The α-d-glc-configured cyclophellitol cyclosulfamidate 4 binds in a competitive manner the human lysosomal acid α-glucosidase(GAA), ER α-glucosidases, and, at higher concentrations, intestinalα-glucosidases, displaying an excellent selectivity over thehuman β-glucosidases GBA and GBA2 and glucosylceramide synthase(GCS). Cyclosulfamidate 4 stabilizes recombinant humanGAA  in cell medium and plasmaand facilitates enzyme trafficking to lysosomes. It stabilizes rhGAAmore effectively than existing small-molecule chaperones and doesso in vitro, in cellulo, and in vivo in zebrafish, thus representing a promising therapeuticalternative to Miglustat for Pompe disease.α-Glucosidase inhibitors are potential therapeuticsfor thetreatment of diabetes, viral infections, and Pompe disease. Herein,we report a 1,6- However, only asmall group of Pompe patients carry mutations that respond to PCT.14 Combining ERT and PCT was demonstrated in murinePompe models and shown to be more efficacious than either of the individualtreatments alone.15 PCT using the iminosugarsdeoxynojirimycin 16 or N-butyldeoxynojirimycin  as enzyme stabilizers is currently in phase2 and 3 clinical trials, either as monotherapy-stabilizing endogenousenzyme or in combination with ERT-stabilizing rhGAA  that might notbe therapeutically relevant.21 A set of invitro rhGAA activators were described by Marugan et al. morethan a decade ago for which the stabilization mechanism and bindingmode remain to be investigated.22 Morerecently, Kato and collaborators described a set of C-branched arabinose-and glucose-configured iminosugars able to inhibit rhGAA invitro at nanomolar concentrations.24 The most advanced inhibitor, 5-C-heptyl-deoxynojirimycin, despitedisplaying a nanomolar IC50, stabilizes rhGAA in Pompefibroblasts at 10 μM, and its selectivity toward related glycosidasesneeds to be determined. This illustrates that high in vitro inhibitory potencies do not necessarily correlate with an optimalenzyme stabilization. In principle, the ideal pharmacological chaperonein a combined chaperone-ERT setting binds, and stabilizes, recombinantenzyme (here rhGAA) in circulation but is outcompeted by its naturalsubstrate  upon reaching its (sub)cellulartarget. Fine-tuning this inhibition/binding versus an optimal stabilizationeffect is not trivial and cannot be predicted by low in vitro inhibitory constants.The only current treatment for PD comprisesenzyme replacementtherapy (ERT), in which recombinant human GAA  is administered intravenouslyto the patient.ng rhGAA 1B.17,18 CjAgd31B, a bacterial homologue of GAA) and catalytic acid/base(Asp616 in GAA and Asp480 in CjAgd31B)  for less electronegativenitrogen(s) may prevent its nucleophilic displacement by the enzyme’scatalytic nucleophile.26 C–N bondsare less polarized than C–O bonds and sulfamidates are thusless electrophilic than sulfates. This α-gal-cyclosulfamidate, also a 4C1 Michaelis complexmimetic, and although less effective than commercial Migalastat, turnedout to be an effective reversible α-galactosidase A inhibitor able to stabilize α-GalA—which in geneticallymutated forms is at the basis of Fabry disease—in vitro and in cellulo at high micromolar concentrations.26 Both GAA and α-GalA utilize an identicalKoshland double displacement reaction mechanism with 4C1 (Michaelis) → [4H3]‡ → 1S3 (intermediate) → [4H3]‡ → 4C1 (product) as substrate conformational itineraries. Consideringthe reversibility and enzyme stabilization effect of the α-gal-cyclosulfamidate and the nanomolar GAA inhibition bycyclophellitol cyclosulfate 3, we envisioned that thecorresponding α-glc-cyclosulfamidates couldalso lead to competitive α-glucosidase inhibitors that couldact as enzyme stabilizers in the context of PD. Herein, we describethe development of epicyclophellitol cyclosulfamidates 4–7 and sulfamide 8 and reveal theirmechanism of action, their potential as inhibitors for lysosomal,ER, and intestinal α-glucosidases, as well as their superiorityas (recombinant) enzyme stabilizers  1A. We haulfate 3 1B as a pbilizers 1C.4–7 started by nucleophilic addition of sodium azide to tetra-O-benzyl-cyclophellitol 9,27 yielding a mixture of trans-azido alcohols 10 and 19, which were purified and subsequently reducedto trans-amino alcohols 11 and 20 . Heating of 13 and 22 resulted in the formation of oxazolidinones 14 and 23 through neighboring-group participation, which after hydrolysisof the cyclic carbamate gave the desired cis-aminoalcohols 15 and 24, respectively. N-Boc protection followed by treatment with thionyl chlorideand subsequent oxidation gave fully protected α-glc-cyclosulfamidates 16 and 25, which after global deprotection yieldedtarget compounds 4 and 6. N-Boc deprotection of 16 and 25 and methylationunder basic conditions with methyl iodide followed by hydrogenationafforded 5 and 7. α-Glc-cyclosulfamide was synthesized from trans-azido alcohol 19. Diazido-compound 29 was obtained through mesylationof the remaining hydroxyl and subsequent nucleophilic substitutionof mesylate 28 with sodium azide. Reduction of the azideswas followed by cyclization of diamine 30 with sulfamideunder basic conditions to obtain benzyl-protected cyclosulfamide 31, which after debenzylation resulted in final cyclosulfamide 8.The synthesis of sulfamidates 1 and 20 1A,B. N-B4–8 in hand, we first evaluatedtheir potency as inhibitors of the human glycoprocessing enzymes,α-glucosidase (GAA), ER α-glucosidase II (GANAB), theretaining β-glucosidases, GBA and GBA2, and glucosylceramidesynthase (GCS), in comparison to the known GAA inhibitors NB-DNJ 2 and α-glc-cyclosulfate 3  4-methylumbelliferyl (4-MU)-d-glucopyranoside fluorogenicsubstrates, whereas GCS inhibition was evaluated in RAW 264.7 cellsby measuring inhibitor-dependent glucosylation of C6-NBD-ceramideas the fluorescent substrate. Cyclosulfamidate 4 provedto be a low-micromolar inhibitor of GAA (IC50 = 5.17 μM)and is 5 times more potent than NB-DNJ 2 (IC50 = 26.7 μM). Furthermore, cyclosulfamidate 4 showedconsiderably more selectivity compared to NB-DNJ 2, whichinhibits GBA2 (IC50 = 957 nM) and GCS (IC50 =43 μM) rather more potently. Methylated analogue 5 proved to be a much weaker inhibitor toward α-glucosidaseGAA (IC50 = 485 μM) and inactive toward GANAB, suggestingthat there is no room for functionalization at the endocyclic nitrogen.In comparison, sulfamidate 6 with an oxygen at the pseudo-anomericposition showed to be a 10-fold less potent lysosomal GAA inhibitor,with comparable activity for the GANAB . Methylation of analogue 6 is somehow tolerated when inhibiting GANAB with methylated cyclosulfamidate 7 showing inhibition of GANAB with an IC50 of 132μM but targeting GBA and GBA2 as well . Finally, the double substitutionof endocyclic oxygens by nitrogens in sulfamide 8 isdetrimental for α-glucosidase activity.Having ulfate 3 1. Appare4 forα-glucosidasesover β-glucosidases and β-galactosidases was further investigatedby competitive activity-based protein profiling (cABPP) in human fibroblasthomogenates using α-glucosidase ABP 34, broad-spectrumβ-glucosidase ABP 35, and β-galactosidaseABP 36 . cABPPin mouse intestine homogenates demonstrated that sulfamidate 4 and iminosugar 2 inhibit intestinal sucrase-isomaltase(SI) and maltase-glucoamylase (MGAM), which are intestinal α-glucosidasesinvolved in food processing that have been extensively targeted toprevent and treat type II diabetes mellitus.29 Inhibition of gastrointestinal α-glucosidases with antidiabeticdrugs has been related to undesired effects, including flatulence,diarrhea, and abdominal pain. Cyclosulfamidate 4 inhibitsSI and MGAM but does so about a 100-fold less potently than it inhibitsrhGAA or GANAB, and we conclude that it has a more beneficial therapeuticwindow in this regard compared to NB-DNJ 2. Sulfamidate 4 and NB-DNJ 2 inhibit neither β-glucosidasenor β-galactosidase activities of lactase-phlorizin hydrolase(LPH)  showed that whilecyclosulfate 6 is an irreversible inhibitor ,cyclosulfamidate 4 inhibits rhGAA in a competitive manner. Kinetic parameters determinedwith increasing 4-MU-α-glucoside concentrations proved thatcyclosulfamidate 4 reversibly inhibits GAA with a Ki of 3.40 μM, approximately 5-fold morepotent than NB-DNJ 2 (Ki 15.2μM) (26 cyclosulfamidate 6 in turn proved to be anirreversible inhibitor of GAA with a KI of 378 μM and kinact of 0.35 min–1.A closer look into inhibition kinetics with inhibitor 15.2μM) 2.Contra21  further demonstrated the binding motifsof cyclosulfamidates 4 and 6. The X-raystructure of human α-glucosidase rhGAA in complex with sulfamidate 4 revealed that it binds the enzyme active site adopting a 4C1 Michaelis-type conformation in a noncovalentmanner , and N-hydroxyethyl-DNJ(NHE-DNJ),21 notably with Arg600, His674,Asp404, and the acid/base Asp616, strengthened by solvent-mediatedinteractions with Asp443, Asp616, Asp645, and Trp481 . The endocyclic nitrogen of the sulfamidatemoiety establishes a tight hydrogen-bonding interaction with the acid/baseAsp616, providing a structural rational for the lack of inhibitionof the methylated analogue 5 due to steric hindrance.The endocyclic nitrogen and the sulfate moieties of sulfamidate 4 overlap spatially with the nitrogen and subsite +1 aglyconeatomic features, respectively, of acarvosine reported previously21 , reinforcingthat sulfamidate 4 is a true substrate Michaelis complexmimetic for GAA. Structural data for the rhGAA-sulfamidate 6 complex clearly revealed that the cyclosulfamidate with the oxygenattached to the pseudo-anomeric position acts as an electrophilictrap, yielding a covalent complex with the enzyme and adopting a final 1S3 conformation in the enzymatic pocket after covalentreaction with the catalytic nucleophile Asp51831 . The energy cost of this structuralperturbation is compensated for by several H-bonding interactionsbetween the sulfamidate and rhGAA active-site residues, pleading forstabilization of the abortive complex.Crystallographic studies with the proteolytically digested formof human recombinant rhGAA (Myozyme)21 2 and thejaponicus (SupportjaponicusS3 furthejaponicusS3 furthe4 to stabilize α-glucosidases in vitro and in situ. Thermostability assays(TSAs) showed that α-glc-cyclosulfamidate 4 stabilizes rhGAA with a maximum shift in inflection temperature(ΔTimax) of 11.2 °C and a half maximal effectiveconcentration (EC50) of 1.16 μM, whereas α-glc-cyclosulfamidate 6 does not prevent enzymeunfolding at increasing temperature (S7). Similarassays with CjAgd31B using a Sypro Orange dye revealedthat α-glc-cyclosulfamidate 4 stabilizesthe bacterial analogue in vitro as well with a ΔTmmax of 5.2 °C and an EC50 of 134 μM . Prompted by these positive results,we also investigated the capacity of cyclosulfamidate 4 to stabilize rhGAA  in cell medium andin fibroblasts from Pompe adult patients with almost no α-glucosidaseactivity. Two different cell passages of Pompe fibroblasts were incubatedwith either NB-DNJ 2 (at 20 μM) or cyclosulfamidate 4 (at 20 μM) in the presence or absence of rhGAA Myozyme(10 nM) for 1–3 days, and activity was measured every 24 hwithout refreshing the media , suggesting that cyclosulfamidate 4 may stabilize the enzyme in the medium and more enzyme is availablefor cell internalization and transport toward the lysosomes. Indeed,rhGAA showed complete degradation in cell medium over 4 days, whichis reduced to 50% degradation by the addition of cyclosulfamidate 4 (20 μM), a significant improvement compared to the5% remaining with NB-DNJ 2 . In contrast to the cell medium, degradationof rhGAA in plasma occurs much faster, with more than 85% being degradedafter just 1 h. Addition of cyclosulfamidate 4 (20 μM)slows such degradation in plasma from healthy and Pompe patients withsimilar efficacy: more than 3-fold increase in rhGAA activity is observedafter 2 h in plasma from an adult Pompe patient  andcorresponding lysosomal forms (70 and 76 