Approximate 70% of cobra venom is composed of cytotoxin (CTX), which is responsible for the dermonecrotic symptoms of cobra envenomation. However, CTX is generally low in immunogenicity, and the antivenom is ineffective in attenuating its in vivo toxicity. Furthermore, little is known about its epitope properties for empirical antivenom therapy. This study aimed to determine the epitope sequences of CTX using the immunoinformatic analyses and epitope-omics profiling. A conserved CTX was used in this study to determine its T-cell and B-cell epitope sequences using immunoinformatic tools and molecular docking simulation with different Human Leukocyte Antigens (HLAs). The potential T-cell and B-cell epitopes were 'KLVPLFY,' 'CPAGKNLCY,' 'MFMVSTPTK,' and 'DVCPKNSLL.' Molecular docking simulations disclosed that the HLA-B62 supertype exhibited the greatest binding affinity towards cobra venom cytotoxin. The namely L7, G18, K19, N20, M25, K33, V43, C44, K46, N47, and S48 of CTX exhibited prominent intermolecular interactions with HLA-B62. The multi-enzymatic-limited-digestion/liquid chromatography-mass spectrometry (MELD/LC–MS) also revealed three potential epitope sequences as 'LVPLFYK,' 'MFMVS,' and ‘TVPVKR’. From different epitope mapping approaches, we concluded four potential epitope sites of CTX as ‘KLVPLFYK’, ‘AGKNL’, ‘MFMVSTPKVPV’ and ‘DVCPKNSLL’. Site-directed mutagenesis of these epitopes confirmed their locations at the functional loops of CTX. These epitope sequences are crucial to CTX’s structural folding and cytotoxicity. The results concluded the epitopes that resided within the functional loops constituted potential targets to fabricate synthetic epitopes for CTX-targeted antivenom production. The cobra (Naja sp.) envenomation is unequivocally a feared snake species due to their cytotoxic venoms, leading causes of physical impairment that engenders lifetime handicaps in most survivors2. The cytotoxic envenomation is manifested as local dermonecrosis, which is a sequelae event due to cell death and subsequent loss-of-function injuries6. The venomics of Naja venom revealed that, the major venom toxins are phospholipase A2s (PLA2s), neurotoxins (NTXs) and cytotoxins (CTXs). PLA2s and CTXs constitute average 20% and 70% of the Naja venoms’ dry weight, respectively10. It is noteworthy that, synergisms of PLA2s and CTXs have been summarized to cause downstream cytotoxic effects upon envenomation11. Nevertheless, CTXs play major roles in venom-induced dermonecrosis due to their abundances in Naja venom12.The World Health Organization (WHO) has declared snakebite envenomation (SBE) as a category A Neglected Tropical Disease13. They are 7 kDa with approximately 59–62 amino acid residues14. They appear in three-finger loops from five antiparallel β-sheets13. These β-sheets make up the hydrophobic core of the toxins, which are stabilized by four conserved disulfide bridges15. The hydrophobic core is also flanked by basic lysine and arginine residues responsible for cytotoxicity16. It allows the toxins to interact with the cell membranes and contributes to the formation of membrane pores, resulting in subsequent cell death cascade events20. Thus, it is said that the CTX’s loops are responsible for dermonecrosis in snakebite victims.Cytotoxins are highly basic and amphipathic polypeptides21. However, they present undesirable drawbacks, especially exposing patients to the risk of hypersensitivity reactions and anaphylaxis23. Besides, the antivenoms exhibit limited neutralizing ability on the CTX-induced necrosis12. The low efficacy of the antivenoms suggests the inadequate immunogenicity of the CTXs to produce CTX-specific antibodies. Several factors affecting the immunogenic capability of CTXs including the molecular structure, and sequence variations24. Small molecular size of the toxins may contribute to the sparse antigenicity25. Moreover, CTXs exhibit low immunogenicity due to their three-finger folded structures that limit its surface exposure for epitope recognition. It is conspicuous that the existing antivenoms are ineffective against dermonecrosis due to their poor pharmacokinetic profiles26. This sheds light in the recruitment of biotechnological tools to expedite the development of next generation antivenom, which allows the immunological characterization of venom toxins responsible for envenomation28.Although SBE poses a significant global health risk, immunotherapy with heterologous antivenoms is the only currently effective intervention. Antivenoms are usually produced by isolating polyclonal antibodies or antibody fragments from the serum or plasma of immunized animals with venom2s (PLA2s), cysteine-rich secretory proteins (CRISP), disintegrins (DIS), Kunitz peptides (KUN), L-amino acid oxidases (LAAO), natriuretic peptides (NP), snake C-type lectins (SNACLEC), snake venom metalloproteinases (SVMP) and snake venom serine proteases (SVSP) 30. Furthermore, high-throughput approaches such as peptide microarray, mass spectrometry (MS) epitope mapping, and phage display methods have also been developed to characterize the toxin-antivenom interaction33.By characterizing the epitopes of venom toxins, the possible immunogenic sites of toxins can be identified to aids in developing appropriate antivenoms specifically targeting the respective toxins. Computational analysis has been reported to examine the immunogenicity of toxins, such as three-finger toxins (3FTX), phospholipase A35. The recognition of epitopes elicits immune responses to produce antibodies that bind to eradicate the antigen and inhibit its toxic effects36. This is the fundamental principle in current antivenom production, which involves the neutralization of venom toxins by the antibody37. Recent research demonstrates innovative venom-independent immunization strategies using synthetic epitopes for antibody production37. Such epitopes have shown more direct and potent protectivity than the immune response conferred by whole protein38. Notably, most ongoing research today focuses on the whole venom instead of toxin-specific antitoxins of interest39. The understanding of immunogenic properties of venom toxins shows the prospects of producing standardized synthetic epitopes for specific and effective antivenoms without causing toxicity to the host.Epitopes are short molecular regions of an antigen recognized by the immune systemNaja species, aiming to overcome the predicaments of species diversity and geographical variation40. We applied immunoinformatic and multi-enzymatic-limited-digestion/liquid chromatography-mass spectrometry (MELD/LC–MS) to determine the immunogenic properties of the conserved CTX can be determined. Furthermore, we performed site-directed mutagenesis to validate the presence of epitope sites at the hydrophobic functional loops of CTX.Despite the promising prospects of synthetic epitopes of toxins, limited knowledge is known on utilizing synthetic epitopes in CTXs-targeted therapeutics. Furthermore, the functional epitopes properties of CTXs remain unclear. Therefore, in this study, epitope characteristics of CTXs were determined to identify the potential epitopes of significant relevance as synthetic epitope leads for next generation antivenoms. This study used a conserved CTX sequence deduced from various CTX isoforms from different The list of predicted T-cell epitopes is summarized in Table The parameters such as frequency of epitope among supertypes, affinity ranking, transporter associated with antigen processing (TAP) efficiency, and combined score were analyzed for ranking statistics Table . The ideEquation (1):The frequency value determined which T-cell epitopes were predominant in the CTX. This can be deduced by ranking the T-cell epitopes according to their frequency of presence.16. Despite some predicted T-cell epitope sequences such as 'CPKNSVK' and 'DVCPKNSLL' resided entirely on the functional loops' amino acids, these sequences somehow demonstrated a lower presence probability value of 0.1111.The sequences ‘NNKLVPLFY’ and ‘KMFMVSTPT’ exhibited the highest probability of 0.3333 across all MHC supertypes Table . Both ep50000(affinity score), whereby log50000 is the logarithm with base 50,000 for the affinity score. The higher the scoring, the higher the binding affinity. Based on the result, 'FMVSTPTKV' exhibited a higher affinity score (Table The affinity (supertype) scoring represents the predicted MHC binding affinity as -logre Table . This ep41. However, before they are presented on the surfaces of HLAs, the transport associated with antigen processing (TAP) recognizes and transports these epitope sequences into the endoplasmic reticulum42. Therefore, the role of TAP must significantly influence the selection of T-cell epitopes. The TAP efficiency ranking showed that 'NNKLVPLFY' and 