L-asparaginase is effective as part of the first line childhood acute lymphoblastic leukaemia (ALL) treatment regimen but suffers the risk of antibody production causing immune-mediated sequelae. This article aimed to describe the clinical implication of L-asparaginase hypersensitivity and review the types of antibodies and genetic polymorphisms contributing to it. Clinical or subclinical L-asparaginase hypersensitivity may lead to suboptimum therapeutic effect and jeopardise the clinical outcome in ALL children. Anti-asparaginase antibodies immunoglobulin (Ig)G, IgM and IgE were identified in the L-asparaginase hypersensitivities. Enzyme-linked immunosorbent assay (ELISA) is commonly used to quantify the IgG and IgM levels. The role of IgE in mediating L-asparaginase hypersensitivity is contradictory. Moreover, the presence of antibodies may not necessarily correlate inversely with the L-asparaginase efficacies in some studies. Patients with specific genetic variants have been shown to be more susceptible to clinical hypersensitivity of L-asparaginase. With the advance of technology, gene polymorphisms have been identified among Caucasians using whole-genome or exon sequencing, but the evidence is scanty among Asians. There is lack of pre-clinical study models that could help in understanding the pathophysiological pathway co-relating the gene expression and anti-asparaginase antibody formation. In conclusion, future research studies are required to fill the current gap in understanding the immune mediated reactions towards L-asparaginase upon its administration and its potential impact to the disease outcome. Acute lymphoblastic leukaemia (ALL) is defined as a malignancy that originates from the failure of lymphoid cells maturation and uncontrolled proliferation of lymphoid progenitors in one’s body. In turn, these immature lymphoblasts outnumber the normal haematopoietic cells and occupy the bone marrow or metastasise to extramedullary sites such as the liver, spleen and lymph nodes through blood circulation . The cliALL is the most prevalent type of cancer among children. Studies showed that ALL constituted approximately 25% of the total malignancy cases in this population (below 15 years old) . It was L-asparaginase, an enzymatic chemotherapeutic drug, has been introduced as the first line treatment regimen for childhood ALL. In the early 1970s, it was evidenced in clinical trials that paediatric patients who received treatment protocols that included L-asparaginase had better clinical outcomes compared to those who did not receive the L-asparaginase treatment . The mecEscherichia coli or Erwinia chrysanthemi. The E. coli L-asparaginase is produced either in native or the pegylated form, the latter has a covalently attached monomethoxypolyethylene glycol polymer. The pegylated E. coli L-asparaginase has been employed in the first-line treatment of childhood ALL since the early 2000s in developed countries. These different formulations can be administered via intramuscular or intravenous routes and they possess similar mechanisms of action towards depleting L-asparagine in ALL patient’s bloodstream. However, a significant difference is seen in their pharmacokinetic properties. It was demonstrated that each L-asparaginase preparation has a distinct half-life in the body. The native E. coli, pegylated E. coli and Erwinia L-asparaginases had half-lives of 1.28 days, 5.73 days and 0.65 days, respectively, when injected intramuscularly  . Other taginases . Formatiaginases .Erwinia L-asparaginase is antigenically distinct from E. coli-based formulation. Hence, there is cross immunoreactivity between the native E. coli L-asparaginase and its pegylated formulation, but not with the Erwinia asparaginase. In this paper, emphasis is placed on reviewing published information and data on hypersensitivity caused by anti-asparaginase antibodies in children with ALL.Identification of hypersensitivity events towards L-asparaginases becomes crucial in cases with subclinical hypersensitivity, which are usually clinically undetected. The Ponte di Legno Toxicity Working Group has classified L-asparaginase hypersensitivity into three major categories, as illustrated in Antibodies in humans are classified into five major isotypes: immunoglobulin (Ig)M, IgG, IgD, IgA and IgE, and all are synthesised from B lymphocytes or plasma cells. Existing publications reveal IgG, IgM and IgE to be the most frequently studied antibodies for L-asparaginase clinical hypersensitivity and silent inactivation  25–29).–29.25–29There are substantial dissimilarities among IgG, IgM and IgE antibodies. Notably, IgM is the first antibody to be expressed in response to new exposure to pathogens, owing to its ability to be generated without very recent isotype switching. These antibodies exist mainly in blood and lymph. The molecular structure of IgM is made up of five interlinked Y-shaped monomers, forming a pentameric structure with 10 antigen-binding sites (therefore has a larger size), possessing a higher overall avidity yet a lower affinity for antigen binding. As for the antibodies IgG and IgE, both are present in monomeric structures, thus having a smaller size and capable of binding to the antigens with a higher affinity than IgM. IgG antibodies constitute approximately 75% of the human body’s total antibodies. They are found abundantly in the blood and extracellular fluid and primarily carry out opsonisation and neutralisation of antigens. On the other hand, IgE antibodies play a vital role in allergic reactions by binding to the mast cell receptors after binding to their respective antigens. Binding of IgE to mast cell receptors triggers mast cell activation which subsequently release the chemical mediators to expel the antigens, either through coughing, sneezing or vomiting .Contradictory reports exist on the role of IgE antibodies in contributing to L-asparaginase hypersensitivity in paediatric ALL patients. Unlike IgE antibodies, established supportive data in the form of enzyme-linked immunosorbent assay (ELISA) studies, demonstrate the tendency of both, IgG and IgM antibodies to act against L-asparaginases in the serum or plasma of paediatric ALL patients . Some reBased on literature review, the indirect ELISA technique is seen to be the most popular and widely used quantification method for anti-asparaginase antibodies (IgG or IgM) using patient serum or plasma. Pharmacogenetic studies have significantly contributed to the understanding of the different inter-patient variations in clinical and subclinical hypersensitivity development towards L-asparaginase during their cancer treatment. Identification of genetic variants can predict the predisposition of the paediatric ALL patients in developing L-asparaginase resistance or adverse reactions, helping establish a more suitable and rational treatment regimen individually tailored for each of the patients, in turn maximising the efficacy of the antineoplastic agent . The polAmong the findings illustrated in NFATC2 gene was inhibited led to the discovery of a new variant of the gene (NFATC2 gene did not develop hypersensitivity towards L-asparaginase after being immunised with the foreign enzyme. Moreover, the levels of IgE in these NFATC2-deficient mice were relatively lower than that in the wild-type mice. The researchers claimed that such gene inhibition outcomes might provide greater insight into generating attenuation against L-asparaginase hypersensitivity in human subjects in the near future (Recently, when studying the extent of hypersensitivity reaction in murine models towards L-asparaginase wherein the the gene . Previour future . More stL-asparaginase hypersensitivity has long been a deterrent in improving the treatment outcome, and the survival rates of children suffering from ALL. Clinicians and medical researchers around the world have made significant efforts towards understanding the roles of anti-asparaginase antibodies and the identification of genetic variants related to it in the hope of addressing L-asparaginase hypersensitivity. The mechanisms of action of how these antibodies target L-asparaginase after the drug administration as well as the specific pathways involved in these reactions remain unclear till date and need to be studied in future. Based on current understanding, further and more thorough studies are anticipated to help optimise the therapy in paediatric ALL patients.