KDa) are due to differencesin glycosylation. as well 4G. The r as well 4B,C corrwell  were injected intravenouslyinto the sinus venous/duct of Cuvier with Myozyme (34 pmol) or a combinationof Myozyme (34 pmol) and either NB-DNJ 2 or cyclosulfamidate 4 at different molar ratios .After 5 dpf, zebrafish embryos were homogenized, and α-glucosidaseactivity was measured using the 4-MU-d-glucopyranoside fluorogenicsubstrate. Zebrafish larvae injected with Myozyme showed a significantincrease in GAA activity (7.5%) compared to nontreated controls. Zebrafishtreated with Myozyme in combination with cyclosulfamidate 4 showed a significantly increased GAA activity (±25% at 1:4ratio) at 5 dpf compared to the zebrafish injected with only Myozyme showed a small decrease in GAA activity, indicatingthat a high concentration of ligand might inhibit Myozyme as wellas endogenous GAA. Of note, this experiment is limited by the amountof volume (2 nL) and concentration of the rhGAA stock that can beinjected in the 2 dpf zebrafish.In order to analyze rhGAA stabilization  Myozyme5. This iepi-cyclophellitol cyclosulfamidate 4 is a selective reversibleα-glucosidase ligand thatbinds through 4C1 conformational Michaelis complexmimicry. Kinetic and crystallographic studies of rhGAA demonstratethat the position of the endocyclic nitrogen dictates the reactivityof these cyclic sulfamidates, and whereas cyclosulfamidate 6 reacts covalently, sulfamidate 4 binds in a competitivemanner, providing a new chemical space for potential α-glucosidasestabilizers. By reversible occupancy of the enzyme active site, cyclosulfamidate 4 stabilizes rhGAA and prevents its degradation invitro in cell medium and plasma from Pompe patients, in situ in Pompe fibroblasts, and in vivo zebrafish larvae, facilitating the enzyme transport toward the lysosome.We conclude that cyclosulfamidate 4 presents a superiorstabilization and selectivity profile compared to NB-DNJ 2, the benchmark compound in clinical combination ERT/PCT trials forthe treatment of Pompe disease.In conclusion, 1,6-


Phosphorylation is one of the most extensively investigated post-translational modifications that orchestrate a variety of cellular signal transduction processes. The phosphorylation of virus-encoded proteins plays an important regulatory role in the infection cycle of such viruses in plants. In recent years, molecular mechanisms underlying the phosphorylation of plant viral proteins have been widely studied. Based on recent publications, our study summarizes the phosphorylation analyses of plant viral proteins and categorizes their effects on biological functions according to the viral life cycle. This review provides a theoretical basis for elucidating the molecular mechanisms of viral infection. Furthermore, it deepens our understanding of the biological functions of phosphorylation in the interactions between plants and viruses. Rapid responses to internal and external cues are vital for complex life on the earth. Whether to maintain optimal conditions for growth or to avoid biotic/abiotic stresses, these responses must occur on a timescale that affords the organisms an obvious survival advantage. Cells have evolved sophisticated regulatory systems that can sense, transmit, sort, store, and interpret information that could enact a coordinated and timely response to the changes in environments. Post-translational modification (PTM) is an elegant transducer of the signals for adaptations of living organisms by the reversible and rapid nature, relatively small expenses, and profoundly function modulation of the target protein. Examples of PTM include phosphorylation , glycosyO-phosphorylation, which mainly occurs at the hydroxyl residues of serine (Ser), threonine (Thr), tyrosine (Tyr), and hydroxyproline  experiments in vivo and in vitro, have been widely used for the identification of viral protein phosphorylation  is transferred to the substrate protein by kinase , and the phosphorylated proteins were detected using X-autoradiography or a phosphorous storage screen . This method is the most direct protein phosphorylation detection assay in vitro. It has the advantages of high sensitivity and accuracy and has been widely applied in the identification of plant viral protein phosphorylation. Using this method, the phosphorylation of a single protein was proven in vitro, and the detailed AA sequences of the substrate protein and catalytic kinase were validated. However, this is a hazardous procedure for operators, especially for first-time and inexperienced researchers.Under kinase catalysis, the radioisotope y kinase . In the in vitro phosphorylation experiment are as follows. A 20 μL phosphorylation reaction system is prepared with 20 mmol.L–1 Tris-HCl buffer (pH = 7.5), 0.1 μg kinase/total protein extractions, 1.0 μg substrate (purified protein), 25 μmol.L–1 ATP, 1 μCi [γ-32P]-ATP , 5 mmol.L–1 MgCl2 , and ultrapure water. After incubation at 30°C for 30 min, 5 μL of 2 × SDS loading buffer  are added. The reaction is terminated by denaturation at 95°C for 5 min. All proteins in the reaction system are then separated via 12.5% SDS-PAGE for 2 h at 80 V. The gel is placed on a filter paper (8 cm × 10 cm), and dried in a gel dryer for 40 min. The dried gel is exposed to X-rays and detected using a phosphorous storage screen or film   . Specifi2+ or Zn2+ to form a relatively stable compound . Specific antibodies with high affinity for the target protein or phosphorylated Ser/Thr/Tyr are used to detect the target protein . SecondaThe western blot assay is relatively easy to perform and has no equipment or location restrictions. Antigen–antibody recognition of the phosphorylation site is specific and has high resolution in western blotting. It is often used for the validation of LC-MS/MS results in phosphorylation assays. However, this method is semi-quantitative, it is difficult to identify new or multiple phosphorylation sites in a single protein because of the problems in preparing the corresponding specific phosphorylation antibody.Phosphorylation/dephosphorylation is involved in multiple biological processes that regulate plant growth, development, and aging, such as plant biotic/abiotic stresses, hormone signal transduction, ion transportation, and material metabolism . DiffereUnderstanding the recognition mechanisms and biological functions of kinases and their substrates is essential. The surrounding phosphorylated AAs (Tyr/Ser/Thr) can be specifically recognized by the modular phosphoprotein-binding domains of kinases, called p-motifs. in vitro methods for validation of phosphorylation  , glycogeArabidopsis, CK1 regulates blue-light signaling and ethylene biosynthesis through phosphorylation (Arabidopsis genome encodes at least 14 CK1-like (CKL) protein kinases located in the cytoplasm, nucleus, endoplasmic reticulum, and vesicular granular structure and participates in the regulation of multiple biological processes (Protein kinase 1 (CK1) is a highly conserved serine/threonine kinase in eukaryotes. Its N-terminal catalytic domain is relatively conserved and functions in the recognition of substrate proteins . The C-trylation . Additiorocesses .in vivo  phosphorylates various plant viral proteins and plays an important role in the regulation of viral infection and host defense. CK2 is a highly conserved serine/threonine-specific kinase with multiple physiological functions in eukaryotes . The CK2in vivo . In planin vivo . Most CKd barley . CK2 is d barley . CK2-medd barley .GSK genes are more diverse (Glycogen synthesis kinase-3 (GSK3) is a serine/threonine phosphokinase that participates in all biological processes . In mamm diverse . Phospho diverse . In plan diverse .Protein kinase A (PKA) is an enzyme family whose catalytic activity depends on the levels of cyclic adenylate (cAMP) . It is ain vivo by phosphorylating the hydroxyl groups and adding a phosphate group to the serine/threonine side chains  and a C-terminal catalytic region (approximately 45 kDa) . It regue chains . PKC cane chains . It playe chains .In vivo, phosphorylation can regulate the interaction between large and small subunits of replicase, viral genomic RNA or DNA catalytic synthesis activity of replicase, stability of the viral replication complex (VRC), the interaction between viral proteins and host factors, and the binding of viral protein to RNA/DNA. This study summarizes the research on the biological functions of phosphorylated plant viral proteins , movement protein (MP), RNA-dependent RNA polymerase (RdRp), and viral suppressors of RNA silencing (VSRs), undergo phosphorylation during viral infection. The phosphorylation state of viral proteins is important for the stable infection of the virus in the host plant. proteins , 4 from Cucumber necrosis virus (CNV) belongs to the genus Tombusvirus (CNV) can be phosphorylated by membrane-related kinases and PKC. The phosphorylation sites of p33CNV are Thr-205, Ser-210, and Thr-211. The replicase cofactor function of p33CNV is completely lost in plant protoplasts and yeast when p33 is mutated to a phosphorylation mimic (T/S to D). Contrastingly, when p33 was mutated to the state of dephosphorylation (T/S to A), replication can occur in plant protoplasts and yeast could occur, but the viral infection efficiency is lower than that of the wild-type virus. These results indicate that in the process of viral infection, the alanine mutants of p33CNV in the viral genome affected the synthesis of subgenomic RNA and reduced the proportions of positive- and negative-strand RNA, ultimately affecting viral replication (CNV attenuates the RNA-binding ability and RdRp activity in plants (busvirus . The p33n plants .Turnip yellow mosaic virus (TYMV) is a member of Tymovirus (TYMV) encodes an RdRp with a mass of 206 kDa, which functions in viral RNA replication. In vivo, ORF1TYMV produces 140 kDa (p140) and 66 kDa (p66) replicase subunits via self-cleavage. The 66 kDa replicase subunits can be phosphorylated in plants, three main phosphorylation sites, thr-64, Ser-80, and Ser-326, have been identified via mass spectrometry. Dephosphorylation or phosphorylation-mimic mutants at the three phosphorylation sites of the 66 kDa replicase subunit have different effects on viral infectivity. The double phosphorylation-mimic mutant p66T64D/S80D and the single mutants p66S326A, p66S326D, and p66S326T have remarkable effects on viral infectivity, thereby significantly reducing the viral genomic RNA and CP protein accumulation levels. Phosphorylation of p66 does not affect its interaction with p140. In the viral infection cycle, the p66 phosphorylation is inhibited by the interactions between p140 and p66 subunits. These interactions mark the initiation of the viral replication complex assembly at the host cell membrane. Ser-326 site of p66 is the key to viral RNA synthesis regulation, and the phosphorylation/dephosphorylation of p66S326 affects the binding and depolymerization of viral genomic RNA and the replication complex. These results indicate that the phosphorylation of p66 is a molecular switch that regulates the speed of viral replication speed in a self-optimized manner and balances the interaction between the virus and the host plant. The speed of adaptive viral genomic RNA replication could guarantee the long-term coexistence of the virus in the host plant in terms of evolution (ymovirus . The ORFBarley stripe mosaic virus (BSMV) γb protein is a typical VSR and viral symptom determinant  phosphorylates and interacts with γb. The antiviral role of NbSTY46 involves kinase activity, accordingly, the NbSTY46T436A mutant lacking kinase activity not only loses the ability to phosphorylate or interact with γb but also fails to sustain virus systemic infection in plants. Self-phosphorylated NbSYT46 directly interacts with and phosphorylates the γb protein, which negatively regulates the viral replication and