'CPAGKNLCY' exhibited TAP efficiency scores of 2.8000 and 2.6060, respectively. The third-ranked TAP efficiency sequence was 'KTCPAGKNL.' There was a drastic difference in TAP efficiency scores among the epitope sequences, presumably due to the strict specificity of TAP binding with peptide sequences. Notably, the amino acid residue 'KT' were found in the epitopes with lower TAP efficiency scores, indicating that these two amino acids significantly reduced the TAP efficiency. Structurally, these two amino acids comprise the β-sheet of loop I in the CTX. Their structural role may be accounting for their stringent non-interactive characteristics. Interestingly, none of the amino acid residues of 'CPAGKNLCY' was found to reside within the functional loops of CTX. This epitope sequence constituted the region that connected loops I and II of CTX, suggesting that this sequence may facilitate CTX-membrane interactions.The ranking analysis of the TAP efficiency of each predicted T-cell epitope sequence was also performed. The interactions of cytotoxic T-cells with other cells abide by a strict and specific recognition process dictated by T-cell epitopes presented on the surfaces of human leukocyte antigens (HLAs)43. To determine a compromise between sensitivity and specificity, a threshold of 0.75 for the combined score of predicted epitope sequences was used for selection. Thus, the predicted four epitope sequences 'FMVSTPTKV,' 'KLVPLFYKT,' 'MFMVSTPTK,' and 'VPVKRGCID' exhibited the highest chances as potential T-cell epitopes of CTX.The next parameter used in predicting T-cell epitopes was combined score ranking. The combined score consists of an integrated peptide binding affinity, C-terminal cleavage affinity, and TAP efficiency scores, which are all significantly involved in the MHC Class I processing pathway13. This epitope sequence also connected the two prominent β-sheets of loops I and II, which are vital to the overall functionality and stability of CTX. Since functional loops of CTX are important for cytotoxicity, and antivenoms have a binding affinity towards functional sites of toxins, it is feasible to deduce that the amino acid sequences that make up the functional loops were potential T-cell epitopes44.Based on the results, 'NNKLVPLFY' and 'KLVPLFYKT' were found within loop I, while 'FMVSTPTKV' and 'MFMVSTPTK' were situated in loop II. 'CPAGKNLCY' constituted the region between loops I and II of CTX. These sequences were the best sequences representing T-cell epitopes of CTX. Although 'CPAGKNLCY' was not situated in the functional loops of CTX, this epitope sequence exhibited a greater TAP efficiency score and presence probability among all MHC supertypes. Furthermore, this sequence was amphiphilic and highly basic, conserved in CTX for interaction with targets45. The B-cell epitope prediction based on the PI score for CTX was summarized in Table The protrusion index (PI) is a simple and effective concept for identifying protein regions that protrude from its surface. A high PI score is often related to sites of antigenicity46. The Parker hydrophilicity prediction of CTX is illustrated in Fig. The basis of utilizing hydrophilicity to predict epitopes is attributed to the rational nature of epitopes on the surface, and they are exposed to be potentially antigenic47. As illustrated in Fig. Kolaskar & Tongaonkar antigenicity prediction analyzes the physiochemical properties and abundance of amino acids within CTX compared to B-cell epitope references. Higher antigenicity scores are associated with a higher potential to induce immune responsesAn essential criterion of a potent epitope is its accessibility to other molecules for interaction. Emini surface accessibility prediction was therefore used to analyze and evaluate the surface accessibility of the amino acid residues. The linear B-cell epitopes were predicted based on their surface accessibility characteristics. As shown in Fig. 48. Karplus & Schulz's flexibility prediction was used to analyze the structural flexibility contributed by individual amino acid residues of CTX. As shown in Fig. Flexibility in protein structures is a vital characteristic that influences a protein’s ability to perform binding, catalysis, and allosteric interactionshttps://www.ebi.ac.uk/Tools/msa/clustalo/)51. Based on the multiple sequence alignment, three potential T-cell epitope sequences of 'KLVPLFY,' 'CPAGKNLCY,' and 'MFMVSTPTK' were selected due to high consensus frequency  between the docking structures. This scoring parameter was used to rank the docking clusters52. A lower HADDOCK score indicates a more reliable docking structure with better energetic stability54. HLA-B62 possessed the lowest HADDOCK score of 257.6 ± 30.1, followed by HLA-B27 and HLA-A02  value is used to assess whether a docked simulation between a ligand and receptor in a specific binding orientation and conformation are reproducible and values of < 2.0 Å indicate successful and appropriate docking56. Only HLA-B07 and HLA-B62 exhibited RMSD values < 2.0 Å, thus implying that they are reliable and reproducible docking structures. Therefore, HLA-B62 exhibited the best binding affinity and stability towards CTX's epitopes.The HADDOCK 2.2 web server clustered 583 structures into 89 clusters, representing 36.44% of the water-refined models. The docking parameters are summarized in Table 57. The RING analysis then revealed four highly interacting regions of 'NKLVPLFYK,' 'AGKNL,' 'MFMVSTPTLVPVK,' and 'DVCPKNSLLV' as the potential epitope sites, within the functional loops of CTX  analysis to determine the interacting residues between CTX and different HLA supertypes. RING analysis enables the identification of non-covalent interactions at the atomic level of a proteinCTX Fig. A.Figure Van der Waals (VDW) forces, hydrogen bonds, and salt bridges  simulations) in a water box for 150 ns at 300 K. The mean RMSD value  approach was first introduced by Morsa et al.60. The formation of immunocomplexes occurred from 3.125 to 50 µg/mL of anti-CTX, whereby the greatest turbidity was measured at 25 µg/mL of anti-CTX.In this experiment, an anti-CTX monoclonal antibody with broad specificity was used to map the epitope sites on CTX. Upon binding to the anti-CTX, the CTX's epitopes were shielded from enzymatic proteolysis, and the exposed regions were digested. The reaction mixture was then subjected to LC–MS to identify the peptide(s) of the undigested region(s) known to be the epitopes. Prior to the MS-based epitope mapping, the immunoturbidimetric assay confirmed the formation of immunocomplexes between the anti-CTX and CTX. Based on the kinetic analysis Fig. A, the foFrom the MELD/LC–MS results, 1:5000 diluted anti-CTX demonstrated a greater coverage of peptides mapping  contained four mutated sites within the potential epitope sequences on the functional loops. The gene sequences of CTXWT and CTXVAR are as follows, underlined residues indicate mutated regions while asterisk (*) represents identical residues:A CTX variant  than CTX wildtype, CTXWT (pI: 9.37). The interaction between CTX and the cell membrane is known to be closely related to the types of CTX, namely S-type and P-type. The insertion of S-type CTX into the cell membrane was primarily relying on the structural adaptation of loop II62. On the other hand, loops II and III of the P-type CTX were found to be more critical in lipid membrane penetration63. Additionally, studies showed that loop I of both S- and P-type CTX was involved in the membrane interaction65. Altogether, no conclusive finding supports a more significant loop among the three functional loops of the CTX during membrane penetration. However, loop II has been reported to involve in the membrane insertion for both S- and P-type CTX, implying that it plays a prominent role in the interaction between the CTX and cellular membranes.The alteration of amino acid residues in CTXWT and CTXVAR was determined in the human skin keratinocytes (HaCat cell lines). Both CTXWT and CTXVAR significantly (p < 0.0001) reduced the viability of the HaCat cell lines . These sequences constitute different CTX isoforms of different Naja species40. The final consensus CTX sequence is shown below:A conserved sequence of cytotoxin (CTX) was constructed in our laboratoryNaja atra cytotoxin  with 95% sequence similarity40.This conserved CTX is highly matched to https://services.healthtech.dtu.dk/service.php?NetCTL-1.2) 43. The HLA supertypes selected for the prediction were A1, A2, A3, A24, A26, B7, B8, B27, B39, B44, B58, and B62; the threshold score for epitope identification was set at 0.50, while the weight on C terminal cleavage and TAP transport efficiency were 0.15 and 0.05, respectively. For the HLA-B62 supertype, a prototypical member of the supertype, HLAB*05:01, was used to represent it69. Statistical