Tectorigenin is a well-known natural flavonoid aglycone and an active component that exists in numerous plants. Growing evidence suggests that tectorigenin has multiple pharmacological effects, such as anticancer, antidiabetic, hepatoprotective, anti-inflammatory, antioxidative, antimicrobial, cardioprotective, and neuroprotective. These pharmacological properties provide the basis for the treatment of many kinds of illnesses, including several types of cancer, diabetes, hepatic fibrosis, osteoarthritis, Alzheimer’s disease, etc. The purpose of this paper is to provide a comprehensive summary and review of the sources, extraction and synthesis, pharmacological effects, toxicity, pharmacokinetics, and delivery strategy aspects of tectorigenin. Tectorigenin may exert certain cytotoxicity, which is related to the administration time and concentration. Pharmacokinetic studies have demonstrated that the main metabolic pathways in rats for tectorigenin are glucuronidation, sulfation, demethylation and methoxylation, but that it exhibits poor bioavailability. From our perspective, further research on tectorigenin should cover: exploring the pharmacological targets and mechanisms of action; finding an appropriate concentration to balance pharmacological effects and toxicity; attempting diversified delivery strategies to improve the bioavailability; and structural modification to obtain tectorigenin derivatives with higher pharmacological activity. It is well known that natural products or botanicals are valuable resources in the treatment of dozens of diseases. Up until now, natural products remain an important basis for the development of emerging therapeutic agents . Newman Tectorigenin is an isoflavone that exists in numerous plant resources, and it is especially abundant in Belamcandae Rhizoma and Puerariae flos ,18, whicBelamcanda chinensis (L.) DC., a traditional Chinese medicine (TCM) named Belamcandae Rhizoma [Iris spuria L.  [Iris tectorum Maxim [Iris japonica Thunb. [Iris dichotoma Pall. [Iris germanica L. [Iris unguicularis Poiret [Iris loczyi Kan. [Iris kashmiriana Baker [Iris crocea Jacq. ex R. C. Foster [Iris ensata Thunb. [Iris germanica L. [Iris hungarica Waldst. et Kit. [Iris confusa Sealy [Iris pseudacorus L. [Pueraria lobata (Willd.) Ohwi [Pueraria thomsonii Benth. [Pueraria thunbergiana Benth. [Dalbergia odorifera T. Chen [Dalbergia parviflora Roxb. [Euchresta formosana (Hayata) Ohwi as well [Codonopsis pilosula (Franch.) Nannf. (Campanulaceae) [Morus alba L. (Moraceae) [Viola hondoensis W. Becker et H. Boissieu. (Violaceae) [Eleocharis dulcis (Burm. f.) Trin. ex Hensch. (Cyperaceae) [Tectorigenin, a methoxy isoflavone with three hydroxyl groups, is chemically noted as 5,7-dihydroxy-3-(4-hydroxyphenyl)-6-methoxy-4H-chromen-4-one . As a bi Rhizoma ,26. The alizona) , Iris teum Maxim , Iris jaa Thunb. ,30, Irisma Pall. , Iris geanica L. , Iris uns Poiret , Iris lozyi Kan. , Iris kana Baker , Iris cr. Foster ,36, Irisanica L. , Iris hu et Kit. , Iris cosa Sealy , and Iricorus L. , containcorus L. . Tectorid.) Ohwi ,41, Pueri Benth. ,40, and a Benth. , which a T. Chen , the heara Roxb. , and the as well . Moreoveulaceae) , Morus aoraceae) , Viola holaceae) , and Eleeraceae) . The pla2) was also used in some studies [Pueraria thomsonii, and an extraction yield of about 0.15 mg/g tectorigenin was obtained from 500 g raw material by countercurrent chromatography and semipreparative liquid chromatography. High levels of glycoside forms of tectorigenin, such as tectoridin, also exist in Belamcandae Rhizoma and Puerariae Flos, so the yield of tectorigenin can be increased by acid hydrolysis during extraction [Pueraria thunbergiana with H2SO4 to a final concentration of 1% (w/v), and the tectorigenin content increased from 17.10 µmol/g before acid hydrolysis to 49.58 µmol/g [The extraction and isolation of tectorigenin from plants with a high tectorigenin content have been widely performed in practical industrial production. For instance, tectorigenin can be prepared on a large scale from Belamcandae Rhizoma and Puerariae Flos ,40,49. T studies . The cru studies , chlorof studies , methyle studies . In most studies , and oct studies . Joung e studies  used the studies  first detraction . Kim et 8 µmol/g .2O  at 85 ℃ under reflux for 4 h in MeOH/H2O with a yield of around 37.5% [In addition to being isolated from plants, there are two approaches to obtaining synthetic tectorigenin : (i) It nd 37.5% ,61, and nd 37.5% , althougnd 37.5% ,64. In 1nd 37.5%  pointed nd 37.5% ,67 propoAs a ubiquitous plant isoflavone, tectorigenin has been widely reported to be effective in many areas, such as anticancer, anti-diabetes, hepatoprotection, anti-inflammation, antioxidation, antimicrobial, etc. . It seemPuerariae thomsonii was found to possess the highest anti-proliferation activity against prostate cancer cells (IC50 = 0.08 μM) [Several epidemiological studies support the role of phytoestrogens, nonsteroidal plant-derived compounds with estrogenic activity, in reducing cancer risk . In the 0.08 μM) . Stettne0.08 μM)  also repBreast cancer is a major life-threatening malignancy that affects women all around the world . MCF-7, 50 of tectorigenin against A2780 cells was 48.67 ± 0.31 μM [Ovarian cancer is another common gynecologic malignancy with a high mortality rate ,80. New  0.31 μM .50 = 776.12 μg/mL) [There are also several studies on the effect of tectorigenin on lung cancer. The ethyl acetate extract of Chinese water chestnut peel, which contains tectorigenin at levels of 12.41 mg/g, showed good inhibitory activities on human alveolar adenocarcinoma cell line A549 (IC2 μg/mL) . Tectori2 μg/mL) . In gene2 μg/mL) . It was 2 μg/mL)  that tecIt has been shown by Jiang et al.  that tecDiabetes is a metabolic disorder characterized by hyperglycemia that has become a global epidemic . Tectori50 = 13.3 mM) and suggested that tectorigenin may ameliorate hyperglycemia through the inhibition of preadipocyte differentiation and adipocytokine secretion [Diabetes is closely linked to the epidemic of obesity . The antecretion . The stu4) and tert-butyl hyperoxide-induced liver damage [The liver plays a pivotal role in bile synthesis, metabolic function, and the degradation of toxins in the body . Severalr damage ,105,106.r damage ,106. Sevr damage ,105,107.r damage . It has r damage ,109. An r damage . Anotherr damage . Additio2 in inflammatory cells. This may be one of the mechanisms of action by which Belamcandae Rhizoma exert an anti-inflammatory effect [Several flavonoids, including tectorigenin, have been reported to inhibit nitric oxide (NO) production, one of the inflammatory mediators . Tectoriy effect ,114,115.y effect , LPS-stiy effect . In the y effect . An in vy effect . Wang ety effect  (2020) ay effect . Tectoriy effect . In addiy effect ,117,120.4 and bromobenzene-induced malondialdehyde formation in the rats [3+/ferricyanide reduction, hydroxyl radical and superoxide anion radical scavenging, DPPH, and lipid peroxidation assays in vitro. Another study evaluated the effects of Belamcandae Rhizoma extract containing tectorigenin on collagen degradation and apoptosis in UV-B-induced HaCaT cells. The extract exhibited good free radical scavenging capability and cytoprotective effects, and mitigated cell apoptosis through diminishing caspase-3 levels and increasing the B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X (Bax) ratio [A loss of balance in biological systems between the production of ROS and antioxidant defense levels can induce oxidative stress, which may result in extensive intracellular damage ,122. Thethe rats ,128. Tecthe rats , maintaix) ratio . Taken tTrichophyton ranged from 3.12–6.25 μg/mL [Helicobacter pylori strains (MIC: 50–100 μg/mL) [S. aureus ATCC 33591, S. aureus ATCC 25923, which were strains of methicillin-resistant Staphylococcus aureus (MRSA), and three clinical isolates of MRSA with MIC values of 125 μg/mL [Clostridium perfringens infection, the biofilm formation rates in the presence of 4, 8, 16, and 32 μg/mL tectorigenin were well below the control in a dose-dependent manner [Clostridium perfringens. However, more research is still needed to further verify the antibacterial activity of tectorigenin in cells and animals [The increasing incidence of microorganisms’ drug resistance makes the search for new antimicrobial agents urgent. In this connection, medicinal plants are promising resources . In 200125 μg/mL , and als0 μg/mL) . In anot25 μg/mL . Furthert manner . In addi animals .Common skeletal diseases, such as osteoarthritis, osteoporosis, fractures, etc., greatly affect the quality of life of older adults . The homUltraviolet radiation (UV) can induce skin photoaging and inflammation, and also lead to free radical and ROS accumulation, collagen loss and degradation, epidermal pigmentation, and abnormal cell death . Several2O2-induced oxidative stress injury by upregulating the PI3K/AKT pathway. In addition, tectorigenin could alleviate cognitive impairment, hippocampal tissue and myelin damage, and inflammation of chronic cerebral ischemia mice [Cardiovascular and cerebrovascular diseases, including angor pectoris, myocardial infarction, stroke, etc., are the leading causes of death globally . Atherosmia mice . In vitrmia mice ,153.Tectorigenin was regarded as an interventional strategy by inhibiting respiratory dysfunction and death in respiratory diseases. Several studies have illustrated the protective effect of tectorigenin in respiratory disorders, including asthma, airway epithelial injury, and pulmonary fibrosis ,154,155.50 values were 1.3–2.3 μM). From the structure-activity relationships of a number of isoflavonoids with inhibitory activity against microglial activation, the methoxyl group at the 6-position of tectorigenin enhanced the activity. Glioblastoma cell viability was dose-dependently decreased after 24 h exposure to tectorigenin, and 200 μM and 300 μM could block the cell cycle arrest at G0/G1 phase [Pueraria thomsonii, and the IC50 value of tectorigenin against MAO-B was 54.36 μg/mL. In addition, pretreatment with tectorigenin exhibited a protective effect against neuronal damage in PC12 cells. Gong et al. (2017) [In neurodegenerative diseases caused by multiple pathological procedures, neuroprotection is an interventional strategy to delay and even stop neuronal dysfunction and abnormal death . MicroglG1 phase . AnotherG1 phase . As a glG1 phase ,162, butG1 phase . TectoriG1 phase . MonoamiG1 phase . Li et aG1 phase  isolated. (2017)  investig. (2017) .Except for the pharmacological activities mentioned above, tectorigenin also has other biological activities. For instance, tectorigenin was verified as a therapeutic strategy for the treatment of obstructive nephropathy. In mice suffering unilateral ureteral obstruction, tectorigenin significantly decreased the levels of the kidney injury index, including creatinine, blood urea nitrogen, and kidney injury molecule-1 (KIM-1), and alleviated pathological damage and renal interstitial fibrosis . In vitr2+ homeostasis in HepG2 cells [2+ concentration may trigger some cellular responses, so caution is needed when using tectorigenin. The results of Cheng et al. [2+ influx might be evoked by protein kinase C (PKC)-insensitive store-operated Ca2+ entry and Ca2+ release from the endoplasmic reticulum via phospholipase C (PLC)-associated pathways. Tectorigenin at concentrations between 10–60 μM exerted renal cytotoxicity, and the IC50 was approximately 45.5 μM. However, using a cytosolic Ca2+ chelating agent did not affect the cytotoxicity of tectorigenin, suggesting that tectorigenin caused cell death in a Ca2+-independent manner [50 of tectorigenin by intragastric administration was 1.78 g/kg in swiss mice, and no significant differences in body weights, food consumption, or hematological or biochemical parameters were observed at tectorigenin’s doses not exceeding 300 mg/kg during a consecutive 28-day treatment [Evidence shows that tectorigenin affected CaG2 cells , HSC-T6 G2 cells , isolateG2 cells , and MadG2 cells . The chag et al.  showed tt manner . In termt manner . In anott manner . In addireatment . In anotreatment . Similarreatment . In strereatment . These sreatment ,143. HowThe pharmacokinetic properties, including absorption, distribution, metabolism, and excretion, are critical for determining the optimal dose of compounds and the frequency of administration as well as for ensuring drug adherence, and all of these play an important role in clinical application . After rmax) of 12.0 ± 0.63 μmol/L, an area under the curve (AUC0–t) of 84.2 ± 8.15 μmol/L × h, whereas the long half-life period (t½) 11.7 ± 5.74 h showed that tectorigenin had a long action time [max of 1.46 ± 0.30 μg/mL and AUC(0–t) of 7.31 ± 1.20 μg/mL × h [(0–t) of 219 ± 94 ng/mL × h [Iris tectorum, also described the pharmacokinetic characteristics of tectorigenin as the main component [Iris tectorum extract, and the same phenomenon was observed in some other studies as well [Nevertheless, due to its low water solubility and low permeability, tectorigenin showed poor absolute bioavailability after oral administration in rats. It was reported that oral tectorigenin (130 mg/kg) administration resulted in a peak concentration  Although various biological activities of tectorigenin are confirmed in vitro and in vivo, the molecular mechanisms of action are still unclear, especially the binding sites of definitive target proteins, which remain undetermined. Hence, it is of great significance to further explore the mechanism of its biological activity at the molecular level. (2) The safety of a new drug is especially important before it is used in clinical practice, and more systematic studies of dosage should be conducted to balance both its pharmacological effect and its toxicity. It is also necessary to carry out toxicity evaluations of multiple organs. (3) At present, there are few studies on pharmacokinetics, which should be conducted on more animals in the future, such as dogs and monkeys. Different methods of administration are also worth trying. (4) Pro-drug design, formulation optimization, and novel drug delivery systems, such as nanotechnology, including liposomes, nanoparticles, phytosomes, dendrimers, polymer-drug conjugates, and microparticles, could be attempted to increase tectorigenin’s water solubility, stability, and bioavailability. (5) Structural modification is still an efficient and promising way to obtain tectorigenin derivatives with higher pharmacological activity and relatively better bioavailability, while some key active substituent groups need to be retained.


A central question is how the elementary chemical steps occurring in the three catalytic sites are coupled to the mechanical rotation. Here, we performed cold chase promotion experiments and measured the rates and extents of hydrolysis of preloaded bound ATP and promoter ATP bound in the catalytic sites. We found that rotation was caused by the electrostatic free energy change associated with the ATP cleavage reaction followed by Pi release. The combination of these two processes occurs sequentially in two different catalytic sites on the enzyme, thereby driving the two rotational sub-steps of the 120o rotation. The mechanistic implications of this finding are discussed based on the overall energy balance of the system. General principles of free energy transduction are formulated, and their important physical and biochemical consequences are analyzed. In particular, how exactly ATP performs useful external work in biomolecular systems is discussed. A molecular mechanism of steady-state, trisite ATP hydrolysis by F1-ATPase, consistent with physical laws and principles and the consolidated body of available biochemical information, is developed. Taken together with previous results, this mechanism essentially completes the coupling scheme. Discrete snapshots seen in high-resolution X-ray structures are assigned to specific intermediate stages in the 120o hydrolysis cycle, and reasons for the necessity of these conformations are readily understood. The major roles played by the “minor” subunits of ATP synthase in enabling physiological energy coupling and catalysis, first predicted by Nath's torsional mechanism of energy transduction and ATP synthesis 25 years ago, are now revealed with great clarity. The working of nine-stepped , six-stepped , and three-stepped (PdF1) F1 motors and of the α3β3γ subcomplex of F1 is explained by the same unified mechanism without invoking additional assumptions or postulating different mechanochemical coupling schemes. Some novel predictions of the unified theory on the mode of action of F1 inhibitors, such as sodium azide, of great pharmaceutical importance, and on more exotic artificial or hybrid/chimera F1 motors have been made and analyzed mathematically. The detailed ATP hydrolysis cycle for the enzyme as a whole is shown to provide a biochemical basis for a theory of “unisite” and steady-state multisite catalysis by F1-ATPase that had remained elusive for a very long time. The theory is supported by a probability-based calculation of enzyme species distributions and analysis of catalytic site occupancies by Mg-nucleotides and the activity of F1-ATPase. A new concept of energy coupling in ATP synthesis/hydrolysis based on fundamental ligand substitution chemistry has been advanced, which offers a deeper understanding, elucidates enzyme activation and catalysis in a better way, and provides a unified molecular explanation of elementary chemical events occurring at enzyme catalytic sites. As such, these developments take us beyond binding change mechanisms of ATP synthesis/hydrolysis proposed for oxidative phosphorylation and photophosphorylation in bioenergetics.F OF1–ATP synthase catalyzes the ATP synthesis/hydrolysis reaction, vital to life in all living organisms  with a conserved subunit stoichiometry α3β3γ δε in all organisms. In E. coli F1, the molecular masses of α, β, γ, δ, and ε measure 55.3, 50.3, 31.6, 19.3, and 14.9 kDa, respectively  in the catalytic cycle, and assign them to specific conformations of the enzyme during catalysis.A definitive solution to the aforementioned longstanding “elusive” problems is attempted in this work. The refined molecular mechanism of ATP hydrolysis by F1-ATPase are deduced in 1-ATPase consistent with physical laws and principles and the body of available biochemical information that goes beyond previous theories , six-stepped , and three-stepped (PdF1) F1 motors and of the α3β3γ subcomplex of F1 is explained by a unified mechanism. The theory is supported by a probability-based calculation of enzyme species distributions and analysis of catalytic site occupancies by Mg-nucleotides and the activity of F1-ATPase. Some novel predictions of the unified theory that are of pharmacological importance are also made in ligand substitution chemistry is proposed, which takes us beyond the binding change mechanism of ATP synthesis/hydrolysis.This article is organized as follows. theories  is formuA mathematical model for estimating economics and opportunity cost in choosing between competing theories is developed in the 1 was prepared from bovine heart mitochondria using standard procedures, as described previously 2SO4 supernatant. The pellets were dissolved in 100 μL Mg buffer, and the enzyme solution was passed through a centrifuge column equilibrated with the same buffer. The enzyme was incubated with Mg buffer for 1 h at 23°C. [γ-32P]ATP was prepared as described previously .MFeviously . The speeviously . The spepparatus . The err1 in Mg buffer was added to a glass reaction vessel containing Mg buffer to determine the maximum equilibrium hydrolysis of preloaded and promoter ATP in cold chase experiments. The solution was stirred at high speed with a magnetic stirrer and stirrer bar. A volume of 20 μL of 15 μM [γ-32P]ATP or ATP was pipetted and incubated for 3 s. Chase solutions were pipetted from 0.5 mM stock solutions of ATP or [γ-32P]ATP, respectively. The final volume measured 1.0 ml in Mg buffer. After the addition of the chase, the solutions were incubated for 10 s, and the reaction was thereafter terminated by adding 0.2 ml of 70% perchloric acid. For the control without adding promoter ATP, the reaction was allowed to continue for 10 s and quenched by adding perchloric acid before the chase. 32Pi was separated from [γ-32P]ATP and counted as described  0.5 μM each and 2) 1 μM each in Mg buffer at 23°C. In other experiments, 10% excess F1 was employed with respect to the substrate to obtain the distribution of bound substrate and product at F1-ATPase catalytic sites. A complex between the two species was allowed to form, and unbound 32P was removed on centrifuge columns. Column effluents were collected at 1, 5, 10, and 15 min in perchloric acid quench, the bound 32Pi was determined, and the fraction 32P present as 32Pi was quantitated at various times.F1 with 0.3 μM [γ-32P]ATP, i.e., under sub-stoichiometric conditions that predominantly favor substrate binding to a single catalytic site of the F1-ATPase , results in the formation of an enzyme–substrate complex. This is followed by the hydrolysis of ATP to ADP.Pi on the enzyme, limited by the slow rate of dissociation of the products  site with intermediate affinity for ATP) results in hydrolysis of 92%–96% of [γ-32P]ATP bound in the highest affinity catalytic site 1. Similar results were obtained when 3 mM ATP is used as the chase when all the three catalytic sites of F1 are expected to be occupied by bound nucleotide. Note that a super-stoichiometric concentration of promoter ATP is essential to ensure that the F1-ATPase undergoes multiple cycles of rotation during the 10 s incubation time, given that a major objective of the work is to detect the rate enhancement (over unisite rates) that increases turnover to Vmax when ATP is allowed to bind at multiple catalytic sites. However, complete  hydrolysis of the added ATP was not observed even with mM concentrations of the cold chase. 32P]ATP is used as the chase, 92%–94% of the promoter ATP is hydrolyzed in site 2 (L) in the same 10 s time period.As shown in step 1 of the reaction scheme  are recruited into the “normal” catalytic pathway of F1-ATPase during rapid multisite Vmax hydrolysis of at least 100 s–1 by the addition of excess ATP, and as a result, the unisite characteristics of F1 are amalgamated. Furthermore, the bound ATP in site 1 is hydrolyzed during the chase at approximately the same rate as the chase ATP bound at a catalytic site with intermediate affinity and not at substantially lower rates as reported  hydrolyze ATP at approximately the same rates and with similar kinetics (the following paragraph and The results described in te on F1 . Howeverreported . We estie higher . In this1 (at different times) can hydrolyze ATP at the so-called highest affinity site 1 (T)  due to binding and subsequent hydrolysis of ATP at site 2 causes site 1 (T) to be converted to (a new) site 2 (L), then similar rates and extents of ATP hydrolysis in the two cases are also logically explained. In other words, the two sites are the same: both are L-sites (site 2). In other words, only site 2 can hydrolyze ATP and release products.The cold chase experimental results have an even more important biological implication: they show that two catalytic sites on Fte 1 (T)  and at t32Pi to total bound 32P at two different concentrations of F1 and [γ-32P]ATP and various incubation times from 1 to 15 min. This ratio was approximately constant at one-third under all conditions tested; i.e., the data shown in 1 over substrate was used. It should be clearly understood that the total bound 32P includes both bound 32Pi and bound [γ-32P]ATP species. The results can be either interpreted in terms of an equilibrium constant for reversible hydrolysis or explained equally well by a quasi-steady state constant for irreversible hydrolysis in which 1-ATPase simply binds ATP in site 2 (L); the enzyme needs to additionally hydrolyze the bound ATP (that had exchanged with ADP in site 2 (L)) (1 side) also changes the conformation