the RFlens was set to 60%. The ion transfer tube temperature was 325 °C,and the vaporizer temperature was set to 350 °C. Internal masscalibration with Easy-IC (fluoranthene) was used.Massspectra were measured using electrospray ionization with a 3.5 kVspray voltage in positive ion mode and a −2.5 kV spray voltagein negative ion mode. The orbitrap resolution was 500 000 (at 3+ peptides (exact masses in Table S2). DDA consisted of an MS1 scan with amass range of m/z 50–1000 and a resolutionof 120 000, followed by MS/MS scans for the cycle time of 2s with a quadrupole isolation window of 1.1 Da, collision-induceddissociation (CID) fragmentation with a fixed collision energy of30%, an orbitrap resolution of 60 000, and a scan range of m/z 50–1000. For MS/MS, AGC was set to accumulate1 × 105 ions with a maximum injection time of 118ms.MS/MS measurementswere performed using a data-dependent acquisitionmethod (DDA) with an inclusion list of possible precursor ions from[M + 3H]3 or H2O and verifiedusing interactive peptide spectral annotator (IPSA)24 for fragments with relative abundance above 1%. To distinguishthe possible loss of phosphorylation during the MS/MS scan, all massspectra were reviewed against nonphosphorylated controls using IPSA.Modifications added to the unmodified peptide sequence in IPSA were+15.9949 Da for tyrosine oxidation to l-DOPA, +79.9663 Dafor phosphorylation, and +95.9612 Da for both phosphorylation andoxidation in the same amino acid.Average MS/MS spectra of the main phosphorylation productswereannotated individually for peptide-specific a, b, y, and x fragmentsand neutral losses of NH29 were downloaded from the MassIVE repository and obtainedfrom collaborators. Protein identification was done with MaxQuant/Andromeda(MaxQuant 2.1.0.0 and 2.1.4.0).32 The search database used wasreviewed human or mouse UniProt/Swiss-Prot33 proteome . A combination of phosphorylationand oxidation (phospho-oxidation) in the same tyrosine residue wassearched using both a dependent peptide (DP) search (that does notrequire a priori assumptions of the PTMs) and a standardvariable modification search method. Methionine oxidation, serine/threonine/tyrosinephosphorylation, tyrosine oxidation, and tyrosine phospho-oxidation(added to MaxQuant modification table with P(1) O(4) H(1) compositionresulting in a mass change of +95.9612 Da) were set as variable modifications.Cysteine carbamidomethylation was set as a static modification. DPsearch was performed alongside the variable modification search usinga DP-specific FDR of 0.01. The largest data set1 was rerun for DP without the same site tyrosine phospho-oxidationas a variable modification to remove these peptide spectral matchesfrom the main or second peptide search and thus permitting unassignedMS/MS spectra to be matched in dependent peptide search. This DP reanalysiswas done using cysteine carbamidomethylation as a static modificationand methionine oxidation and serine/threonine/tyrosine phosphorylationsas variable modifications.Raw phosphoproteomicsdata8H11NO4P+, m/z 216.04202) and the novel phosphorylated l-DOPA immonium ion  reported in this workwere searched. The quantification method (label-free/TMT/iTRAQ) waschosen according to the technique used in each study. Besides these,no changes were made to the recommended search settings.Modification site and dependent peptidetables were used for data analysis. Decoy database matches and potentialcontaminants were removed. DP results were parsed to residue locations,location probability, and individual PSM. DP results containing “peptide:”prefix in annotated modification column were removed. Stringent identificationand filtering criteria were applied: false discovery rate (FDR) <0.01was applied for both peptide and protein identification, and onlymodification sites with a localization probability of above 0.999and posterior error probability (PEP) <0.01 were considered reliableidentifications in the variable modification search. DP result binswith the highest localization on tyrosine were parsed to their individualpeptide spectrum matches. The known diagnostic phosphorylated tyrosineimmonium ion (Cl-DOPA phosphorylation(tyrosine phospho-oxidation) is shown in Table S1, and samples in phosphoproteomics search results are in l-DOPA phosphorylation was performed with IR peptidesindividually and as a mixture . Relative comparison of reaction rate wasdone using IR peptide mixture samples with 1, 5, 30, and 60 min incubationtimes . l-DOPA phosphorylation results were revised against matrix-matchedcontrol samples of eluent, blank with kinase, and peptides withoutkinase individually and as a mixture . Furthermore, the reagent ratios were optimized,and phosphorylation was replicated with additional standards and phosphorylatedstandard, individual, and mixture of IR peptides (similar to samples1–15 in Table S1). These also serveas process replicates and complement LC-MS and LC-MS/MS results. Dataanalysis was performed as described above, and Microsoft Excel wasused for statistical analysis.Eachsample used for the verification of invivo, and in vitro), quantification methods(label-free and label-based), and research questions. In total, 223raw files were searched. Experiment designs and the numbers of samples,replicates, and controls are described in the respective publications.No quantitative analysis was performed on the modified peptides thatwere identified.Raw phosphoproteomics data setswere chosen to represent different sample types  withthe identifier MSV000090106. Raw data for tyrosine oxidation and tyrosinephospho-oxidation in published phosphoproteomics results may be, accordingto 34 or MassIVE, or inquiredfrom original authors.29The original phosphorylated l-DOPA residues eluted typically after the phosphorylated peptides. The EICs of thephosphorylation products  show that the main product was the singly phosphorylated productfor each of the IR peptides . IR0 includes three tyrosine residues,which in the other peptides were replaced with one (IR1), two (IR2),or three (IR3) residues 1. The expeptides 2. The repeptides 2.Only sTables S2–S4 present the high-resolutionpositive and negative ion mass spectra of the phosphorylation reactionproducts with accurate masses, mass errors, and relative intensities.All mass spectra featured a charge state distribution from [M + 2H]2+ to [M + 4H]4+, the [M + 3H]3+ ionbeing the most intensive in positive ion mode  and [M – 2H]2– the onlyion in negative ion mode. The mass accuracies of the multiply chargedions were below 2.6 mmu, and those of the deconvoluted protonatedand deprotonated molecules were below 5.2 mmu, with all phosphorylationproducts of IR0, IR1, IR2, and IR3, confirming the identificationof the products.l-DOPA , the formation of singly phosphorylated IR3 (IR3+ 1p) was clearly slower than with IR0 (IR0 + 1p), in which none ofthe three tyrosines is oxidized. Also, the formation of IR2 + 1p wasslower than the formation of IR0 + 1p. However, it is clear that oxidizedtyrosine (l-DOPA) was phosphorylated but at a slightly lowerrate than tyrosine.Because the singly phosphorylated product wasthe most abundantwith all IR peptides, we studied the formation of singly phosphorylatedIR peptides as a function of time using positive and negative ionmodes 3. There3+ ions were the most abundant for all nonphosphorylatedand phosphorylated IR peptides , they were selected as the precursor ions for the MS/MS analyses.No major coeluting and coisolating impurities were present in totalion chromatograms of LC-MS/MS  or in LC-HRMS data . The annotated MS/MS spectra of the main LC-MS/MS chromatographicpeaks with accurate masses, mass errors, and relative abundances arepresented fully in File S1 and as a representativeexemplary spectrum in LC-MS/MSwas used to verify phosphorylation sites of the IR peptides.As the [M + 3H]peptides S3, they 2O and NH3. Also, the known phosphorylated tyrosine immonium ion  was observed with phosphorylatedtyrosine (<0.5 ppm mass accuracy and 10.4% average relative abundance),and a novel phosphorylated l-DOPA immonium ion  was observed specificallyin the MS/MS spectra of peptides containing phosphorylated l-DOPA . The productions y4 and y5 of singly phosphorylated peptides include a phosphate group,but y3 does not, indicating that the main site of phosphorylationis Tyr9 and l-DOPA9 in the case of IR3 + p. This is supportedby the fact that neither tyrosine nor l-DOPA of the singlyphosphorylated IR peptides at position 5 is phosphorylated, as phosphorylatedb5 ions are not present. The MS/MS spectra of the doublyphosphorylated IR peptides  show y3 with one phosphate group and both y4 and y5 ions with two phosphate groups, indicating thatthe residues Tyr9 and Tyr10 of IR0 and IR1, Tyr9, and l-DOPA10of IR2, and l-DOPA9 and l-DOPA10 of IR3 are phosphorylated.The MS/MS spectra of all triply phosphorylated IR peptides  show doubly phosphorylated y4 and y5 ions and singly phosphorylated b5 ions, indicating that all tyrosine or l-DOPA residues arephosphorylated. The IR peptides also show small abundances of differentphosphorylation combinations (Tyr and l-DOPA), but theseare not typically chromatographically separated or abundant enoughfor confident annotation. Furthermore, small abundances  of correspondingnonphosphorylated sequence diagnostic b ions and y ions besides thephosphorylated diagnostic ions were observed. Examples of these arey4 and y5 ions in Table S5: singly phosphorylated peptides show singly phosphorylatedfragments with a high relative abundance and unphosphorylated fragmentswith low relative abundance. Likewise, doubly phosphorylated peptidesshow doubly phosphorylated fragments with a high relative abundanceand singly phosphorylated fragments with low relative abundance. Theseare likely caused by typical phosphate neutral loss, as peptide phosphorylationis a labile modification.35The MS/MS spectra of the main chromatographicpeaks of singly,doubly, and triply phosphorylated IR peptides show very similar sitesof phosphorylation .The same site phosphorylation and oxidation were rare and in the sameorder of magnitude as the stochastic probability