analyses were performed on each T-cell epitope candidate and ranked by four categories: frequency of epitope presence across supertypes, affinity score, transport associated with antigen processing (TAP) efficiency score, and combined score.T-cell epitope (CTL) prediction of the cobra venom cytotoxin was performed using the NetCTL 1.2 webserver  70, IEDB analysis resource (http://tools.immuneepitope.org/bcell/) 71, antigenic peptide prediction method (http://imed.med.ucm.es/Tools/antigenic.pl) 72 and ABCPred (http://www.imtech.res.in/raghava/abcpred/) 74. Whereas the discontinuous B-cell epitopes were predicted using ElliPro webserver (http://tools.iedb.org/ellipro/) 75, provided by the Immune Epitope Database (IEDB) based on each amino acid residues' protrusion index score. Karplus & Schulz flexibility prediction , Kolaskar & Tongaonkar antigenicity , and Parker hydrophilicity prediction , was applied for the prediction. The predicted sequences from the outputs were aligned, whereby amino acid sequences with more than three overlapping frequencies were potential B-cell epitopes.The potential linear B-cell epitopes of CTX were identified using four different prediction algorithms, namely BepiPred-2.0 (http://tools.iedb.org/mhci/). NetMHCpan EL 4.1 was selected as the prediction method recommended by the IEDB for the binding prediction analysis across all alleles76. The peptide length was set as nonamer. The peptide-MHC I combinations with the highest scoring were filtered out from each peptide sequence input. A higher score indicates a higher affinity for the interaction between the peptide and MHC molecules. Prediction of the T-helper cell (HTL) epitopes for the CTX was performed using the peptide binding to MHC class II molecules tool from IEDB (http://tools.iedb.org/mhcii/). IEDB recommended NetMHCIIpan 4.1 EL was selected as the prediction method using specific MHC II alleles76. A 15-mer peptide length was selected. The peptide-MHC II interactions with the highest percentile rank in each set, indicating the strongest binding affinity, were filtered out for further analyses.The T-cell epitopes (CTL) were also used for the HLA distribution analysis as the epitopes were also MHC ligands. The binding prediction of the epitopes to MHC Class I was evaluated using the peptide binding to MHC Class I molecules tool from the Immune Epitope Database , HLA-A02 (1AO7), HLA-A03 (3RL1), HLA-A24 (4F7P), HLA-B07 (5EO1), HLA-B27 (2BST), HLA-B58 (5IM7) and HLA-B62 (3C9N). CTX was set as the first molecule with functional loops' residues  as active residues. The second molecule input was HLA supertypes, and all amino acid residues were set as active residues. The best docking clusters were determined by assessing each structure's HADDOCK score, Z-score, and RMSD value. Apart from that, the chosen docking cluster was accepted if the cluster maintained its native conformation, i.e., β-strands that are responsible for the stability of the structure. Later, the PDB of the best docking clusters was subjected to Residue Interaction Network Generator 2.0.1 (RING 2.0.1) analysis (http://old.protein.bio.unipd.it/ring/) 57 to determine the types and strength of both intra- and inter- non-covalent interactions of the docked structural clusters. The intermolecular non-covalent interactions between amino acids of CTX and HLA supertypes were mapped based on the aligned T-cell and B-cell epitopes.According to the predicted T-cell epitopes list, molecular docking analysis of T-cell epitopes with different HLA supertypes was performed on HADDOCK 2.2 to determine the interaction80. The MD simulations of all complexes were carried out for 150 ns in a solvated TIP3P water box using CHARMM 36 m force field, whereby the trajectory and energy files were written every 10 ps. Three chloride ions were added to the complex to neutralize the overall system. The protonation states were checked at pH 7 using the H +  + web server for His, Lys, Arg, Asp, and Glu residues. Minimization was carried out for 5000 steps using Steepest Descent Method, and the convergence was achieved within the maximum force < 1000 (KJ mol-1 nm-1) to remove any steric clashes. All three systems were equilibrated at NVT and NPT ensembles for 100 ps  and 1000 ps , respectively, using time steps 0.2 and 0.1 fs, respectively, at 300 K to ensure a fully converged system for the production run. The production runs for simulation were carried out at a constant temperature of 300 K and a pressure of 1 atm or bar (NPT) using weak coupling velocity-rescaling (modified Berendsen thermostat) and Parrinello-Rahman algorithms, respectively. All bond lengths involving hydrogen atoms were kept rigid at ideal bond lengths using the Linear Constraint Solver (lincs) algorithm, allowing for a time step of 2 fs. The Verlet scheme was used for the calculation of non-bonded interactions. Periodic Boundary Conditions (PBC) were used in all x, y, and z directions. Interactions within a short-range cutoff of 1.2 nm were calculated in each time step. Particle Mesh Ewald (PME) was used to calculate the electrostatic interactions and forces to account for a homogeneous medium outside the long-range cutoff.The stability of CTX-HLA supertype complexes was further examined by molecular dynamic (MD) simulations using the GROMACS simulation packageWT and CTXVAR”) in 1:1 (v/v) for 2 h at room temperature to form immunocomplexes. Subsequently, the immunocomplexes were concentrated in PBS, and the unbound CTX was removed with a 10 kDa AmiconⓇ Ultra Centrifugal Filter Spin Column . The immunocomplex was then reduced with dithiothreitol  and alkylated and iodoacetamide . This was followed by proteolytic digestion with multiple endoproteases. The endoproteases used in MELD were trypsin , chymotrypsin , and Lys-C  in a 1:20 enzyme-to-protein ratio by mass. The reaction mixtures were incubated at 37 °C overnight before desalting using ZipTipⓇ pipette tips . The digested peptides were then vacuum-dried before LC–MS analysis.In this experiment, MELD was used to prepare the immunocomplexes for peptide mapping in epitope analysis. In brief, an anti-CTX monoclonal antibody , was diluted according to the ratio of 1:5000, 1:10,000, and 1:30,000 in phosphate-buffered saline (PBS). The anti-CTX was mixed with pure CTX  was added into each well, resulting in a final concentration of 12.5 µg/mL. Absorbance readings were taken every minute at wavelength 340 nm using the Tecan microplate plate reader at 37 °C for 40 min. The kinetics of the immunocomplexes formation were then plotted and examined.The immunoturbidimetric assay protocol was modified from a reported study by O’ Leary and colleaguesm/z) range of 50–3000. Solvent A was used as a blank to eliminate peptide contamination from other sources. The data was then processed using the Peaks X + software, searching against the Serpentes protein database (Taxid: 8570).The identification of the MELD-peptides was conducted using the Agilent 1200 HPLC-Chip/MS Interface, coupled with Agilent 6550 iFunnel Q-TOF LC/MS instruments. The lyophilized samples were reconstituted in solvent A (0.1% formic acid in water). Subsequently, 1 µL of the sample was injected into the C18 enrichment and analytical column  at a flow rate of 4 µL/min from Agilent 1200 Series Capillary pump and 0.5 µL/min from Agilent 1200 Series Nano Pump with solvent B (90% of acetonitrile in water with 0.1% formic acid). The peptide mixtures were separated for 50 min under an increasing gradient of solvent B from 5 to 70%, followed by an 8-min post-run analysis. The elution of peptides was configured as follows: positive ion mode, capillary voltage 1.9 kV, fragmentor voltage 360 V, gas temperature 325 °C, drying gas flow 5.0 L/min. Mass spectra were acquired in an MS/MS mode over a mass  and variant (CTXVAR) genes by Economy Gene Synthesis . Both genes were inserted into pET-22b ( +) plasmids at the NdeI/XhoI restriction sites  was used to synthesize wildtype CTX  following the manufacturer’s protocol. The CTXWT and CTXVAR genes were amplified by touch-down PCR with their respective forward and reverse primers (Supplementary File 1-Table The pET-22b ( +)-CTXWT and CTXVAR, pET-22b ( +) plasmids harboring the respective genes were first transcribed into RNA using the HiScribe™ T7 Quick High Yield RNA Synthesis Kit . The transcribed RNAs were then expressed using NEBExpress® Cell-free E. coli Protein Synthesis System  for in vitro protein translation, according to the manufacturer's protocol. After that, CTXWT and CTXVAR were purified using a HisTrap™ HP, 1 mL affinity column on ÄKTA Pure protein purification system . The HisTrap column was equilibrated with buffer A  before the samples were injected into the column. CTXWT and CTXVAR were purified with a gradient elution from 4 to 100% of buffer B  at a flow