inhibits viral infection (erminant . The γbBBarley yellow striate mosaic virus (BYSMV) is a member of the genus Cytorhabdovirus, which elicits chlorotic striate and mosaic symptoms in the leaves of cereal plant species (189SASRPSSIAS198) that were phosphorylated. The viral dephosphorylation mutant (BYSMV-PS5A) enhanced viral replication, whereas the transcription was facilitated by the phosphorylation-mimic mutant (BYSMV-PS5D). In vivo, the two forms of P proteins (PS5A and PS5D) were preferentially associated with the nucleocapsid protein-RNA template and large polymerase protein, providing optimal replication and transcription of BYSMV, respectively. Biochemical assays demonstrated that plant and insect CK1 proteins can phosphorylate the SR motif of the P protein. Superphosphorylation of SR induces conformational changes in proteins. Moreover, the overexpression of CK1 or a dominant-negative mutant impairs the phosphorylation/dephosphorylation balance between p42 and p44, thereby compromising viral infection in barley. These observations indicate that BYSMV recruits the conserved CK1 kinases to finely regulate the replication-transcription phase during the viral life cycle and achieve optimal cross-kingdom infection of plants and insect vectors ( species . BYSMV g species . In BYSM vectors .Hordeum vulgare) MAPK-MPK3 (HvMPK3) and the planthopper ERK (LsERK) proteins can interact with the viral structural protein nucleoprotein and directly phosphorylate this N protein at Ser-290. Overexpression of HvMPK3 could inhibit BYSMV infection, and barley plants treated with the MAPK pathway inhibitor U0126 are more susceptible to BYSMV. The dsRNA was synthesized and microinjected into the planthopper to knock down the LsERK, then the virus infection was promoted. A phosphomimetic mutant of the nucleoprotein Ser-290 (S290D) completely abolished virus infection because of the impaired self-interaction of BYSMV N and the formation of unstable N–RNA complexes. These results demonstrated that the conserved MAPK and ERK directly phosphorylate the viral nucleoprotein to trigger immunity against cross-kingdom immunity to BYSMV in host plants and its insect vectors ( vectors .Beet black scorch virus (BBSV) belongs to the genus Necrovirus in the family Tombusviridae (BBSV can be phosphorylated at Thr-41 by the cAMP-dependent protein kinase (PKA)-like kinases in vivo and in vitro. The non-phosphorylated state of CPT41 does not affect the initial viral RNA replication stage, however, viral genomic RNA accumulation is affected as the infection progresses. CPBBSV phosphorylation is inferred to be involved in the later stages of genomic RNA replication, effectively interfering with the host defense system. RNase-sensitivity assays revealed that the BBSVT41A and BBSVT41E mutant viruses are easily degraded by endogenous ribonucleases in Nicotiana benthamiana. Electron microscopy revealed that the BBSVT41A and BBSVT41E mutants are abnormal compared to the wild-type BBSV particle  is a member of the genus Potexvirus and possesses a positive single-stranded RNA genome (N. benthamiana (NbCK2). Studies revealed that the main phosphorylation site is Ser-241 and both CPBaMV and NbCK2α subunits are colocalized on the plasmodesmata (PD). These results suggest that phosphorylation of CPBaMV may be involved in the viral cell-to-cell movement. Although in vitro binding assays showed that the RNA-binding ability of CPS241A mutant was not impaired, the cell-to-cell movement ability of mutant BaMV-CPS241A was reduced. If the Ser-241 site is mutated to D , its RNA-binding ability is significantly weakened, and the cell-to-cell movement ability of the corresponding mutant virus is blocked. Further deletion of the AA from 240 to 242 in CPBaMV enhances the RNA-binding ability of CP, but the cell-to-cell movement ability of the corresponding mutant virus is affected. These results indicate that the NbCK2 regulates the RNA-binding ability of CPBaMV by phosphorylating the AA 241 , belongs to the genus of Pomovirus and the family Virgaviridae  is mutated to alanine (A). These results demonstrated that the Tyr  is the actual phosphorylation site of TGB3.aviridae . GenomicPMTV enhances the interaction intensity between TGB3 and TGB2 in yeast. However, the intercellular movement ability of the corresponding viral mutant (PMTV-TGB3T87–89A) is weakened in N. benthamiana. Although the TGB3Y120A mutant does not affect the interaction with TGB2 protein, it cannot infect N. benthamiana. This demonstrates that tyrosine phosphorylation modification exists in plant viral proteins and is also a method of regulating plant virus movement and infectivity  is a representative species of hordeivirus . The mutant viruses can systematically infect barley and wheat, however, the infection efficiency and viral accumulation levels decreased dramatically. In N. benthamiana, only the mutant virus BSMV-TGB1T395A can establish a systemic infection. Together, these results demonstrate that the phosphorylation/dephosphorylation status of TGB1 at Thr-395 and Thr-401 affects the viral systemic movement ability differently in monocot and dicot hosts. This may be a consequence of the architecture of host-specific vasculature. Furthermore, the phosphorylation of TGB1 promotes viral infection by enhancing the interaction intensity with TGB3 proteins, which is apt to form the viral ribonucleoprotein complex (BSMV intracellular movement complex). However, the phosphorylation modification of TGB1 is not sufficient to substantially alter the TGB localization patterns visualized via laser confocal microscopy (deivirus . The tricroscopy .Chinese wheat mosaic virus (CWMV), a member of the genus Furovirus and family of Virgaviridae, is an RNA virus with two positive-sense single-stranded genomic RNAs (CWMV) are the two phosphorylation sites catalyzed by wheat SAPK7 protein in vivo and in vitro. Mutational analyses have shown that the double-site phosphorylation-mimic mutant virus (CWMV-CRPS162/165D) enhances viral infectivity in plants because of the suppressed cell death activity and hydrogen peroxide production by the CRPS162/165D protein. Furthermore, the CWMV-CRPS162/165D interacts with the RNA-binding protein UBP1-associated protein 2C (TaUBA2C) in wheat, whereas the phosphorylation-deficient mutant virus (CWMV-CRPS162/165A) does not. Silencing of TaUBA2C promotes viral infection in wheat plants, while the overexpression of TaUBA2C inhibits viral infection. TaUBA2C recruits the pre-mRNAs of TaNPR1, TaPR1, and TaRBOHD to induce plant cell death and hydrogen peroxide production, ultimately inhibiting viral infection. This effect can be suppressed by phosphorylated CRPS162/165D through direct binding of TaUBA2C, resulting in changes in the chromatin-bound status and the attenuation of the RNA- or DNA-binding activity of TaUBA2C in plants (mic RNAs . Ser-162n plants .Tomato yellow leaf curl virus (TYLCV) is a member of Geminivirus with a single genomic component that is composed of a single-stranded circular DNA (DNA-A) and satellite DNA (DNA-B)  and in vivo bimolecular fluorescence complementation (BiFC) revealed that βC1TYLCV interacts with tomato SNFI-related kinase (SnRK1). Phosphorylation experiments showed that βC1TYLCV is phosphorylated by SnRK1 kinase, and Ser-33, Thr-78, Tyr-5, and Tyr-110 are the main phosphorylation sites. The symptoms of phosphorylation-deficient mutant viruses  are aggravated, and the viral genomic DNA accumulation levels are significantly higher than those of the wild-type virus. The phosphorylation-mimic mutant viruses  delay infection, and the viral DNA accumulation levels are completely opposed to those in the phosphorylation-deficient mutant viruses. These results indicate that SnRK1 interacts with and phosphorylates βC1TYLCV protein to weaken viral infectivity in tomatoes. Compared with those of the double mutant virus TYLCV-βC1-2D infection, the symptoms of TYLCV-βC1-2D/2E (S33D/T78D/Y5E/Y110E) infection are further attenuated, whereas the VSR activity of βC1-2D/2E is significantly weakened. Furthermore, phosphorylation of the βC1 protein by SnRK1 greatly affects its interaction with N. benthamiana ASYMMETRIC LEAVES 1, and regulates the induction of symptoms ( (DNA-B) . The βC1 (DNA-B) . βC1TYLCsymptoms .Yunnan tomato leaf curl virus (TLCYnV) is a member of the Geminivirus family with a single-component genomic DNA (TLCYnV) is a symptom determinant in plants  are significantly reduced. Furthermore, subcellular localization revealed that the C4T51A mutant confines the NbSKη protein to the cell membrane, and myristoylation of the C4 N-terminal Gly-2 position directly results in the cell membrane localization. The pathogenicity and viral genomic DNA accumulation levels of the mutant TLCYnV-C4G2A virus are significantly reduced, respectively. During TLCYnV-C4G2A virus infection, NbSKη cannot localize to the cell membrane. Chemical inhibition of N-myristoyltransferases or exportin-α enhances the nuclear retention of C4TLCYnV, and mutations in the putative phosphorylation (T51A) or myristoylation sites (G2A) of C4TLCYnV result in increased nuclear localization and reduced severity of the C4-induced developmental abnormalities. These results suggest that the nucleocytoplasmic shuttling of C4TLCYnV via protein modifications, including NbSKη-mediated protein phosphorylation, protein myristoylation, and the interaction with exportin-α, is critical for viral pathogenicity in plants  and the O-linked N-acetylglucosamine hydrolase (OGA), respectively   and coexisting (coordinately regulated)  of CPPPV has profound significance for a deeper understanding of the plant virus life cycle.In addition to phosphorylation, the side chain hydroxyl group on the serine and threonine residues of the target protein often undergo ectively . The O-Gcription , epigenecription , intracecription , and vircription . The samresidues , 2011. On humans . ATK is n humans . The O-GSer-473) . These oproteins . In wintgulated) . In planlication . Seven Anfection , whereasnfection . The corin vitro. The finding is not representative of the in vivo conditions because the total protein obtained from plant contained many kinases, and phosphorylation can occur between the kinase and substrate that would not normally be in contact in vivo. Different protein kinases can phosphorylate multiple sites of the same and different viral proteins encoded by the same virus, and these modifications have different regulatory roles in plant–virus interactions.More than 20 viral proteins are phosphorylated during viral infection cycle , 4. Do oin vivo. In addition, the substrate of the target kinase is often cell-specific. Explaining the distinctive effects of various stimuli on different tissues requires further consideration. Hence, powerful methods, coupled with further exploitation of important methodological and technical advances, are needed to identify the substance protein and the corresponding target kinase. Of greater importance is research to control the outbreaks and devastation caused by plant viruses, develop tissue and phosphor-specific antibodies, certain cell-type-specific permeant inhibitors of protein kinase, and design new therapies via taking advantage of the distinct cell types not expressing a particular protein kinase or phosphatase.In plant virology, the core question is how to coordinate plant resistance with yield and quality. The phosphorylation studies discussed here are promising. Massive progress in the identification of the major substrates of each protein kinase and phosphatase has been achieved in recent years using the modern MS-based proteomics. However, major gaps in our knowledge still exist regarding the identities of the key substances and protein kinases, and how their phosphorylation contributes to changes in cell physiology in response to particular stimuli. A certain genome size determines a finite number