of both oxidationand phosphorylation together. Due to the low numbers of identificationsfor tyrosine residues that are both phosphorylated and oxidized, falsepositives from the proteomics search engine cannot be ruled out. Therefore,we chose to further investigate the issue using a dependent peptidesearch and specific immonium ions.In each data set, both oxidized tyrosine modifications and phosphorylatedtyrosine modifications were identified with variable modificationsearch in similar abundance and in numbers about 2 orders of magnitudelower than unmodified tyrosine residues in detected peptides. We alsoidentified tyrosine residues that carried both oxidation and phosphorylationas a modification, albeit in low numbers  with the highest localization in tyrosine residues were only foundin the largest data set in which phosphotyrosines were specificallyimmunoenriched.1Dependent peptide (DP) searchis a ModifiCombFigure S5). Most of them  were within −100 and 100 Da and equalwith highly abundant modifications such as phosphorylation, dephosphorylation,oxidation, and reduction. Notably, a modification with mass changeequal to phosphorylation and oxidation (+95.96 Da) was detected .In these DP results,1223 individual modifications of 535 uniquemass changes were observed and localized to tyrosine (File S3).This data set was rerun for DP without the same site phosphorylationand oxidation as a variable modification to remove these peptide spectralmatches from the main or second peptide search and thus permittingunassigned MS/MS to be matched in DP search. This increased the matchesto 99 MS/MS scans with PEP <0.01 in 19 peptides (File S3). Nine peptides were common between reanalyzed DPsearch and variable modification search, and reanalysis results includedalso PSMs of all peptides found in the first DP search . Some initial DP search PSMs withPEP >0.01 were decreased to PEP <0.01 after reanalysis. Of thesepeptides, five had the novel phospho-l-DOPA immonium ion, 12 peptideshad the known phosphotyrosine immonium ion , and three peptides had both immoniumions in MS/MS. The annotated mass spectra of two PSMs with high sequenceion coverage and pl-DOPA immonium ion, unambiguously showingthe pl-DOPA modification, are presented in 37 along with the DP result information in File S3.Tyrosine residues that carriedboth oxidation and phosphorylationas a modification were observed, again in low numbers. The same sitephosphorylation and oxidation were matched to 22 MS/MS scans withPEP <0.01 in six peptides (P search S6. Some l-DOPA immonium ion peak were detected (File S3).Given the previous research on endogenous tyrosine oxidation42 and the inhibitory role of tyrosine phosphorylation on its oxidation,19 as well as the findings presented in this work,tyrosine oxidation and its interplay with phosphorylation may be ofbiological significance.According to these results, tyrosineoxidation can be detectedin various biological samples, in some rare cases together with phosphorylationon the same residue. Although false positives likely still persist,also excellent matches with sequence diagnostic ion series and p44 terms wasperformed using Enrichr46 on the MaxQuant variablemodification search results (proteins in which modified peptides wereidentified). Lists of proteins in which oxidized and oxidized andphosphorylated tyrosine residues were identified (File S2) were used as the input, and GO terms with adjusted p-value <0.01 for enrichment were considered significant.For both phosphorylation combined with oxidation and oxidation alone,no significantly enriched GO terms were shared between the data sets(File S2); rather, in studies where anyGO terms were significantly enriched, they represented the sampletype in the particular study. These results suggest that the potentialbiological significance of tyrosine oxidation and tyrosine oxidationand phosphorylation is not similar in different organisms, tissues,and biological functions but depends on the biological context.Despite the fact that many of the peptideidentifications withthe variable modification search of both phosphorylated and oxidizedtyrosine residues may be false positives, we decided to tentativelyinvestigate the potential biological role of these modifications.Enrichment analysis of Gene Ontologyl-DOPA has the additional capacity to reduce, bind, and chelatetransition metals and inflict secondary damage to other biomoleculesby itself.47 While phosphorylation is oneof the most prominent and investigated PTMs, little is known aboutthe susceptibility of modified protein amino acid residues, such asoxidized tyrosine, to phosphorylation.Oxidative damage to proteins can cause proteincross-linking, fragmentation,and modification of amino acid residues, potentially resulting ininactive proteins. Oxidation of tyrosine residues mostly to protein-bound l-DOPA) canbe phosphorylated, contrary to a previously postulatedhypothesis,17 but the l-DOPA phosphorylationrate appears to be slower than with tyrosine. We also showed, usingraw phosphoproteomics data from various experimental settings, thatboth tyrosine oxidation to l-DOPA and l-DOPA phosphorylationcan be identified in biological samples, although in low numbers.As phosphorylated l-DOPA abundance is low, searching multiplevariable modifications with low abundance can produce false positiveand negative results.36 Thereby, complementaryconfirmation methods such as diagnostic ions or unrestricted searchmethods are required. The currently detected numbers of tyrosine residuescarrying both phosphorylation and oxidation PTMs are orders of magnitudebelow phosphorylation or oxidation, and further research would benefitfrom efficient enrichment, MS/MS optimization for immonium ion formation,or ultradeep proteome approaches.Here, we have shown thatoxidized tyrosine residues  analysis ofprotein-bound l-DOPA.48 Giventhe low abundance of proteins with oxidized tyrosines, such toolsare crucial for elucidating the biological role of tyrosine oxidationin proteins.Based on bioinformatics enrichmentanalysis, these modificationsmay have biological functions, but the details of such functions remainto be elucidated. Recently, a proteome-wide spectral library of peptideswith tyrosine residues enzymatically oxidized into l-DOPAinstead of tyrosinecould affect the stability of this prominent reversible PTM, proteinfolding, and interactions. Phosphorylated protein-bound l-DOPA could alter the tyrosine kinase signaling networks. The observeddifference in phosphorylation rate could impede the dynamic natureof PTM-mediated and phosphorylation kinetics-sensitive signaling networksor oscillating phosphorylation systems such as those in circadiancontrol.51Phosphorylation of protein-bound


A two-dimensional (2D) polar monolayer with a polarization electric field can be used as a potential photocatalyst. In this work, first principle calculations were used to investigate the stability and photocatalytic properties of 2D polar monolayer SiTe as a potential promising catalyst in water-splitting. Our results show that the 2D polar monolayer SiTe possesses an indirect band gap of 2.41 eV, a polarization electric field from the  Renewaby crisis . It is ay crisis ,6.3N4 [2 Nanosheet [To improve the efficiency of photocatalysis, researchers have focused on addressing the issue of carrier annihilation ,8,9,10. 3N4 , group-I3N4 , WS2 Nananosheet , MXene [anosheet , and g-Zanosheet , were reanosheet ,18,19,202-VI3 group monolayer In2Se3 [2OS [2Se6, [In the recent literature, much research has suggested that 2D polar photocatalysts, which possess a polarization electric field, may be more effective at quickly separating photogenerated carriers ,22,23,24r In2Se3 ,33,34,35Se3 [2OS , and monS [2Se6,  as well S [2Se6, ,39,40, PS [2Se6, ,42, PtSOS [2Se6, , and WSeS [2Se6, ) for useS [2Se6, ,47,48,49S [2Se6, ,31,50, eHere, we used the first principles to systematically calculate the stability and photocatalytic properties of 2D polar monolayer SiTe. Our goal was to attain a better comprehension of the potential of this material for use in photocatalytic water-splitting and other applications. Our results show that 2D polar monolayer SiTe exhibits high dynamic, mechanical, and thermal stability. The band gap, band edge positions, and surface potential difference of 2D polar monolayer SiTe are suitable and helpful for water-splitting. Ultraviolet and visible light can be effectively absorbed by 2D polar monolayer SiTe. Furthermore, we found that the properties of 2D polar monolayer SiTe, including its band gap, surface potential difference, polarization electric field, absorption, and photocatalytic activity, can be tuned and enhanced by strain engineering. These findings suggest that 2D polar monolayer SiTe is a promising photocatalyst.To verify the dynamic stability of the 2D polar monolayer SiTe, we used the density functional perturbation theory method to calculate the phonon dispersion. This allowed us to identify any potential phonon instabilities that could indicate a lack of structural stability in the material. As shown clearly in Γ point. The conduction band minimum (CBM), on the other hand, is situated between the Γ and M points, indicating that 2D polar monolayer SiTe is an indirect semiconductor. Moreover, the band gap of 2D polar monolayer SiTe is 2.41 eV, calculated using the HSE06 functional. This result suggests that ultraviolet light and visible light can be efficiently absorbed by 2D polar monolayer SiTe. As shown in p orbitals of Te and Si atoms, whereas the CBM above the Fermi energy level is primarily provided by the p orbitals of Si atoms. The band gap shown in the PDOS is consistent with that shown in the band structure.The band structure is a significant factor that regulates the photocatalytic activity of a material. The band gap of a photocatalyst should be noteworthy. As shown in Γ point, and the CBM is situated between the Γ and M points, similar to the band structure without strain engineering. However, when the tensile strain is in the range from +1% to +5%, the VBM is located between the K and Γ points. As shown in To tune the band structure properties of 2D polar monolayer SiTe effectively, the strain engineering method ,58,59,60As previously mentioned, 2D polar monolayer SiTe has an unsymmetrical structure along the vertical direction, with a bottom layer of Si atoms and a top layer of Te atoms. As shown in ng group , the fold (Eeff) .