rate of 500 uL/min. The presence of his-tagged expressed CTX and its purity were visualized by western blot.To express CTXWT and CTXVAR was determined using the cell-counting kit-8 (CCK-8) assay . Briefly, 100,000 cells/mL of human skin keratinocytes, Hacat cell lines , were seeded in a 96-well plate at 37 °C, 5% CO2 overnight. The cells were treated with 100 µL of 8 µg/mL CTXWT and CTXVAR in serum-free cell culture media for 24 h. Ten microliters of CCK-8 reagent were added to each well and incubated for 1 h. The level of cell viability was quantified through the formation of orange-colored products upon reduction of the WST-8 salts present in the CCK-8 reagent by dehydrogenase in viable cells. The absorbance readings were obtained using the Tecan microplate reader at wavelength 450 nm. The percentage (%) of cell viability was calculated.The cytotoxicity of CTX82 to determine if site-directed mutagenesis produced any structural changes to CTXVAR. The DOPE per residue profile of each amino acid residue within the CTXWT and CTXVAR was plotted on Gnumeric for comparison. CTXWT and CTXVAR models were subjected to further visual evaluation using BIOVIA Discovery Studio Visualizer 2021 by superimposing both models. The corresponding root-mean-squared deviation (RMSD) value was also determined for CTXWT and CTXVAR models.We performed homology modeling on MODELLER 9.21Van der Waals interactions, and salt bridges. The crucial amino acid residues on CTX, namely L7, G18, K19, N20, M25, K33, V43, C44, K46, N47, and S48 were involved in intermolecular interactions with HLA-B62. The epitope-omic analysis revealed three highly potential epitope sequences ' LVPLFYK,' 'MFMVS,' and ‘TVPVKR,’ which were also located at the functional loops I and II. Different mapping approaches finalized the potential epitopes of CTX as ‘KLVPLFYK’, ‘AGKNL’, ‘MFMVSTPKVPV’ and ‘DVCPKNSLL.’ Furthermore, site-directed mutagenesis of these four epitope sites altered the physicochemical properties and cytotoxicity of CTX albeit the overall structure of CTX was retained. This concluded that, the identified epitope sequences were located at the functional loops of CTX. Since the functional loops of CTX form its hydrophobic three-finger folded core, our findings provide a justification of low immunogenicity of CTX, which results in inadequate efficacy of existing antivenoms targeting CTX. In conclusion, the CTX’s functional epitopes represented consensus epitope properties of different CTX isoforms. These functional epitopes can be fabricated as synthetic epitopes to produce toxin-targeted cross-reactive antivenom that antagonize the cytotoxic effects in dermonecrosis.Potential T-cell and B-cell epitopes of CTX were identified to be 'KLVPLFY,' 'CPAGKNLCY,' 'MFMVSTPTKVP,' and 'DVCPKNSLL’, whereby 'KLVPLFY,' ‘MFMVSTPTKVP,' and 'DVCPKNSLL’ were found to reside within the functional loops of CTX. 'CPAGKNLCY' constituted the region between loops I and II, which is vital for CTX’s structural folding and stability. HLA-B62 supertypes was identified to possess the highest binding towards CTX. The intermolecular bonds between the HLA-CTX complex were hydrogen bonds, Supplementary Information.


In order to actively promote green production and address these concerns, there is an urgent need for new packaging materials to replace traditional plastic products. Starch−based packaging materials, composed of starch, fiber, and plasticizers, offer a degradable and environmentally friendly alternative. However, there are challenges related to the high crystallinity and poor compatibility between thermoplastic starch and fibers, resulting in decreased mechanical properties. To address these challenges, a novel approach combining plasticizer optimization and response surface method (RSM) optimization has been proposed to enhance the mechanical properties of starch−based packaging materials. This method leverages the advantages of composite plasticizers and process parameters. Scanning electron microscopy and X-ray crystallography results demonstrate that the composite plasticizer effectively disrupts the hydrogen bonding and granule morphology of starch, leading to a significant reduction in crystallinity. Fourier transform infrared spectroscopy results show that an addition of glycerol and D−fructose to the starch can form new hydrogen bonds between them, resulting in an enhanced plasticizing effect. The optimal process parameters are determined using the RSM, resulting in a forming temperature of 198 °C, a forming time of 5.4 min, and an AC content of 0.84 g. Compared with the non−optimized values, the tensile strength increases by 12.2% and the rebound rate increases by 8.1%. Packaging materials derived from petroleum−based plastics are non−biodegradable, leading to severe pollution of the environment upon which humans depend ,2,3 and Among various biomass materials, starch has gained recognition as an environmentally friendly material due to its easy availability, economic benefits, and ability to completely decompose into carbon dioxide and water under natural conditions ,12. StarCurrently, the most common method of disrupting the hydrogen bonding mechanism and modifying the structure of starch is through the use of plasticizers, which enhance the thermoplastic processing performance and ensure the compatibility of starch with other polymers ,19,20. AThe investigation of plasticizers has facilitated the development of thermoplastic starch (TPS). However, in the actual preparation and production of starch−based packaging materials, it is necessary to incorporate natural fibers into the starch matrix. Nevertheless, the high crystallinity and poor biocompatibility between the two components significantly impact their mechanical properties ,27,28. IThe previous research demonstrates that the predominant focus has been on optimizing plasticizers. However, there have been limited studies concerning the amalgamation of plasticizer optimization and the optimization of process parameters for thermal cavity forming. Ararsa et al.  employedIn this current study, a blend of glycerin and D−fructose was incorporated into corn starch to create a novel thermoplastic starch (TPS). The intention was to disrupt the crystal structure of TPS and diminish the crystallization peak, thereby augmenting the plasticizing effect. Subsequently, the formulated TPS was combined with pre−fabricated sisal fibers and processed using a thermal cavity forming machine. The investigation delved into the impacts of forming temperature, forming time, and foaming agent concentration on tensile strength and resilience during the thermal cavity forming process. Through response surface analysis, optimal process parameters were determined. Consequently, the objective of this study was to produce an innovative composite material employing a starch matrix, wherein thermoplastic corn starch serves as the matrix and sisal fibers function as reinforcing agents. Furthermore, the study aimed to fine−tune the processing parameters using response surface analysis.In this study, NCS  was used as the substrate. Sisal fibers  with an average length of 5 mm and an average diameter of 60 μm were used as reinforcement material. GLY  and D−fructose  were used as the plastic agents. The molecular structures of GLY and D−fructose are shown in The preparation process for the starch−based packing structure is provided in It is worth noting that the moisture content and relative humidity may adversely affect the gelatinization and crystallization of the starch. To avoid these effects, a dehumidifier was used to keep the indoor humidity below 40%. The resulting gelatinized starch was dried in a vacuum drying oven at 80 °C for 24 h to obtain TPS, as shown in The foaming process is depicted in −1. Scanning was recorded with a resolution of 2 cm−1. X-ray diffraction (XRD)  was utilized to analyze the changes in crystal types before and after starch modification. The scanning speed was set at 5°/min, and the analysis angle ranged from 10° to 80°. Scanning Electron Microscopy (SEM)  was employed to examine the impact of plasticizers on the micromorphology of starch.A portion of the TPS prepared in 2.1 was subjected to drying in a drying box at 60 °C for 24 h to remove moisture. After thorough grinding, it was strained through a 200−mesh screen and left at room temperature for 30 days. A sample of 1 mg of TPS was taken and mixed with 150 mg of potassium bromide, then pressed into a transparent sheet. Fourier Transform Infrared Spectroscopy (FT−IR)  was employed to detect and analyze the effect of plasticizers on the hydrogen bonds of starch within the spectral range of 500–4000 cmPrior to tensile and compression tests, the samples were dried in an oven at 86 °C for 24 h to remove moisture. Tensile specimens were prepared