of kinases and phosphatase in plants, and there are multiple potential phosphorylation sites within a single protein, which implies that the kinase and phosphatase isoforms may have overlapping specificities. These kinases are involved in the phosphorylation of several hundreds of substrates in vitro phosphorylation experiments. The corresponding kinases can also be identified in the follow-up research using reverse genetics techniques. Furthermore, it would be beneficial to explore the impact of certain types of kinase and viral mechanism, including viral replication, viral transcription, and the host’s defense response. However, several proteins encoded by one certain plant virus can be phosphorylated by several types of kinases. The elucidation of specific kinases and their substrates and determination of the corresponding relationship are essential aspects that should be considered in future studies of biological function. If the common pathways involved in the broad-spectrum antiviral immunity conferred by kinases can be identified, we could cultivate new antiviral crop germplasm through transgenic or CRISPR-Cas9-mediated gene editing methods. Moreover, small-molecule activators or inhibitors could be designed according to the advanced structures of target kinases, which could be used as novel pesticides for spraying crop leaves in the field for virus control.In future plant–virus protein phosphorylation studies, the phosphorylation sites on viral proteins can be identified in detail by mass spectrometry combined with the KZ and XZ contributed substantially to the conception and design of this review article and co-wrote the manuscript. KZ and ZH reviewed the manuscript before submission for its intellectual content. All authors gave final approval of the published version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Impaired adipogenesis is associated with the development of insulin resistance and an increased risk of type 2 diabetes (T2D). GATA Binding Protein 3 (GATA3) is implicated in impaired adipogenesis and the onset of insulin resistance. Therefore, we hypothesize that inhibition of GATA3 could promote adipogenesis, restore healthy fat distribution, and enhance insulin signaling. Primary human preadipocytes were treated with GATA3 inhibitor (DNAzyme hgd40). Cell proliferation, adipogenic capacity, gene expression, and insulin signaling were measured following well-established protocols. BALB/c mice were treated with DNAzyme hgd40 over a period of 2 weeks. Liposomes loaded with DNAzyme hgd40, pioglitazone (positive), or vehicle (negative) controls were administered subcutaneously every 2 days at the right thigh. At the end of the study, adipose tissues were collected and weighed from the site of injection, the opposite side, and the omental depot. Antioxidant enzyme  activities were assessed in animals’ sera, and gene expression was measured using well-established protocols. In vitro GATA3 inhibition induced the adipogenesis of primary human preadipocytes and enhanced insulin signaling through the reduced expression of p70S6K. In vivo GATA3 inhibition promoted adipogenesis at the site of injection and reduced MCP-1 expression. GATA3 inhibition also reduced omental tissue size and PPARγ expression. These findings suggest that modulating GATA3 expression offers a potential therapeutic benefit by correcting impaired adipogenesis, promoting healthy fat distribution, improving insulin sensitivity, and potentially lowering the risk of T2D. Obesity is at epidemic proportions with a steadily increasing prevalence that is expected to reach 300 million patients by 2025 . In addiAlthough the association between insulin resistance and obesity is well established ,8, the lPrevious studies have shown that GATA3 suppresses the transition from preadipocytes to adipocytes by inhibiting the expression and activity of PPARγ2 and C/EBPs ,21,22,23We previously showed that GATA-3 inhibition causes reduction in GATA-3 expression in 3T3L-1 mouse preadipocytes after 48 h of treatment . GATA3 ip-value 0.020246) increase in animal weight, with positive and treated groups showing a 1.03-fold , and 1.04-fold  increase, respectively. When comparing the weight difference between the right (treatment site) and left (opposite control side) SAT, there was a significant increase in the right adipose tissue weight from the GATA3 inhibitor-treated group of 1.3-fold , but not in the control group . However, the positive control group showed a significant decrease in the weight of the right adipose tissue of 0.9-fold  , and between the GATA3 inhibitor-treated and positive control groups of 0.7-fold  , Positive Control (N = 8), and GATA3 inhibitor-treated (N = 12) Group. Repeated treatment of animals with 10 µg/0.1 mL of DNAzyme over a period of 2 weeks showed a significant increase in animal total weight at the end of the study compared to the animal weight at the beginning of the study A in all e 0.004) B. As repe 0.004) B 26]. F. Fp-valuue 0.02) C. Howeveue 0.02) C. There was a significant increase in SOD activity in the GATA3 inhibitor-treated group compared to the vehicle control group, but no significant difference between the positive control and vehicle control groups A. MoreovIn terms of gene expression from the SVF derived from different adipose tissue sites , we found that GATA3 inhibition decreased the expression levels of MCP-1 in the right (injection site) compared to opposite SAT site A. In addVarious studies have suggested that non-obese individuals could equally become insulin resistant and develop T2D if left untreated, with the term metabolically obese apparently healthy individuals being used to describe insulin-resistant non-obese individuals ,8,28. MoRecent reports have suggested that both preadipocytes and mature adipocytes play an equally important role in the maintenance of adipose tissue hemostasis and the development of insulin resistance when dysfunctional . These rImpaired adipogenesis could affect the levels of different proteins and kinases involved in insulin signaling pathway . Our in In order to validate the effect of GATA3 inhibition in vivo, we carried out a proof-of-concept study utilizing normal-weight mice. Our findings demonstrate that there was an increase in the total animal weight at the end of the experiment from the GATA3 inhibitor-treated group. The increase in the animal weight was accompanied by an increase in the weight of the tissues dissected from the right site (injection site) compared to the left-site tissues. Inhibiting GATA3 in the injection sites might have enhanced the mobilization, recruitment, and differentiation of adipocyte progenitor cells to the right site, therefore promoting adipogenesis and causing an increase in the tissue weight. In contrast, the GATA3 inhibitor-treated group showed a reduction in the omental adipose tissue weight compared to the other groups, perhaps accounting for the increased weight of fat tissue at the injection site in treated animals. Such reduction was marked by a decrease in the expression levels of PPARγ. These data suggest a protective effect associated with GATA3 inhibition, which is in agreement with previous studies associating the omental adipose tissue mass/size with the amplified inflammatory status and insulin resistance . MoreoveImpaired adipogenesis is characterized by having an imbalance between the oxidative and antioxidative markers. SOD, a major antioxidant enzyme that protects adipocytes during proliferation and differentiation, was found to be elevated in tissues and cells undergoing active adipogenesis and cellular differentiation . ImpaireThe proinflammatory profile of dysfunctional adipose tissue plays a critical role in lowering the adipogenic capacity of preadipocytes, leading to a reduction of the lipid storage space and elevation in ectopic lipid accumulation . To valiHowever, this study has potential limitations, including the need to verify whether the increased adipose tissue weight at the injection site is due to increased fat content or adipocyte number. In addition, since this in vivo study is a proof of concept, the used animal models were healthy mice with normal insulin signaling. Hence, the effect of GATA3 inhibition on activation (phosphorylation) of insulin signaling was not investigated. These limitations must be addressed in future studies using diseased models with insulin resistance to explore the effect of GATA3 inhibition effect on reversing IR in vivo. In conclusion, our in vitro and in vivo data indicate that inhibiting GATA-3 expression restores adipogenesis and fat distribution, improves adipocytes differentiation, and enhances insulin signaling. Our data therefore suggest the potential utilization of GATA-3 modulation for preventing the development of insulin resistance in non-obese as well as obese individuals. However, despite holding great potential and being a promising modulator, such approach requires further investigation and validation in relevant animal disease models.4/cm2. The cells were then maintained in a humidified incubator at 37 °C with 5% CO2. The media was changed every 2–3 days until the cells achieve 80–90% confluence. When confluent, cells were either harvested or induced to differentiate by changing the medium into differentiation medium , 1 µM dexamethasone, 0.25 mM IBMX, 0.2 µM Insulin, Biotin (66 µM), Rosiglitazone (PPARγ agonist) (5 µM))) for 3–7 days, followed by 9–10 days in maintenance medium containing the same components as the differentiation medium, excluding IBMX and rosiglitazone as described previously [Recruitment criteria of participants were previously described . Approvaeviously . To inveeviously . BrieflyCells were fixed with 4% formaldehyde  and stained with DAPI  and Lipidtox  as previously described . Total nLiposomes were prepared using the ethanol-based proliposome technology by adapting a previously published protocol . Brieflyhttps://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6285.pdf (accessed on the 17 September 2022).Insulin signaling was measured by assessing the phosphorylation levels of IRS-1, GSK3B, IGF1R, Akt, Mtor, p70s6k, IR, PTEN, GSK3, TSC2, and RPS6 in total cell lysates  as previously described , using a commercial Bio-Plex Pro™ Cell Signaling Akt Panel  using Luminex 200 technology  following the manufacturer’s instructions. The Bio-Plex Pro cell signaling assays sensitivity, as well as the intra- and inter-assay %coefficient variation per species, can be found at Adult normal weight male (12–16 weeks old) BALB/c mice were provided by the Laboratory Animal Research Center (LARC) at Qatar University (QU). Animals were housed in individually ventilated cages (IVC) under standard husbandry conditions , provided with normal chow diet and drinking water ad libitum. All animal procedures were performed according to approved institutional ethical rules and regulations and were approved by Qatar University-Institutional Animal Care and Use Committee (QU-IACUC 024/2020). A total of 28 animals was used in this study and divided into three groups: (A) Vehicle Control Group, with 8 animals that were treated with 100 µL of DNAzyme-free liposomes; (B) Positive Control Group, with 8 animals that were treated with liposome-loaded with 1 µM of pioglitazone (40 mg/Kg); and (C) GATA3 inhibitor-treated Group, with 12 animals treated with liposome-loaded DNAzyme . Treatments were administered subcutaneously to the right flank region (site of injection), twice a week for 2 weeks. The mice were housed under standard animal husbandry conditions with 12 h dark and light cycle and were provided standard rodent chow and water ad libitum. Animals were weighed at the beginning and the end of the study. All the animals were euthanized as per AVMA guidelines, and the subcutaneous adipose tissues from right flank (site of injection), left flank (opposite site), and omental were collected from scarified mice, weighed. Blood was drawn via a cardiac puncture. Oxidative stress was assessed by measuring the activity of antioxidant enzymes, including catalase and superoxide dismutase (SOD), in serum samples prepared from collected blood using the Catalase Assay Kit (Merck Millipore), and the SOD kit  following the manufacturer’s instructions. Measurements and data analysis was performed using the Cytation 5 Cell Imaging Multi-Mode Reader .For the in vivo experiments, SVFs were isolated from adipose tissue biopsies collected from right/left thighs and omental depots using established protocol ,44,45. Bt-test, one-way ANOVA, two-way ANOVA, or linear.Comparisons were performed using a The authors declare a patent involved in the reported work .