(1)ΔΦ=ee is the elementary charge constant, and d is the distance between the Si atomic surface and Te atomic surface.Here, The larger equation ,(2)EG>1These photocatalysts, such as 2D polar monolayer SiTe, can absorb more solar energy in the infrared and visible region for photocatalytic water-splitting. Therefore, tuning the surface potential difference As To appraise the suitability of 2D polar monolayer SiTe for photocatalytic water-splitting, we calculated its band alignment using the method described in previous studies ,22. The Here, Here, +/H2) and the oxidation potential (H2O/O2) are −4.44 eV and −5.67 eV, respectively. Since the ΔΦ is 0.441 eV in 2D polar monolayer SiTe, the energy level of the 2O is reducesed by the electrons according to the following equation,As shown in 2O is oxidized the holes according to the following equation,In contrast, on the +/H2 (−4.44 eV), and the VBM is 0.415 eV lower than the energy level of H2O/O2 (−5.67 eV). Moreover, under the effect of an electric field The CBM of 2D polar monolayer SiTe is 1.208 eV higher than the energy level of HAs we can see from G) in the hydrogen reduction was determined using the following equation to investigate the dependence power of total water-splitting [The Gibbs free energy difference ΔG=ΔE is the adsorption energy, ΔzpeE is the difference in zero-point energy, T is the system temperature , and ΔS is the entropy difference.Here, ΔG is 1.28 eV. To control and decrease the value of ΔG, strain engineering is adopted to tune the value. As shown in G decreases from 1.24 eV to 0.92 eV as the compressive strain decreases from 0 to −3%. For tensile strain, when the strain is +2%, the ΔG is 1.33 eV. As the strain increases from +2% to +5%, the band gap decreases from 1.33 eV to 1.27 eV regularly. Notably, the value of ΔG can be effectively tuned via strain engineering.As shown in g,E obtained by the optical properties is 2.38 eV, similar to the band gap calculated by the band structures using the HSE06 method. This confirms that the solar light, which has energy larger than the band gap, will be absorbed efficiently by the 2D polar monolayer SiTe. This indicates that 2D polar monolayer SiTe has the potential to be an efficient photocatalyst with high solar energy conversion efficiency. The band gap of 2D polar monolayer SiTe, which is approximately 2.41 eV, plays a role in improving solar energy conversion efficiency. As shown in The following equation was used to determine the absorption coefficient −6 eV and 10−3 eV/Å, respectively. The first integration of the Brillouin zone was performed using the Gamma center method in KPOINTS [We used the Vienna ab initio simulation package (VASP)  with the KPOINTS . Structu KPOINTS  to inves KPOINTS  softwareIn conclusion, our study, which used first principle calculations, has shown that the 2D polar monolayer SiTe is a potential promising catalyst for water-splitting using visible light. According to the results, the 2D polar monolayer SiTe possesses an indirect band gap of 2.41 eV. The 2D polar monolayer SiTe can absorb ultraviolet and visible light in a wide range. What is more, in the 2D polar monolayer SiTe, the polarization electric field helps to reduce the likelihood of photoinduced electron–hole pair recombination and lower the band gap necessary for water-splitting. The direction of the polarization electric field is from the


Trimming is routinely performed in horses, with direct effects on hoof structures; however, the immediate effect on forelimb joints is unknown. Thus, we investigated the immediate effect of trimming on hoof structures and angulation of the thoracic limb joints through linear and angular measurements. Differences were found in hoof length, toe angle, heel angle, medial heel height, and metacarpophalangeal angle after the procedure, proving the immediate effect of the procedure. Correlations were found between trimming and the proximal joint angles.p < 0.05) were found in hoof length, toe angle, heel angle, medial heel height, and metacarpophalangeal angle. Before trimming, correlations were found between frog length and scapulohumeral angle (SH) , and between toe length and shoulder-ground angle (SG) . A correlation was also seen between the distance from the frog to the lateral wall and the SH angle . After trimming, there was a correlation between humeroradial (HR) and SH joint angles , and the SG and SH angles . This study showed an immediate effect of trimming on the toe angle and heel angle and on the metacarpophalangeal joint angle, in addition to correlations between the hoof and proximal joint angles, following trimming, thus evidencing the relevance of trimming not only in hoof morphology, but also in the conformation of the appendicular skeleton of horses.It is important to understand the effects of hoof trimming on hoof and limb conformation to maximize its benefits on the health of the appendicular skeleton of horses, thus promoting improvements in athletic performance and sporting longevity with regard to athletic horses. There is little information on possible changes in the angulation of the thoracic limb joints after hoof trimming and correlations between the angulation of the thoracic limb joints with hoof measurements. To that purpose, nineteen Mangalarga mares received routine hoof trimming. Visual recordings (photographs) were taken before and after the procedure. Differences ( Hoof trimming and shoeing are routine procedures with significant effects on the health of horses and may influence the performance of athletic horses. They alter the external and internal structures of the hoof, directly influencing the entire limb . Good faTrimming and shoeing influence not only the morphology of the hooves ,5,6 but Since hoof trimming can impact the entire limb , changesAccurate hoof measurements can be made from photographs and radiographs using commercially available software . LikewisChanges in hoof conformation due to routine trimming and its effect on proximal and distal thoracic limb joint angles have not been previously reported in Mangalarga horses. In so, we ought to evaluate the immediate effects of trimming on hoof morphometry, and proximal and distal joint angles, as well as to investigate existing correlations between hoof measurements and joint angles before and after trimming.The project was approved and supervised by the institution’s animal use and care committee (protocol 018789/17). Nineteen healthy Mangalarga mares, embryo donors, with a mean age of 10.37 ± 4.55 years and a mean body weight of 500.75 ± 47.07 kg, were included in this study. All animals lived under a semi-extensive regime and were distributed in two distinct farms. None of the horses performed mounted or guided work, and they were used only as embryo donors. None of the mares showed obvious lameness at pace or trot previous to the study. Regular hoof trimming was performed with an interval between six and eight weeks, and none of the mares had been shod in the previous six months. The animals were kept in paddocks, where hay and commercial feed were offered twice a day, with free access to potable water. Two evaluations were performed. The first before trimming and the second immediately after the procedure. The solar surface of the thoracic limb hooves was cleaned before data collection to remove all the solar dirt. Anatomical landmarks were used to place circular markers on the left forelimbs to ensure that the same anatomical regions were measured in all animals . SphericIn addition to the circular markers, adhesive tape was applied to aid digital measurements of the hooves . The tapThe digital camera (Nikon Coolpix L820 (ISO-200) was positioned after adjusting the static positioning of the animals. For the photographic record, the camera was positioned on a leveled tripod, with the center of the lens 1.0 m from the ground, and 1.90 m from the animals. By convention, the equipment was always kept to the side of the horse . After capturing the image of the animal in a forced static position, the camera was removed from the tripod, and each thoracic hoof was photographed.Three images were taken for each thoracic digit under evaluation  . Images Following the image capture, all four hooves were trimmed by a single professional, following the precepts of geometric balance previously described ,21, keepImages were visualized using Image J software . All measurements were performed by the same evaluator. Morphometric and angular measurements are listed in Hoof measurements and joint angles were determined in the software described above; all linear measurements were performed with the aid of the straight-line tool. The toe angles (TA), heel angle (HA), and the shoulder-ground (SG), humeroradial (HR), scapulohumeral (SH), and metacarpophalangeal (MCP) joint angles were determined using the angle tool.p < 0.05); data regarding joint angles were submitted to paired t-test, and they were carried out in a using a computational statistical program .Data regarding hoof measurements were submitted to a two-way analysis of variance , followed by Tukey’s post hoc test .Correlations between angular measurements and hoof measurements were evaluated using scatterplots and Pearson’s correlation coefficient. The statistical significance of the correlation coefficient (r) was set at Differences were observed after trimming for the variables HL, TA, HA, and HMH. There was an increase in HL in the left hooves, an increase in TA for the left hoof and a decrease in the right hoof, and a decrease in HA and HMH values in both antimeres. The TA measurement differed between limbs before trimming .There was a decrease in the MCP angle after trimming . No furtA total of 39 correlations were found. Of these, 19 were observed before trimming and 31 after the procedure .The animals