of site 1 (T) to a new site 2 (L), after which the bound ATP hydrolyzes in the new site 2 (L) to ADP.Pi, and Pi subsequently leaves and donates energy for a further ∼40° rotation, as described in great detail previously )  to ADP.Pe 2 (L))  in site eviously , and mateviously  , with a very low dissociation constant Kd—vis-à-vis site 2 (L) such that ATP is differentially stabilized on the enzyme surface relative to ADP + Pi by eq close to 1. Historically, these concepts have greatly influenced the interpretation of experimental measurements and catalytic mechanism in F1-ATPase and muscle myosin. However, are these concepts correct?According to the basic tenets of the binding change mechanism , the prid of 1 pM for catalytic site 1 , site 2 (L), and site 3 (O) of 0.02, 1.4, and 23 μM, respectively  relative to (ADP + Pi) . The enzThe aforementioned general principle of energy transduction also explains why non-hydrolyzable analogs of ATP cannot perform useful external work. This primarily arises because the free energy available by reversal of the conformational change due to the binding of the ATP analog is used for the release of the bound analog. Hence, it cannot perform useful mechanical work. However, in the case of ATP hydrolysis, the products ADP and Pi are more stable in solution  is cleaved, the binding of Pi to the catalytic site is reduced, and the Pi allowed to move away to infinity from bound ADP; this energy can be used for the performance of useful work by F1 and by biological systems in general  in F1. The enzyme also needs to hydrolyze the bound ATP in site 2 (L)  to ADP.Pi. Subsequently, Pi needs to move away and be released from L, as explained in detail in E. coli F1-ATPase, i.e., the difference between the binding energy of MgATP in L (36 kJ/mol) and the binding energy of MgADP in L (27 kJ/mol). This 9 kJ/mol energy released weakens the binding of bound Pi formed upon ATP hydrolysis in site 2 to approximately zero . The efnd MgADP . As Pi mnd MgADP , it rele hexamer .o rotation of the top of γ, site 2 (L) changes to a closed conformation Cʹ. Upon the above 80o rotation, the top of γ interacts with the β-catalytic site 1 ATP cold chase experiments (The present work shows that site 1 (T) contains bound ATP that does not hydrolyze by itself in T. In other words, site 1 contains tightly bound, non-exchangeable MgATP. Hence, “unisite” hydrolysis cannot take place in site 1, contrary to the original view of eriments ; Table 11 enzyme does not hydrolyze ATP  is also in full agreement with the finding of an important recent cryo-EM structural study performed under various reaction conditions  can be modeled by a simple probabilistic approach. If The filling of multiple FThe probability that the site is not occupied by bound ligand is given by1-ATPase, respectively, then the fraction of the enzyme species with all three sites occupied  is given as follows:If The fraction of species with sites 1 and 2 occupied by MgATP (but with site 3 unoccupied) is given by1 for the three sites have not been reliably measured. However, we can simulate the system with the 1. It is known from previous studies that EF1 is very similar to MF1 in terms of nucleotide exchangeability and other biochemical properties, except that the binding at the lowest-affinity catalytic sites is less tight  does not alter the results presented in the next paragraph to any appreciable extent.Dissociation constant values for MFss tight . Using cectively . Use of OF1-ATP synthase , even at sub-stoichiometric conditions of 0.3 μM ATP and 1 μM F1 (–1) rate of ATP hydrolysis. At higher ATP concentrations (super-stoichiometric conditions), events due to cold chase by [110] species are integrated into multisite catalysis  rotary mechanism operates for Fllimolar .1-ATPase are logically explained.We have thus shown in 1-ATPase   , in cleatalysis) , we consportance   can reasynthase .1-ATPase. This view has been shown to be incomplete and incorrect. Fresh insights into the mechanism are obtained by measuring the maximum extents and kinetic rates of hydrolysis of preloaded bound [γ-32P]ATP and promoter [γ-32P]ATP in cold chase experiments. Mechanistic implications arising from the experiments and the first law of thermodynamics have been spelled out, and general physical principles for biological free energy transduction have been formulated. The question of how ATP acts as an energy source and performs useful external work has been asked and answered. A molecular mechanism of steady-state trisite ATP hydrolysis by F1-ATPase, which is consistent with the formulated physical principles and the body of available biochemical information, has been constructed. Only such a detailed mechanism can truly explain the function of F1-ATPase as a molecular energy machine. The detailed mechanism of the 120o catalytic cycle (E. coli EF1 (1 (o (or 120o) ATP-binding dwell compared to the Leslie–Walker structures , six-stepped , and three-stepped (PdF1) F1 motors and of the α3β3γ subcomplex of F1 is explained by the unified theory without invoking additional assumptions or postulating different coupling schemes. The aforementioned developments have wide ramifications (The formulated general physical principles of free energy transduction  and Sect and the 1-ATPase  have imp1-ATPase . Severalications  for a wh


The fluorescence quenching studies indicate that the intrinsic fluorescence of α-glucosidase was quenched via a static quenching process and only one binding site was present between the hesperidin and α-glucosidase. The interaction between them was spontaneous and mainly driven by hydrogen bonds, as well as hydrophobic forces. Furthermore, the molecular docking results suggest that hesperidin might bond to the entrance or outlet part of the active site of α-glucosidase through a network of five hydrogen bonds formed between hesperidin and the four amino acid residues  of α-glucosidase and the hydrophobic effects. These results provide new insight into the inhibitory mechanisms of hesperidin on α-glucosidase, supporting the potential application of a hesperidin-rich orange product as a hypoglycemic functional food.The α-glucosidase inhibitor is of interest to researchers due to its association with type-II diabetes treatment by suppressing postprandial hyperglycemia. Hesperidin is a major flavonoid in orange fruit with diverse biological properties. This paper evaluates the effects of hesperidin on α-glucosidase through inhibitory kinetics, fluorescence quenching, and molecular docking methods for the first time. The inhibition kinetic analysis shows that hesperidin reversibly inhibited the α-glucosidase activity with an IC Diabetes mellitus (DM) is a metabolic illness presently influencing 425 million individuals around the world, and the overall number is expected to rise to 642 million by 2045 . In addiAnoectochilus roxburghii  and [I] represent the concentrations of the substrate pNPG and the inhibitor hesperidin, respectively.The inhibition kinetic analysis was performed by the same procedure as that described for the in vitro α-glucosidase inhibition assay. For the reversibility test, the concentration of substrate s method .(2)1V=K−5 M for hesperidin, respectively. The corresponding enzyme solution without the addition of hesperidin was used as a blank control. At each concentration after the titration of hesperidin, the intrinsic fluorescence of the α-glucosidase enzyme was scanned using a fluorescence spectrophotometer at a wavelength of 300–500 nm after being excited at 280 nm. The emission and excitation slit width was set to 2.5 nm. The fluorescence spectra were recorded at three different temperatures . All the fluorescence data were corrected based on Equation (5) to subtract the internal filtering effect caused by the ultraviolet absorption , suggestive of the type of uncompetitive inhibition [The reversibility of the inhibitory effect was investigated by the plot of the α-glucosidase concentration ([α-glucosidase]) versus the velocity (V) of the enzymatic reaction. As shown in hibition . Km and Vmax values acquired from Equation (2) gradually decreased with the increase of the hesperidin concentration, which is in line with the uncompetitive inhibition kinetics of cinnamic acid amide on α-glucosidase  is the concentration of the inhibitor; Kq and KSV represent the bimolecular quenching rate constant and quenching constant, respectively; and τ0 represents the fluorophore lifetime of a free biomacromolecule, which is approximately 10−8 s for α-glucosidase  exhibits good linearity at all three temperatures , indicating that the quenching process by hesperidin was a single static or dynamic quenching  and [Qt] were the concentrations of α-glucosidase and hesperidin, respectively.By using the fluorescence quenching data, the binding constant (tion (7)  was alsoKa at 298, 304, and 310 K were calculated. As shown in Ka values had a magnitude order of 104 L mol−1, suggesting a strong binding affinity between α-glucosidase and hesperidin [Ka value decreased with the increase in the reaction temperature, indicating that the stability of the inhibitor–enzyme complex decreased with the increase in temperature, and this result further confirms that the fluorescence quenching by hesperidin was a static process. Furthermore, the n values  obtained at three different temperatures were all close to 1 (According to Equation (7), the values of ose to 1 , suggestH°), entropy change (ΔS°), and the Gibbs free energy (ΔG°) of the binding process were calculated according to the Equations (8) and (9) (van’t Hoff equation), where R represents the gas constant (8.314 J mol−1 K−1), and Ka is the binding constant at the corresponding temperature.To further identify the driving forces for the formation of the α-glucosidase–hesperidin complex, the thermodynamic parameters involving enthalpy change (ΔH°) can be considered as a constant [G° of the present study was around −25 KJ mol−1 at all three temperatures, less than zero (ΔG° < 0), indicating that the binding of hesperidin with α-glucosidase was a spontaneous procedure [H° < 0 and ΔS° > 0 (In the present study, since the variation range of the temperature was narrow, the enthalpy change (Δconstant . The mairocedure . Further ΔS° > 0  suggest G° < 0. As shown in Molecular docking was used to further determine the interaction between α-glucosidase and hesperidin . The lowAs an uncompetitive inhibitor, hesperidin only binds with the enzyme–substrate complexes, showing no competition to the active center of the enzyme. Therefore, the present results allow us to propose that the hesperidin might bond to the entrance or outlet part of the active center, thereby obstructing the release of the substrate and catalytic reaction product from the active center, eventually inhibiting the enzymatic activity of α-glucosidase. Similarly, uncompetitive inhibitors, like cinnamic acid amide , corosolThe present research elucidates the inhibitory mechanism of hesperidin on α-glucosidase through enzyme kinetics, thermodynamics, and a molecular modeling approach for the first time. Hesperidin was found to exhibit a significant inhibitory effect on the enzymatic activity of α-glucosidase through a reversible and uncompetitive inhibition mode. The fluorescence quenching results indicate that hesperidin spontaneously bonded to α-glucosidase with one binding site by non-covalent interactions, which mainly consisted of hydrogen bonds and hydrophobic effects. The molecular docking studies suggest that hesperidin might bond to the entrance or outlet part of the active center of the enzyme, thus blocking the release of the substrate and catalytic product. Further research via in vivo experiments involving the efficacy and safety evaluations would be much more supportive of the antidiabetic application of hesperidin. Meanwhile, some of the other major flavonoids present in orange fruit, such as narirutin, didymin, sinensetin, and nobiletin, should be assessed for their synergistic effects alongside hesperidin for inhibiting the α-glucosidase effect.