Cyclooxygenase (COX) and Lipoxygenase (LOX) are essential enzymes for arachidonic acid (AA) to eicosanoids conversion. These AA-derived eicosanoids are essential for initiating immunological responses, causing inflammation, and resolving inflammation. Dual COX/5-LOX inhibitors are believed to be promising novel anti-inflammatory agents. They inhibit the synthesis of prostaglandins (PGs) and leukotrienes (LTs), but have no effect on lipoxin formation. This mechanism of combined inhibition circumvents certain limitations for selective COX-2 inhibitors and spares the gastrointestinal mucosa. Natural products, i.e. spice chemicals and herbs, offer an excellent opportunity for drug discovery. They have proven anti-inflammatory properties. However, the potential of a molecule to be a lead/ drug candidate can be much more enhanced if it has the property of inhibition in a dual mechanism. Synergistic activity is always a better option than the molecule's normal biological activity. Herein, we have explored the dual COX/5-LOX inhibition property of the three major potent phytoconsituents  from Indian spices using in silico tools and biophysical techniques in a quest to identify their probable inhibitory role as anti-inflammatory agents. Results revealed the dual COX/5-LOX inhibitory potential of curcumin. Gingerol and capsaicin also revealed favorable results as dual COX/5-LOX inhibitors. Our results are substantiated by target similarity studies, molecular docking, molecular dynamics, energy calculations, DFT, and QSAR studies. In experimental inhibitory (in vitro) studies, curcumin exhibited the best dual inhibitory activities against COX-1/2 and 5-LOX enzymes. Capsaicin and gingerol also showed inhibitory potential against both COX and LOX enzymes. In view of the anti-inflammatory potential these spice chemicals, this research could pave the way for more scientific exploration in this area for drug discovery. Reducing risk factors might be considered an approach to tackling such chronic illness2. As evidence links many chronic diseases to inflammatory events, modulation of the inflammatory cascade appears to be a critical new approach to preventing these conditions3. Inflammation may be caused by various agents such as pathogens , external body injuries, chemicals or radiation effects, and a diseased condition . Severe inflammation, in some cases, might cause allergic reactions. Mostly all diseases are connected to inflammation, viz. arthritis (autoimmune disease), HIV (human immunodeficiency virus), psoriasis, TB (Tuberculosis), cancer, pneumonia, asthma, hypersensitivities, inflammatory bowel diseases, interstitial cystitis, vasculitis, chronic prostatitis, and many more4.Non-communicable diseases (NCDs) lie in the epicenter among all diseases as they represent the most prominent class in the death rate. The global chronic disease epidemic is responsible for the premature deaths of about 17 million people (80%) annually5. Eicosanoids, such as leukotrienes, prostaglandins, and thromboxane, are major inflammatory mediators, and their physiological production requires these enzymes.COX-1 is an essential enzyme that catalyzes the biosynthesis of eicosanoids which in turn promotes platelet aggregation, thromboxane, and vasoconstriction. It is mostly found in the kidney, stomach, and platelets6. Inhibition of this enzyme could help reduce biomarkers of inflammation. Non-steroidal anti-inflammatory drugs (NSAIDs), whether selective or non-selective, are commonly used as the first line of treatment in inflammatory diseases, because cyclooxygenase isoenzymes (COX-1 and COX-2) are responsible for the production of prostaglandins that play an important role in inflammation. They suppress the formation of pro-inflammatory eicosanoids. However, the long-term usage of these inhibitors may lead to bleeding, kidney failure, severe gastrointestinal disorders, and bronchospasm7. To avoid side effects, scientists had focused on developing selective COX-2 inhibitors in response to mounting evidence that this particular isoenzyme is over-expressed in inflammatory conditions, as opposed to COX-1 or combined COX-1/COX-2 inhibitors , which are associated with a wide range of unpleasant side effects8. Eicosanoids are released by the second essential biosynthetic pathway, activated by 5-lipoxygenase (5-LOX), an isozyme of LOX. Leukotriene B4, a mediator of atherosclerosis, cancer, and cardiovascular disease, is produced at the termination of the 5-LOX pathway. Thus, lowering leukotrienes by inhibiting 5-LOX may mitigate the risks of selective COX-2 and COX-1 inhibitors on the cardiovascular and gastrointestinal systems. With this perspective, it was anticipated that co-inhibition of cyclooxygenases (COX-I and COX-II) and 5-lipoxygenase (5-LOX) could lessen the cardiovascular and gastrointestinal side effects, while preserving the drugs' primary effectiveness against cyclooxygenase, particularly COX-210. Dual COX/5-LOX inhibitors offer numerous therapeutic benefits over traditional NSAIDs; firstly, they act on the two major arachidonic acid metabolic pathways and possess a wide range of anti-inflammatory activity. Secondly, dual inhibitors provide enhanced gastric protection and a safer cardiovascular profile, which is COX inhibitor’s most cumbersome side effect13. As a result, there has been significant effort in the field of dual-acting COX/5-LOX inhibitors in recent years, with very promising results. Darbufelone and Licofelone were developed and tested in humans as COX/5-LOX inhibitors. High toxicity and low efficacy prevented their commercialization, though. Hence, the need is to discover or repurpose drugs that could have dual COX/5-LOX inhibitory nature as both COX and LOX derivatives  are involved in other diseases than inflammation such as cancer proliferation wherein the use of dual inhibitors could be an interesting approach. In light of these ideas, it has been proposed that inhibiting the production of prostaglandins and leukotrienes could have synergistic effects and lead to the greatest reduction in inflammatory reactions possible12. Figure Many human diseases can be traced back to arachidonic acid cascade inflammatory mediators produced by the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. Arachidonic acid (AA) is metabolized by a series of enzymes known collectively as cyclooxygenases COX-1/2) and lipoxygenases (LOX)/2 and li13. Numerous studies on plant whole crude extracts have been conducted over the past few decades, and it has been discovered that several plants have anti-inflammatory properties. Multiple inflammatory conditions can be effectively treated with compounds derived from plants. Alkaloids, flavonoids, polyphenols, and terpenoids are the few chemical classes linked to anti-inflammatory effects. Some well-known examples include andrograpanin, azadirachtin, Curcumin, embelin, resveratrol, and quercetin. Polyphenols are essential anti-inflammatory compounds found in spices that help improve health and decrease undesirable medication reactions. The discovery of flavonoids as a valuable substitute for traditional analgesics and anti-inflammatory agents with dual-inhibitory action, especially on COX-2 and 5-LOX, can reduce or resolve this issue and has attracted much attention. The anti-inflammatory compound flavocoxid, which contains the flavonoids catechin and baicalin, works by simultaneously inhibiting the COX 1/2 and 5-LOX enzymes14. Flavonoids are potent antioxidants that may regulate the oxidative generation of arachidonic acid from phospholipids, hence decreasing lipid peroxidation processes and activation of inflammatory pathways15. Flavocoxid suppresses inflammation not only by inhibiting cyclooxygenase (COX 1/2) and lipoxygenase (5-LOX), but also by lowering NF-B-induced production of pro-inflammatory cytokines16.In this regard, phytomedicine offer an excellent source of bioactive compounds that could serve the purpose above. Their bioactive constituents or natural products are resonably safer and more active than synthetic compounds17. Ginger and its phenolic compounds , sesquiterpenes have shown potent activity in reducing lung inflammation19. It is highly effective in inflammations associated with alimentary channel such as colitis and inflammatory bowel disease20. It also has clinical evidence in reducing the pain associated with osteoarthritis21. Polyphenols in green tea have anti-inflammatory effects. Green tea has been found in clinical research to lower inflammation related with cardiovascular disease, arthritis, and other inflammatory disorders22. It has also been demonstrated to be useful in lowering obesity-related inflammation and Alzheimer’s disease23. Capsaicin has anti-inflammatory activities24. In addition to these there are many other natural compounds which have clinically proven anti-inflammatory effects i.e. colchicine, resveratrol, epigallocatechin-3-gallate (EGCG), and quercetin25.Many natural products have shown proven efficacy towards preventing/curing inflammation associated with various diseases in clinical investigations. Turmeric and its curcumin-enriched preparations have been used to treat arthritis and alleviate its symptoms i.e. morning stiffness of joints30. Medical practitioners have used them to treat various ailments, including inflammation31. As per reports, culinary herbs and spices exert anti-inflammatory activities by activating PPARα and PPARγ, thereby causing inhibition the activation of NF-κB and enhancing the expression of anti-inflammatory cytokines32. Studies on animal inflammation models have demonstrated that curcumin and capsaicin delay the development and minimize the incidence and severity of arthritis by inhibiting TNF-α, IL-1β, IL-6, and NF-κB (inflammatory cytokines)33. Curcumin is superior to aspirin in this situation because it selectively inhibits the development of the anti-inflammatory TXA2 without side effects. Capsaicin down regulates the expression of the COX-2 enzyme and represses the inducible nitric oxide synthase (iNOS) to reduce PGE2 synthesis. It suppresses the synthesis of pro-inflammatory mediators, TNF-α and IL-6, following the elevation in the production of anti-inflammatory mediator IL-1034. It has been demonstrated that capsaicin modulates the NF-B signaling pathway bythe formation of pro-inflammatory cytokines. It exerts an anti-inflammatory effect by blocking the signaling path, coupling pro-inflammatory stimuli with cyclooxygenase activation, including inhibiting NF-κB nuclear translocation35. Gingerol, the bioactive constituent of ginger, is anti-inflammatory, as it inhibits PGE2 generation and reduces nitric oxide production. They ultimately activate the endogenous antioxidant defense system by enhancing the generation of anti-inflammatory cytokines and inhibiting the formation of pro-inflammatory cytokines36. According to the meta-analysis, ginger consumption reduces C-reactive protein (CRP), high sensitivity C-reactive protein (hs-CRP), and tumor necrosis factor(TNF-α) considerably37. A wide array of spices and their phytoconstituents remain unexplored for their pharmacological activity due to limited studies.This shifts our attention to the common culinary spices, as since time immemorial, they have played a very significant role due to their traditional importance in fields like Ayurveda and homeopathyCurcuma longa), ginger  and red pepper (Capsicum annuum) and identifying the possible mechanistic pathway (COX/5-LOX inhibition) through in silico and in vitro studies39. The need for new anti-inflammatory agents having dual mechanisms with improved safety profiles that prevent the release of both prostaglandins and leukotrienes encouraged us to repurpose a few anti-inflammatory agents  for their dual inhibitory activity to reclassify them