in this study showed poor dorsal palmar balance, which significantly contributes to the long toe/heel underrun conformation. With the growth of the hoof, there was a consequent decrease in contact in the palmar region; this alteration increases the tension in the deep digital flexor tendon and can affect associated structures . TrimminThere was an increase in TA in the left hoof and a decrease in TA in the right hoof; this difference was attributed to a longer toe on the left hoof when compared to the contralateral hoof in the study animals. The farrier during trimming, using the precepts of geometric balancing ,21, matcAlthough it is reported in the literature that the ideal TA for the thoracic hooves is 45 degrees , this vaThe toe angle (TA) is defined as the angle formed by the junction of the hoof wall and its weight-bearing surface  and is related to the hoof pastern axis (HPA) . TherefoIn addition to the differences found in hoof measurements immediately after trimming, there was a significant decrease in the MCP angle. The HPA “broken backward” leads to hyperextension of the interphalangeal joint . AdequatThe ImageJ software is easy to use, free, has excellent accuracy , and is Trimming has an immediate effect on the toe angle  and heel angle  in Mangalarga horses, improving dorso/palmar balance and realigning the HPA. Trimming changes not only the morphometry of the hoof, but also the metacarpophalangeal angle immediately after the procedure, resulting in a decrease in this angle.The effect of hoofing differs between shoed and barefoot animals. Barefoot animals show greater toe wear, affecting linear and angular measurements, differing from studies evaluating shoed animals .Assessments carried out in a population of horses of a specific breed with the same physical activity reduce the degree of inter-individual variation and can reveal whether the trimming interval is adequate for that specific population, since each breed has particularities in the conformation of the hooves ,37.Longer intervals of trimming result in decreased AT by increasing palmar loading, resulting in weakening and collapse of the heels, which increases the load on the suspensory apparatus, which leads to increased susceptibility to injury ,38, so a


Cisplatin-based combination chemotherapy is the foundation for treatment of advanced bladder cancer (BlCa), but many patients develop chemoresistance mediated by increased Akt and ERK phosphorylation. However, the mechanism by which cisplatin induces this increase has not been elucidated. Among six patient-derived xenograft (PDX) models of BlCa, we observed that the cisplatin-resistant BL0269 express high epidermal growth factor receptor, ErbB2/HER2 and ErbB3/HER3. Cisplatin treatment transiently increased phospho-ErbB3 (Y1328), phospho-ERK (T202/Y204) and phospho-Akt (S473), and analysis of radical cystectomy tissues from patients with BlCa showed correlation between ErbB3 and ERK phosphorylation, likely due to the activation of ERK via the ErbB3 pathway. In vitro analysis revealed a role for the ErbB3 ligand heregulin1-β1 (HRG1/NRG1), which is higher in chemoresistant lines compared to cisplatin-sensitive cells. Additionally, cisplatin treatment, both in PDX and cell models, increased HRG1 levels. The monoclonal antibody seribantumab, that obstructs ErbB3 ligand-binding, suppressed HRG1-induced ErbB3, Akt and ERK phosphorylation. Seribantumab also prevented tumor growth in both the chemosensitive BL0440 and chemoresistant BL0269 models. Our data demonstrate that cisplatin-associated increases in Akt and ERK phosphorylation is mediated by an elevation in HRG1, suggesting that inhibition of ErbB3 phosphorylation may be a useful therapeutic strategy in BlCa with high phospho-ErbB3 and HRG1 levels. Recommended treatment for MIBC patients is neoadjuvant chemotherapy (NAC) using cis-diamminedichlorideplatinum (II) (cisplatin)-based chemotherapy  or gemcitabine plus cisplatin (GC)) followed by radical cystectomy (RC)4. In practice, though, patients , often do not receive NAC7 and are considered for adjuvant chemotherapy (AC) with the same regimens. Recurrence occurs frequently and a number of assays have been introduced for early detection and monitoring of recurrence8. While AC increases time to recurrence, 50% of patients ultimately relapse9.Muscle invasive bladder cancer (MIBC) constitutes about 25% of all initial diagnoses of bladder cancer (BlCa) but 80% of deaths from this disease10 that reaches an objective response rate (ORR) < 30%11, but ICIs are known to also cause autoimmune diseases12. Erdafitinib, a small molecule inhibitor of fibroblast growth factor receptor (FGFR), is active in platinum refractory BlCa expressing FGFR3 mutations and FGFR2/3 fusions13. Antibody drug conjugates (ADCs) enfortumab vedotin, a nectin-4-directed antibody and microtubule inhibitor conjugate15, and sacituzumab govitecan, an anti-Trop-2-antibody conjugated to a topoisomerase inhibitor16, also demonstrated activity in BlCa. RC48-ADC (disitamab vedotin), conjugating the anti-HER2 antibody hertuzumab to monomethyl auristatin E (MMAE)18, had activity in patients with HER2-amplified or -mutated BlCa that failed chemotherapy22. However, relatively few patients benefit from these treatments. Therefore, understanding the mechanism of cisplatin resistance and developing therapeutic strategies to overcome them are of utmost importance23.Recent developments indicate a number of additional treatments for patients who fail chemotherapy. Nivolumab, an immune-checkpoint inhibitor (ICI) targeting programmed death-1 (PD-1), is an established adjuvant treatment of MIBC26, while phosphorylation of protein kinase B (PKB/Akt)28 was also increased by cisplatin treatment. The epidermal growth factor receptor (EGFR), and its effector Src, was shown to mediate cisplatin-induced upregulation of ERK phosphorylation. This caused acquired chemoresistance in non-small cell lung cancer (NSCLC) via phosphorylation of paxillin resulting in increased Bcl-2 expression and cell survival26, and increased Akt/mTOR phosphorylation via activation of p38MAPK29. However, this increase in Akt and ERK phosphorylation was also observed in systems where the EGFR/Src pathway was not active, indicating the presence of alternate mechanism by which the ERK and Akt pathways are activated by cisplatin, that has not previously been identified.One of the causes of cisplatin failure has been traced to cisplatin-induced activation of extracellular regulated kinase 1/2 (ERK1/2) 31. RTKs are stimulated by ligands, including epidermal growth factor (EGF) for EGFR, and heregulins (HRG1/2) for ErbB332, while HER2 is a constitutively-active orphan receptor33. Two other heregulins, HRG3-4, are ligands for ErbB4, which is not highly expressed in BlCa. HRG1-4  are encoded by the genes neuregulin (NRG1-4), also referred to as NEU. HRG1 has been known to be expressed in six different forms stemming from alternative splicing34—here we will use the most common form, heregulin1-β1 (type I). The RTKs of the EGFR family undergo homo- or heterodimerization following ligand binding, stimulating autophosphorylation, and signaling to downstream targets including the phosphatidylinositol 3-kinase (PI3K)/Akt and Ras/MAPK pathways31. ErbB3 contains six binding sites for the p85-regulatory subunit of PI3K, including Y128935; while Y1328 mostly binds Shc, signaling to the MAPKs ERK1/236. However, the trigger that activates these pathways downstream of cisplatin treatment has not been identified.The EGFR family of receptor tyrosine kinases (RTK)  plays a significant role in the growth and progression of MIBC31, trials of EGFR inhibitors were relatively unsuccessful. The EGFR inhibitor panitumumab failed to reach efficacy in combination with MVAC37, while a Phase III trial of the dual EGFR/HER2 inhibitor lapatinib following chemotherapy showed no significant benefit38. However, trastuzumab-deruxtecan (targeting HER2 and topoisomerase-I respectively) with nivolumab showed activity in HER2-overexpressing BlCa39, while disitamab-vedotin  was granted US-FDA breakthrough-therapy designation for patients with HER2-positive BlCa following platinum-based chemotherapy40. Significantly, disitamab-vedotin was also efficacious (ORR = 26.3%) in HER2-negative BlCa patients21. Since HER2 most commonly dimerizes with ErbB3 in BlCa41, these results may indicate parallel effects of ErbB3 in this disease. In support of this hypothesis, the pan-ErbB inhibitor Afatinib demonstrated significant activity in patients with platinum-refractory BlCa with HER2 or ERBB3 alterations42. These results indicate an increased role of ErbB3 in BlCa progression.Although multiple ErbB inhibitors have been tested in clinical trials for BlCa43. The humanized monoclonal antibody, seribantumab (MM-121/SAR256212), that blocks the ligand binding domain of ErbB3, completed Phase II studies in breast46, ovarian48, pancreatic49, and NSCLC50, and was found to be effective in combination with trastuzumab51, and the EGFR inhibitors erlotinib49, gefitinib52, and cetuximab53. However, few studies investigated ErbB3 in cisplatin-resistant MIBC54.A number of specific ErbB3 inhibitors have been developed, including patritumab deruxtecan that reached Phase III in NSCLC; and seribantumab, lumretuzumab, elgemtumab, duligotuzumab, and istiratumab that reached Phase IIHere, we demonstrate for the first time that cisplatin increases the levels of the ErbB3 ligand, HRG1, in BlCa cell lines and animal models, which in turn increases Akt and ERK phosphorylation. Inhibition of HRG1 binding to its receptor, and ErbB3 activation, with seribantumab, reduced tumor growth in immunocompromised mice bearing patient-derived xenograft (PDX) tumors that expressed high HRG1 levels and an activated HER2/ErbB3 axis. The current results indicate correlation between cisplatin-resistance and both ErbB3 expression and activation in the context of advanced BlCa.55. The study identified the EGFR family as some of the most highly mutated/activated genes in MIBC. Hence, we compared the protein levels of this family in 6 PDX models that varied in