Proteins in the thioredoxin superfamily share a similar fold, contain a -CXXC- active site, and catalyze oxidoreductase reactions by dithiol-disulfide exchange mechanisms. Protein disulfide isomerase (PDI) has two -CGHC- active sites. For in vitro studies, oxidation/reduction of PDI during the catalytic cycle is accomplished with glutathione. Glutathione may act as electron donor/acceptor for PDI also in vivo, but at least for oxidation reactions, GSSG probably is not the major electron acceptor and PDI may not have evolved to react with glutathione with high affinity, but merely having adequate affinity for both glutathione and folding proteins/peptides. Glutaredoxins, on the other hand, have a high affinity for glutathione. They commonly have -CXFC- or -CXYC- active site, where the tyrosine residue forms part of the GSH binding groove. Mutating the active site of PDI to a more glutaredoxin-like motif increased its reactivity with glutathione. All such variants showed an increased rate in GSH-dependent reduction or GSSG-dependent oxidation of the active site, as well as a decreased rate of the native disulfide bond formation, with the magnitude of the effect increasing with glutathione concentration. This suggests that these variants lead to competition in binding between glutathione and folding protein substrates. Proteins belonging to the thioredoxin superfamily share a similar fold and catalyze oxidoreductase reactions by dithiol-disulfide exchange mechanisms. They contain a -Cys-X-X-Cys- active site motif. The reactions catalyzed by individual members are determined by the redox equilibrium of the disulfide-dithiol state of the active site cysteines and their substrate specificity . ThioredSince the glutathione redox potential in the ER is more oxidizing than in the cytoplasm  and is cGlutaredoxins, on the other hand, have a high affinity for glutathione and primarily catalyze deglutathionylation reactions, i.e., reduction of protein-GSH mixed disulfides . Both PDIn this study, the PDI active site motif was mutated to contain either phenylalanine or tyrosine in the place of histidine to make it more glutaredoxin-like. These mutations did not cause any gross structural defects, as determined by far-UV CD. Mutating the PDI active site to more glutaredoxin-like motif increased its reactivity with glutathione. All such variants showed an increased rate in GSH-dependent reduction or GSSG-dependent oxidation of the PDI active site, as determined by stopped-flow experiments, as well as the ability to catalyze deglutathionylation reactions. The effect of variants on the ability to catalyze both oxidative foldings (oxidation and isomerization reactions) was studied using bovine pancreatic trypsin inhibitor (BPTI) refolding assays. Variants that increased the rate of reaction with glutathione decreased the rate of native disulfide bond formation, with the magnitude of the effect increasing with glutathione concentration. The results suggest that generating a more glutaredoxin-like active site in PDI leads to a competition between binding to glutathione and to folding proteins. These results are consistent with a functional role for glutathione in physiological native disulfide bond formation in the ER with glutathione acting as a redox buffer and that PDI has evolved to have an adequate and appropriate affinity for both glutathione and folding proteins.a domain of human PDI, were generated by site-directed mutagenesis according to the instructions of the QuikChange Site-Directed Mutagenesis protocol . All genes of interest cloned into vectors were checked for correctness by DNA sequencing. Proteins were expressed in the E. coli strain BL21(DE3) pLysS grown in LB medium at 37 °C and induced at an A600 of 0.3 for three hours with 1 mM IPTG. Expressed PDI variants were purified by immobilized metal affinity chromatography and ion exchange chromatography as described for the a domain of PDI [All constructs used are listed in n of PDI . Pure frn of PDI . The conn of PDI .Far-UV circular dichroism (CD) spectra were recorded with a Chirascan Plus CD Spectrometer  as described previously . The HT The BPTI refolding was carried out as described previously  with theBefore the assays described below, the proteins were either reduced with a 10-fold molar excess of DTT or oxidized with a 10-fold molar excess of GSSG for 30 min at room temperature, followed by removal of the excess of DTT/GSSG and buffer exchange into desired reaction buffer using centrifugal filter device (Amicon Ultra with MWCO 10 K).a domain constructs by glutathione were determined using a KinTek SF-2004 stopped-flow fluorometer  as described previously [The rates of oxidation and reduction of the active site of different PDI eviously  except aThe rate of oxidation by DHA was determined as described previously  (Saaranea domain constructs towards Ellman’s reagent (DTNB) was analyzed by measuring the change in absorbance at 412 nm using KinTek SF-2004 stopped-flow instrument as described previously [2HPO4, 0.1 M citric acid, 0.1 M boric acid, 1 mM EDTA, pH 7.0 with 184 µM DTNB and 10 µM reduced enzyme.The reactivity of active site cysteines of the different PDI eviously  in 0.1 MThe deglutathionylation activity of the enzymes was analyzed based on a method described previously . BrieflyMolecular Biology I, students generated individual point mutations in the active site of an enzyme. In Protein Chemistry I, they purified their protein variant. In Biochemical methodologies II, they characterized the activity of the variant and undertook biophysical characterization, and in English for Biochemists II, the students compiled the data from all of the variants and wrote a report in the style of a scientific publication. Neither students nor staff would know the expected results as the mutations made were random. The enzyme chosen was the catalytic a domain of human PDI, as it was expected that a large number of different variants could be obtained that decreased one or more activities. Furthermore, it was anticipated that these variants could be clustered by the students during their analysis e.g., mutations affecting accessibility or reactivity or pKa of the N-terminal active site cysteine, in mutations affecting pKa of the C-terminal active site cysteine or mutations which destabilized the protein, etc. While large numbers of variants with such expected activity modulating effects were obtained, unexpectedly, several variants were obtained, which increased the activity of this domain in oxidative folding of a peptide substrate [As part of the renewal of the Biochemistry BSc curriculum at the University of Oulu the practical components of several second-year courses were linked together to provide the students a more comprehensive overview of workflows in biochemical research. In ubstrate  and in ra domain as well as the double active site mutations H55F/H399F and H55Y/H399Y in full-length mature human PDI. All variants were analyzed by far UV circular dichroism, and no significant differences were observed in the spectra compared to that of the wild-type protein  was monitored by stopped flow using changes in absorbance at 412 nm [To discriminate between these two options, the reactivity of the active site of the variants towards other chemical species was examined. The reaction of the reduced t 412 nm . As expeIncreasing the affinity of the active site of PDI for glutathione would be predicted to increase the activity of PDI catalysis of deglutathionylation reactions. Using changes in fluorescence of a model glutathionylated peptide ; Figure D, the reTo examine the effect of the mutations on other PDI activities, we examined the refolding kinetics of the model protein bovine pancreatic trypsin inhibitor (BPTI) by mass spectrometry. Under standard refolding conditions, with 2 mM GSH and 0.5 mM GSSG, both the H55Y/H399Y and H55F/H399F variants behaved similarly, with both resulting in a slightly slower rate of attainment of the native three disulfide (3S) state of BPTI compared with the wild-type enzyme A. The kip = 0.003), neither variant showed a significant change in rate with increasing glutathione concentration.The reduced rate of catalysis of late-stage folding events of BPTI by the PDI variants is not due to reduced rates of redox-exchange with glutathione, as both variants showed increased rates of oxidation by GSSG and reduction by GSH . It is pTo function PDI must interact with a wide range of molecules, both low molecular weight and macromolecules. PDI catalyzes oxidation, reduction, and isomerization of disulfide bonds in folding substrates in the ER. Catalysis of reduction and isomerization requires the active site of PDI to be in the reduced state (dithiol), while in contrast, catalysis of oxidation requires the active site to be in the oxidized state (disulfide). Cycling between these two states is an essential part of the physiological function of PDI.Oxidation of the active site of PDI can be done via a wide range of pathways (,4, incluIn contrast, only three pathways are known to allow the reduction of the active site in vivo. One recently characterized pathway is based on transferring reducing equivalents derived from cytosolic thioredoxin system across the ER membrane via a still unknown transmembrane protein ,30. The a domain of PDI towards GSSG up to 2.3-fold (a of either active site cysteine or due to altering their accessibility. Instead, the results suggest that these variants increase the affinity of PDI towards glutathione in both the oxidized and reduced states.Here we report that variants in the active site, in particular mutations in the histidine in the CGHC motif to tyrosine or phenylalanine can increase the reactivity of the catalytic 2.3-fold D and tow2.3-fold B. These 2.3-fold B or modu2.3-fold A, and thThe effects of increased affinity towards glutathione would be predicted to be two-fold. Firstly, these variant enzymes would be expected to show increased affinity to glutathionylated species, increasing the rate of deglutathionylation reactions. Both the H55F and H55Y variants do show significantly increased rates of deglutathionylation catalysis E. SecondIt is reasonable to assume that PDI has evolved to have an adequate and appropriate affinity for both glutathione and folding protein substrates. The ER contains high levels of glutathione and creates an optimal redox potential for protein folding, at least in vitro. PDI needs to bind efficiently to the folding protein substrate, thus too high affinity for glutathione might be detrimental to its function, while too low would prevent it from utilizing the glutathione redox buffer. Another member of the thioredoxin superfamily, glutaredoxins, on the other hand, have a high affinity towards glutathione and glutathionylated substrates. In this study, we demonstrated that the introduction of a more glutaredoxin-like active site to PDI interferes with the optimal affinity of PDI towards different substrates by increasing the affinity towards glutathione, thus resulting in competition between binding glutathione and the folding protein substrate.