The oxygen isotope data allow to discriminate the different freshwater processes showing that in the subpolar region, the freshening is largely driven by the increase in net precipitation (by a factor two) while the decrease in sea ice melt is largely balanced by the contribution of glacial meltwater at these latitudes. These changes extend the growing evidence for an acceleration of the hydrological cycle and a melting cryosphere that can be expected from global warming.The hydrological cycle is expected to intensify in a warming climate. However, observational evidence of such changes in the Southern Ocean is difficult to obtain due to sparse measurements and a complex superposition of changes in precipitation, sea ice, and glacial meltwater. Here we disentangle these signals using a dataset of salinity and seawater oxygen isotope observations collected in the Indian sector of the Southern Ocean. Our results show that the atmospheric water cycle has intensified in this region between 1993 and 2021, increasing the salinity in subtropical surface waters by 0.06 ± 0.07 g kg Based on concurrent salinity and oxygen isotope observations, the authors find that amplification of the atmospheric water cycle is the main contributor to changes in surface salinity in the Indian Southern Ocean over the past three decades. The temperature changes are accompanied with a near-surface freshening in the subpolar Southern Ocean and extending at depth into the Antarctic intermediate waters8. These patterns of changes have important consequences for regional and global climate. The large warming north of the ACC indicates the capacity of the Southern Ocean to take up large amounts of excess heat associated with climate change and to store it in intermediate layers, thereby buffering global atmospheric warming9. Changes in salinity mirror shifts in the water cycle that strengthen the static stability of the subpolar upper ocean, with consequences for the large-scale circulation12, sea ice regime13, and surface subpolar ocean cooling15. However, despite their widespread importance, the processes that cause changes in Southern Ocean temperature and salinity remain not fully understood. A number of studies have suggested that the widespread subpolar Southern Ocean surface freshening is consistent with an amplification of the global hydrological cycle associated with an increase in net precipitation at high latitudes18. Other studies have proposed different mechanisms including intensified freshwater flux from increased ice shelf and iceberg melting20, or from intensification of sea ice freshwater transport21. While climate models suggest that changes in Southern Ocean salinity over the past decades can be attributed in part to human-induced forcing22, these simulations are known to not include a realistic representation of changes in meltwater from Antarctic ice shelves, as well as having questionable representation of Antarctic sea ice cover and trends, and a highly biased representation of net precipitation27.Over the past several decades, the Southern Ocean has experienced a complex array of hydrographic changes. These include large areas of the subpolar sector, south of the Antarctic Circumpolar Current (ACC), that have slightly cooled at the ocean surface∘E and 90∘E, Fig. 28 and which provides a regional sampling of surface salinity and δ18O (relative proportion of 18O to 16O referenced to Vienna Standard Mean Ocean Water) measurements; and additional hydrographic sections in the same sector of the Southern Ocean that span over the period 1993 to 1998. This dataset gives access to concurrent δ18O observations, allowing for the investigation of long-term changes in the freshwater sources that have distinct δ18O signatures. In the open ocean, away from the poles, the δ18O of surface waters is predominantly controlled by atmospheric processes —increasing from evaporation and decreasing from precipitation. At the poles, however, δ18O is also impacted by continental ice melt, which is very depleted in δ18O29, while salinity is impacted by both continental ice melt and sea ice formation and melt. As a result, δ18O is a useful tracer to differentiate freshwater signals coming from meteoric  or oceanic (sea ice) sources32.In this paper, we explore long-term observations of seawater oxygen isotope and salinity in surface waters of the Indian sector 33 and associated with the ACC. North of the SAF-N (north of ∘S), the two properties actually follow a clear linear slope depending on the evaporation and precipitation characteristics of the region34  that the water parcel encountered during its lifetime, which tends to be advected by ocean currents. Conversely to the northern region, but still consistent with the mean fields, both parameters exhibit a substantially weaker relationship, inferred from low linear correlation coefficients, in the region south of ∘S  fluxes.Based on the zonally averaged meridional profiles, we define the local spatial anomaly for each individual observation with respect to the climatologies in Fig. entclass1pt{minimaentclass1pt{minimaδ18O. For this purpose, we first construct time series of annual medians of salinity and δ18O anomalies in the northern and southern regions —positive P-E is a flux into the ocean—trends over the Southern Ocean  is limited to the subpolar region and does not considerably influence the subtropical region. The surface water influenced by sea ice in the subpolar sector is actually advected northward through meridional Ekman transport, but most of it subducts into intermediate and mode waters along the ACC fronts38, and does not remain in the surface layer in the subtropical sector. Consequently, we aim to explain the observed changes in surface salinity and δ18O by a combination of changes in P-E, sea ice, and glacial meltwater inputs in the subpolar sector, and solely by changes in P-E in the subtropical sector (see Eqs. (P−E) from observed changes in salinity (ΔS) and δ18O (Δδ18O):SP−E and δ18OP−E are the salinity and δ18O of precipitation, S0 and δ18O0 the mean mixed layer salinity and δ18O. We repeat the computation of each of these two equations 50 000 times, but at each iteration we randomly perturb estimates of ΔS, Δδ18O, SP−E, δ18OP−E, within their error ranges . Our best estimate and associated error of ΔFP−E is the mean ± standard deviation of these sub selected solutions. We also compute the corresponding best estimate and error of δ18OP−E with the same strategy .Qualitatively, long-term salinification in the subtropics can be explained by a decrease in P-E, i.e., a decline of freshwater flux into the ocean. In contrast, long-term freshening in the subpolar sector could be explained by an increasing P-E, i.e., an amplification of freshwater flux into the ocean, a shift in sea ice regime, an increasing rate of ice sheet mass loss, or any combination of these. The weak correlation between salinity and ics Fig. d suggestsee Eqs.  and 5)  δ18O in entclass1pt{minima39 and sea ice40 ))5)). The18. However, the source of these changes has remained a conundrum. Salinity observations alone are not able to delineate the different elements involved in the freshwater cycle in the Southern Ocean. Here, in addition to salinity, we use δ18O observations and focus on surface water properties in the Indian sector of the Southern Ocean between 1993 and 2021 during summer months (December to February). We investigate the changes in two sectors, south and north of ∘S, which corresponds to the northern branch of Subantarctic Front (SAF-N) of the ACC33, separating Antarctic Surface Waters in the south from Subtropical Surface Waters in the north. We attempt to minimize the aliasing of spatial variability by splitting measurements based on their respective dynamical region, as estimated by the climatological mean dynamic topography. We report a multidecadal salinification in the northern region of 0.06 ± 0.07 g kg−1 per decade, associated with an increasing δ18O of 0.03 ± 0.04‰ per decade from 1993 to 2021. In contrast, the southern sector is freshened by −0.02 ± 0.01 g kg−1 per decade, and δ18O decreased at a rate of −0.01 ± 0.02‰ per decade from 1993 to 2021. These results are consistent with the latest IPCC report that assessed with high confidence that human-induced climate change has driven detectable changes in the global water cycle since the mid-20th century, with enhanced contrasts in P-E patterns over the oceans, which are projected to continue in a warmer world41. We demonstrate that the observed surface ocean changes result from an acceleration of the hydrological cycle between 1993 and 2021, with a decrease in P-E of −71 ± 47 mm yr−1 per decade in the subtropical sector, contrasted by an increase in P-E of 33 ± 11 mm yr−1 per decade in the subpolar sector. Our multidecadal trend estimates are based on a mean isotopic composition of precipitation of −7 ± 2‰ in the northern sector and −11 ± 3‰ in the southern sector, consistent with previously reported estimates and their associated errors42 reported in Table Long-term salinity observations in the Southern Ocean have revealed changes over the past decades, which are among the most pronounced in the global ocean44 and enhanced flux at the grounding line45 mostly drive changes on the continental shelf5. We find that the enhanced glacial meltwater discharge from the Antarctic ice shelves only has a very limited influence  on the open ocean salinity changes in the Indian sector of the Southern Ocean between 1993 and 2021. This result is consistent with refs. 17, stating that glacial freshwater flux increase is insufficient to explain the historical changes observed in the open ocean, away from the continental shelf. We note that as ice sheet mass loss is projected to continue in the future46, the signature on surface ocean salinity and δ18O might become more pronounced. In contrast, sea ice decline (implying less formation and volume) induces a positive salinity trend (0.008 ± 0.002 g kg−1 per decade) in the northern part of the sea ice sector and further north in the subpolar Indian sector of the Southern Ocean. This positive trend is compensated and surpassed by a negative trend induced by P-E (−0.02 ± 0.008 g kg−1 per decade), leading to net surface ocean freshening. This feature is consistent with estimates from ref. 21 for this region, showing a decline in the northward sea ice transport in the Indian sector of the Southern Ocean and upstream of this region. The ocean’s salinity response to this signal in a model5 is a clear salinification, consistent with the slight negative salinity trend accompanied by the strong negative δ18O trend south of ∘S. Despite large regional variations, the total Antarctic sea ice cover slightly increased since the late 1970s47 and sharply decreased in 201648, likely affecting the salinity anomalies towards the end of our time series . The use of the approach here whose strength comes from the concurrent observations of oxygen isotopes and salinity to disentangle the processes at play in surface salinity changes should be replicated in other regions of the Southern Ocean to address the spatially complex pattern of changes in surface water fluxes and their imprint