following the guidelines outlined in GB/T9641−88. The tensile test was conducted using the Electronic Universal Material Experimental machine  at a constant speed of 5 mm/min. To minimize experimental errors, five trials were performed for each composite material. The compressed sample was prepared in accordance with the GB/T 8186−2008 standard, with a sample size of 25 mm (length) × 25 mm (width) × 5 mm (height). The compression test machine was set to a speed of 12 mm/min. The original thickness of the sample was maintained, and the load was gradually applied in the direction perpendicular to the thickness. After the sample underwent a 30% deformation, a stable load was sustained for a duration of 3 min. Afterward, the sample was unloaded and left for 30 s. The thickness of the compressed specimen was measured using a spiral micrometer, and the rebound rate was calculated. Each group of experiments was repeated three times, with an interval of 1 min between each experiment. The average result from the three repetitions was considered the rebound rate for that particular sample group. The formula for calculating the rebound rate is as follows :(1)tj=TjA starch−based biomass packaging material with a bubble cell structure was prepared using TPS as the matrix, alkalized sisal fiber as the reinforcement, and azodicarbonamide (AC) as the blowing agent. The thermal cavity−forming process was employed for fabrication. The RSM was utilized to optimize the process parameters, with the variables being forming temperature, forming time, and AC content. The response values considered for optimization were rebound rate and tensile strength. A regression model was fitted, and variance analysis was conducted to optimize the forming process. Each factor was designed with three levels, as presented in The three input variables were optimized based on the following steps.(1)Establishing a general mathematical relationship between the input variables and response, which can be expressed as follows:(2)Looking for mathematical models. Based on mathematical analysis, the existing McLaughlin or Taylor expansions can meet convergence conditions. According to Samir Charola et al. , the exp(3)p value. The actual values are the tensile strength and rebound rate measured by experiments. Based on the second−order polynomial model, the response surface and the contour plot, which describe the influences of two variables on a single target, can be obtained. The third input variable remains unchanged at the center point. Then, the effects of input variables on the response will be analyzed to obtain the highest rebound rate and tensile strength simultaneously.Using analysis of variance (ANOVA) to evaluate the significance of the prediction model. The reliability of the model can be determined by loss of fit (LOF) and Starch is considered a natural polycrystalline system. Crystalline, submicron crystalline, and amorphous structures alternate to form the granular structure of starch. Under the combined action of plasticizers, temperature, and moisture, the inherent crystalline structure of starch is disrupted, resulting in a lower glass transition temperature and obtaining thermoplastic processing properties. The plasticization process of starch can be divided into four stages, as shown in Stage I: Starch absorbs water and swells. Natural starch aggregates in the form of granules. When subjected to distilled water, the starch granules absorb water and swell, which is also known as reversible water absorption and swelling. During this process, water absorption occurs in the amorphous regions of the starch, while the crystalline regions remain intact. Once the water disperses, the starch can revert back to its original granular structure.Stage II: Starch granules break. As the ambient temperature increases, under the combined action of plasticizers, mechanical agitation, and moisture, the starch granules begin to break.Stage III: A new hydrogen bonding mechanism forms. Under the action of thermal and continuous mechanical agitation, plasticizer molecules enter the interior of starch granules, disrupting the existing hydrogen bonds. The entire molecules undergo relaxation, resulting in a destruction of the crystalline structure and an increase in the number of free branching chains, thereby increasing the free volume within the starch. At the same time, the plasticizer molecules provide a large number of –OH groups, making them more prone to interact with the –OH groups in starch. The resulting starch−plasticizer hydrogen bonding mechanism replaces the original starch−starch hydrogen bonding mechanism.Stage IV: Plasticized starch forms. Under the influence of the new hydrogen bonding mechanism, starch molecules undergo rearrangement, leading to increased molecular chain mobility and a decrease in melting temperature. As a result, starch acquires processability.In the plasticization process, plasticizers play a crucial role by primarily disrupting the hydrogen bonding in starch. The mechanism of their action is illustrated in −1. With the increase in GLY content, the frequency of the absorption peak gradually decreases to 3441.1 cm−1, 3416.5 cm−1, 3404.6 cm−1, and 3423.7 cm−1, indicating that a new hydrogen bond is formed between GLY and NCS molecules. When the mass ratio between NCS and GLY is 10:3, the vibration frequency of the –OH group is the lowest, and the spectrum is wide, indicating that the hydrogen bond is the strongest and the plasticization effect is the best at this mass ratio. When the GLY content increases until the mass ratio between NCS and GLY reaches 10:4, the absorption peak of the –OH wave tends to shift to a higher frequency, indicating that an excessive GLY addition was not conducive to the plasticization process of NCS. This might be attributed to the fact that the excess GLY itself also forms hydrogen bonds, of which one –OH and the other –OH form a hydrogen bond, thus enhancing the stability of the GLY and reducing the plasticization effect.The infrared spectrum of NCS  is known for its high efficiency, processing speed, and simplicity and is widely utilized in industrial research. 1), and rebound rate (Y2) is established by second−order polynomial fitting of experimental data. Y1 and Y2 can be expressed as follows:The quadratic polynomial regression model (code) of the forming temperature (A), forming time (B), AC content (C), tensile strength  represents the ratio of the difference between the maximum and minimum and predicted values to the average standard deviation of all predicted values. It serves as a measure of the signal−to−noise ratio, and an AP value higher than four is considered desirable . In thisFurthermore, the normality of the residuals is assessed through a normal probability graph of the residuals. When the number of models is adequate, the overall shape of the residual graph should resemble a straight line if the residuals follow a normal distribution . Figure In In The optimization process is conducted using the Design Expert software. The optimal process parameters are as follows: the forming temperature (A) is 198.13 °C, the forming time (B) is 5.39 min, and the AC content (C) is 0.84 g. Based on the fitted second−order polynomial mathematical model, the predicted tensile strength is 4.28 MPa, and the rebound rate is 96%. Considering equipment limitations during actual processing, the specific process parameters selected for material preparation are as follows: forming temperature (A) is 198 °C, forming time (B) is 5.4 min, and AC content (C) is 0.84 g. The mechanical properties are then tested. The test results show that the actual process parameters result in a tensile strength of 4.22 MPa and a rebound rate of 95.7%, which are similar to the predicted values generated by the software and mathematical model. This demonstrates the reliability of the RSM for optimizing the forming process of starch−based packaging materials. The process parameters of group 3 in In this study, processable TPS is prepared by adding plasticizers. It has been observed that in the presence of plasticizers, the hydrogen bonds of starch are broken, leading to a reduction in crystalline peaks and disruption in the granule morphology. The use of composite plasticizers resulted in better effects. Subsequently, the preparation of starch−based packaging materials is carried out in a thermal cavity forming machine, and the optimization of the forming process parameters, including forming temperature, forming time, and AC content, is studied using RSM. It is observed that both the tensile strength and rebound rate exhibit an increasing trend followed by a decreasing trend with the increase of the parameters. The optimal mechanical properties are achieved at the process parameters of 198 °C forming temperature, 5.4 min forming time, and 0.84 g AC content. Compared to the non−optimized mechanical properties in the experimental design stage, the tensile strength increases by 12.2%, and the rebound rate increases by 8.1%.