as potent anti-inflammatory agents against both targets with a different probable mechanism of action. Their dual inhibitory activity and binding modes have been investigated using in silico tools41. Such a viewpoint opens the door, identifying culinary spices and herbs as a potential source of potent phytochemicals of the COX-1/2 and 5-LOX dual-route and offering it as an option in inflammation associated with various diseases. In view of the anti-inflammatory potential these spice chemicals, this study could pave a path for the scientific community to explore further in this direction, ultimately benefiting humanity.In order to explore the role of phytoconstituents of common culinary spices in preventing/curing inflammation associated with various diseases, herein we aim to examine the role of culinary spice phytochemicals like curcumin, gingerol, and capsaicin from turmeric 43.Target similarity represents the first step towards identifying dual inhibitors for any class. It helps identify structurally/functionally similar regions within proteins, which can justify it’s common evolutionary descent. Further, it supports the concept of dual inhibition by predicting the location and function of protein-coding and transcription-regulation regions in the genomic DNA by identifying conserved regions® Core™ i7-11,800 CPU@2.30 GHz processor, 16 GB RAM). All co-crystallized protein structures were obtained from the Protein Data Bank (PDB) database44. Various modules of Schr 45. The QSAR study was carried out usingHyperChem Professional 8.0.3 program.All simulation studies were performed on a DELL workstation running Ubuntu 20.04.3 LTS 47, and 5-LOX(PDB ID:3V99)48. The X-ray crystal structures of all proteins were downloaded from the Protein Data Bank49. The proteins were in complex with an inhibitor . The protein was prepared using the protein preparation wizard module of Schrödinger software50. The steps were addition of missing polar hydrogens, deletion of water molecules (beyond 5 Å) from hetero groups, ionization,generation of tautomeric statesat pH of 7.4 , optimization of hydrogen bonds [predicted by PROPKA (pKa prediction)], and finally energy minimization.Three pro-inflammatory receptors/proteinswereemployed in this study. These are COX-1 (PDB ID:3N8Y)Three phytocompounds, viz., curcumin, capsaicin, and gingerol were used as ligands for screening against COX-1, COX-2, and 5-LOX to determine their triple inhibitory potential. These ligand molecules are shown in Table All molecular docking simulation studies were performed using the Ligand docking program of the Maestro 12.3 module of Schrodinger. Before the initiation of docking, the active site coordinates on the target proteins were generated employing the Receptor Grid Generation module. The van der Waals radii scaling factor and partial charge cutoff were kept at 1.0 and 0.25. The remaining parameters were kept as default. The Extra Precision (XP) mode was used to perform molecular docking of the three phytocompounds in the active site of three respective proteins. The binding affinities and molecular interactions were analyzed, and data were recorded. Lastly, the protein–ligand complex interactions were visualized in 2D/3D, and images were saved for representation.https://www.swissadme.ch) webserver, wasemployed to compute the ADME values51. Predicting the in silico toxicity of a substance is essential for evaluating its safety profile. They not only aid in reducing the number of animal testing, but also help determine the maximum tolerable levels in animals. In this study, pkCSM web server (https://biosig.lab.uq.edu.au/pkcsm/prediction) was used to predict the pharmacokinetic properties/toxicities of our small molecules using graph-based signatures52. This server database provides various toxicity parameters, i.e. AMES toxicity, maximum tolerated dose, hepatotoxicity, skin sensitization, and hERG I and II inhibition. In both servers, the first step involves either uploading or writing the SMILES string of the particular molecule in the given space. The second step is to allow the server to run the program. Following this, the predictive data can be gathered and analyzed.The pharmacokinetic process known as absorption, distribution, metabolism, and excretion (ADME) describes how the body responds to a drug. Predictive in silico ADME data aid the drug development process since it aids lead optimization. Several software programs were used to forecast the in silico ADMET attributes for all the molecules. In this study, the Swiss ADME, developed and maintained by the Swiss Institute of Bioinformatics (SIB) (54. MDS was carried out using the Desmond module of the Schrödinger Suite developed by the D.E. Shaw research group (Academic license)55. The system builder panel was used to build the orthorhombic simulation box with Simple Point-Charge (SPC)56. The periodic boundary conditions were set at 10 Å from the outer part of the protein surface. The TIP3P water model was used to solvate the system. To the solvated system, sufficient counter ions were added to neutralize the system. The 0.15 M NaCl was added to the simulation panel maintained an isosmotic state. OPLS_AA force field was applied to the protein–ligand complex. Energy minimization was carried out till the system reached a stable condition . This equilibrated system was used for the final production of the MD simulation for 100 ns at 310.15 K temperatures at 1.0 bar pressure with NPT  ensemble while using default settings for relaxation before simulation. The results of the MD simulation were analyzed by generating a simulation interaction diagram. RMSD, RMSF, protein–ligand interaction diagram, interacting amino acid residues with the ligand in each trajectory frame, and the trajectory of different ligand properties were analyzed.Molecular dynamics simulation (MDS) helps study the protein–ligand complex’s structural stability and flexibility. In this study, MDS was performed for the top hit compounds to legitimize the protein–ligand complex (PLC) and measure the ligand-binding constancy in the active site of the selected targetthermal_MMGBSA.py script of the Prime/Desmond module of the Schrödinger suite was used. The post-simulation MM-GBSA analysis of free binding energy calculation was carried out with the generation of 0–1000 frames. Two hundred frames were processed and analyzed throughout the MM-GBSA calculation of 100 ns MDS data. The binding free energies  were then calculated.Post-simulation MM-GBSA analysis was carried out. The 58. Following the optimization, analysis was done using the Gauss View 6.0.16 program .As one of the most reliable theoretical methods, density functional computations were used to conduct DFT experiments using the Gaussian09 suit programs45. In this study, calculations were performed using B3LYP and exchange–correlation functional with a basic set of 6-31G  for the atoms of carbon, nitrogen, oxygen, and hydrogen, and a theoretical geometry was established. For the ligands under study, DFT/B3LYP 6–31 + G  basis sets were used to assess single-point energy and dipole moment (D) values61. Calculated variables included the ionization energy, electron affinity, electronegativity, electronic chemical energy, chemical hardness, chemical softness, and electrophilicity index. Using DFT analysis, the energy difference between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies were used to study the interactions of the ligand/s with the proteins62. The study considered the active site of the protein and examined the binding energies of the interacting residues.DFT calculations help to investigate the electronic/nuclear structure of various systems. Based on quantum mechanical concepts, DFT computations enable predicting and estimating the system’s behaviorThe QSAR study was carried out using HyperChem Professional 8.0.3 program. The ligands were optimized by applying force field (MMþ) using semi-empirical PM3 methods. Energy minimization was carried out using the Fletcher–Reeves conjugate gradient algorithm. In addition, various other parameters like volume, hydration energy, Log P, refractivity, polarizability, mass, total energy, dipole moment, and RMS gradient were also computed for all the ligands under study.65. The inhibitory activity was measured and reported as IC50 value (µM). Data are expressed as IC50 ± SEM for triplicate studies.The inhibitory activities of the test compounds against COX-1 and COX-2 enzymes were determined by enzyme immune assay (EIA) kits  using COX-1 (ovine) and COX-2 (human recombinant) according to reported methods50 values were measured in µM according to the manufacturer’s instructions and reported method65. Data are expressed as IC50 ± SEM for triplicate studies.The ability of the test compounds to inhibit the 5-LOX enzyme was evaluated using the Human Lipoxygenase Inhibitor screening assay (EIA) kit . The ICFor dual inhibition of COX-1/2 and 5-LOX, all the major proteins involved in the inflammation pathways were selected based on literature review as we searched for significant proteins that influence inflammation. Keeping in pace with the concept of dual inhibition, three targets (COX-1/2 and 5-LOX) were selected,as presented in Table Sequence alignment was done for all three targets under study. Figure 66. The presence of multiple electropositive micro-domains being close to each other could favor the binding of small molecule inhibitors. The active site pocket of the COX-2 binding site is 20% larger than that of COX-1, whereas, in the case of 5-LOX,the active site is fully encapsulated. In the latter case, the amino acid residue Phe177plays a critical role and helps in the substrate/inhibitor binding in the active site67.To validate the results of target similarity, it is very important to locate the binding sites on the proteins to access the ligand interaction. For this, we evaluated the electrostatic surface potential (ESP) of the three studied proteins, as shown in Fig. 3Further, His600 helps in substrate catalysis. Hence, the structural motif of 5-LOX is termed a “twist-and-pour cap”. Thus, from the above images and discussion, it can be interpreted that these three proteins share a high degree of similarity. Hence for our study, these three targets represent the primary class of proteins involved in the inflammatory pathways. This also justifies the rationale behind the design of dual inhibitors targeting COX-1/2 and 5-LOX.68.The validation/re-docking studies Fig.  on the cThe ligand docking study with COX-1/2 and 5-LOX enzymes revealed favorable binding energies. The binding affinities/docking scores of all molecules under investigation have been presented in Table Structurally, there were relative correlations among the number of H-bonds. Upon binding with 1CVU, curcumin had two H-bonds with Ser530 and Tyr355, gingerol had two H-bonds with Met522 and Tyr335, and capsaicin had two H-bonds with Val349 and Met522 Figs. ; Table 469. Moreover, the position of the co-crystallized ligand and the three test compounds inside the catalytic site of COX-2 was stabilized by surrounded hydrophobic interactions.Due to the high homology of binding site residues, various interactions were conserved. The high docking score of curcumin could be attributed to the formation of H-bonds and different other interactions, i.e., hydrophobic and polar. Apart from conserving the H-bond with Ser530, curcumin was able to make a new H-bond with Tyr355. This new H-bond formation might allow deeper penetration into COX-2 protein. It has also been observed that the COX-2 binding site was 20% larger than that of COX-1. Apart from establishing H-bonds, it also showed varied hydrophobic interactions with a variety of amino acid residues . These interactions play a major role in stabilizing