on the ocean54. The different responses of local regime to direct or remote forcing are important to consider in order to investigate surface changes in the Southern Ocean as a whole.Our study quantifies the contribution of changes in freshwater forcing that influence surface water properties in the Southern Ocean directly from observations. Here we analyze the ocean response to both local and non-local freshwater forcing perturbations as signal from sea ice and meteoric waters is advected northward. Furthermore, the process of northward freshwater transport by sea ice contribute also to non-local contribution of meteoric waters through snow accumulated on top of sea ice. The results have important implications. First, we demonstrate the strength of the OISO long-term monitoring program recording concomitantly δ18O observed trends and the best estimate of glacial meltwater and sea ice changes ; Entr corresponds to the entrained salinity flux at the base of the mixed layer; and Diff is the diffusive salinity flux across the mixed layer boundary, which can involve either vertical or horizontal mixing. Integrating the salinity budget over an entire seasonal cycle, and assuming that entrainment and diffusion have not changed, the long-term salinity change ΔS can be written as:∫ FWF is the integrated freshwater flux over one year, and Δ∫ FWF is its long-term change. Decomposing the freshwater component in P-E flux (∫ FWF = FP−E), sea ice induced flux (∫ FWF = FSI), and glacial meltwater flux (∫ FWF = FIS) gives:To assess how the changing atmospheric freshwater flux affected the surface ocean properties in the Indian sector of the Southern Ocean, we compute a possible range of P-E changes from salinity, δ18O budget integrated over the mixed layer leads us similarly to:Repeating the same exercise but for a 56. Any small potential southward shift of fronts would in fact lead to a signal of opposite sign than what we observe, i.e., higher salinities towards the south, and are thus unlikely to affect the overall conclusion of our study. In addition, the possibility of a slight change in the fronts position or any other approximation made in these equations are, to some extent, taken into consideration by repeating 50 000 times the computation of Eqs. (P−E when computed by Eq. (−1 per decade). The uncertainties in Δδ18O and ΔS are inferred from the standard errors of the computed trends, and all other terms and associated uncertainties are summarized in Table Here we use Eqs.  and 5)  5) to de of Eqs.  and (5),d by Eq.  comparedδ18O) and salinity , from underway sampling at a depth of about 5–7 m from the sea surface and from stations sampling between a depth of 0 and 50 m, in the Indian sector of the Southern Ocean  database for those from 1993 to 199834. All data flagged as “bad" or “probably bad" are discarded. Most of the observations were processed at the Laboratoire d’Océanographie et du Climat Expérimentations et Approches Numeriques (LOCEAN) in Paris, France. The seawater samples were analyzed using two different instrumental methods; (i) most data prior to 2010 were analyzed using isotope ratio mass spectrometry (Isoprime IRMS) coupled with a Multiprep system , (ii) whereas most data since 2010 (and a few earlier data) were obtained by cavity ring down spectroscopy (CRDS) using a Picarro instrument (model L2130-i Isotopic H2O) to measure for δ18O. Seawater samples were analyzed directly with the CRDS system using a stainless-steel liner from Picarro that is inserted in the injection port to avoid salt accumulation in the vaporizer. The use of a liner has the advantage of preserving the accuracy of the seawater isotope analyses as compared to direct injections into the vaporizer and the mesh traps about 80% of the seawater salt57. Once the database was calibrated, it was then corrected for the analyzer-dependent “sea salt effect"57. The δ18O database is corrected so that deep water properties, which should be stable over the time period considered in this study, are constant across the different datasets. Typically, we focused over a range of neutral densities spanning over 28.15 and 28.3 kg m−3γn which corresponds to the old and stable Circumpolar Deep Water and encompasses water masses with typical time-scales of ventilation of the order of hundred years. This approach allowed us to correct for nonphysical offsets coming from different analytical techniques, different instruments and changes in standards. δ18O measurements uncertainty is on the order of 0.06‰ including instrumental and systematic errors associated with combining different datasets. In addition, we use concomitant salinity measurements from samples analyzed on a salinometer, calibrated CTD (Conductivity Temperature Depth) casts, and calibrated thermosalinograph data.In this study, we use surface observations of oxygen-18 isotopic composition , (i) we first compute local anomalies from the zonal-mean meridional profile by removing the mean of all observations in the streamwise coordinate bins; (ii) we then compute the annual 25, 50, 75 percentiles of anomalies in the large sector; (iii) finally, we fit a weighted linear regression model to the annual medians, weighted by w = 1/IQR2, where IQR is the interquartile range (percentile 75 minus percentile 25), which provides an estimate of the long-term trend. Its associated uncertainty is then estimated as the standard error of the trend calculated using a bootstrap approach .Trends are estimated by fitting linear regression models with their associated standard errors. The confidence level is established by computing the standard error of the regression. For trends performed in streamwise coordinate bins, we indicate trend values that are (i) less than half the computed standard errors, (ii) between half and one standard errors, (iii) between one and two standard errors, and (iv) more than two standard errors, to estimate the trend robustness with regards to the standard error. For trends produced in large sectors . The sea ice freshwater flux trends inferred from these published estimates in our region of interest, between 40∘E and 90∘E, is −31 ± 6 mm yr−1 per decade corresponding to the mean over the entire sector, i.e. it tends to increase surface ocean salinity. The mean sea ice freshwater flux trend over the latitude band we are considering as the subpolar sector in this study, between 60∘S and 46∘S, correspond to −12 ± 4 mm yr−1 per decade.Sea ice. The freshwater flux induced by the sea ice seasonal cycle and its interannual changes are obtained over the period 1993–2008 from ref. 46. In accordance with this assessment, the change in ice discharge is approximately equivalent to the change in mass balance. The Antarctic Ice Sheet mass loss rate was 49 ± 51 Gt yr−1 from 1992 to 1999, 70 ± 59 Gt yr−1 from 2000 to 2009, and 148 ± 54 Gt yr−1 from 2010 to 201646. In consequence, assuming no change from 1992 to 1993, and that the rate of change was the same from 2016 to 2021 as from 2005 to 2013 (10 ± 14 Gt yr−2), we infer that the ice discharge increased by a total of 317 ± 118 Gt yr−1 from 1993 to 2021, which translates into a mean acceleration of ice discharge corresponding to 113 ± 42 Gt yr−1 per decade during this period. The latest IPCC assessment also reports estimates of basal melt related to ice shelf thinning with, however, substantial spatio-temporal variability46. They estimated an increase from 1100 ± 150 Gt yr−1 to 1570 ± 140 Gt yr−1 from the early 1990s to the late 2000s, but a decrease from 1570 ± 140 Gt yr−1 to 1160 ± 150 Gt yr−1 in the late 2010s. The reconstruction of time-varying total glacial freshwater fluxes from the Antarctic Ice Sheet is a challenge when considering changes between the early 1990s and the early 2020s only, with evidence for significant interannual variability60 and a reduction in melt rates in the last decade44. Based on these published results, we consider that the best estimate of plausible changes from 1993 to 2021 due to ice shelf thinning ranges between 20 and 200 Gt yr−1 per decade. In addition to these two components, ice mass loss associated with ice front retreat has been recently estimated as approximately equal to net ice mass loss due to ice shelf thinning61. Our best estimate of changes in total (grounding line plus double ice shelf thinning) Antarctic glacial meltwater input therefore ranges from about 150 to 550 Gt yr−1 per decade from 1993 to 2021, consistent with the estimate discussed in ref. 62. Assuming that this glacial meltwater is evenly spread within the mixed layer over the entire subpolar region , it converts into a freshwater flux trend ranging between 5 and 17 mm yr−1 per decade from 1993 to 2021. However, we note that those changes in glacial meltwater flux are likely not evenly spread within the mixed layer over the entire subpolar region, and most likely have an impact to a greater extent in the coastal Antarctic region than in the northern edge of the subpolar region of the Indian sector of the Southern Ocean. We therefore consider this rate of changes as an upper range estimate for the region considered in the present study.Ice sheet. Continental ice meltwater changes can come from either increased ice discharge at the Antarctic grounding lines, or from ice shelf thinning. Here we use the assessment of the latest IPCC report to estimate these two components63 and as we can observe between three different global atmospheric reanalysis products, ERA564, JRA5565 and GPCP coupled with OAFlux67, in our region of interest and at the circumpolar scale (Supplementary Note Precipitation and Evaporation. As a result of the sparseness of in situ observations, atmospheric reanalyses have been widely used as a tool to report P-E over the Southern Ocean—the sign convention used here is that positive values of P-E refer to precipitation dominating over evaporation. However, long-term trends in P-E are highly uncertain due to the lack of measurements both spatially and temporally in the Southern Ocean in order to validate satellite products and modeled precipitation. Cautious use of reanalyses for climate change evaluation is thus needed as inhomogeneities between different products have been shownSupplementary InformationPeer Review File