Vision is thought to play a substantial role in hitting and fielding in cricket. An understanding of which visual skills contribute during cricket play could inform future clinical training trials. This paper reviews what has been reported thus far regarding the relationship of visual skills to cricket performance and reviews the results of clinical trials in which the impact of visual skills training on cricket performance has been addressed. Fundamental or low-level visual skills, with the exception of color vision and perhaps near stereopsis and dynamic visual acuity, are similar between cricket players and the general population. Simple reaction time has been found to be shorter in cricket players in some but not all studies. While there is mixed or no evidence that the aforementioned visual skills are superior in cricket players compared to non-players, comparisons of eye and head movements and gaze tracking have revealed consistent differences between elite cricket batters and sub-elite batters. Future training studies could examine whether teaching sub-elite batters to emulate the gaze tracking patterns of elite batters is beneficial for batting. Lastly, clinical trials in which visual skills of cricket players have been trained have in many cases resulted in positive effects on visual skills, or judgments required in cricket, or cricket play. However, clinical trials with larger and more diverse groups of participants and correlations to on-field metrics and on-field performance  are necessary before conclusions can be drawn regarding the efficacy of vision training. Resolving detail;Estimating depth;Tracking moving objects;Visuomotor integration;Visual information processing.A wide array of vision-related attributes are likely to play a role in cricket. A number of investigators have proposed schemes by which to group visual skills that pertain to sports. For example, Ciuffreda and Wang  listed fOther investigators have proposed similar schemes, including Kirschen and Laby  and mostThe goal of sports vision is to determine which visual skills influence sports performance, and to train those visual skills to maximize performance during competition. From the categorization schemes described above, one could first develop a list of specific visual skills that may impact cricket batting and fielding performance, and then indirectly (ex situ or laboratory assessments) or directly (in situ or on-field assessments) determine whether these visual skills could or do influence in-game performance.There are multiple methods that have been used to determine which visual skills may be important for a particular sport. One of these methods is to compare a particular visual skill between expert performers and novice or less-expert performers. If a visual skill is shown to be better in experts, this could imply that this skill plays a role in performance, requiring further sport-specific study of the skill. A second method is to compare or correlate the visual skill with performance metrics from competitions. This method only indirectly suggests a relationship between the visual skill of interest and performance in competition. This is because regression analyses correlating visual skills and performance reflect associations between these variables, but they do not necessarily imply that visual skills lead to better performance. Finally, a third method to assess the impact of a visual skill on sport performance is to perform a placebo-controlled clinical trial. In this scenario, a visual skill or group of skills are trained. Pre-training and post-training assessments of either visual skills thought to be related to sports performance or of skills directly related to performance may be compared. Ideally, the outcome of clinical trials would include a comparison of pre-training and post-training on-field performance metrics. Clinical trials involving pre-training and post-training measures of game-related skills  are most definitive in establishing the efficacy of the training paradigms.Hodges and colleagues divided visual skills for interceptive sports into fundamental skills , low-level visual skills , high-level visual and attentional skills , and cognitive skills . Under each of these skills, it is possible to draw a distinction between domain-specific and domain-general training methods and assessments . In thisFor the various categories of skills proposed by Hodges et al., both domain-specific and domain-general methods have been used to compare athletes with different skill levels . While sBecause there are published studies on vision in cricket that encompass both low-level and high-level visual skills as well as domain-specific and domain-general visual skill assessments, the sport of cricket provides an interesting opportunity to examine questions such as whether one group of skills or one group of assessments (low-level or high-level) correlate better with the skill level of players in the context of just one sport. The focus on one sport is significant, since it is unclear whether the assessment and training of visual skills in one sport transfers to another sport ,8.In this narrative review, we first summarize studies in which the visual skills of cricket players at different levels are compared and studies in which the visual skills of cricket players and the general (non-playing) population are compared. The purpose of this first section of the paper is to determine which visual skills have thus far been shown to be better in cricket players, to describe the method of assessment of these skills , and to determine whether particular groups of visual skills  are more likely to be different for cricket players at different levels or in cricket players versus non-players .In the second section of the paper, we review studies in which the impact of vision training on cricket performance is assessed. This part of the review includes 12 studies, which to our knowledge represents the most comprehensive review on this topic in cricket thus far. The visual skills trained in each of these twelve studies will be compared to the visual skills shown to be important for cricket performance in the first section of the paper to determine whether the training paradigms are in fact targeting skills that are expected to enhance performance. Then, the outcome measures of each training study will be evaluated to determine if they are domain-specific or domain-general measures. The review will inform future studies on vision in cricket.Papers used in the following sections were identified using four sources. The first source was PubMed , the second source was Google Scholar, and a third source was SPORTDiscus. The keywords “Cricket” and “Vision” were used in all of these searches. Finally, a general search was performed using the “Google” search engine. This latter search was primarily executed using the search term “vision in cricket”. Reference lists in the publications yielded through these searches were also examined for relevant papers and book chapters. The majority of these resources were research articles.To begin the discussion of those visual skills that have been compared between cricket players and non-cricket players or between cricket players at different levels, in this section, we discuss those non-visual and visual cues that cricket players may use to intercept projected balls. Much of our understanding of these cues is derived from temporal occlusion studies, in which the vision of the observer is occluded at different times prior to and after pitch release . SpatialAdvance cues, which include both contextual and kinematic cues, are those cues available prior to release of an approaching ball. Contextual cues are sources of information about a projected ball’s speed or trajectory that are known to the batter or fielder prior to any action on the part of a bowler or batter. These cues can help in predicting the trajectory of the ball. In the case of cricket batters, examples of contextual cues could be the batter’s knowledge of a bowler’s tendencies, or the batter’s knowledge regarding the most likely ball types associated with the current placement of the fielders . KinematMüller and colleagues have also examined the integration of contextual and kinematic cues . In one Müller and Abernethy suggested that the trajectory information available from a ball’s flight included the rotation, “apex”, and “drift and dip” of the approaching ball . Regan hTo estimate the time at which an approaching object will reach an observer, the ratio of the object’s instantaneous angular subtense to the rate of change