response to cisplatin treatment 55. IHC analysis demonstrated that EGFR expression was extremely high in BL0382, moderately high in BL0269 and BL0479, while levels in BL0293, BL0515 and in BL0440 were low  with the goal to analyze cisplatin’s effects on ErbB3 signaling. First, we determined cisplatin’s effect on viability of J82, TCCSUP, T24, and RT4 cell by MTT assay. IC50 values showed that J82 cells are the most sensitive (IC50 65.2 nM) and RT4 cells are the least sensitive (IC50 653.9)  were constitutively phosphorylated but were further stimulated only by EGF    containing three representative cores from each patient were stained for total ErbB3, phospho-ErbB3, or phospho-ERK as described in ion Fig. A. None oion Fig. A. ERK ph01) Fig. B.Figure 60     = 26.3%) in HER2-negative BlCa patients21. Thus, it is worthwhile to consider other closely related targets in BlCa, like ErbB3. Here, we show for the first time that cisplatin upregulates HRG1 in BlCa. These findings support previous studies indicating correlation between cisplatin resistance and NRG1/HRG166, labelled an oncogene that stimulates the dimerization of ErbB3 with HER2 or EGFR67.Cisplatin is the foundation of first-line combination chemotherapy used in standard-of-care treatment for MIBC in both NAC and AC for primary BlCa, as well as in treatment-naïve metastatic BlCa68. In the case of ErbB3, phosphorylation at Y1289, the p85PI3K-binding site33, and at Y1328, the Shc binding site36, responded to the increase in HRG1 by cisplatin. Activation of the receptor is complete when it binds to its dimerization partners EGFR and HER2 (as most BlCa do not express ErbB455). Remarkably, in RT4 cells, HRG1 stimulated HER2 and ErbB3 phosphorylation (indicating the formation of HER2/ErbB3 dimers), and not EGFR phosphorylation. On the other hand, EGF stimulated phosphorylation in EGFR and HER2, but not ErbB3 (suggesting the formation of EGFR/HER2 heterodimers and EGFR homodimers). These RTKs then activated their downstream targets, Akt and ERK. Our data demonstrate that in human BlCa tissue, ERK phosphorylation is correlated with ErbB3 phosphorylation, suggesting the significance of the Y1328 site, and PDX models also demonstrated correlation with Akt phosphorylation. These results explain how an increase in HRG1 by cisplatin may upregulate ERK and Akt phosphorylation.Receptor desensitization following prolonged ligand exposure, despite initial activation, typically is followed by autophosphorylation at sites promoting endosomal encapsulation and eventual degradationneuregulin 1 (NRG1) and our data indicate that cisplatin increases NRG1 transcription rates. It is important to note that cisplatin crosslinks with purine bases on DNA, interfering with DNA repair mechanisms, thereby inducing apoptosis69. Several transcription factors have been associated with cisplatin resistance, including ATF2, E2F1, NR4A and Sp170. Some of these transcription factors, including ATF2 and Sp1, are also transcriptional regulators of NRG1. While outside of the scope of the current article, it may be speculated that cisplatin resistance may activate these transcription factors resulting in NRG1 transcription.HRG1 is transcribed by the gene Seribantumab, which prevents HRG1 binding, reduced tumor growth in both cisplatin-sensitive BL0440 and cisplatin-resistant BL0269 PDX models. Our data demonstrate significant HRG1 expression in these tumors, which was further increased by cisplatin treatment. These results suggest that seribantumab was able to suppress the growth of the tumor by inhibiting the intrinsic expression of HRG1 in them, whereas cisplatin was ineffective due to high HRG1 levels. In contrast, J82 cells express low levels of HRG1—therefore, seribantumab was ineffective in these cells as a monotherapy. However, as demonstrated, cisplatin treatment increases HRG1 levels thereby allowing seribantumab efficacy. Therefore, in these cells, the combination of seribantumab and cisplatin was highly effective.55. Additionally, BL0440 had high HER2 and ErbB3 levels but low EGFR levels, suggesting that ErbB signaling may take place through HER2/ErbB3 dimers. Our data shows that under conditions of high HRG1 and active ErbB3, ErbB3 inhibition reduces BlCa tumor growth. In high-HER2-expressing BL0440 tumors and high-EGFR-expressing BL0269, inhibition of HRG1-ErbB3 binding reduced tumor volume. Despite cisplatin increasing HRG1 levels, combining cisplatin and seribantumab had no benefit above seribantumab monotherapy in a chemoresistant PDX model. Development of ADCs which conjugate ErbB3 antibodies with payload chemotherapeutic agents may have utility in urothelial carcinoma, especially in patients who are refractory to platinum-based therapy.Note that BL0440, although cisplatin sensitive, was gemcitabine resistantIn conclusion, HER2/ErbB3 signaling may be impactful in cisplatin-resistant BlCa, but both cisplatin-sensitive and -resistant BlCa positive for HRG1 may be susceptible to ErbB3 inhibitors like seribantumab if they express high HRG1 levels. Overall, our data suggest that ErbB3 inhibition may be a viable treatment option for patients with MIBC that express HRG1 and rely on HRG1/ErbB3 signaling whether cisplatin-sensitive or -resistant Fig. . To our 72 using a monoclonal anti-ErbB3 antibody, anti-HRG1 antibody and anti-phospho-Erk1/2 antibody , UC Davis  and all experiments were performed in accordance with University of California Davis (UCD) IRB guidelines and regulations. All patients provided informed consent to participate under the IRB-approved protocol. Tissue microarrays (TMA) representing a total of 55 BlCa patients in quadruple were obtained in a deidentified manner from the UCD Comprehensive Cancer Center (UCDCCC) biorepository that collected the tissues. Annotated data from the 55 patients are presented in Supplementary Table T24, TCCSUP, RT4, and J-82 were purchased from American Type Culture Collection (ATCC), Manassas, VA , while cisplatin was purchased from Fisher Scientific  and Selleck Chem . Seribantumab was pre-prepared in a proprietary solution from Merrimack Pharmaceuticals  and cisplatin was dissolved in platinum binding buffer (CBB) (3 mM NaCl and 1 mM sodium phosphate). Epidermal Growth Factor  and Heregulin-beta1β-1  was purchased from Peprotech and used to stimulate cells 15 min before collection or across a time-course. Cell viability in the presence of inhibitors was estimated by 3--2,5-diphenyl-tetrazolium bromide (MTT) assay as described before72. Immunohistochemistry73 and Immunofluorescence62 was performed as previously described. The antibodies used in this study can be found in Supplementary Table Western blotting55. When tumors reached 150–200 mm3, mice were randomly assigned (no apriori set criteria were used for the assignment) and treated twice weekly—vehicle , 2 mg/Kg cisplatin, 10 mg/Kg seribantumab by intra-peritoneal (ip) injection and the combination of cisplatin and seribantumab . Isoflurane (1–5%) by inhalation was used for anesthesia, if needed. For combination treatments, seribantumab was delivered first followed by cisplatin. All animals were included in the final calculations. Treatments continued for 3 weeks after which the mice were observed for a total of 40 days. Tumor volume (lengthXwidth2X0.5) and animal body weight were recorded twice weekly, in no particular order. Mice were euthanized by pentobarbital overdose followed by cervical dislocation when at least one of the following humane endpoints were reached: (i) tumor volume of 1.5 cm3, (ii) 15 mm tumor length towards any direction, (iii) tumor ulceration, (iv) more than 20% reduction in animal body weight. Tumors were collected and formalin fixed, and paraffin embedded, then sectioned and stained with antibodies as described elsewhere73. The sections were read by a blinded Veterinary Pathologist and scored as described73.Clinical information and cancer specimens from BlCa patients for PDX development was collected under the University of California Davis IRB Protocol No. 218204. Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC), UC Davis and all experiments were performed in accordance with UC Davis IACUC #17794. Experimental descriptions in this manuscript complies with the ARRIVE guidelines. PDX models (BL0269 (JAX# TM00015), BL0293 (JAX# TM00016), BL0382 (JAX# TM00020), BL0440 (JAX# TM00024), BL0479 (JAX# TM00026), BL0515 (JAX# TM00031)) were implanted subcutaneously into the flanks of 6–8 week old female NOD/scid/gamma (NSG) mice50 was calculated from MTT results performed in triplicate at multiple doses of drug using GraphPad Prism software (v9.0.0). Normal distribution was assessed using Shapiro–Wilk test and data were analyzed accordingly. Significance between two sets of data in mouse tumors or in vitro assays were calculated by paired t-test using at least three independent values. For in vivo experiments, a sample size of at least n = 6 was chosen assuming α = 0.05, β = 0.2, to achieve 80% power. FlowJo software was used to analyzed flow cytometry experiments and GraphPad Prism software (v9.0.0) was used for the rest of the analyses in animal and cell line studies. P value < 0.05 was considered statistically significant. Western blots were scanned by Image J software and band intensities were reported as intensity of the immunoblot developed using the specific antibody normalized to that of the corresponding band in the loading control (e.g. GAPDH).ICTMA IHC data were summarized across positions by taking the median of non-missing values. The correlation between age and marker expression was calculated using Spearman (nonparametric) correlations. Marker expression was compared between levels of categorical variables with two levels using Wilcoxon rank-sum tests. Marker expression was compared among levels of categorical variables with more than two levels using Kruskal–Wallis tests (nonparametric ANOVA), followed by Dunn tests in the case of a significant global test. Analyses were conducted using R version 4.0.2 (2020–06-22), with Dunn tests conducted using the R package FSA, version 0.8.30.Supplementary Information.Supplementary Figures.