the ligands at the binding interface. Moreover, the strong binding affinity could be attributed to the presence of polar residues, namely Arg120, Ser353, Tyr385, Gly526, and Glu524 that enhance the chances of squeezing the molecule into the active site. To sum up, curcumin’s selectivity to COX-2 was primarily due to the binding to the hydrophobic region and extending to the lobby region near the entrance of the COX-2 binding site, thereby forming hydrogen bonds with Ser53070.To understand the binding modes of ligands in COX-1, it is crucial to have a detailed analysis of the various interactions at the residues level. Figures 69. Capsaicin and gingerol displayed less binding energies and interactions with the COX-1 binding site. This could be attributed to the incompatibility between the narrow groove (Arg120 to Tyr355) and the bulkiness of the molecules. There might be an inconsistency in their positions and interaction patterns concerning curcumin and internal ligand. Based on the analysis of interactions, curcumin seems to inhibit COX-1 prominently.The curcumin ligand’s backbone makes various hydrophobic contacts, viz. Val349, Leu352, Trp387, Leu384, Phe381, Leu531, Met522, Ala527, Ile523, Val116, Leu93, Pro86, and Ile89, and few polar interactions viz. Ser353, Tyr355, Tyr385, Ser530, Gly526, and Glu524. This encloses the curcumin molecule in a cage-like structure, justifying its COX-1 inhibition and increasing its selectivity71. Figures +2) which forms the center of the active site. The active site cavity appears to have a partial U-shaped cavity lined with highly conserved Leu and Ile residues. However, 5-LOX protein has a unique catalytic cavity with no substrate access site due to the shielding effects of outer solvents. It comprises the side chains of residues Phe177 and Tyr181 positioned to impede access into the catalytic cavity. The presence of catalytic iron in 5-LOX acts as both an electron donor andacceptor72.The 5-lipoxygenase (5-LOX) enzyme is responsible for the initiation of biosynthesis of pro-inflammatory leukotriene lipid mediators and the synthesis of anti-inflammatory lipoxins+2 cofactor of the 5-LOX enzyme was bound to capsaicin and gingerol, which causes the chelation of these molecules. Besides, all ligands were mostly enclosed around hydrophobic residues. These hydrophobic interactions  were primarily conserved in all cases, along with polar interactions (histidine and serine residues). Though all ligands bind effectively with 5-LOX, the number of H-bonds made by curcumin is more than other ligands under study. It was stabilized by surrounded hydrophobic and polar interactions, contributing to a slightly higher 5-LOX inhibition than different ligands. Hence, curcumin effectively binds to the active site and inhibits 5-LOX activity that might reduce leukotriene production thereby reducing the inflammatory responses.Compared to the internal ligand, wherein hydrogen bonds were formed, curcumin, capsaicin, and gingerol were interactive enough to establish H-bonds. Curcumin had three H-bonds with Lys409, Ala672, and Phe177, capsaicin had two H-bonds with Val671 and Phe177; and gingerol had two H-bonds with Asn554 and Phe177. These interactions help the ligands access the active site via the α2 helix. The H-bond with Phe177 is critical in providing a fully functional active site.FeTo conclude, among all three ligands under study, curcumin could be considered the best-fit molecule for all three proteins. The geometry of curcumin allows its insertion into the active site of all three proteins, with hydrogen bonding potential, i.e. acceptor/donor groups that improve the affinity for COX-1/2 and 5-LOX. From the above discussion, it is evident that curcumin has the potential to block both COX-1/2 and the 5-LOX enzymes. It generally acts by blocking the formation of both prostaglandins and leukotrienes, but does not affect lipoxin formation. Such combined inhibition might avoid some disadvantages of selective COX-2 inhibitors and spares the gastrointestinal mucosa.http://www.swissadme.ch/). The obtained ADME properties are presented in detail in Table 75. Results indicated that the molar refractivity, which accounts for the overall polarity of the molecules, was 102.80, 90.52, and 84.55 for curcumin, capsaicin, and gingerol, respectively; these values were in the normal range (30–140)76.The topological polar surface area (TPSA) was 93.06, 58.56 and 66.76 Å2 for curcumin, capsaicin, and gingerol, respectively. This refers to the molecule’s ability to permeate cells, i.e. molecules with TPSA > 140 Å2 are poor at cell permeation. A TPSA < 90 Å2 is usually preferred for molecules to permeate the BBB and act on CNS76. This suggests that curcumin cannot cross the blood–brain barrier (BBB), while the other two can.Regarding drug properties that affect ADMET, “solubility class lipophilicity” refers to a molecule's ability to dissolve into a lipophilic medium77. These properties include permeability, ADME, solubility, plasma protein binding, and toxicity. Results of iLOGP78 and SILICOS-IT suggested that the iLOGP values of all the molecules were in the acceptable range (− 0.5 to + 5.4), while SILICOS-IT values for all the identified leads were in the most favorable range (− 10 to 0). Water solubility is a significant factor determining a drug’s distribution and absorption. Log S calculations show the molecule’s solubility in water at 25 °C. The ESOL model’s calculated log S values shouldn’t be more significant than six for appropriate solubility79. Curcumin, capsaicin, and gingerol showed log S values of − 3.94, − 3.53, and − 2.96, accounting for a good solubility profile. According to the findings above, all of the lead compounds showed a good balance between permeability and solubility and may exhibit acceptable bioavailability when taken orally. All the molecules revealed a high predicted GI absorption80. Understanding the results of ADMET and cell-based bioassays is aided by permeability predictions81. Results indicated that the permeability over human skin was -6.28, -5.62, and -6.14 cm/s for curcumin, capsaicin, and gingerol, respectively. These predicted values were in the acceptable range81.Drug interactions caused by metabolism can occasionally reduce a drug’s bioavailability. Drug-metabolizing enzymes can only bind to the drug in its free form. As the most significant class of metabolizing enzymes, cytochrome P450 enzymes (CYPs) must be studied to understand the metabolic behavior of studied molecules. The CYPs (CYPs of human liver microsomes (HLM)) inhibitory activity of all the lead compounds was evaluated82. Only curcumin showed a good profile in this regard. A molecule's drug-likeness indicates whether it has the potential to develop into an oral medication.All three ligands under study were evaluated for their ADMET profile using online web server i.e. Swiss ADME  to 10 (very tough)).Thus, from the above data, it is evident that all molecules predicted ADME data are within the recommended values, with curcumin being the best.In our investigation, the drug-likeness was determined using five distinct filters. All molecules displayed no violation of drug-likeness rules and had a bioavailability score of 55% (good bioavailability). The Abbot Bioavailability score, which can be determined by a feasibility score of 11%, 17%, 56%, or 85%, forecasts the fate of a chemical for an experiment including quantifiable Caco-2 cell line permeability or 10% oral bioavailability (in rats)https://biosig.lab.uq.edu.au/pkcsm/prediction). The AMES test, which uses microorganisms to determine a chemical compound's projected mutagenic potential, produced positive results for all molecules, revealing no AMES toxicity. The most significant dose that most patients can accept is technically known as the maximally tolerated dose (MTD). Herein the maximum tolerated dose (human) was 0.081, 0.46, and 0.0635 Log mg/kg/day for curcumin, capsaicin, and gingerol, representing a moderate dosage level per protocols, with curcumin being the most potent one. hERGI and II (human Ether-a-go-go-Related gene) codes for proteins that regulate ion channels responsible for the cardiac electrical action potential of the heart. Hence, drugs under study should prohibit their inhibition during drug development. Only curcumin showed no hERGI and II inhibition, negating ventricular arrhythmias' probability. For curcumin, capsaicin, and gingerol, the Oral Rat Chronic Toxicity (LD50) values were 1.833, 2.065, and 1.958 mol/kg, respectively, while the Oral Rat Chronic Toxicity (LOAEL) values were 2.228, 1.827, and 1.631, respectively, indicating a good safety profile. All molecules (curcumin and gingerol) except for capsaicin were predicted to be non-hepatotoxic. All molecules showed no skin sensitization as well. T. Pyriformis toxicity and Minnow toxicity were at acceptable levels. Results of the predicted toxicity of all molecules under study are shown in Table All three molecules  under study were evaluated for their toxicity profile using an online web server, pkCSM . MD studies are usually implemented to study the nature of macromolecules or analyze the physical movements of atoms and molecules. The obtained simulation findings are discussed below.The RMSD values were substantial for all protein–ligand conformations. RMSD analysis indicates whether the simulation has equilibrated around a fixed value. In complex A, the protein RMSD was stable throughout the simulation with minor fluctuations between . The variation was in the range of 1.6–2.8 Å.How stable a ligand is in relation to a protein and its binding pocket is shown by the ligand RMSD (right Y-axis). Ligand RMSD was highly unstable as it showed significant fluctuations with 2.0–3.5 Å from 0 to 40 ns which then stabilized, showing 5–8 Å till 100 ns with some minor deviations Fig. A. For coFor describing local variations along the protein chain, the RMSF is helpful. Peaks on this diagram represent regions of the proteins that vary most during the simulation. Usually, the protein’s N- and C-terminal tails move more than any other area. Because they are often more rigid than the protein's unstructured portion, parts of secondary structures like alpha helices and beta strands change less than loop sections. The RMSF of 3N8Y protein Fig. A looks sThe trajectories of different properties of co-crystal ligand-enzyme complexes  during the 100 ns of MD simulation are depicted in Fig. Protein–ligand contacts were monitored throughout the simulation. H-bonds are essential for ligand binding. Because they have such a significant impact on drug specificity, metabolization, and adsorption. Hydrogen-bonding properties must be taken into account while developing new drugs. Herein complex A Fig. A showed The interactions and contacts  presented andare shown in the form of a timeline in Fig. The average change in displacement of a particular set of atoms for a given frame relative to a reference frame is calculated using the RMSD values. For each frame in the trajectory, it is computed. The RMSD values are aligned with the protein-internal ligand confirmations. In complex A Fig. A, the prRMSF analysis helps in understanding the local variations along the protein chain. Usually, peaks represent the residues that fluctuate the most during the simulation. The primary structures of a protein typically move more than the secondary structures as they are more rigid.The fluctuations in the protein-curcumin complex were almost the same as in the protein-internal ligand case. Hence this shows the complexes’ overall stability. Herein the RMSF of 3N8Y protein Fig. A looks sThe trajectories of different properties of curcumin-enzyme complexes  during the 150 ns of MD simulation are depicted in Fig. Protein–ligand contacts refer to the