Lactobacillus acidophilus LA-5, Lacticaseibacillus rhamnosus GG, and Bifidobacterium animalis subsp. lactis in yogurt ice cream after in vitro digestion and compared α-amylase and α-glucosidase inhibition activities. Lacticaseibacillus rhamnosus GG had the highest viability after in vitro digestion . Lactobacillus acidophilus LA-5 and Lacticaseibacillus rhamnosus GG exhibited the highest percentages of α-glucosidase (16.37% ± 0.32%) and α-amylase (41.37% ± 0.61%) inhibition. Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus acidophilus LA-5 showed the highest antioxidant activities via the α,α-diphenyl-β-picrylhydrazyl free radical-scavenging method and ferric-reducing antioxidant power assay, respectively. These findings suggest that yogurt ice cream can provide a suitable matrix for the delivery of probiotics from dairy culture to promote intestinal homeostasis with probiotic benefits in the host as well as a potential functional food to help reduce postprandial hyperglycaemia.Probiotics can ameliorate type 2 diabetes mellitus (T2DM) via several mechanisms such as by decreasing inflammatory cytokines and increasing pancreatic β-cell functions. Another targeted mechanism for managing T2DM involves inhibiting α-amylase and α-glucosidase, which exhibit antioxidant activity and affect carbohydrate metabolism by delaying carbohydrate digestion, thus mitigating glucose in the circulation. Dairy products are effective matrices for delivering probiotics through the gastrointestinal tract. We compared the viability and antioxidant activity of the probiotics  Type 2 diabetes mellitus (T2DM) is among the most common non-communicable diseases and a serious public health concern. A current report from the International Diabetes Federation indicated that the global prevalence of T2DM in adults was 10.5% (536.6 million people) in 2021 and that 783.2 million people (12.2%) worldwide would be living with diabetes by 2045  of Lactobacillus acidophilus LA-5 and 109 CFU of Bifidobacterium animalis subsp. lactis BB-12 had lower HbA1C : 6.61 ± 0.02 vs. 5.12 ± 0.02 log CFU/g, yogurt ice cream Bifidobacterium animalis subsp. lactis BB-12 [YI-BB-12]: 6.23 ± 0.03 vs. 4.53 ± 0.57 log CFU/g, and yogurt ice cream LGG [YI-LGG]: 7.68 ± 0.04 vs. 6.35 ± 0.09 log CFU/g, all p < 0.05). Several factors in yogurt ice cream production, including the freezing process, thermal shock, osmotic pressure within the cells, incorporation of oxygen into the mixed ice cream, and mechanical action during air mixing, can reduce the probiotic viability [Lactobacillus strains are more resistant to hazardous conditions during the ice cream manufacturing process than are bifidobacterial strains [Inoculating 108 CFU/g probiotics into mixed ice cream provided yogurt ice cream with predigestion viable counts of ≥ 6 log CFU/g of Lactobacillus acidophilus LA-5 and Bifidobacterium animalis subsp. lactis BB-12 decreased by approximately 1 log, and viable counts were <106 CFU/g. However, the differences were significant only in Bifidobacterium animalis subsp. lactis BB-12 . Conversely, LGG did not decrease significantly compared with that in the oral phase (6.35 ± 0.09 vs. 6.23 ± 0.08 log CFU/g). For the duodenal phase, YI-BB-12 and YI-LGG exhibited the highest and lowest sensitivity, respectively, to simulated intestinal stress  reported a survival rate of 50.82% for Bifidobacterium animalis subsp. Lactis BB-12 after simulating in vitro digestion by adding 109 CFU/g of Bifidobacterium animalis subsp. Lactis BB-12 to ice cream mixtures [After the gastric phase, the viability of l stress . Viable et al. 20 reportedLactobacillus acidophilus, Lacticaseibacillus rhamnosus, and Bifidobacterium animalis subsp. lactis, which were the most common species of LAB used in probiotic ice cream for fermented dairy products. Effects of the overrun process can lead to decreased viable cell counts of probiotic bacteria. A previous study found that inclusion of 106–108% overrun decreased the viability of probiotics  but by not more than 10% after freezing and hardening [Factors affecting probiotic survival in ice cream included probiotic strain, osmotic pressure, freeze injury, pH, overrun, oxygen scavengers, packaging, and frozen storage . In thisardening . In addiInhibiting α-amylase and α-glucosidase is the main mechanism of controlling postprandial hyperglycemia. We evaluated the inhibition of α-amylase and α-glucosidase via probiotic yogurt ice cream. Acarbose was used as a positive control because it functions as a carbohydrate digestion enzyme inhibitor by competing for binding sites and diminishing the enzymes’ affinity for oligosaccharides from dietary starch along with the monosaccharide production rate . Figure The probiotics in the YIEs significantly differed in their ability to inhibit both enzymes . The inhSeveral factors may be responsible for α-amylase and α-glucosidase activities, including pH, different substrate, temperature, and the presence of any inhibitors or activators , and alsLactobacillus subsp. strains through dipeptidyl peptidase IV (DPP-IV) inhibitory and antioxidant activities. The results of the experiment demonstrated the significant DPP-IV inhibitory activity and probiotic qualities of L. acidophilus KLDS1.0901 in vitro. In T2D mice, administration of L. acidophilus KLDS1.0901 could improve oxidative stress and insulin resistance while preserving blood glucose homeostasis [Lactic acid bacteria (LAB) can ferment sugar into lactic acid and produce proteolytic enzymes. By supplying nitrogen molecules, these enzymes play important roles in LAB growth. Protein in yogurt ice cream may also play a role in LAB growth and influence the inhibitory effects on α-amylase and α-glucosidase activities. Variations in the α-amylase and α-glucosidase inhibition percentages can be explained by the total protein contents in different types of milk. LAB possess a complex system of proteinases and peptidases that allow them to break down milk casein into small peptides. Peptides possess inhibitory potential against α-glucosidase and α-amylase, and their probable mechanism of action involves forming hydrophobic bonds at the enzyme’s active site ,30. In aeostasis .Some probiotic strains possess antioxidative activity and reduce free radical accumulation by modulating the redox status of the host via an antioxidant system . We measLacticaseibacillus rhamnosus, Lactobacillus acidophilus, Lactiplantibacillus plantarum and Lacticaseibacillus casei) had higher percentages of scavenging activity than did the unfermented control [Lactobacillus acidophilus had a higher FRAP value than did fermented milk supplemented with Bifidobacterium animalis subsp. lactis and Lacticaseibacillus rhamnosus [Lactobacillus acidophilus LA-5 with FRAP and DPPH methods in a dose-dependent manner [Our finding is similar to that of Zahrani et al. (2023), who observed that all the fermented soy- and almond-based milks that they prepared by inoculation with single probiotic strains ( control . Further control . YIE-LA-hamnosus . Severalhamnosus . Najgebat manner .Raw milk antioxidant activity is meaningful for consumer health and can be enhanced by the antioxidant potential of bioactive peptides from probiotics and antioxidant properties of the probiotics themselves . Casein,In vitro digestive systems are widely used to investigate and analyze changes, interactions, and the bioaccessibility of nutrients and non-nutritive compounds. This model is multipurpose, adaptable, accurate, and reproducible  and reprLacticaseibacillus rhamnosus had a higher probiotic content than 6 log CFU/g and therefore contributed evidence to support the concept that yogurt ice cream could provide a suitable matrix for the delivery of probiotics from dairy culture to enhance intestinal homeostasis with potential to promote probiotic benefits in the host. For antidiabetic properties, Lactobacillus acidophilus and Lacticaseibacillus rhamnosus showed higher levels of inhibition of α-glucosidase and α-amylase activities than Bifidobacterium animalis subsp. lactis. The beneficial effects of the three probiotics in yogurt ice cream as determined by DPPH and FRAP assays were higher than that of the control yogurt ice cream. These findings show that probiotic yogurt ice cream should be investigated in further studies as a potential functional food to help reduce post-prandial hyperglycaemia. Furthermore, the limitations of this investigation concerning overrun and pH effects, which impact the viability of probiotics in yogurt ice cream, should be assessed in conjunction with a comparative analysis of the inhibition of α-amylase and α-glucosidase by acarbose.The present study indicated that in the small intestine phase, YIE with


An 84-year-old man with medical history of T1 bladder cancer underwent radical cystectomy, with subsequent complication of surgical site abscess. Abdominal computed tomography revealed free gas at the surgical site and a rectal wall defect suggestive of fistula. Despite conservative treatment, the patient had persistent penial and anal discharge and was referred for endoscopic closure.Colonoscopy showed a 6-mm fistulous tract between the cystectomy surgical site and the rectal wall . InitiaAfter fluoroscopic characterization of the defect , a 16/4/12 mm CSDO was chosen . After Video 1 Use of a cardiac septal defect occluder for endoscopic closure of a fistula between the rectum and post-cystectomy surgical site.The patient remains well, without clinical recurrence.CSDO is an off-label device for closure of gastrointestinal fistulas and should be considered for chronic fistulas refractory to conventional endoscopic treatmentsEndoscopy_UCTN_Code_TTT_1AQ_2AG


Chimeric small moleculesthat induce post-translational modification(PTM) on a target protein by bringing it into proximity to a PTM-inducingenzyme are furnishing novel modalities to perturb protein function.Despite recent advances, such molecules are unavailable for a criticalPTM, tyrosine phosphorylation. Furthermore, the contemporary designparadigm of chimeric molecules, formed by joining a noninhibitorybinder of the PTM-inducing enzyme with the binder of the target protein,prohibits the recruitment of most PTM-inducing enzymes as their noninhibitorybinders are unavailable. Here, we report two platforms to generatephosphorylation-inducing chimeric small molecules (PHICS) for tyrosinephosphorylation. We generate PHICS from both noninhibitory binders and kinase inhibitors  using cysteine-based group transfer chemistry.PHICS triggered phosphorylation on tyrosine residues in diverse sequencecontexts and target proteins and displayed multiple bioactivities, including the initiation ofa growth receptor signaling cascade and the death of drug-resistantcancer cells. These studies provide an approach to induce biologicallyrelevant PTM and lay the foundation for pharmacologic PTM editing of target proteins using abundantly availableinhibitors of PTM-inducing or -erasing enzymes. Generation and validation of proximity-inducing chimericmolecules that induce tyrosine phosphorylation using inhibitory ornoninhibitory kinase binders. The human genome codes 90 tyrosinekinases,6 107 tyrosine phosphatases,8 and over hundreds of pTyr-recognition domains,11 and the immune system can produce antibodies thatselectively recognize pTyr over phosphorylated serine or threonine.These attributes have led to suggestions to consider pTyr a functionallyseparate PTM class.12 While molecules thatinhibit tyrosine phosphorylation exist and constitute one of the largestclasses of drugs,15 there are no small molecules that can induce tyrosinephosphorylation on a given target of interest, even though these compoundswill be of immense utility in both basic research and biomedicine.16 PHICS-induced phosphorylation can rewire cell-signaling,change protein conformations and function,17 evoke an immune response,18 induce phaseseparation,20 and alter protein–proteinand protein–RNA/DNA interactions.22 Thus, PHICS can be useful in diverse settings, including basic researchand biomedicine.Protein phosphorylation is the most frequentand well-studied post-translationalmodification (PTM),25 These PHICS were formedby