For the BCD-TA-TPC@PSf membrane, the flux was increased from 8 LMH (L/m2.h) to 36 LMH as the transmembrane pressure was increased from 5 bar to 25 bar.Given the huge potential of thin film composite (TFC) nanofiltration (NF) membranes for desalination and micro-pollutant removal, two different sets of six NF membranes were synthesized. The molecular structure of the polyamide active layer was tuned by using two different cross-linkers, terephthaloyl chloride (TPC) and trimesoyl chloride (TMC), reacted with tetra-amine solution containing β-Cyclodextrin (BCD). To further tune the structure of the active layers, the time duration of interfacial polymerization (IP) was varied from 1 to 3 min. The membranes were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), water contact angle (WCA), attenuated total reflectance Fourier transform infra-red (ATR-FTIR) spectroscopy, elemental mapping and energy dispersive (EDX) analysis. The six fabricated membranes were tested for their ability to reject divalent and monovalent ions followed by rejection of micro-pollutants . Consequently, terephthaloyl chloride turned out to be the most effective crosslinker for the fabrication of membrane active layer with tetra-amine in the presence of β-Cyclodextrin using interfacial polymerization reaction for 1 min. The membrane fabricated using TPC crosslinker (BCD-TA-TPC@PSf) showed higher % rejection for divalent ions (Na Clean water is an enormous challenge for a rapidly growing human population and industrialization . A few tThin film composite (TFC) membranes have been fabricated through interfacial polymerization (IP) on an ultrafiltration (UF) support . IP reac2 was loaded with Ag nanoparticles and the resulting Ag@UiO-66-NH2 was incorporated in the polyamide active layer during IP. The resultant Ag@UiO-66-NH2 decorated membrane showed excellent antifouling performance with a flux recovery ratio of 95.6%. Moreover, due to the presence of Ag metal, the membrane showed antibacterial rate >95%. Due to the porous nature of Ag@UiO-66-NH2, the membrane showed a permeate flux of 47.3 LMH [Many strategies, such as tuning the active layers of the polyamide membranes during IP, have been explored in the literature. Many studies have shown that use of different materials, such as MOFs , COFs 1, nanomat47.3 LMH . 2SO4, MgCl2 and MgSO4, respectively [Similarly, many macrocyclic compounds have recently been used during fabrication of NF membranes. The most commonly used macrocycles are crown ethers, cyclenes , cyclodeectively . 2H5 side chains [Another important aspect of NF membranes is tuning the polyamide active layer by altering the chemistry of the monomers used during IP. The pore structure of the NF membranes has been tuned by selecting different combinations of aqueous amines and non-aqueous cross-linkers. The microstructure of the polyamide active layer can be tuned by enhancing the inter-pore connectivity of the polyamide active layer. In a work carried out by Livingston and co-workers, a highly dense polyarylate layer had bisphenol monomers with a rigid structure instead of PIP. The resultant membrane showed higher organic solvent permeate flux and gas permeation . In anote chains . The hyde chains . AlthougThe current study was focused on altering the molecular structure of the polyamide active layer by including BCD, N,N′-bis(3-aminopropyl)ethylenediamine (BAPEDA), terephthaloyl chloride (TPC) and trimesoyl chloride (TMC) in membrane fabrication. An aqueous solution of BAPEDA containing BCD was used during IP. Two sets of different membranes were fabricated by using two different cross-linkers TPC and TMC. Furthermore, the effect of crosslinking time during IP was also studied by varying the time of IP, measuring at 1, 2 and 3 min, leading to two sets of three polyamide TFC NF membranes. Following characterization, the membranes were used for desalination and micro-pollutants (antibiotics) removal.2, CaCl2, MgSO4, Na2SO4, NaCl) and pharmaceutically active compounds  were also bought from Sigma.Beta-cyclodextrin (BCD), terephthaloyl chloride (TPC), Trimesoyl chloride (TMC), polysulfone, triethylamine (TEA) and N,N′-bis(3-aminopropyl)ethylenediamine (BAPEDA) were purchased from Sigma Aldrich, St. Louis, MO, USA. For the filtration test, different salts , a scanning electron microscope , an atomic force microscope  and water contact angle (KRUSS DSA25). The feed and permeate solution were tested using a conductivity meter  for salts and a JASCO V-750 UV-Vis spectophotometer for pharmaceutically active compounds. The membranes were tested for their performance using the Sterlitech CF042 Membrane test system, United States of America.w/v) % and 4 (w/v) % TEA, and 2 (w/v) % BAPEDA. The resulting solution was probe sonicated for 15 min to homogenize the contents. Two crosslinker solutions were prepared by dissolving TMC or TPC at a concentration of 0.2 (w/v) % to n-hexane for interfacial polymerization. Initially, the polysulfone (PSf) layer was cast onto polyester terephthalate (PET) nonwoven fabric via wet phase inversion methodology. Later, it was dipped into the BCD/amine aqueous solution and impregnation was carried out for 10 min using a Cole-Parmer mini rocking shaker. After removing the membrane from the aqueous amine solutions, the rubber roller was used to sweep the extra solution. The membranes were then dipped into the crosslinker either TMC or TPC for 1, 2 or 3 min. This pattern resulted in 6 membranes of different typologies that were denoted as BCD-TA-TMC@PSf-X , while the other set was named as BCD-TA-TPC@PSf-X . The extra crosslinker solution was washed out by rinsing the membrane with 10 mL of n-hexane. The membranes resulting from the crosslinking of TMC were kept inside an oven at 80 °C for 10 min, where the TPC crosslinked membranes were kept for 1 h at a similar temperature. Before beginning the filtration test, membranes were soaked inside distilled water. Different stages of membrane fabrication are listed in To evaluate the effect of the crosslinker (TMC vs. TPC), the addition of BCD and the interfacial polymerization time , six membranes typologies were fabricated. One amine aqueous solution was prepared by keeping the BCD amount constant at 0.1 (2) and acid chloride (–COCl) led to the formation of amide linkage (–CONH), leading to polyamide synthesis. Moreover, hydroxyl groups (–OH) of BCD also reacted with –COCl leading to the covalent linkage of BCD in the active layer of the membrane. The proposed reaction between different reacting monomers is given in the following The active layer was generated by reacting tetra-amine with TPC and TMC while BCD was added as an additive during IP. The reaction between amine  linkage in the active layer. Furthermore, the hydroxyl (–OH) groups of the BCD are also overlapped by –N-H stretching of amide linkage. The presence of several other peaks in the fingerprint region is almost like in the case of all membranes. The peak located at around 1650 cm−1 to 1680 cm−1 can be attributed to carbonyl (>C=O) functional groups of the ester (–COOR) and amide linkages (–CONH). Similarly, the presence of a strong peak at around 1200 cm−1 is due to –S=O (sulfone) group of PSf. Hence, the ATR-FTIR spectra of all the membranes confirmed the presence of all the participating functional groups in the structure of the membranes. In order to establish the structure of the best performing membranes (BCD-TA-TPC@PSf and BCD-TA-TMC@PSf), a thorough characterization is extensively described here. The identification of different functional groups present in different fabricated membranes was recorded by an ATR-FTIR spectrum of each membrane, as shown in Surface hydrophilicity of fabricated membranes is highly essential in understanding the filtration performance of the membranes during filtration experiments. For the sake of measuring surface hydrophilicity of the newly developed membrane, WCA of all the membranes along with PSf support was recorded, as given in aR) and root mean square roughness (qR) of the membranes are given in aR (6.46 nm) and qR (8.16 nm) are low compared to BCD-TA-TPC@PSf and BCD-TA-TMC@PSf. The aR and qR values of BCD-TA-TPC@PSf were found to be 41.60 nm and 46.6 nm, respectively, while in the case of BCD-TA-TMC@PSf these values were found to be aR = 7.16 nm and qR = 8.43 nm. The higher values of aR and qR in the case of BCD-TA-TMC@PSf have confirmed that the membrane surface has valleys and ridges providing greater amplitude during roughness measurements. This ridge and valley conformation is ideal for providing appropriate channels during filtration experiments, leading to the rejection of salts and the permeation of pure water. Another highly useful surface feature of fabricated membranes is surface roughness as it helps in understanding the performance of the membrane during passage of permeate through the membranes. AFM images of the newly fabricated membranes have been given in To further explore and understand the surface features of the fabricated membranes, the surface morphologies of the membranes were studied through SEM micrographs of the membranes which were recorded at different magnifications, as shown in Another salient feature of the membrane surface is the elemental distribution and composition of the membrane. EDX analysis of the PSf support and newly fabricated membranes was carried out, as given in The distribution of elements in all the membranes detected by EDX analysis is given below in 2, MgSO4, CaCl2 and Na2SO4), monovalent (NaCl) salts and micro-pollutants  given in The nanofiltration performance of the newly fabricated membranes was studied by using different feeds containing divalent (MgCl−2.h−1 (LMH) to 115 LMH when the transmembrane pressure was increased from 5 bar to 25 bar, respectively (2SO4 was found to be the highest (93%) compared to other salts, followed by MgSO4 (92%), which was followed by MgCl2 (91%), which was followed by CaCl2 (84%). The lower rejection of CaCl2 can be attributed to the smaller hydration shell (0.334 nm) of Ca2+ ions [2+ and SO42− ions which have hydration radii of 0.86 nm and 0.76 nm, respectively. The rejection of salts depends upon the hydration radii of the permeating ions [2 and Na2SO4 is higher than CaCl2. In the case of monovalent salt, the rejection of NaCl was found to be 85%. The rejection of salts was found to be lower for all the salts in the case of the BCD-TA-TMC@PSf membrane. The rejection and flux performance of membranes suggested that the BCD-TA-TMC@PSf membrane has a relatively loose polyamide active layer with a slightly higher concentration of BCD, as confirmed by EDX analysis. The incorporation of BCD led to higher permeate flux, while a slightly loose polyamide active layer of the BCD-TA-TMC@PSf membrane resulted in lower salt rejection. On the other hand, BCD in the dense polyamide active layer of the BCD-TA-TPC@PSf membrane equip the membrane with reasonable flux and high salt rejection. The increase in permeate flux due to BCD is attributed to outer hydrophilic sphere of hydroxyl groups on the pyranose moieties of BCD.Prior to the filtration experiments with the above mentioned feeds, the membranes were installed on a crossflow filtration setup and compacted for an hour using distilled water as feed. Initially, the effect of pressure on permeate flux was studied where it was found that the permeate flux increased in a linear manner with increasing transmembrane pressure. The best performing membranes, BCD-TA-TPC@PSf and BCD-TA-TMC@PSf, are described in detail in the upcoming sections. The BCD-TA-TMC@PSf membrane showed higher flux compared to BCD-TA-TPC@PSf a. The vaectively a. In thea2+ ions  compareding ions . Hence, The concentration of different micro-pollutants, especially pharmaceuticals, is increasing in water bodies at an alarming pace, leading to multidrug resistant bacterial strains. The rejection of the model drugs  a have beThe performance of the BCD-TA-TPC@PSf membrane was also compared to the performance of membranes in the published literature. The BCD-TA-TPC@PSf membrane has shown either improved or comparable results as shown in 2SO4, MgSO4, MgCl2 and CaCl2, to be 93%, 92%, 91% and 84%, respectively. In the case of BCD-TA-TMC@PSf, the rejection of all of the salts Na2SO4, MgSO4, MgCl2 and CaCl2 was nearly 80%. Similarly, in the case of pharmaceutical micropollutants in the BCD-TA-TPC@PSf membrane, the higher permeate flux was achieved through the integration of BCD in the polyamide active layer. The BCD-TA-TMC@PSf membrane showed higher permeate flux of 115 LMH compared to the BCD-TA-TPC@PSf membrane, which had a flux of 36 LMH at 25 bar. This might be due to the formation of a slightly loose polyamide active layer with TMC and TA in the presence of BCD. However, due to the formation of a dense polyamide active layer with TPC and TA in the presence of BCD, the BCD-TA-TPC@PSf membrane showed resonable flux and a high level of salts and drug rejections.The BCD-TA-TMC@PSf and BCD-TA-TPC@PSf thin film composite nanofiltration membranes were successfully fabricated by interfacial polymerization using two different cross-linkers, terephthaloyl chloride (TPC) and trimesoyl chloride (TMC), reacted with tetra-amine (TA) solution containing β-Cyclodextrin (BCD) for desalination and micro-pollutants removal. ATR-FTIR, SEM, EDS and mapping analysis of the fabricated membranes demonstrated the effective formation of the polyamide active layer on the surface of polysulfone (PSf) support. The thin film composite nanofiltration membranes fabricated using TPC as crosslinker in the presence of TA and BCD showed a better performance for desalination and micro-pollutant removal compared to the thin film composite nanofiltration membranes fabricated using TMC as crosslinker. BCD-TA-TPC@PSf showed rejection of salts, such as Na


Antarctic sea ice is mostly seasonal. While changes in sea ice seasonality have been observed in recent decades, the lack of process understanding remains a key challenge to interpret these changes. To address this knowledge gap, we investigate the processes driving the ice season onset, known as sea ice advance, using remote sensing and in situ observations. Here, we find that seawater freezing predominantly drives advance in the inner seasonal ice zone. By contrast, in an outer band a few degrees wide, advance is due to the import of drifting ice into warmer waters. We show that advance dates are strongly related to the heat stored in the summer ocean mixed layer. This heat is controlled by the timing of sea ice retreat, explaining the tight link between retreat and advance dates. Such a thermodynamic linkage strongly constrains the climatology and interannual variations, albeit with less influence on the latter. Processes controlling the onset of the Antarctic sea ice season remain unclear. Here, analyses of observations show that ocean solar energy storage and sea ice drift are key drivers, providing insights to understand variations in sea ice season duration. Over the last 40 years, changes in the timing of Antarctic sea ice advance and retreat have been documented from satellite-based passive microwave sensors10. The changes, highly variable regionally, were attributed to wind-driven changes in ice transport and seasonal thermodynamic ice-ocean feedbacks15. Yet interpretation is complicated: strong interannual variability dominates the trends in the last two decades16 and drivers involve multiple oceanic and atmospheric processes18, in a context of limited understanding of the drivers of sea ice advance and retreat.The seasonal cycle of Antarctic sea ice is marked by two key transitions between open water and ice-covered conditions: advance and retreat. Advance or retreat are defined as the first day in the year when smoothed-in-time sea ice concentration exceeds or falls below 15%22. Freezing starts ultimately once the entire mixed layer, having warmed up from solar absorption in spring and summer then cooled down through fall, approaches the freezing temperature24. Close to the winter sea ice edge, freezing can, however, be inhibited by entrained25 or advected20 oceanic heat into the mixed layer.Sea ice advance and retreat are influenced by different processes implying specific observational needs and problems. In this paper, we focus on the fundamental drivers of the sea-ice advance date and on links to the sea-ice retreat date. Sea-ice advance can be controlled either by the freezing of seawater or by sea-ice drifting from already frozen areas26 to recently available observational datasets. They include passive microwave-based sea ice concentration budget diagnostics that split sea ice changes into dynamic  and thermodynamic  process contributions22; thermal infra-red radiance satellite sea surface temperature27 (SST); compilations of in situ hydrographic measurements28 which now provide a detailed climatological view of the upper oceanic thermohaline structure under Antarctic sea ice, thanks to animal-borne sensor records29. Combining these sources, we highlight a strong overarching contribution of upper ocean thermodynamics in setting the climatological date of sea ice advance and an important role for ice drift in an outer band, with a width of a few degrees of latitude. We then provide evidence that these mechanisms also contribute to a certain extent to observed interannual changes in the timing of sea ice advance.As such, one can hypothesize that the date of sea ice advance is controlled by the upper ocean heat content, surface fluxes, sea ice thermodynamics and drift. To understand these contributions, we relate climatological dates of sea ice advance derived from passive-microwave sea ice concentration18, is used to assess how thermodynamic  and dynamic  processes control the spatial variability of climatological dates of advance (da)  and dynamic (Dy) contributions to the total sea ice concentration tendency over the 30 days following da, as well as their ratio (Dy/Th)  Fig. . The sea30. We refer to the region south of the |Dy/Th| = 1 contour, as the inner zone and north of this contour, as the outer zone. The latter represents 32% of the seasonal ice zone area. These zones are robust to the choice of the time window over which the budget is integrated following da, being weakly sensitive to the window size from 15 to 60 days , evaluated from an infrared satellite SST climatology (2003–2010)27, is consistent with our analysis of the sea ice concentration budget  in the outer zone (median +/− IQR: 0.6 + /− 0.3 °C). Also, the 5% highest values of SSTda- Tf  than in the outer zone, however this difference is not significant, which may reflect uncertainties in the exact position of the inner-outer zone boundary, or in SSTda. Nevertheless, these findings are robust to the choice of alternative SST products . Maps of climatological da and SSTmax, shown respectively in Fig. da to SSTmax (R2 = 0.81), suggesting that SSTmax could be a proxy of the mixed layer heat gained in spring and summer, which is then lost before sea ice advance. However, a nonlinearity in the relationship appears when representing the spatial distributions of da anomalies versus SSTmax anomalies on a 2D histogram  than SSTmax does  grid point over the open ocean cooling period, a direct link between da and MLHmax anomalies arises:da is the mixed layer depth evaluated in the month of da. The average net heat loss during the cooling period <Q-> sets the rate of mixed layer heat loss between the date of maximum SST and da. A linear MLHmax-da relationship over the whole seasonal ice