interactions between the protein and ligand during the simulation period. They can be classified under four major headings: H-bonds, hydrophobic interactions, ionic interactions, and water bridges. The bars represent the % of the simulation time the specific interactions were maintained. Herein complex A Fig. A showed The interactions and contacts  presented and shown in the form of a timeline in Fig. The three ligands were subjected to a MM-GBSA analysis to determine the binding energies between the ligands and the proteins. It helps determine the protein–ligand complex’s stability after it is bound to the active site. Herein, the dG value represents the difference in energy between the optimized protein–ligand complex (prime) energy and a combination of optimized free ligand and protein energy. The detailed MM-GBSA dG binding values have been presented in Table The MM-GBSA analysis revealed that curcumin had the best binding energy with all the three proteins under study . These binding energies were relatively better than the co-crystallized protein–ligand complex, highlighting curcumin’s potential in binding and inhibiting all three proteins under study.The DFT analysis investigated the binding affinity pattern of the ligands under investigation as it revealed electronic structural characteristics of the ligands. Using Koopman’s theorem, all three ligands were optimized to the B3LYP/6-311G level, and their molecular orbitals were investigated. Table Furthermore, positive chemical hardness and chemical softness values enhance compound’s molecular interactions. Other metrics, such as polarizability (r) and electrophilicity (x), quantify the tendency to take an electron from its surroundings as a positive amount. Figure The QSAR analysis showed several critical aspects related to the chemical structures of the phytocompounds. Table 50) was determined using serial dilution method  for each compound. Ibuprofen, celecoxib, and zileuton were used as reference drugs for COX-1, COX-2, and 5-LOX, respectively. From results presented in Table Colorimetric enzyme immunoassay (EIA) was used to assess the inhibitory potential of the test compounds against COX-1, COX-2, and 5-LOX enzymes. The test concentration which produces 50% inhibition  are comparable to some extent with ibuprofen and celecoxib (COX-I and COX-2 inhibitors) and zileuton (5-LOX inhibitor). However, curcumin possesses the best dual inhibitory activity towards COX-1/2 (IC50 = 18.58 ± 0.44 µM/13.42 ± 0.92 µM) and 5-LOX (IC50 = 24.08 ± 0.63 µM) enzymes. The in vitro inhibitory activities thus confirm the predicted inhibition of COX-1/2 and 5-LOX enzymes in molecular docking studies. Experimental investigation thus validates the in silico inhibition study.Among the three compounds, curcumin exhibited the best inhibitory activity against COX-1/2 and LOX enzymes. The inhibitory activities of capsaicin and gingerol were also prominent, but the extent of inhibition was less as compared to curcumin. When compared with the standard drugs, the ICThe currently available therapeutics, i.e. NSAIDs that target various enzymes in the inflammation pathways, suffer from side effects. There is an urgent need to rediscover a single molecule with intense synergistic anti-inflammatory activity. This could only be possible through “dual inhibition of enzymes” in the inflammation pathway. In this perspective, we focused on exploring the role of culinary spice chemicals as dual inhibitors of key enzymes, COX and LOX in inflammatory pathways. There are strong evidences that several culinary spices could exert health benefits with strong antioxidant and anti-inflammatory properties.40. For example, several studies have shown that the combination of curcumin (major bioactive from turmeric) and piperine (major bioactive from black pepper) results in a synergistic inhibition of cancer cell growth. This is thought to occur because piperine enhances the bioavailability of curcumin, allowing it to more effectively target cancer cells. Another example is the combination of berberine  and metformin (a synthetic drug used to treat diabetes). It has been reported that the combination of these two compounds results in a greater reduction in blood sugar levels than either compound alone, suggesting a synergistic inhibition of diabetes. On this note, the concept of synergistic inhibition of natural products holds promise for the development of more effective and efficient therapies for a range of diseases and conditions87.This paves the way to employ the phytoconstituents of culinary spices as potent inhibitors of the enzymes involved in the inflammation pathways. Developing new and selective COX inhibitors requires knowledge of COX structure and isoform specificity. COX’s active site comprises a lengthy hydrophobic channel with a limited entry at the membrane-binding domain. Despite the fact that their active sites are quite similar, their binding cavities differ, with COX-2 having a bigger binding cavity than COX-188. This created opportunities for the creation of specific COX inhibitors. The selectivity of COX inhibitors is determined by interactions with three distinct areas of the enzyme’s active site: the Arg120/Tyr355-containing entrance region, the hydrophobic pocket directly below the haem group, and the side pocket. Highly conserved sections include the active site entrance and the hydrophobic pocket; however, the side pocket is non-conserved, with a few different amino acid residues that give rise to an additional pocket in COX-2.COX-1 and COX-2 have highly similar active sites89 However, COX-2 has a side pocket just above the COX binding site’s entrance (Arg120 and Tyr355). The amino acids that comprise the 5-LOX binding site are primarily hydrophobic and form a deep-bent shape. Reports have indicated that Phe177 and Tyr181 in the cleft's top region form part of the 5-LOX binding site, with Trp599 and Leu420 at the cleft's base90. In addition, the polar amino acid Lys409 is present at the entrance of 5-LOX. The research suggested numerous other amino acid residues, including Tyr181, Leu414, Asn425, Arg411, and Phe421, are crucial for the interaction between 5-LOX and its substrate67. Hence, dual inhibition could be achieved by simultaneous inhibition of these two enzymes11.Synergistic inhibition occurs when two or more bioactive compounds work together to enhance each other’s inhibitory effects, resulting in a greater overall effect than would be achieved by either compound alone. This can occur through a variety of mechanisms, including increased potency, increased bioavailability, and enhanced target specificityπ-π interactions between the ring B of the flavanone and Tyr355 at the entrance to the COX binding site are responsible for the COX-2 selectivity of curcumin. The hydrophobic contacts between the COX enzyme’s hydrophobic region and the lobby region further promoted effective binding. Curcumin’s expected activity in blocking 5-LOX comes from the hydrogen bond interactions withLys409, Ala672, and Phe177. This H-bonding was absent in the case of co-crystallized ligand. The hydrophobic and polar interactions were the primary contributors to the effective binding of curcumin. Molecular dynamics data further strengthened the results of molecular docking. Results revealed the high stability of curcumin-protein complexes despite minor acceptable deviations. Upon binding of curcumin in the protein’s active site, the complex displayed minor RMSD values. MMGBSA binding energies were quite better than the co-crystallized protein–ligand complex, highlighting curcumin’s potential in binding and inhibiting all the three proteins under study68. The other two spice chemicals, capsaicin and gingerol, displayed good binding modalities with the proteins under study. This study also explored the pharmacokinetics (ADMET) and the chemical interactions of the ligands under study, with various proteins and curcumin being the good among all. The present investigational data is also supported by DFT and QSAR studies.As per our study, curcumin, the active ingredient of turmeric, displayed significant dual COX-1/2 and 5-LOX inhibitory activity. The results of molecular docking and dynamics studies can justify this. Hydrogen bond interactions with critical amino acids, such as Ser530 in the COX active site and There are some limitations regarding the analysis of natural products for inflammation associated with various diseases: (1) Complexity of natural products: Natural products are often complex mixtures of various compounds, which makes it difficult to isolate and identify the active compounds responsible for their anti-inflammatory effects. This complexity can also make it challenging to determine their mechanisms of action. (2) Lack of standardization: There is often a lack of standardization in the preparation and use of natural products, which can lead to variability in their potency and efficacy. This makes it difficult to compare results across different studies and to establish consistent dosing regimens. (3) Limited understanding of mechanisms of action: While natural products have been used for centuries to treat inflammatory conditions, the exact mechanisms by which they exert their anti-inflammatory effects are often poorly understood. This limits our ability to predict their efficacy in different disease contexts and to develop new drugs based on their mechanisms of action. (4) Limited clinical evidence: While there is a growing body of preclinical evidence supporting the anti-inflammatory effects of natural products, there is often a lack of well-designed clinical trials to support their use in humans. This makes it difficult to establish their safety and efficacy in clinical settings and to obtain regulatory approval for their use. (5) Interactions with other drugs: Natural products can interact with other drugs and medications, which can lead to unintended side effects or reduced efficacy. These interactions can be difficult to predict and may vary depending on the specific natural product and the individual patient.Overall, while natural products show promise as potential treatments for inflammatory conditions, there are still many limitations to their use and further research is needed to fully understand their mechanisms of action and potential clinical applications.Our study pioneered investigating the role of culinary herbs as a potential inhibitor of the enzymes (COX and LOX) associated with inflammation. Herein our study identified a spice chemical, curcumin as a potential dual inhibitor of COX-1/2 and 5-LOX. This perspective identifies curcumin as a possible phytoconstituent of the COX-1/2 and 5-LOX dual pathway and offers it as potential drug inflammation-associated diseases. Overall, the curcumin has the potential for the dual inhibition of COX-2 and 5-LOX. In experimental inhibitory (in vitro) studies, curcumin exhibited the best dual inhibitory activities against COX-1/2 and 5-LOX enzymes. Capsaicin and gingerol also showed inhibitory potential against both COX and LOX enzymes. This study also highlights the important role of culinary herbs and Indian spices in inflammation associated with various disorders. In addition, our study also provides compelling support for the idea that consuming a diet rich in certain anti-inflammatory spices  can help reduce inflammation and protect against diseases associated with chronic inflammation. Although these spices have been used for millennia to alleviate inflammation, more research is needed to determine whether or if they may be used therapeutically to prevent or treat inflammatory conditions. In view of the anti-inflammatory potential these spice chemicals, this research could pave the way for more scientific exploration in this area for drug discovery.