To reduce the degradation of polylactic acid (PLA) during processing, which reduces the molecular weight of PLA and its properties, prior studies have recommended low processing temperatures. In contrast, this work investigated the impact of four factors affecting shear heating  on the degradation of PLA. The polylactic acid was processed using a quad screw extruder (QSE) and a comparable twin screw extruder (TSE), two screw configurations, higher screw speeds, and several feed rates. The processed PLA was characterized by its rheological, thermal, and material composition properties. In both screw configurations, the QSE (which has a greater free volume) produced 3–4 °C increases in melt temperature when the screw speed was increased from 400 rpm to 1000 rpm, whereas the temperature rise was 24–25 °C in the TSE. PLA processed at low screw speeds, however, exhibited greater reductions in molecular weight—i.e., 9% in the QSE and 7% in the TSE. Screw configurations with fewer kneading blocks, and higher feed rates in the QSE, reduced degradation of PLA. At lower processing temperatures, it was found that an increase in melt temperature and shear rate did not significantly contribute to the degradation of PLA. Reducing the residence time during processing minimized the degradation of PLA in a molten state. The large quantity of plastic waste produced by non-degradable polymers is a global concern ; therefoA promising alternative to petroleum-based polymers is the biodegradable polymer, polylactic acid (PLA) ,2,3,4,5.−1, which is linked to carbonyl (C=O) stretching, 1183 cm−1, and 1085 cm−1, both of which are attributed to the asymmetric vibration of the ester group (C–O–C).The degradation of polylactic acid during melt processing has been evaluated using rheological measurements, differential scanning calorimetry (DSC), and Fourier-transform infrared radiation (FTIR) ,21,22. RSeveral research groups have evaluated the effect of melt temperature on the degradation of polylactic acid during twin screw extrusion ,22,23. MResearchers have also investigated the degradation of PLA using very low screw speeds (25 rpm to 250 rpm) and low processing temperatures. Taubner and Shishoo  stated tMost commercial compounds of biodegradable polymers, such as PLA, occur in twin screw extruders (TSEs) ,20. OtheThis work investigated the impact of four factors affecting shear heating  on the degradation of polylactic acid. Higher screw speeds were used to determine the balance between shear rates and shearing time . Two screw configurations provided the effect of kneading blocks compared to conveying elements. The impact of greater free volume in a QSE was compared to the tighter spacing in a similar TSE. Feed rates for the QSE were increased to evaluate the effects of greater filling of the screw channels and reduced shear on the PLA. Following this, degradation was measured by studying changes in zero-shear viscosity, melt index, chemical composition, and crystallinity of the PLA. The results unexpectedly contradicted prior assumptions about viscous dissipation in the extruders.TM Biopolymer 2003D, USA, Minnetonka, WI, USA). This PLA is considered to be of “general purpose extrusion” grade. The reported density was 1240 kg/m3 and melt flow rate (210 °C/2.16 kg) was 6.0 g/10 min. Prior to extrusion, the PLA was dried using a desiccant drier  at a temperature of 55 °C for 24 h to prevent hydrolysis. The moisture levels in the dried PLA were measured using a moisture analyzer ; these levels were consistently 0.021% which was within the supplier-recommended range of <0.025%.This work was conducted using commercial grade polylactic acid  and a QSE . Both extruders had 15-mm diameter screws, length-to-diameter ratios of 45:1, and the same screw configurations. The two screw configurations are shown in The PLA pellets were fed at the beginning of zone 1. Melt exiting the extruders passed through a single strand die in the TSE and a double strand die in the QSE. The extruded strands were cooled in a water bath and then pelletized . The processing and equipment used for the extrusion trials are listed in Melt temperature was measured using a type K immersion thermocouple , by extending 6 mm into the melt stream which was located in the die adaptors. Although immersion thermocouples produce somewhat high melt temperature readings due to shear heating, this thermocouple provided a relative measurement of the increase in melt temperature associated with changes in extrusion parameters. Residence time was determined using a pigment as a tracer. Multiple melt temperature measurements were recorded during extrusion and these values were averaged. This pigment was fed directly into the feed port with the PLA pellets, and residence time was measured as the time required to detect visual changes in the color of the extrudate.To evaluate the degradation, the dynamic rheological characterization in the molten state of PLA was accomplished using a parallel plate rheometer . The plates had diameters of 25 mm and a gap of 1.5 mm. The specimen disks were prepared by micro-injection  with a soak time of 3 min and a barrel temperature of 180 °C. Prior to the molding and the rheology trials, the samples were dried in a vacuum oven at 55 °C for 24 h. The rheological characterization was carried out at 180 °C. A strain sweep was used to verify that the strain used in frequency sweeps was within the linear viscoelastic region. Frequency sweeps were then conducted from 0.02 to 15.92 Hz at 2% of strain while recording the complex viscosity (η*). To ensure accurate results, 2 replicates were conducted for each sample and the data are presented as the mean ± the standard deviation.o) and relaxation time (λ) were determined using Trios 5.0 software  to fit the viscosity–shear rate data to the Carreau–Yasuda model [n is a power-law coefficient, a is the adjustable exponent, and λ is the relaxation time.Since polylactic acid generally obeys Cox–Merz rules , the comda model :(1)η(γ˙)o) were reported.The melt flow index (MFI) measurements were performed in accordance with ASTM D1238-20, Procedure A, using a temperature of 210 °C and load of 2.16 kg. Five measurements were carried out in the extrusion plastometer  and the average values were calculated. The ratios of average MFI of the reprocessed sample to the average MFI of the virgin sample  stretching), 1183 cm−1, and 1085 cm−1 (which are linked to the asymmetric vibration of the ester group (C–O–C)) [Peak X and HPeak Ref are the heights of the study peak and reference peak, respectively (1455 cm−1). These peak heights were calculated using:Fourier-transform infrared (FTIR) spectra of the virgin and processed PLA from Trials 1 and 2 were obtained using an FTIR spectrometer  in ATR mode. The room temperature measurements were performed at 4 cm(C–O–C)) . The pea(C–O–C)) :(2)Peak venumber . These pg), melt temperature (Tm), and percent crystallinity of the virgin and processed PLA samples.Differential scanning calorimetry (DSC) was used to determine changes in thermal behavior of the processed polylactic acid. The tests were carried out using a Mettler Toledo instrument . Material samples of approximately 5.0 to 9.0 mg were placed in aluminum crucibles with pierced lids. In an inert nitrogen atmosphere, these crucibles were heated to 200 °C at a rate of 10 °C/min, held in the molten state for 5 min, and then cooled to 20 °C at a rate of 10 °C/min. The heating/cooling cycle was repeated twice to erase the thermal history of the material and to evaluate the DSC curves from the second melting step. Calculations employed a melting enthalpy of 93.0 J/g reported for 100% PLA . STARe s3 and 273 cm3, respectively [ectively . The greectively ,32. Due ectively . The preAs shown in r) could be modeled as tr = 51.8 s–5.5 (Qf) where Qf is the feed rate. The coefficient of determination was 0.99.When the QSE was operated at a screw speed of 400 rpm, increasing the feed rate did not significantly increase the melt temperature. As illustrated in The complex viscosity–angular frequency curves are shown in o) and the relaxation times (λ), which were determined when the viscosity–shear rate data were put into the Carreau–Yasuda model. The virgin PLA exhibited a zero-shear viscosity of 5672 Pa.s. Processing the PLA reduced the zero-shear viscosity, with the QSE and screw configurations with kneading blocks in the mixing zone producing greater reductions in zero-shear viscosity than the TSE and screw configurations with only convey elements, respectively. This behavior may have been due to the greater shear applied by the kneading blocks and the three intermeshing zones of the QSE, but it may also have been due to the increase in residence time. PLA extruded at 1000 rpm, however, showed higher zero-shear viscosities than when the screw speed was 400 pm. This significant reduction in degradation was attributed to the shorter extrusion residence time with the higher screw speed. For all processing configurations, the trend for residence time (tr) could be modeled as:r):In addition, melt relaxation behavior can provide an indirect indication of changes in the molecular weight distribution of thermoplastic polymer materials . The vir 250 rpm  and poly 250 rpm . For thiw) were calculated from the zero-shear viscosities in −15 and 3.4) had been determined for PLA. The weight-average molecular weights and their relationship to the weight-average molecular weights of the unprocessed PLA are presented in w for the unprocessed PLA  was consistent with the molecular weight of polylactic acid (grade: 2003D) in material data mentioned by Shojaeiarani et al. [w values than the TSE. The reductions in Mw ranged from 5.31% to 9.42% for the QSE, whereas they were 1.81% to 7.47% for the TSE. The presence of kneading blocks in the mixing zone and the use of lower screw speeds also reduced the weight-average molecular weight. Since the QSE produced only a minor increase in melt temperature, the degradation in molecular weight was attributed to the longer residence time that materials were exposed to shear during extrusion. Surprisingly, the significant rise in melt temperature (~25 °C) that occurred with the higher screw speed in the TSE did not substantially contribute to the reduction in molecular weight for both screw configurations. Overall, when the processing temperature was relatively low (180 °C), degradation of the molecular weight depended primarily on residence time during the process.The weight-average molecular weights . MFI/MFIo values increased from 1.04 to 1.26, indicating degradation of PLA. The flowability of the processed PLA increased more with lower screw speeds, kneading blocks in the mixing zone, and the QSE. This behavior suggests that extruder residence time was controlling the degradation. Indeed, the MFI/MFIo exhibited as a linear relationship with residence time (tr):o/MFIo) and the slope, b, was 0.0052 s−1. The coefficient of determination was 0.89. These findings were in a good agreement with the results from the rheological measurements discussed in The melt flow index (MFI) was measured to validate the rheological analysis. The melt flow index provides an indirect assessment of the polymer’s molecular weight based on flowability at relatively low shear rates . Figure −1 and provided several insights. Firstly, upon processing PLA, the peak at 1630 cm−1 (which corresponds to H-O-H bending vibrations) was not visible in the infrared spectra. This result could have been due to thermal chain scission at the C–O bond [−1, due to the asymmetric vibration of the ester group (C–O), and at 1750 cm−1, which is associated with carbonyl stretching (C=O) [−1 region, which is attributed to changes in crystallinity [Changes in the rheological properties of PLA melts subjected to different processing conditions clearly exhibited degradation in the molecular weight of the polymer chains. Therefore, ATR-FTIR was used to assess the effects of these processing conditions on the chemical structure of PLA. C–O bond . Secondlng (C=O) ,42. Thirallinity .