joining small-molecule ligands of targeted protein and a kinasevia a linker. Our previously reported PHICS brought Ser/Thr kinases(AMPK or PKC) in proximity to a target protein, inducing the phosphorylationof the latter even for nonsubstrates  of thekinase. However, these PHICS did not recruit endogenous tyrosine kinasesor induce a downstream signaling event. Furthermore, these PHICS requirenoninhibitory kinase binders that are unavailable for most kinases,limiting the recruitment of only two of ≈538 human kinases.26 Beyond phosphorylation, heterobifunctional moleculesthat can induce or remove other PTMs, including acetylation and glycosylation,by recruiting PTM-inducing or -erasing enzymes are needed.27 However, the necessity of noninhibitory ligands for these enzymeshas been a major roadblock in the development of such heterobifunctionalmolecules. Finally, the previously reported PHICS did not explorethe covalent binders for the target protein or kinase which will havea relatively simple two-body equilibrium vs the complex three-bodyequilibrium displayed by noncovalent binders.32Recently, we reported on an approach to inducephosphorylationusing heterobifunctional small molecules termed phosphorylation-inducingchimeric small molecules (PHICS) that recruit endogenous Ser/Thr kinasesto a target protein of interest.Herein, we report tyrosine PHICS that recruitAbelson Kinase (ABL)or Bruton’s Tyrosine Kinase (BTK) to induce the phosphorylationof neo-substrates in cells. We report five noninhibitory molecularscaffolds to recruit ABL to a target protein, and PHICS generatedfrom these scaffolds display hallmarks of contemporary heterobifunctionalmolecules, including hook effect, turnover, and ternary complex formationin cells. Importantly, we showed that ABL PHICS was able to initiatea signaling pathway by inducing phosphorylation of the epidermal growthfactor receptor (EGFR). Furthermore, we report a new design paradigmof a heterobifunctional molecule consisting of a BTK inhibitor connectedto the target binder through a linker that covalently labels BTK withthe target protein binder while releasing the inhibitor from BTK andenabling phosphorylation of the target by BTK. We demonstrated thisgroup transfer chemistry with two commonly employed covalent inhibitorscaffolds . Using BTK or ABLPHICS, we phosphorylated tyrosine residues in a diverse sequence context,including those surrounded by neutral, acidic, or basic residues.Finally, we generated ABL-BTK PHICS using a covalent inhibitor ofBTK  to induce its ABL-mediated phosphorylation;these PHICS effectively kill ibrutinib-resistant cancer cell linesand inhibit the downstream survival signaling of BTK, highlightingthe therapeutic potential of this class of molecules.36 We selected four scaffolds that competitively bind to the myristoylpocket of ABL: dihydropyrazole , thiazole , pyrazole ,36 and hydantoin .35 Using molecular dockingstudies, we identified the solvent-exposed 3-amino group as the linkerattachment site for the dihydropyrazole, pyrazole, and thiazole scaffolds.36 For the hydantoin scaffold (PDB ID: 3PYY),35 linkers were grown from the ortho, meta, or para positionsof the solvent-exposed aryl ring . Previous reports for synthesizing hydantoin scaffold35 involving the Vilsmer–Haack reactionprovided poor yield37 and were sensitiveto substitutions on the aryl ring  preventing exploration of linker growth from -ortho, -meta, or-para positions from aryl ring. We developed a new synthetic routewith fewer steps to rapidly access hydantoin scaffold precursors byinvolving Cu-mediated high-yielding C–N bond coupling38 between pyrazole and appropriately substitutedphenylboronic acids . By applyingthis synthetic strategy, we were able to rapidly generate a libraryof hydantoin-based PHICS with linkers through the -ortho, -meta, and-para positions of the aryl ring.For the tyrosinekinase recruited by PHICS, we first chose ABL,as it can phosphorylate wide-ranging target sequences and has severalnoninhibitory, selective, and allosteric binders with high affinitiesthat also have cocrystal structures to aid the rational design ofPHICS.S)-JQ1 , which binds our proof-of-concepttarget bromodomain-containing protein 4 (BRD4).39 With these compounds in hand, we measured PHICS-inducedphosphorylation using recombinant ABL and BRD4 proteins. Dihydropyrazoleinduced the most BRD4 phosphorylation, followed by PHICS based onthiazole, while the pyrazole scaffold (an oxidized form of the dihydropyrazole)induced significantly less phosphorylation . Hydantoin-derived PHICS induced less phosphorylation thanthe dihydropyrazole-derived PHICS with the meta-substituted analogueshowing the highest phosphorylation levels compared to the ortho-and para-substituted analogues . These studies identified the dihydropyrazole scaffoldas the most efficient for inducing BRD4 phosphorylation.To assess the efficacy ofthese scaffolds, we synthesized four different PHICS, each containingone of the four binders of ABL, a linker composed of four poly(ethyleneglycol) units, and the small molecule (1 (2 generatedby replacing (S)-JQ1 in 1 with inactiveenantiomer (R)-JQ1, 3 formed by replacingthe dihydropyrazole in 1 with a pyrazole scaffold, and 4 a dihydropyrazole scaffold with a short linker. In biochemicalsettings, we confirmed that tyrosine phosphorylation of BRD4 occurredonly in the presence of all components of the ternary complex, namely, 1, BRD4, and ABL ,40 wherein 1, but not 2, showed a “hookeffect” which is a hallmark of 3-body equilibrium29 defined as the ratio of affinity constants (KD) of binary to the ternary complex30 suggested negative cooperativity between BRD4 and ABL .Tomore deeply characterize the ability of the dihydropyrazolescaffold to induce BRD4 phosphorylation, PHICS 1 1A was se and ABL 1B. We thibrium29 1C. Here,BRD4 lacking an intrinsic nuclearlocalization signal41 and incubated HEK293Tcells transfected with both HA-BRD4 and ABL-FLAG plasmids with bifunctional molecules and controls. Here, HA-basedimmunoprecipitation (IP) showed significantly higher coimmunoprecipitationof ABL-FLAG with 1 compared to the control compounds, suggestingternary complex formation is specific to 1 , confirming that PHICS exhibitsturnover, like our previously reported Ser/Thr PHICS.23To demonstrate BRD4 phosphorylation in cells usingPHICS, we useda construct of fic to 1 1D. Probiontrol 2 1E. Here,44 Upon EGF binding, EGFR oligomerizes and autophosphorylates tyrosineresidues, which acts as docking site of proteins with phosphotyrosine-bindingSH2 or PTB domains leading to a series of molecular events and cellularresponse.45 We were interested in determiningif ABL PHICS can trigger signaling by phosphorylating relevant EGFRtyrosines (F36V (iEGFR-FKBPF36V-FLAG) and synthesizedPHICS 5 using the FKBPF36V binder AP1867 (S8).46 HEK293T cellstransfected with iEGFR-FKBPF36V-FLAG and ABL-HA were treatedwith 5 or dimethyl sulfoxide (DMSO). Immunoblotting ofcell lysates and probing with site-specific phosphotyrosine antibodiessuggested that ABL PHICS could phosphorylate tyrosines present indiverse sequence environments , pointing to the generality of the designed PHICS.EGFR is a tyrosinekinase receptor that responds to the epidermal growth factor (EGF)binding to initiate a signaling pathway that regulates the growth,proliferation, survival, and differentiation of cells.yrosines 2A. Some r AP1867 2B and S8ronments 2C in linon EGFR 2Care re5 ondownstream EGFRsignaling, we used a previously described luciferase-based reportergene assay with serum response element (SRE).53 Here, HEK293T cells were cotransfected with iEGFR-FKBPF36V-FLAG and pGL4.33[luc2P/SRE/Hygro]vectors, treated with PHICS 5, or controls (DMSO or 4); the activation of EGFR signaling was quantified usingPromega’s ONE-Glo Luciferase Assay System. PHICS 5 induced a 2-fold higher luciferase activity than DMSO or ABL binder 4, indicating activation of EGFR by 5 .56 To confirmthe cell permeability of these compounds and their ability to efficientlylabel HaloTag, we used a competition assay wherein HEK293T cells stablyexpressing HaloTag were treated with compounds 7, 9, 11, or 12 and controls (10 and 4), and the lysates were subsequentlylabeled with tetramethylrhodamine (TMR)-HaloTag, a fluorescent probe.57 We found that the lysates from cells pretreatedwith HaloTag-PHICS were not labeled by TMR-HaloTag, whereas control-treatedlysates (10 and 4) showed efficient TMR-labeling, indicating that HaloTag-PHICSare cell-permeable and efficiently label the HaloTag protein. To assessthe relative ability of these covalent PHICS to induce tyrosine phosphorylation,we transiently expressed the ABL-HA construct in HEK293T cells stablyexpressing the HaloTag protein and treated these cells with variouscovalent PHICS  and controls . We observed significanttyrosine phosphorylation on HaloTag in the presence of active PHICScompared with control-treated samples  near the inhibitor-binding pocket caninitiate a group transfer chemistry70 resulting in appending the target protein binder to the kinase whilereleasing the inhibitor from the kinase’s active site . We also synthesized inactive controls -JQ1 and testedwhether 17 or 19 can rewire the BTK’sspecificity and induce the phosphorylation of BRD4. Indeed, we observedmuch higher BRD4 phosphorylation in the presence of 17 or 19 (18 or 20) in cells .Allof the reported PHICS were developed using noninhibitory allostericbinders, which are rare, though kinase inhibitors are plentiful andform an important class of therapeutic.ive site 4A. The k , the Cu-catalyzedclick reaction was performed on the lysates using sulfo-Cy5.5 azide;BTK labeling was confirmed via in-gel fluorescence (74 between a nanoluciferase (nanoLuc)and a fluorophore probe that binds to the ATP pocket; a higher inhibitoroccupancy in this ATP pocket will prevent the binding of the tracerand lower the BRET signal75  did not affect the binding of tracerat the working concentrations of PHICS . These studies demonstrate that PHICS can be generated from cysteine-targetingcovalent inhibitors, which are available for many kinases76 with diverse cellular and tissue localization, sequence preferences,and successful clinical outcomes.Next, we performed target engagementand labeling studies designedto support the mechanism outlined in 17 or 19 4B covalerescence 5A. To designal75 5B. As exof PHICS 5C,D. Agr24 Wedesigned compound 21, connecting the reversible binderof ABL with the covalent binder of BTK via a PEG3 linker (S14) and used this PHICS along with a mixtureof the separate binders (22 and 4) for thetreatment of HEK293T cells cotransfected with ABL-HA and BTK-FLAG plasmids. Western blotting with apan antiphosphotyrosine antibody revealed increased levels of BTKphosphorylation in the presence of 21 compared to thecontrol (mixture of binders 22 and 4) , in agreement with our expectation based on the unaffected phosphorylationof ABL in HEK293T-based studies (22 and 4) nor ibrutinibinduced the same effect of cell viability in BTK-dependent and ibrutinib-resistantcancer cells as compound 21. These results with ibrutinibare especially interesting, as this BTK inhibitor is an approved therapeuticagent for several B-cell cancers. However, the