The East Antarctic Ice Sheet (EAIS) is currently surrounded by relatively cool water, but climatic shifts have the potential to increase basal melting via intrusions of warm modified Circumpolar Deep Water (mCDW) onto the continental shelf. Here we use an ice sheet model to show that under the current ocean regime, with only limited intrusions of mCDW, the EAIS will likely gain mass over the next 200 years due to the increased precipitation from a warming atmosphere outweighing increased ice discharge due to ice-shelf melting. However, if the ocean regime were to become dominated by greater mCDW intrusions, the EAIS would have a negative mass balance, contributing up to 48 mm of SLE over this time period. Our modelling finds George V Land to be particularly at risk to increased ocean induced melting. With warmer oceans, we also find that a mid range RCP4.5 emissions scenario is likely to result in a more negative mass balance than a high RCP8.5 emissions scenario, as the relative difference between increased precipitation due to a warming atmosphere and increased ice discharge due to a warming ocean is more negative in the mid range RCP4.5 emission scenario. The East Antarctic Ice Sheet is currently surrounded by relatively cool water but changes in ocean dynamics may lead to warmer ocean water on the shelf in the future. This has the potential to dramatically increase its future sea level contribution. Despite this large potential for mass loss, most estimates of its recent mass balance give close to zero , but some suggest large gains  and others suggest large losses , e.g. Fig. The East Antarctic Ice Sheet (EAIS) is the single largest potential contributor to future global mean sea level rise, containing 52.2 m of sea level equivalent (SLE)5 found that, whilst models consistently predict a negative mass balance for the West Antarctic Ice Sheet in the period leading up to 2100, there is a much greater disagreement amongst models about whether the EAIS will have a positive or negative mass balance over the same period7. The most recent simulations show a general trend for future East Antarctic mass balance to be accumulation dominated, with increasing emission scenarios leading to a gain in mass, although this is not found for all models, particularly when using low emission scenarios9. A warming climate is expected to lead to greater precipitation over Antarctica10 due to the greater moisture-holding ability of warmer air, and hence a positive change in mass balance unless there is a dramatic increase in ice discharge to compensate.The ISMIP6 project12. A warming climate is likely to affect the ocean temperature surrounding the EAIS via indirect means, with the strongest observed warming trends currently occurring in water to the north of ice shelf cavities rather than in their direct vicinity14. However, there remains the potential for substantial future warming due to climatic conditions forcing a shift in the particular water mass driving ice shelf melt15.Ice sheets contribute to sea level rise via surface melting and via ice discharge over the grounding line, a process enhanced by ocean-driven melting of floating ice reducing the buttressing effect of ice shelves17, Vincennes Bay18, Shirase19 and Amery ice shelf20. The Antarctic Slope Front (ASF) has been shown to regulate these intrusions, with a strong ASF limiting cross-shelf intrusions whilst a weaker ASF leads to more intrusions21. The strengthening (positive phase) and weakening (negative phase) of the band of westerly winds around Antarctica is known as the Southern Annular Mode (SAM). A positive SAM has been shown to weaken the ASF via wind-sea surface interactions, allowing increased intrusion of CDW over the continental shelf22. Observations of the SAM have shown a positive trend since the 1970s, with this trend predicted to continue with a warming climate23. Numerical modelling studies have also shown that the position of coastal currents and the upwelling of warm CDW would shift southward with a more positive SAM25. A poleward shift in the southern boundary of the Antarctic Circumpolar Current has been observed and modelled over the last 30 years off East Antarctica, potentially leading to warming shelf water26. In addition, ocean water freshening due to increased ice-sheet melt-water production has been shown to enhance CDW transport towards the continental shelf via a strengthening of the surface stratification near the Antarctic coast27, causing a positive feedback loop with increased basal melting28. A previous study found there to be spatial variation in this freshwater feedback loop, with the Western Antarctic region seeing a cooling of ocean temperatures and the Ross Sea region in East Antarctica a warming in ocean temperatures29. A comparison of two different coupled models found one undergoing oceanic warming whilst the other experienced cooling due to freshwater feedbacks30. Recent ocean observations, however, have noted a poleward shift in CDW in East Antarctica since 201015. Paleoenvironmental and paleoceanographic records from the Wilkes Subglacial Basin also find evidence for past grounding line retreat coinciding with periods of increased oceanic warming during late Pleistocene interglacials, most likely a result of CDW intrusions31. Evidence for a past retreat of the grounding line in the Wilkes Subglacial Basin of the order of 700 km has also been shown to correspond to ocean temperatures 1–2 °C warmer than present conditions, equivalent to the relative difference between shelf water and CDW32. If changing climatic conditions lead to CDW intrusions becoming more common in the future, as predicted by Herraiz-Borreguero and Garabato15, East Antarctic melt rates could grow to resemble the far higher CDW driven melt rates seen in West Antarctic ice shelves like Pine Island Glacier33 and Thwaites Glacier34.At present, the primary water mass in contact with East Antarctic ice shelves is relatively cool (≈−1.5 °C), but recent observations show intrusions of warmer (≈0 °C) Circumpolar Deep Water (CDW) in the area surrounding Totten Glacier25. To achieve this, we use a numerical ice sheet model driven by parameterised atmospheric and oceanic forcings to determine the future mass balance of the EAIS. This represents the first study to examine the potential impact such a shift would have on the EAIS as well as identify the particular regions that are most at risk to rapidly increasing oceanic melting.Here, we seek to quantify the sensitivity of EAIS mass balance over the next 200 years to a shift to more frequent CDW intrusions driven by a change in ocean circulation when compared to a future emissions scenario with no change in large scale ocean dynamics. We are primarily concerned with determining the effect such a shift would have upon the future mass balance of the EAIS rather than explicitly simulating the mechanisms by which these intrusions would occur, thus seeking to ascertain the spatial and temporal impact of such a switch across the EAIS. We consider the effect of both an intermediate  and extreme (RCP-8.5 emissions scenario) warming scenario on both ocean and atmospheric forcing, as they have both been shown to produce conditions likely to lead to increased intrusions of CDW over the continental shelf35 and the potential, relatively warm, oceanic regime that has become dominated by CDW intrusions mixing with shelf waters as modified Circumpolar Deep Water forcing (mCDW). We also assume, for the sake of simplicity and ease of comparison between different forcing regimes, that the general pattern of future atmospheric and oceanic forcing over the next 200 years will be an increase to a maximum value followed by a period of relatively constant forcing at this new maximum. As such, we apply these forcings in a manner that increases linearly from present conditions to a maximum after 100 years with these maximum values held constant for a further 100 years. This allows us to investigate the response of the EAIS to both increasing atmospheric and oceanic warming as well as a period of committed atmospheric and ocean warming36. A full description of our numerical modelling framework as well as the atmospheric and oceanic forcings applied can be found in the Methods section.We simulate the effects of both a continuation of the present day ocean regime and a shift towards an ocean regime dominated by CDW intrusions combined with an intermediate (RCP4.5) and extreme (RCP8.5) emissions scenario). We define the current, relatively cool, oceanic regime from here on as Shelf Water forcing (SW)We simulate four distinct climate scenarios, namely; (1) RCP4.5 atmospheric and oceanic warming, with ocean properties representative of a cooler SW ocean regime (RCP4.5-SW); (2) RCP8.5 atmospheric and oceanic warming, with ocean properties representative of SW (RCP8.5-SW); (3) RCP4.5 atmospheric and oceanic warming, with ocean properties representative of a warmer mCDW regime (RCP4.5-mCDW) and (4) RCP8.5 atmospheric and oceanic warming, with ocean properties representative of mCDW RCP8.5-mCDW. To account for model uncertainty associated with parameters for which no observational constraint exists results are given relative to a reference run with present day forcing held constant for 200 years.We first consider the effect of how ice volume in the domain changes throughout the model simulations by considering both the total ice volume in the domain Fig. a, b as wTotal ice volume relative to the reference run Fig. a for theTotal VAF relative to the reference run Fig. c increasTotal floating ice relative to the reference run Fig. e shows a2 for the RCP8.5-mCDW and −47174 km2 for the RCP4.5-mCDW scenario. The SW forced scenarios have losses in grounded area equal to −3835 km2 for the RCP8.5-SW and −4701 km2 for the RCP4.5-SW scenarios. The rate of change of grounded area  and +181 Gt a−1 (−0.5 mm a−1 SLE), respectively. All individual regions have a positive mass balance, with the exception of the D–D region in the RCP4.5-SW scenario which is approximately in balance. The RCP4.5-mCDW scenario . All regions except A–B, C–C and D–E have a negative mass balance, with D–D having the most negative at −34 Gt a−1. While the RCP8.5-mCDW  after 100 years, four regions, K–A, A–A, B–C and D–D, have a negative mass balance. A shift to mCDW forced melting has the potential to increase ice discharge by a large amount  and +143 Gt a−1 (−0.4 mm a−1 SLE), respectively. Individual regions maintain this trend of a positive mass balance that is smaller in magnitude than the first 100 years of simulation. The one exception is the D-D region  in the RCP4.5-SW scenario which now has a negative mass balance of −14 Gt a−1 in the RCP4.5-SW scenario, making it the only region to experience a negative mass balance change with SW forcing. Due to increased ice discharge over the final 100 years of model simulation the RCP4.5-mCDW scenario  whilst the RCP8.5-mCDW scenario  by the