zone would then suggest spatially uniform <Q->, which seems to hold overall  but is likely dominated by air-sea fluxes32. Such net air-sea heat loss is consistent with reanalysis-based estimates of net surface fall heat loss in Antarctic ice-free waters .Based on the climatological mixed layer depth and SSTpth Fig. . Strikinity Fig. . MLHmax del9 see . Integraall Fig. . The scadel Fig. , we esti34; Supplementary Fig. dr). Consistently, we find a remarkably strong linear link between climatological dr and MLHmax  than in the inner zone (R2 = 0.89), but still explains a large part of the da variance. Similarly, the dr-MLHmax link is weaker  in the outer zone than in the inner zone (R2 = 0.80). This general weakening and the associated larger regression errors might reflect a larger spatial variability in net heat fluxes in the outer zone , and does not explain as much of the da variance there.The statistical relationships between zone see , possiblzone see . The depdr strongly affects the climatological da in the inner zone only. By contrast, the climatological MLHmax determines the climatological da throughout the seasonal ice zone, regardless of the processes (freezing or ice import) increasing sea ice concentration at that time.In summary, the climatological 7 disclosed significant correlations between detrended dates of retreat and subsequent advance over 1980–2010. An ice-ocean thermodynamic feedback was hypothesized to explain this link. The same mechanism was also identified in the Arctic35. However, observations and CMIP5 model analyses suggest that thermodynamic processes are less effective at explaining interannual variations than they are for the mean state9. Based on what precedes, we question to which extent our findings on the mean state can be applied to interannual variations.Thermodynamic processes in ice-free waters provide strong constraints to the climatological date of advance. Whether such mechanisms also apply at interannual time scales is not straightforward. Stammerjohn et al.dr-MLHmax-da framework: an earlier retreat on a given year would lead to a higher maximum MLH and a later advance. We examine these links at the interannual time scale, using the SST as a proxy for the MLH, due to the limited spatial coverage of interannual mixed layer depth data. Based on detrended time series over 1982-2018, we find significant and relatively strong negative links between anomalies of dr and subsequent SSTmax  over 1982–2018 . For the sea surface temperature (SST), we use a daily satellite product available from 1982, based on thermal infra-red radiance measurements, and taken from the global L4  European Space Agency (ESA) SST Climate Change Initiative (CCI) analysis with a resolution of 0.05°27, provided with an estimate of the analysis uncertainty on the SST.We assess the relationships between the date of sea ice advance and the state of the underlying ocean based on a number of observational data sets. We use daily passive microwave sea ice concentration (SIC) from the EUMETSAT Ocean and Sea Ice Satellite Application Facility28. Conductivity–temperature–depth , Argo floats  and marine mammal-borne sensor profiles  were included. Generalized least squares linear-regressions of individual in situ profiles are performed around each grid point to produce gridded maps of climatological mean fields.We also use a gap-filled monthly 1979–2018 climatology of mixed layer depth and stratification, based on in situ observations47, available over 1982-2016 (ISCCP). Finally, we use NOAA Advanced very High Resolution Radiometer (AVHRR) Optimum Interpolation (OI) 0.25° daily SST v2.0 analysis data31, also referred to as Reynolds’ SST, to ensure the robustness of our analysis.Other datasets are used to support our analysis. To evaluate the radiative heat fluxes during the open water season, we derive a daily climatology of surface shortwave and longwave radiative fluxes from the FH-series data of the International Satellite Cloud Climatology ProjectAll data were interpolated on the OSI-SAF Equal-Area Scalable Earth 2 (EASE2) 25 km grid.dr) and sea ice advance (da) were derived from OSI SAF sea ice concentration, on which we applied a 15-day temporal filter to avoid retaining any da or dr reflecting short events9. These dates are defined consistently with previous work48. dr is defined as the first day filtered sea ice concentration drops below 15% while da is the first day filtered sea ice concentration exceeds 15%. To ensure dr and subsequent da of the same yearly seasonal cycle are retained, we looked for da (dr) starting on a month where no sea ice advance (retreat) occurs, on average over 1982–2018. We selected January 1 of the current year as the start date for da, and May 1 of the previous year for dr, since the majority (>99%) of da and dr occurs after those dates.Climatological mean dates of sea ice retreat  is less than one third of the total number of years in the considered period, following ref. 9.To obtain a meaningful 1982–2018 climatological average of max and date when this maximum is reached, dSST were identified during the open water season, between dr and da of the corresponding year. We also calculated the yearly SST on the days of advance (SSTda) and retreat (SSTdr). Then, the 1982–2018 average of each of the four ocean seasonality diagnostics was obtained following the same method as for climatological dr and da.Other climatological diagnostics were calculated to diagnose the seasonality of upper ocean thermodynamics, using the ESA CCI satellite SST over 1982–2018. For each year, the seasonal maximum of SST, SSTda, we evaluate the dynamic and thermodynamic contributions to the sea ice concentration budget at the time of advance. We identify regions of ice import/export, ice melt/growth and regions of dominant dynamic/thermodynamic contributions. We use the sea ice concentration budget decomposition outputs from ref. 22 available at daily frequency between 2003 and 2010. We also use a 2003–2010 climatology of da, for temporal consistency. These outputs are obtained based on the technique developed by ref. 11 from daily sea ice concentration  and ice drift fields derived from AMSR-E brightness temperature by a cross-correlation algorithm51. The governing equation for the sea ice concentration, is decomposed between a dynamic term and a residual:To explore the respective role of ice dynamics and thermodynamics in setting The ice concentration flux divergence represents the effects of advection and divergence of sea ice caused by ice drift. The residual term includes both thermodynamic processes (melting/freezing) and mechanical redistribution through ridging and rafting. However, mechanical redistribution should not intervene in the budget at the time of sea ice advance, as it usually occurs for high sea ice concentration. Thus, we consider the residual as purely thermodynamic.∆t following da.Evaluating the different terms of the previous equation at the time of advance requires analyzing the output of the sea ice concentration budget for sea ice concentration below 15%. However, the budget is not defined at such low sea ice concentration because of missing ice drift data and large sea ice concentration errors near the ice edge. To overcome this limitation, we diagnose total sea ice concentration increase (∆SIC), as well as percent dynamic (Dy) and thermodynamic (Th) contributions to sea ice concentration tendency during a period on length The diagnostics are defined as such:∆t of the contours delimiting our regions of interest  was assessed  and dominant dynamic contribution (|Dy/Th| > 1) are consistent both in location and percentage of total seasonal ice zone area, strengthening our confidence that they are a close representation of the sea ice concentration budget prior to da. Hence, our regions of interest should be at similar location and have a similar area at the time of sea ice advance than in any of the time periods ∆t within the 2 months following da. We choose ∆t = 30 days as a compromise between a low proportion of missing values in the considered seasonal ice zone and the proximity in time to da.To choose the most suitable upper bound of integration, the sensitivity to 9 in the context of Arctic sea ice, based on the heat budget in the mixed layer. We define the heat stored in the mixed layer, termed mixed layer heat content (MLH) as:h is the mixed layer depth, T, the mixed layer temperature, cp, the specific heat of seawater.The mathematical description of the simple thermodynamic framework used to explain spatial variations in the timing of advance is an updated version of the framework developed by ref. 52, which writes as:Qt, the total net heat flux in the mixed layer, accounting for surface heat fluxes, entrainment, diffusion and advection. Now, integrating the MLH budget during mixed layer heating (from dr to dMLH) and cooling (from dMLH to da) periods we get:dMLH is the date of maximum MLH. <Q + > and <Q−> are respectively the mean total net heat flux during the heating and the cooling periods:The model is based on the temperature balance equationdr and da:Combining Eqs.  and 13)13), we oT is at the freezing point on dr and da, then MLHdadr and rMLHIf da and MLHmax anomalies, MLHmax and dr anomalies would respectively suggest uniform spatial distributions of <Q−> and <Q + >. Resultingly, the relationship between da and dr anomalies would also become linear , we estimated the observational MLH for any date, t, during the open water season as:t is the monthly mixed layer depth evaluated on the month of the given date, t . The SST is in degrees Celsius. Using this observational estimation of the MLH, we obtain:Using the monthly climatology of mixed layer depth from ref. Supplementary InformationPeer Review FileSource Data