−1 were used to analyze chemical changes  caused by processing. Increases in the carbonyl group peak have been attributed to the appearance of new carbonyl groups both in the middle and at the end of the molecules, which is a sign of reductions in molecular weight [−1 which is assigned to the asymmetric bending of the methyl group (CH3). These absorbance ratios are shown in As the changes in the hydroxyl band for the processed PLA were not significant, the degradation occurred mainly by chain scission. The peaks at 1090, 1185, and 1750 cmr weight . The absg), melt temperatures (Tm), and crystallinity values (χc). In all processing conditions, glass transition temperatures were 57.5 °C to 58.5 °C and melt temperatures were approximately 143 °C to 146 °C. As multiple samples with the same processing conditions showed changes in Tg and Tm of 1 °C and 2 °C, respectively, these temperatures were not affected by extrusion conditions. The crystallinity varied from 0.16% to 1.37%, with multiple samples in the same processing conditions exhibiting variations of 0.06% to 0.14%. Therefore, only the QSE with KBs in zone 5 and a speed of 400 rpm produced samples with a significant change in crystallinity.The crystallization behavior of PLA can be influenced by its molecular weight . TherefoThe low crystallinity values are better understood by examining the results from the virgin PLA pellets. During the first cycle, the glass transition temperature was 59 °C and the melt temperature was 146 °C. As the pellets had crystallized during manufacturing, there was no crystallization peak during the first heating ramp . The cryThe crystallinities of the processed PLA were approximately 0%, because the absolute values of the cold crystallization and melting enthalpy are almost the same due to the slow crystallization rate of PLA and the relatively fast cooling rate (10 °C/min). The cooling rate was comparable to that used in other studies ,35,41. MThis work investigated the effect of two comparably sized compounding systems—a TSE and a QSE—on the degradation of polylactic acid (PLA). Compounding parameters included screw speed and screw configuration. Reductions in zero-shear viscosity and melt flow index indicated that the molecular weight of PLA decreased linearly with increasing residence time. Similarly, reductions in relaxation time suggested that narrowing of the molecular weight distribution decreased linearly with increasing residence time. Unexpectedly, the greater free volume and lower viscous dissipation of the QSE (compared to that of the TSE) were overridden by the longer residence time, as melt flowed across the four parallel screws. Factors that increased residence time, like the use of kneading blocks rather than conveying elements and the use of lower screw speeds, produced higher levels of degradation. Increased feed rates in the QSE reduced the residence time and, therefore, degradation of PLA. FTIR-ATR measurements confirmed that the major degradation mechanism was chain scission, and that greater chain scission occurred with longer residence times. The molecular weight reductions, however, were insufficient to produce major changes in the crystallinity of the processed PLA.


Because it is grounded below sea level5, Thwaites Glacier is thought to be susceptible to runaway retreat triggered at the grounding line (GL) at which the glacier reaches the ocean7. Recent ice-flow acceleration8 and retreat of the ice front10 and GL12 indicate that ice loss will continue. The relative impacts of mechanisms underlying recent retreat are however uncertain. Here we show sustained GL retreat from at least 2011 to 2020 and resolve mechanisms of ice-shelf melt at the submetre scale. Our conclusions are based on observations of the Thwaites Eastern Ice Shelf (TEIS) from an underwater vehicle, extending from the GL to 3 km oceanward and from the ice–ocean interface to the sea floor. These observations show a rough ice base above a sea floor sloping upward towards the GL and an ocean cavity in which the warmest water exceeds 2 °C above freezing. Data closest to the ice base show that enhanced melting occurs along sloped surfaces that initiate near the GL and evolve into steep-sided terraces. This pronounced melting along steep ice faces, including in crevasses, produces stratification that suppresses melt along flat interfaces. These data imply that slope-dependent melting sculpts the ice base and acts as an important response to ocean warming.Thwaites Glacier represents 15% of the ice discharge from the West Antarctic Ice Sheet and influences a wider catchment Thwaites Eastern Ice Shelf observations from a new underwater vehicle show that high melt rates occur where ice is sharply sloped at the ocean interface, with lower melt where the ice is comparatively flat. Thwaites Glacier extends seaward from the Walgreen Coast, forming the Thwaites Glacier Tongue (TGT) to the west and the TEIS that rests on a prominent sea-floor pinning point  onto the Amundsen Sea continental shelfint Fig. . Warm CD10), resulting in breakup of the TGT and propagation of rifts across the TEIS10. Recent GL retreat has varied from 0.6 to 1.2 km year−1 (ref. 12). Ocean melting, dynamic thinning and ice-flow rates influence this retreat12, but exactly how these factors operate is difficult to constrain with generally poor observations below the ice. Satellite observations, which measure the surface elevation of the glacier, suggest that the TEIS is thinning on average 25 metres per decade12, whereas airborne ice-penetrating radar that directly measures ice thickness estimates rates up to 45 metres per decade18.Changes in the Thwaites system have accelerated over the past 20 years -Natural Environment Research Council (NERC) International Thwaites Glacier Collaboration (ITGC), a comprehensive field campaign was carried out over two austral summers, with an ice-shelf-drilling campaign in 2019–2020 to access the ocean cavityel Figs. .Fig. 2Se−1 to 34.40 g kg−1 . We interpret this feature as the sediment wedge produced when the ice shelf was grounded at this position, approximately 1,250 m from the end of the T1 survey and 1,500 m from the furthest upstream 2017 GL location. We observe no other clear evidence of GL wedges in this region. Thus, the bathymetry suggests that the GL retreated smoothly up the prograde sea floor, with only one stable location since at least 2011.The sea floor was primarily characterized by bedform ridges oriented north–south parallel to glacier flow Fig. . Sea-flo12 Fig. . Upstrea12 Fig. ; this is38. Nearest the GL, the ice base comprises a system of short-wavelength ridges  influence melting through modulation of near-ice ocean-density gradients (stratification) and small-scale turbulence that control ocean heat and salt fluxesges Fig.  that havges Fig.  that oveges Fig.  and integes Fig. . Small t33. Terraces are also observed in crevasses. Conversely, the downstream ice under the TEIS is extremely flat, with surface slopes less than 5°  to near zero close to the interface  in recesses along terrace roofs are not fully turbulent, as salinity and dissolved oxygen exhibit much larger signatures than temperature, suggesting a regime in which diffusive processes control heat and salt flux43. The thicknesses of these fresher layers are on the order of tens of centimetres and probably reflect the transition between the fully turbulent outer and viscous inner portions of the ice–ocean boundary layer44.In the terraces, dissolved oxygen increases with decreasing temperature and salinity, consistent with input from melting ice. Some of the strongest stratification we observed was in a shallow terrace formed along the roof of another large terrace, at which the salinity of the boundary layer was 20 g kgT–S) slope of 2.05 °C (g kg−1)−1 than the melt mixing line (roughly 2.5 °C (g kg−1)−1) and decrease in dissolved oxygen with freshening  and originated as SGW that was accreted in the overdeepened basin further upstream , with a dissolved oxygen signature distinct from elsewhere in the region. These data have a shallower temperature–salinity .Although stratification suppresses melting upward , higher lateral turbulent mixingrvations , whereas−1, which matches measured melt rates on similar interfaces from three long-term ApRES and mooring data28 and are consistent with the historical estimates from ice-penetrating radar18. Nearest to the GL along each survey line, melt rates average 2 m year−1 but range from 1 to 10 m year−1  and transect 2 (T2); T1 consists of missions 1 (about 1.2 km south), 2 (about 1.9 km south) and 3 (about 1 km north) and T2 is mission 4 (about 1.6 km southeast). Missions 1-4 were conducted with the navigation module down and mission 5 with the navigation module up. Multipath in the clear basal ice reduced vehicle positioning accuracy in mission 5. T2 intersected the GL at 104.780° W, 75.214° S. For missions 1 and 3, the vehicle conducted survey segments alternating between pitching up towards the ice and down towards the sea floor at about 20–30° to acquire hydrographic profiles while making forward progress, similar to ocean gliders.C–T), pressure (P) and DO sensors. All sensors were factory calibrated before the fieldwork. C–T and DO sensors were field calibrated. Pressure measurements (1 Hz) were interpolated to match the 5-Hz C–T data to derive hydrographic