emergence of resistancehas resulted in a need for next-generation therapeutics;78 the promising activity of BTK-ABL PHICS in ibrutinib-resistantcell lines opens such an alternative therapeutic strategy.Aftervalidating two classes of Tyr-PHICS (ABL- and BTK-derived), we exploredthe functional capabilities of bifunctional molecules consisting ofbinders of both kinases since these kinases are often overexpressedin cancer cells, and we have reported that Ser/Thr PHICS can induceinhibitory phosphorylation on BTK.2 and 4) 6B. Surprectively 6C.77 In 62 cells S15, in a80 has furnished technologies forfacile editing of the DNA or RNA, but methods for facile editing ofpost-translational modifications  on agiven protein of interest is still challenging. We report an approachto induce phosphorylation on the target protein by generating PHICSthat utilizes kinase inhibitors and cysteine-triggered group transferchemistries. Since inhibitors for several PTM-inducing or -removingenzymes are available, and group transfer chemistry can be implementedusing other nucleophilic residues ;82 these studies lay the foundationfor pharmacologic editing of PTMs on proteins. Overall, these studiesfurther highlight the power of bifunctional molecules to endow neo-functionsto proteins in cells with value in basic research and medicine.To address the lack of technologies forthefacile editing of PTMs on a given protein of interest, we reporta new class of bifunctional molecules that induce functionally relevanttyrosine phosphorylation by recruiting an ABL or BTK. PHICS triggeredactivation of EGFR, which belongs to the receptor tyrosine kinasesuperfamily that includes the insulin receptor. A PHICS that similarlyactivates insulin receptor signaling may furnish an orally available,small-molecule substitute for the insulin that requires injectionand may offer an alternative therapeutic modality for patients withinsulin resistance. Furthermore, PHICS induced the death of drug-resistantcancer cells, offering a potentially novel and alternative therapeuticmodality to kinase inhibitors against which resistance has developed.The CRISPR-Cas system


Marine heatwaves are triggering coral bleaching events and devastating coral populations globally, highlighting the need to identify processes promoting coral survival. Here, we show that acceleration of a major ocean current and shallowing of the surface mixed layer enhanced localized upwelling on a central Pacific coral reef during the three strongest El Niño–associated marine heatwaves of the past half century. These conditions mitigated regional declines in primary production and bolstered local supply of nutritional resources to corals during a bleaching event. The reefs subsequently suffered limited post-bleaching coral mortality. Our results reveal how large-scale ocean-climate interactions affect reef ecosystems thousands of kilometers away and provide a valuable framework for identifying reefs that may benefit from such biophysical linkages during future bleaching events. Ocean currents enhance upwelling and promote coral survival during El Niño heatwaves. Oceanographic processes that originate beyond the coastal margins of coral reefs strongly influence the physical, chemical, and biological conditions of these dynamic ecosystems  in September 2012, 2014, and 2015 to examine interannual variation in coral trophic ecology. We used Bayesian analysis of isotopic niches  was reduced by ~70% relative to non–El Niño years (P. meandrina across the Atoll. This compression was driven by a 2 per mil (‰) decline in average δ15N values and >40% reduction in the observed δ13C range in the coral host and endosymbionts .We sampled the reef-building coral, he areas  associatño years , and allP. meandrina as the percentage overlap between coral and endosymbiont standard ellipse areas corrected for sample size (SEAc)  collected at 10 m depth from our sampling sites revealed a substantial shift in the dominant nitrogen source on Palmyra during the 2015–2016 El Niño   ratios increased rather than decreased during 2015, implying that the change in dominant nitrogen source did not result in substantially elevated nutrient concentrations on the reef at our sampling depth (fig. S5 and table S4). This is further supported by in situ measurements of dissolved inorganic nitrogen (DIN) at 10 m depth at our four sampling sites in 2012  and at 13 sites spanning the circumference of Palmyra’s outer reef slope in 2015  (An 8-year times series of δ El Niño . The δ150.02 SD) . While cP. meandrina host tissue δ15N (9.3‰ ± 0.5) was reduced by 2‰ relative to non–El Niño years but remained 3 to 4‰ above the primary producer baseline , consistent with theory that predicts an enrichment between successive trophic levels . Under elevated nutrient concentrations and without substantial heterotrophy, the δ15N value of P. meandrina would likely have remained similar to non–El Niño years while endosymbiont C:N ratios would have strongly declined, resulting in δ15N values more similar to Halimeda  in Palmyra’s lagoon that might help explain reduced δ15N values . Rather, our results are consistent with the hypothesis that the cessation of equatorial upwelling occurred alongside a regional decline in surface ocean primary production (15N = ~7‰) , our observations imply that cold pulses were influencing corals and macroalgae at 10-m-depth atoll wide . Coupled with the strong correlation between cold pulse occurrence at 25 and 15 m over this period  to examine temporal changes in regional oceanographic conditions in the central Pacific. Increased upwelling during the 2015–2016 El Niño was associated with a shallower mixed layer depth, estimated as the bottom of the isothermal layer (0.8°C difference from the surface) (We used 40 years (1980–2020) of temperature data, surface isothermal depth estimates, and zonal current velocities from the National Centers for Environmental Prediction (NCEP) Global Ocean Data Assimilation product   and 6B. surface)  reduced surface) . The intsurface) . This reLocalized upwelling driven by island-current interactions also influences coral communities at the nearby islands of Kiritimati and Jarvis  . RegionaA central challenge in coral reef science is disentangling how natural physical drivers and anthropogenic drivers interact across scales and background conditions to influence differences in ecosystem response to bleaching (P. meandrina colonies at four long-term monitoring sites (10 m depth) on Palmyra Atoll, central Pacific (fig. S1A). We sampled in September 2012 , 2014 (n = 10 from two sites), and 2015 . In 2015, nine additional sites were sampled  (P. meandrina relative to non–El Niño years (fig. S3). Samples were transported on ice and frozen at −20°C. In the laboratory, the coral host and endosymbiont were separated via centrifugation  were measured via EA-IRMS with Costech elemental analyzer  coupled to a Thermo Scientific Delta XP Plus isotope ratio mass spectrometer . Isotopic data are reported as delta values (δ) and expressed in ‰ relative to the international standards (Vienna Pee Dee Belemnite) and atmospheric N2 for carbon and nitrogen, respectively. Within-run SD of in-house reference material was <0.2‰ for δ13C and δ15N.To examine temporal changes in coral trophic ecology, we collected top-central branch tips from unshaded H. opuntia, can reliably distinguish on-reef nitrogen sources at intra-island scales  or all four of the coral collection sites . The low δ15N value in 2008 may be an underestimate of fore reef δ15N values due to inclusion of samples collected in back reef or reef terrace habitats  versus nonacidified (13.5‰ ± 0.3 SD) samples of Halimeda sp. collected at 10 m depth on Starbuck Island, Southern Line Islands (n = 5). See Supplementary Materials and Methods for information on lagoon transplants to address the possibility of N2 fixation on Palmyra during 2015.In all years, n = 13) and the four main sites plus nine additional sites spanning the entire north and south shores of Palmyra in 2015 . Individual samples were filtered through GF/F filters (0.7 μm) and frozen at −20°C before analysis. Samples were analyzed for nitrate and nitrite in the Analytical Lab at the Marine Science Institute, UC Santa Barbara (2012) and the Analytical Core Lab at King Abdullah University of Science and Technology (KAUST) (2015).Discrete water samples were collected over multiple days at 10 m depth at the four main sampling sites in 2012 (a (mg m−3) product derived from the Moderate Resolution Imaging Spectroradiometer (https://modis.gsfc.nasa.gov) , at 1-to-4-month lags before sample collection, and to the average surface chlorophyll a concentration over 4 months before sampling.To examine temporal changes in surface ocean production across the Northern Line Islands, we used the 8-day 0.0147° (~4-km) spatial resolution chlorophyll Upwelling events were quantified using a TSI developed on Palmyra  .To extend the cold pulse record to 2009 and compare with interannual variation in mixed layer depth and current velocity, we calculated monthly mean DCH between 15 and 25 m using all available temperature measurements from the outer reef slope of Palmyra . While we cannot explicitly calculate how much cooling was provided to the corals at 10 m by upwelling (our sampling depth and observations of bleaching), this analysis revealed a stark reduction in cold pulse influence on seawater temperature between 25 and 15 m. We also quantified the total percentage of pulses of each magnitude for the 3 months with the greatest upwelling  to contrast the overall mean conditions cold pulses induced at different depths.https://psl.noaa.gov/data/gridded/data.godas.html to assess variation in the mixed layer depth and zonal current velocity near Palmyra. We averaged the monthly means of potential temperature, isothermal depth, and zonal current velocity from the grid cells on the east and west side of Palmyra (available at a resolution of 0.33° latitude × 1° longitude) between 1980 and 2020. Palmyra lies at the center of the two grid cells, which we averaged together to better reflect regional conditions. The isothermal depth estimates the bottom of the surface mixed layer and is best approximated as a temperature difference of 0.8°C from the surface  using linear mixed effects models with year as a fixed factor and site as a random effect  and El Niño (2015) conditions. Data for each tissue fraction and year were randomly sampled with replacement to calculate the difference in mean values between 2012 and 2015. This process was repeated 10,000 times to generate a distribution of mean differences between years, from which we estimated the mean change ± 95% CI. We quantified changes in the isotopic niche of Pocillopora using Stable Isotope Bayesian Ellipses (b) and the standardized ellipse area corrected for sample size (SEAc). Furthermore, we calculated proportional changes in the six Layman metrics (n = 38 per fraction) to 2015 (n = 39 per fraction).We compared annual changes in the bulk tissue δc  to control for correlated error structure, but this did not improve on the null model . All statistical analyses were performed in R version 4.0.2 (We modeled interannual variation in