in the ball’s angular subtense (termed tau) might be used. A binocular ratio to estimate the time of arrival of an approaching object has also been derived, and it involves the rate of change in the object’s horizontal disparity.Given that there is detailed visual information associated with a cricket ball’s flight that can be used in estimating the ball’s trajectory, a reasonable suggestion would be that low-level visual functions such as visual acuity could impact on cricket performance. However, if characteristics such as making better use of contextual cues or early kinematic cues or more accurately directing attention to locations where the most specific information related to ball trajectory are found have a greater influence on cricket performance compared to low-level visual skills, then low-level skills may not need to be any better in cricket players than in the general population. The following section looks at what is known thus far about the impact of low-level visual functions on cricket performance.As mentioned above, Hodges and colleagues recently published an extensive scheme to classify perceptual-cognitive skills involved in interceptive sports . FundameThe literature search identified four papers in which fundamental and lower-level skills in cricketers were studied using domain-general assessments. Barrett and colleagues examined two groups of cricketers and a group of rugby players . The focThe results for tests of visual function were compared to previously published values on young adults. Overall, distance visual acuity in the cricket players was no better than that published for young adults, and for the elite cricketers, visual acuity was statistically worse than that expected from published values. One of the 16 elite players and 2 of 23 near-elite players showed improvement in distance acuity greater than or equal to one line with the pinhole, suggesting that there was uncorrected refractive error for these players. Near visual acuity was also similar to that expected from the published literature. For the near-elite cricket players, stereoacuity was similar to the expected value while stereoacuity was better than expected in the elite cricket players. Color vision was normal except for two near-elite cricketers. Other visual problems, referred to by the authors as “residual visual issues” were found in three (18.8%) of the elite cricketers and eight (35%) of the near-elite cricketers. A number of these residual issues were due to uncorrected refractive error, or because players did not wear their refractive correction during games. Of the 16 elite cricketers, 69% (11) had received an eye examination within the last two years, 25% (4) had received an examination within 2–5 years, and 6% (1) had received an eye examination more than 5 years ago or never. Of the 23 near-elite cricketers, 52.2% (12) had received an eye examination within the last two years, 17.4% (4) had received an examination within 2–5 years, and 30.4% (7) had received an eye examination more than 5 years ago or never, suggesting that the rate of eye care utilization was modest.Overall, Barrett and colleagues concluded that the fundamental and low-level visual skills they assessed were not generally better than those expected in the population except for near stereoacuity in the elite players, and that perfectly clear vision as measured clinically is not required for successful performance in cricket or rugby. Much like other sports vision researchers have concluded ,21, the In a second study of fundamental and low-level visual skills in cricket players, Brown and Couper examined 100 cricket players at different levels, some who played for district clubs and some who played for sub-district clubs . All of In a third study of fundamental and low-level vision in athletes, Sapkota and colleagues examined 95 national football (soccer) and cricket players from Nepal . All of Finally, in a fourth study involving low-level skills in cricket players, Kelly and Roberts compared the dynamic visual acuity of male cricket players to male non-cricket players . DynamicWhile such differences in dynamic visual acuity between athletes and nonathletes have also been documented in other studies ,26, in aIn agreement with the domain-general assessments in the studies described above ,22,23, sIn a second paper, Mann and his collaborators pointed out and addressed some issues with their original study . First, Finally, Mann and colleagues published a study in which ten male cricket batters who had played in the “local regional first-grade competition” within the 12 months prior to the study viewed balls bowled by live bowlers . The parTaken together, the results of these three studies suggest that blur of as much as +2.00 D has little influence on performance in hitting balls in cricket. These results are somewhat similar to those in baseball, where Reuscher and colleagues showed that the performance of batters hitting balls from a pitching machine was not affected with +1.00 D of blur, but performance was reduced with +2.00 D of blur .The fact that blur less than +3.00 D does not negatively impact on cricket batting at least for slow to medium speed deliveries (less than 110 kph) might suggest that batting is largely regulated by the dorsal visual system (the vision-for-action system) rather than the ventral visual system. The dorsal visual system is said to be relatively insensitive to blur .While the conclusion from Barrett and colleagues’ visual acuity assessments  and from Mann and colleagues’ studies of blur and visual performance (domain-specific) is that perfect vision is not necessary to successfully bat a cricket ball, Mann and colleagues make the point that these results do not argue against providing the “full” refractive correction for cricket players. What the results do suggest is that blur resulting from uncorrected refractive error may not negatively impact on performance as much as one might expect.Another fundamental visual skill related to athletic performance mentioned by Hodges and colleagues is the visual field. A study by Wilkins and colleagues regarding the visual field of cricket batters should be mentioned here . AlthougAnother low-level visual attribute that has been examined in cricket players is color vision. These have been domain-general assessments. Because cricket has been played with a red ball against a green background, it could be that deficient color vision may lead to difficulties in playing cricket . There aScott and colleagues examined slip-catching in five male professional cricketers . Balls wReaction time is another skill related to vision that has been examined in cricketers. Reaction time can be considered a higher-level skill, in that it requires the respondent to direct their attention to and respond to a visual stimulus. Early studies suggest that the reaction time of cricketers may be no better than the overall population. The reaction time of Sir Donald Bradman, an international cricketer, was found to be slightly less than that of an “average university student” . SandersIn a different study, Thomas and colleagues measured visual evoked potentials (VEPs) and choice visual reaction time in cricket players and controls . These wVarious advantages have been ascribed to the dominant eye compared to the non-dominant eye. One of these advantages is that monocular visual reaction time is shorter when measured with the dominant eye ,44. CrosEye movements can be considered higher-level visual skills. Eye movements have been assessed with both domain-general and domain-specific methods.In a recent paper by Murray and colleagues, computerized measurements of visual-motor control were made and compared to cricket batting and bowling statistics . Domain-2 from stepwise regression analyses ranged from 39.4% to 85%) with batting and bowling performance. The only performance metric that was not related to the oculomotor variables was ECON. Overall, these results are comparable to findings in baseball and other interceptive sports [Murray et al. studied 59 Australian male professional cricket players who participated in T20 leagues. T20 is a shorter version of cricket. A total of 30 study participants were bowlers, and 29 were batsmen. The computerized RightEye device  was used to assess a number of oculomotor functions including fixations, saccadic eye movements, and pursuit eye movements. Metrics related to the oculomotor variables were calculated by the RightEye device. Oculomotor performance from the RightEye metrics were then compared to batting metrics (runs and strike rate) or bowling metrics ) using multiple regression analyses. Overall, individual RightEye metrics and combinations of these metrics were modestly or highly correlated  of cricket batters have been examined using