variables. DO measurements (1 Hz) were not interpolated.Hydrographic data come from three sensors on Icefin: conductivity–temperature ,Remove outliers ±2 standard deviations from mean for C–T, P and DO,Apply three-point weighted mean filter to C and T measurements with time lag (0 s lag produced the best results),Align C, T and DO data for vehicle speeds <5 cm s−1,Remove 50),Derive hydrographic variables  using TEOS-10  and retrieves water-column velocities in 2-m bins at a variable start distance from the vehicle. The minimum altitude from an interface for current profiling to occur is 10 m; gradients in vehicle pitch, roll, heading and speed dictate the distance of the first bin from the vehicle and sampling frequency (maximum 5 Hz). We subsample velocities to 1 Hz. Water-column X velocities are differenced from the vehicle velocity, resulting in an uncertainty of ≤1 cm s−1. Vehicle Y and Z velocities are substantially lower than X, so Y and Z velocity uncertainties are likewise lower. Here we only analyse X and Y velocities.Ocean-current speeds are derived from the onboard DVL/ADCP, which calculates the Remove data in out-of-range bins ,−1 or 32,767 m s−1,Remove spurious data: exactly 0 m sRemove measurements when vehicle pitch or roll >+/−30°,Convert from vehicle reference frame to geographic reference frame,Apply 30 s mean filter,Filter for gradients <1 standard deviation from mean vehicle speed, pitch, roll and individual bin velocity,Collate bins by each up/down vehicle swoop into 1-m vertical bins, removing data >1 standard deviation of the mean for that range.Data post-processing:Ice-base and sea-floor elevations are derived from DVL and altimeter data and bathymetric sonar data. The DVL takes into account pitch, roll and heading when producing ranges and the altimeter and sonar data are corrected for these attitudes. Bathymetric sonar was processed in Qimera, in which obvious outliers were filtered or cleaned by hand.Remove data >2 standard deviations of the mean gradient in interface elevation (ice base or sea floor),Manually remove outliers.Data post-processing :After post-processing, 94% of ice-base measurements had horizontal resolution 26 ± 14 cm, with minimum and maximum spacings of 1.4 mm and 3.38 m, respectively. Ninety-three percent of sea-floor data had horizontal resolution 29 ± 20 cm and minimum and maximum spacings of 2.3 mm and 4.72 m, respectively.35 to estimate concentrations of water masses below the shelf, which assumes that hydrographic properties  reflect a mixture of three water masses:Θ = −0.21 °C, SA = 34.50 g kg−1, DO = 4.48 ml l−1,A source water mass (SRC) responsible for driving melt: Θ = −92.50 °C, SA = 0 g kg−1, DO = 25.20 ml l−1,GMW from local ice-shelf melt: 52: Θ = −0.34 °C, SA = 0 g kg−1, DO = 1.61 ml l−1.SGW discharged from upstream beneath the grounded glacierWe use a three-point endmember partition37. Subsets of the data not in a fully turbulent mixing regime are excluded from the partition. These data are easily identified by double-diffusive characteristics—large changes in SA (and DO) relative to Θ that result from faster molecular diffusion of heat than salt44.This partition uses conservative tracers that only vary as a result of a physical mixture of water masses and mix under fully turbulent conditionsT–S and DO–S space that fits the GMW mixing line on which our data lie: T–S and DO–S slopes of −2.49 °C (g kg−1)−1 and 0.60 ml l−1 (g kg−1)−1, respectively. SRC generally resides 10 m below the ice base (outside crevasses). Warmer, saltier and more oxygen-depleted data −1) and DO–S (0.35 ml l−1 (g kg−1)−1) space, suggestive of mixing processes not derived from local glacial modification. Thus, SRC hydrographic properties represent the local water mass melting the ice shelf, referred to as a composite tracer35. SRC is a derivative mixture of modified CDW and winter water that resides around the depth of the highly variable pycnocline separating these water masses in Pine Island Bay. Relatively weak inflow of about 3 cm s−1 into our study region and at a distance of about 75 km from Pine Island Bay28 results in a ventilation period of around one month for SRC. During this time Θ, SA and DO properties could be altered through interaction with the ice base along other sections of the TEIS, consumption by organisms or mixture with other water masses. We forgo choosing hydrographic properties for the absolute source water mass to ensure that the properties used in our three-point water-mass partition are conservative for local ice–ocean interactions.The source water mass , beyond which molecular diffusion operates57. Near-ice ocean-current measurements exhibit shear  parameterization for heat and salt transfer between the ocean and iceear Fig. , which a58, order several millimetres56. We only observed such conditions in two small regions along the roofs of two terraces with extremely fresh layers (S ≈ 20 psu) tens of centimetres thick −1, b = 9.39 × 10−2 and c = −7.53 × 10−8 °C Pa−1 are constants and TB is always at the freezing point. TB and SB are not directly observed and are estimated below.Equation  represenEquation  represenρw and T represent the density and temperature, respectively, of seawater outside the BL/VSL. For seawater, cp = 3,974 J kg−1 °C−1, the friction velocity u* is the kinematic stress at the ice–ocean interface and the heat transfer coefficient ΓT describes turbulent mixing of heat across the BL. The density of ice is ρi = 918 kg m−3, LF = 3.34 × 105 J kg−1 is latent heat of fusion and −1), in which u* to near-ice current speeds61:CD = 2.2 × 10−3 is a dimensionless drag coefficient assumed to be constant27 and U is the current speed. Without a vertical-ice-column temperature profile, we vary 62).In equation , ρw and S is salinity outside the BL/VSL, SB is the ice–ocean interface salinity and SI is the salinity of the ice shelf (considered 0 here). The salt transfer coefficient ΓS is much smaller than ΓT, owing to slower molecular diffusion of salt than heat in the VSL63. We consider a range of published values for ΓS between ΓT (refs. 59) and choose the ratio that produces optimal agreement between melt rates derived by the heat flux and salt flux equations.Equations  and 8)  8) resemT, S and U  for U because observed ocean currents increased towards the ice  by varying SB from the minimum observed salinity to the 75th percentile of S for each subregion and then converted to TB using equation  from observations beneath ice shelves55 by CD, yielding ΓT = 4.60 × 10−3–2.35 × 10−2, then considering that ΓS ranges between ΓT. We vary ΓT and ΓS from their minimum to maximum values based on the sine of the ice-base slope, from 0° to 90°, because slope/roughness of the ice interacts with stratification to produce variable melting65, and rising buoyant GMW also destabilizes stratification along steep ice slopes. The equations by which we calculated this are as follows:We estimate 38: T1 distance 2,250–2,857 m; region 2: T1 distance 1,810–1,904 m and T2 distance 1,480–1,608 m; and region 3: T1 distance 0–960 m. The set of conditions that produced the best fit to the observed melt rates along the flat (slope <5°) ice base were the 75th percentile of T and S, the 100th percentile of U and nearly the freshest SB and warmest TB, with heat conduction ΓS = ΓT = 1.03 × 10−4–5.21 × 10−4. The best-fit melt-rate estimates are: region 1: 3.41 m year−1 versus 3 m year−1 observed; region 2: 4.80 m year−1 (T1) and 4.65 m year−1 (T2) versus 5 m year−1 observed; region 3: 2.37 m year−1 versus 2 m year−1 observed.We compared calculated melt rates to ApRES data at three locations47, considering kT = 1.40 × 10−7 m2 s−1 is the molecular diffusion (conduction) of heat, ks = 1.30 × 10−9 m2 s−1 is the molecular diffusion of salt and t is the amount of time that diffusive convection has driven melting. For t = 1 h, the diffusive-convective melt rate gives 2.26 m year−1 for region 1, 2.35 m year−1 for region 2 and 2.06 m year−1 for region 3. At t = 1 day, the melt rate decreases to 0.46 m year−1 for region 1, 0.48 m year−1 for region 2 and 0.42 m year−1 for region 3. At t = 1 week, the melt estimate decreases to 0.17 m year−1 for region 1, 0.18 m year−1 for region 2 and 0.16 m year−1 for region 3. Although diffusive convection melt rates initially resemble those observed, they abate to <10% of the observations within a week as the BL grows. The ApRES melt rate time series do not exhibit this decrease, showing that, although stratification inhibits turbulence, current shear still determines the melt rate along flat and low-sloped regions of the survey area.We compared stratified, shear-driven turbulent estimates to those for an assumed diffusive-convective melt rate28; Fig. SB (22.14 psu) and therefore high TB reduces assumed thermal forcing (T − TB): the ApRES at the borehole measured lateral melt rates of 70 m year−1 on the terrace wall (mean slope 79°) at 1,800 m along T1, compared with 26.35 m year−1 estimated. Changing to a salinity of 34.28 psu (75th percentile for T1 subregion 3) obtains a melt rate of 45.97 m year−1, closer to observed.Tuning the melt rate uniformly over the survey region overestimates melt along portions of the ice base that are in diffusive regimes and possibly in the unmapped tops of crevasses; however, this is a small fraction of the region surveyed. Ocean properties will cool/freshen with height in the crevasses and current speeds may change compared with the lower 10% surveyed , the buoyancy-driven melt-rate estimate is 18.43 m year−1, considerably lower than that observed and 45.97 m year−1 estimated from shear-driven turbulence, and does not account for We also compared shear-driven melt rates to those from a buoyancy-driven turbulent melt-rate parameterizationAll statistics were performed in MATLAB. The above sections provide the details of these statistical analyses, with the underlying mean and standard deviation functions being native to MATLAB.Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-022-05691-0.Supplementary Video 1Forward-looking video of Icefin’s approach to the GL of the TEIS shows visual evidence of melting and ice–bed interactions. Clear basal ice with embedded pebbles and stones and laminated sediments is observed through the region (video clips from Icefin top forward camera over the last 200 m towards the GL of the TEIS). Variable melting causes particles and pebbles to fall from the ice. The rough, ridged ice base shows topography inherited from scraping along the sea floor as well as the initiation of small, sinuous terraces with scalloped interfaces. At the end of the video, the water column was less than 50 cm thick and the ice is observed resting on the bed in the distance in front of the vehicle.