end of the simulation. The majority of regions in both simulations have a negative mass balance, with the exception of the D–E, A–B and the C–C region. A summary of these results is given in Table When the maximum atmospheric and oceanic forcing that was reached after 100 years of model simulation is maintained for another 100 years (representing the period 2100–2200) there is time for the ice velocities to adjust to the increased forcing, generating ice discharges that are higher when compared to those after 100 years whilst surface precipitation remains constant Fig. . As befoWe can break down each regions response to ocean warming into three different categories Fig. . The fir5 which found a majority of models predicting a positive mass balance for the EAIS as well as other estimates for future East Antarctic mass balance38. Pollard and DeConto39 find Totten glacier (basin C–D) beginning to undergo severe retreat and thinning by 2100, with our simulations only showing comparable results for the mCDW driven scenarios. Their model framework includes parameterisations of surface melt induced hydro-fracturing of ice shelves as well as marine ice cliff instability that our model framework does not, perhaps explaining the discrepancy. We find that the mass gain from increased precipitation outweighs the mass loss from increased ice discharge due to ice-shelf melt in SW forced simulations. It should be noted, however, that this is not necessarily a steady state. All our simulations show a reduction in floating ice as well as grounded area, even when they show an increase in VAF or total ice volume. If this were to continue into the future, East Antarctic ice shelves are likely to be greatly reduced in both thickness and extent compared to their current configurations. Simulations that directly impose an instantaneous collapse of East Antarctic ice shelves have found this to lead to the destabilisation of the Wilkes and Aurora Subglacial Basins over the next 500 years40. More transient simulations of the EAIS find that the removal of grounded ice or atmospheric driven surface melting are required to make the EAIS susceptible to collapse, with only the Recovery catchment in the Weddell Sea region (an area that lies outside of our model domain) proving susceptible to ocean driven collapse41. Model simulations of the Wilkes Subglacial Basin have found it to be stable in response to increased ocean warming, however if this increased ocean driven melt were to remove a plug of ice then the Wilkes Basin would become destabilised42.Our simulations show that if the cooler SW regime continues to be the primary driver of ocean-induced melting then the EAIS is likely to have gained VAF after 200 years for both the RCP4.5-SW  and RCP8.5-SW  scenarios. This is in broad agreement with the ISMIP6 results43 and Wilkes Subglacial Basin32 show this process has occurred in the past, linking indicators of mCDW intrusions with ice-shelf retreat.In contrast, if the water masses around East Antarctica were to switch to a mode that is dominated by enhanced intrusion of mCDW, our simulations reveal that the EAIS will have a negative overall change in VAF after the 200 years of model simulations for both the RCP4.5-mCDW  and the RCP8.5-mCDW  scenarios. Whilst the RCP8.5-mCDW scenario has a positive mass balance after 100 years this becomes negative after 200 years due to maximum ice discharge lagging behind the application of maximum oceanic forcing at year 100. The RCP4.5-mCDW scenario has a negative mass balance for the entirety of the simulation, as the relative increase of ice discharge compared to precipitation is greater throughout the entire 200 years of simulation time. Our simulations show that a switch to mCDW becoming the primary water mass driving ice-shelf melting is sufficient to more than counteract any increased mass gained from precipitation, leading to positive sea level rise contribution from the EAIS over the next 200 years (+4–48 mm SLE contribution). The reduction in both floating ice and grounded area is more pronounced in both the mCDW forced climate scenarios than in the SW forced scenarios, indicating an increased likelihood of ice shelf collapse and run away grounding line retreat that might occur in catchments with reverse-sloping beds inland of present grounding lines. Paleoenvironmental and paleoceanographic records records in the Amundsen sea sector−1 in the RCP4.5-SW scenario after 200 years. It is also the most strongly affected region in the mCDW forced scenarios and the susceptibility of this region to both past32 and future44 climatic shifts has been highlighted by other studies. Findings from the ISMIP6 project5 found Wilkes Land to be the most strongly affected region of East Antarctica, with Totten Glacier to be particularly susceptible to future climatic warming, albeit with a spread of results that our simulations fall within. Our results show a thinning of the ice sheet interior in George V land that we do not see in Wilkes land leading to a more muted effect on sea level contribution from Wilkes Land in our results despite a comparable, albeit slightly greater, reduction in grounded area (a good approximation to grounding line retreat) in George V Land compared to Wilkes land. In contrast, we find that region A–B, C–C and D–E to be least affected by increased oceanic melting. Indeed, these regions are the only regions to show a consistently positive mass balance throughout all climate scenarios. This is most likely a result of a lack of large ice shelves in theses regions combined with the prevalence of inland, terrestrial ice limiting their susceptibility to warming ocean conditions. In contrast, multi-millennial simulations  for the next 10,000 years of the EAIS41 have found that the entirety of the EAIS covered by our domain is negligibly affected by ocean warming of a comparable forcing to our mCDW scenarios whilst Recovery basin (outside our model domain) was strongly affected by a warming Weddell Sea. When subjected to atmospheric warming of 8 °C (greater than the 4 °C warming assumed by our RCP8.5 scenarios) they find that Wilkes Land and George V Land were the most susceptible to surface melting, a process our model framework does not include.Our simulations consistently find that region D-D, and in particular Cook and Ninnis Glacier, is the most susceptible to ocean driven increased ice discharge. It is the only region to show a negative mass balance under an SW forced climate scenario, losing −14 Gt a45 that highlight the potential for increased sea level contribution beyond 2100 from the EAIS. This is in broad agreement with a recent review of future EAIS mass balance predictions out to 25009 that found a greater rate of sea level contribution in the period after 2100 than before it, albeit with correspondingly larger errors. In our results, for a given increase in oceanic melting there is an acceleration in ice discharge rather than an instantaneous increase to a greater velocity. This is demonstrated, particularly for the mCDW forced scenarios, by the rate of change in total ice volume  can have a large potential contribution to sea level rise from East Antarctica over the next couple of hundred years.46, which has previously been used for large scale, transient simulations of Antarctic future mass balance47 as well as smaller, more localised studies such as an investigation into the past behaviour of Cook glacier44 or rift propagation on the Brunt Ice Shelf48. The model domain has been deliberately chosen to encompass the drainage basins corresponding to the IMBIE 2016 definition of East Antarctica49, with the exception of the J’-K and E-E’ basins which drain into the Filchner-Ronne and Ross ice shelves, respectively. Our work chooses to focus on the potential for future mass loss from the comparatively less studied parts of the EAIS. Including these basins would also have necessitated the inclusion of much of West Antarctica to fully model the Ross and Filchner Ronne ice shelves, thus drastically increasing the computational expense of our simulations. The resulting model domain is shown in Fig. 50), similar to that used by Hill et. al.38 whilst precipitation is provided by the RACMO  version 2.3 data set51. A schematic representation of the model framework is shown in Fig. We conduct experiments using the finite element, ice dynamics model Úa52 results. Each of our future climate scenarios assumes a linearly increasing temperature which reaches a maximum 100 years from the present. This new maximum is then held constant for a further 100 years to explore the committed or long-term response to the first 100 years of forcing. Ice thickness and velocity are then used to determine ice discharge, which is combined with surface precipitation to give the overall mass balance, defined here asMB is the mass balance, P the surface precipitation and D the ice discharge over the grounding line. A positive MB indicates that the EAIS is gaining mass whilst a negative MB indicates that the EAIS is losing mass. Note that all results are shown in reference to a baseline simulation with constant forcing representing present day conditions. This has the effect of limiting model bias during initialisation, with results showing the relative effect of imposing a given climate scenario rather than absolute values.Both increasing ocean melt and surface precipitation are driven by a single representative temperature values that increase to represent future climate scenarios, with the increase derived from analysis of the model mean CMIP5We consider our choice of single, representative values for both the increase in oceanic and atmospheric forcing to be valid for the purpose of comparing and isolating the relative impacts on EAIS mass balance of a shift in ocean forcing from the relatively cool, current regime dominated by shelf water to a warmer regime dominated by modified Circumpolar Deep Water. As such our results should be considered in the context of what is likely to happen if modified circumpolar deep water dominates ice shelf melting rather than an exact representation of a particular simulation of a future climate scenario from an individual CMIP model. A more in depth description of the model framework, calibration and forcing can be found in the accompanying Supplementary InformationPeer review file


Glenoid component loosening is a potential complication of reverse total shoulder arthroplasty (rTSA), occurring in part due to lack of adequate screw purchase in quality scapular bone stock. This study was to determine the efficacy of a surgeon-designed, 3D-printed patient-specific instrumentation (PSI) compared to conventional instrumentation (CI) in achieving longer superior and inferior screw lengths for glenoid component fixation.A multi-centre retrospective analysis of patients who underwent rTSA between 2015 and 2020. Lengths of the superior and inferior locking screws inserted for fixation of the glenoid baseplate component were recorded and compared according to whether patients received PSI or CI. Secondary outcomes included operative duration and incidence of complications requiring revision surgery.P < 0.001) and inferior  mean screw lengths, as compared to CI. A greater proportion of maximal screw lengths for the given rTSA construct (48 mm) were observed in the PSI group . Operative duration was not statistically significantly different between the PSI and CI groups . No patients had radiographic loosening of the glenoid component with an average of 2-year follow-up.Seventy-three patients (31 PSI vs. 42 CI) were analysed. Average glenoid diameter was 24.5 mm (SD: 3.1) and 81% of patients had smaller glenoid dimensions compared to the baseplate itself. PSI produced significantly longer superior (44.7 vs. 30.7 mm; PSI facilitates longer superior and inferior screw placement in the fixation of the glenoid component for rTSA. With sufficient training, PSI can be designed and implemented by surgeons themselves. Reverse shoulder arthroplasty (rTSA) is indicated for a number of conditions, including rotator cuff tear arthropathy, glenohumeral arthritis, and three- and four-part proximal humerus fractures in the elderly . rTSA haPatient-specific instrumentation (PSI) using three-dimensionally (3D) printed guides is an increasingly popular tool for improving surgical accuracy across various contexts. PSI has been shown to produce more accurate glenoid component placement in rTSA and total shoulder arthroplasty , 12, 13.This was a retrospective, multi-centre comparative study conducted using data from eight hospitals. Ethical approval was sought and satisfied (HKU/HA HKW reference no. UW 21–111). Consecutive patients who had undergone rTSA using the Delta Xtend Reverse Shoulder System  between 2015 and 2020 were recruited. The inclusion criteria were age 50 years or older, indicated for rTSA for a non-reconstructable proximal humerus fracture, glenohumeral arthritis, or rotator cuff arthropathy with good premorbid function. Patients were excluded if they had axillary nerve deficit, active infection, local neoplastic conditions, or severe bone loss leading to the need for customised implants or two-stage reconstruction procedures. Historical control patients operated before 2018 by the same surgeons had CI performed prior to implementation of 3D-printed PSI. After 2018, both PSI and CI were used depending on centre and surgeon preference.Patient demographics, operative details, and computed tomography (CT) scans were retrieved from a centralised patient database. Pre-operative glenoid dimensions were measured for comparison and assessment of the difficulty for screw insertion. Using a 3D reconstruction of the glenoid in en-face view, the glenoid anterior–posterior (AP) dimension was measured as the diameter of the best-fitting circle for the glenoid centre; glenoid height was measured as the distance between the highest and lowest points on the low axis of the glenoid cavity Fig.  11]. Us. Us11]. t-tests for continuous variables and Fisher’s exact tests for categorical variables. A two-tailed P-value of < 0.05 was considered statistically significant.Baseline characteristics were compared between groups. The superior and inferior glenoid component screw lengths were recorded. Secondary outcomes included operative duration and incidence of complications requiring revision surgery. Statistical analysis was performed using SPSS Statistics version 26 . Patients were analysed according to whether they received PSI or CI during operation. Data were analysed using independent samples https://www.youtube.com/watch?v=bwGJMU_j_WI.The PSI jigs consisted of two components: a guide for the central metaglene peg placement and a guide for the superior and inferior locking screws Fig. . All jigCT scans were obtained in Digital Imaging and Communications in Medicine (DICOM) format with 1.0 mm or finer slice thickness and segmented using Mimics v.21 software . The central peg guide was first created using the coracoid base and the anteroinferior and posterior glenoid rim as surface reference points Fig. . Two diaIn the conventional method, the operational procedure follows the manufacturer’s specifications. In the PSI method, the glenoid is exposed with circumferential release of glenoid and coracoid base soft tissue to ensure correct jig placement. The central peg jig is fitted to the bony anatomy of the coracoid base. A diathermy burn mark is made through the full depth of the articular cartilage along the vertical axis. The burn mark is then reamed until barely visible to ensure proper jig seating and additional marking on the subchondral bone is made as needed. The metaglene is implanted, and superior and inferior drilling and screw placement is performed using the screw jig. The lengths of the two screws are determined using a standard depth gauge inserted to the far cortex. Screw lengths are available in increments of 6 mm, and screw lengths in between sizes will be rounded to the nearest number or rounded up if in the middle. Anterior and posterior screws are placed only if the surgeon feels that there is inadequate purchase of the initial two screws.A total of 73 patients  were included in the analysis, of whom 31 received PSI and 42 received CI (Table P < 0.001) and inferior  screw lengths, as compared to conventional instrumentation (Table P = 0.229), with PSI trending towards shorter operative times. None of our patients had radiological loosening of the glenoid component with an average of at least two years follow-up in the PSI group. There were, however, two cases of complications  requiring re-operation. One patient in the PSI group suffered an early post-operative dislocation of the rTSA. Revision was performed with a thicker polyethylene liner to increase stability. One patient in the CI group had an early periprosthetic joint infection. Two debridement operations were performed to relieve the infection, and the implant was retained after a full course of antibiotics. Additionally, one patient in the PSI group had screw penetration of the inferior scapular neck discovered on post-operative X-ray due to inadequate seating of the PSI screw guide. This was not considered a complication since the screw purchase was excellent and no revision was required in the absence of neurovascular injury or impingement symptoms.Patient-specific instrumentation produced significantly longer superior  of glenoid diameters smaller than the manufacturer baseplate themselves , 11. DifFrom the literature, glenoid component loosening occurs in 1.16% of all reverse shoulder arthroplasties and accounts for up to 13% of the indications for revision surgery , 15. ThiUntil now, no study has examined the effect of PSI on absolute screw length, which remains clinically relevant since longer screw length independently contributes to stronger glenoid baseplate fixation. Previous biomechanical studies have shown superiority of longer screw lengths with cyclical loading and baseplate displacement but further long-term clinical studies are required to determine baseplate loosening in vivo . OptimalThe number of screws required for stable glenoid baseplate fixation has been a topic of recent interest. Several authors have reported achieving stable fixation, at least in the short term, using three or even two screws , 20, 22.Computer navigation (CN) has been introduced as a means of improving glenoid component placement in rTSA. While initial results have been promising, CN has inherent limitations that may preclude its popularisation. In addition to the initial cost and learning curve, the potential for machine inaccuracy and extra intra-operative steps may contribute to increased surgical duration , 25, 26.The PSI guide developed at our centre differs from commercially available designs in several regards. First, most commercially available PSI guides for rTSA utilise either coracoid base referencing, such as the Stryker Truesight  and Affinis Architec PSI guide , or rim referencing via the anterior and posterior glenoid rim, such as the Virtual Implant Positioning Glenoid Targeter  and the Signature One . Coracoid base referencing requires less extensive dissection but accuracy and placement may be more difficult compared to rim referencing. Our design utilises both coracoid base referencing and rim referencing to improve guide placement and accuracy. The aforementioned commercially available guides are designed for baseplate peg placement only. These are useful for situations with glenoid deformity or bone loss. However, the majority of our patients indicated for rTSA involve proximal humerus fractures without glenoid pathology. Thus, providing accurate screw trajectory for longer screw lengths to improve stability was of greater clinical importance in our population. The PSI guides from our centre consist of two components, one for the glenoid base plate and another for screw trajectories. The screw guide utilises the implant metaglene which is first scanned and rendered into a 3D object so that the guide can fit firmly onto the curvature and screw holes. Screw lengths can be predetermined with software measurements during the planning process for jig fabrication, which is analogous to computer navigation. However, these are not provided by commercially available PSI jigs.For the clinician, there are several considerations worth noting for the use of PSI in rTSA. First, the accuracy of the PSI system is dependent on several factors, including the acquisition of an accurate 3D scapular model and ability of the surgeon to ream to the proper depth line . AdequatThis study had several limitations. First, no post-operative CT scans were available to confirm the final screw trajectories and analysis of final screw position. Our analysis relied on the assumption that surgeons followed the standard practice of aiming for maximal screw length in the conventional method. This was a retrospective analysis and further long-term randomised control trial should be conducted to exclude bias and co-founders for implant loosening. Furthermore, this study did not attempt to assess functional outcomes at follow-up. Long-term survivorship data including registries or large population studies are needed to determine the effect of PSI and screw length on implant loosening and longevity.In conclusion, PSI facilitates longer superior and inferior screw placement in the fixation of the glenoid component in rTSA as compared to conventional instrumentation. Furthermore, it does not appear to significantly alter operative time or complication rates. Future studies are required to compare different PSI systems and to classify different PSI guides to aid clinical practice for specific shoulder pathologies. PSI is a useful alternative to computer navigation for improving baseplate fixation in rTSA, and with sufficient training, can be implemented by surgeons themselves.