domain-specific assessments. These investigations have revealed some key differences between expert players and less expert or novice players. There are several reasons why studies of eye and head movements in cricket batters are thought to be important. In general, gaze may be directed toward locations where important information related to performance is gathered. Further, head movements may provide specific advantages in batting as described below. Finally, earlier timing and greater accuracy of predictive saccades that place gaze ahead of the approaching ball may reflect better anticipation of ball trajectory, and these saccades may allow batters to use information after ball bounce to fine-tune interceptive movements.The first of these eye and head tracking studies was published by Land and McLeod . VerticaFollowing the predictive saccade, Land and McLeod reported that there was a period of relative gaze stability around the time the ball bounced. This was followed after the ball bounce by a period of gaze tracking. The initial predictive saccade occurred earlier for the two better batsmen, and later for the less skilled batsman. In addition, for the “best” batsman, the initial saccade was intermixed with pursuit eye movements.Land and McLeod did not address the batting performance of their subjects in terms of bat–ball contact, but they did remark that the two better batsmen played “attacking shots” for the short- and full-length deliveries and “defensive” shots for the good-length balls. The other, less skilled batsman played an attacking shot only for the full-length deliveries.In 2010, Croft and colleagues published a paper on vertical gaze tracking in cricket batting . Gaze trThere was no relationship between the gaze tracking patterns and the percentage of poor bat–ball contacts.In 2013, Mann and colleagues published a paper on vertical head and eye movements of male cricket batters . Two of The elite players initially maintained their gaze on the ball or ahead of the ball, while the club players maintained their gaze on the ball or behind the ball. All of the subjects made predictive saccades. The elite batters made earlier (just as Land and McLeod had reported) and larger predictive saccades compared to the club players. The elite batters often produced two saccades . One of these was to the predicted bounce point of the ball, and the second was to the predicted location of bat–ball contact. The club batters tended to make just one saccade to the predicted bounce point regardless of ball length. Around the time of the bat–ball contact, the gaze was ahead of the ball for the elite players and behind the ball for the club-level batters.In terms of head movements, elite batters moved their head with the ball, while the club batters aligned their eyes with the ball. Mann and colleagues suggested that the purpose of rotating the head with the ball was to maintain the ball in a constant egocentric direction relative to the head, thereby reducing the computation of where the ball would arrive at the batter. As mentioned earlier, Regan provided another explanation as to why head tracking might be beneficial in cricket batting (and fielding) . SpecifiIn 2017, Sarpeshkar and colleagues published a paper in which they examined head and eye movements of 43 male cricket batters in significant depth . A ProBaIn examining the results from the straight trajectory trials , for elite batters, gaze was further ahead of the ball compared to the club batters, as Mann et al. had reported previously . This suDifferent from the results of Land and McLeod, in which predictive saccades occurred earlier for the more experienced players , SarpeshIn a second analysis, head and eye movements and gaze tracking behaviors were compared under conditions where batters knew that the ball would follow a straight trajectory and under conditions when the ball might not follow a straight trajectory . In the random trials, changes in gaze behavior occurred that the authors described as “novice-like”. Specifically, the gaze was further behind the ball, there were fewer predictive saccades, and those saccades that were predictive occurred later, and the gaze at bat–ball contact was less commonly in the direction of the ball when the bat contacted the ball. These findings are interesting and similar to those from Sarpeshkar and colleagues  and thosA third analysis compared gaze tracking when the batters attempted to hit balls that swung versus balls that did not swing. Differences in the behavior of elite and novice batters were exaggerated in the swinging trials compared to the straight trials in that elite batters were better able to maintain their gaze in the direction of the ball in the swinging trials. The frequency of predictive saccades to the location of ball bounce was similar for the swinging and straight trials. However, only the elite players were more likely to make oblique saccades in the swinging trials. The quality of bat–ball contact was reduced when batters attempted to hit swinging trajectories.Finally, in a fourth analysis, Sarpeshkar and colleagues compared visual-motor behavior when projected balls swung toward the batter (in-swing) and when these balls swung away from the batter (out-swing). The batters’ gaze was less likely to be ahead of the ball when the ball swung out. While the frequency of predictive saccades did not depend on the direction of ball swing, batters made earlier saccades in the out-swinging trials that the authors suggested were the result of the batters mistaking the out-swinging trajectories for straight trajectories. The quality of bat–ball contact was reduced for the out-swinging trajectories compared to the in-swinging trajectories.In summary, there is variability in the eye and head movement and gaze tracking findings in these studies ,54,58,59The impact of training upon cricket has been explored in a number of studies. These studies, summarized in In 1996, West and Bressan conducted a study and assessed the impact of two types of visual training on the ability of cricket batsmen to judge the length-of-ball . SelecteA study by Stretch utilized 16 cricketers at a cricket academy (referred to as batsmen and bowlers) aged 17–19 years, who were divided into an experimental and control group . The plaA study by Balasaheb and colleagues examined 30 competitive, club-level male cricket batsmen aged 16 to 25 . Particihttp://www.sportsci.org/news/news9705/hockeyvision.html, accessed on 9 August 2023). The training was completed for three sessions per week for four weeks. Assessments of domain-general visual skills tests  and domain-specific cricket skills tests  were completed before and after the software was used. Those using the computer training showed significant improvements between the pre- and post-assessments for 13 visual skills and six sport-specific skills. Speed and accuracy of catching was statistically different when comparing the improvement from pre- to post-testing and the experimental group compared to the control group. The experimental group had a statistically higher number of successful catches when comparing pre- to post-training, although the improvement was not statistically better than in the control group. Throwing accuracy, directionality, and distance were improved statistically when comparing pre-testing to post-testing in the experimental group. Comparison of throwing distance between the experimental and control groups was also significantly different. Batting was not assessed in this study. The use of cricket (domain-specific) skills in the assessments before and after vision training were useful. Further testing with a larger study population would add more validity to the findings in these small groups.A study by Calder and Kluka evaluated 30 male high school cricket players with at least three years of experience, dividing the participants into two groups of players with matching numbers of participants who played each position . One groA study by Kruger and colleagues recruited 13 under-19 cricket players to complete an 8-week visual skills training program that included two training sessions each week . WhetherThe occlusion technique was used in a training study by Hopwood and colleagues. Twelve senior level male cricket players completed in situ and perceptual testing of fielding before and after six weeks of training . TraininEdgar and colleagues assessed dynamic visual acuity in 18 male cricket players under the age of 19 and 18 male non-cricket players . While tA study by Shunmuganathan evaluated batting performance in 36 male cricket players, dividing them into three groups: one group that completed skills training and visual training, one group that completed only skills training, and one group that served as a control with no additional training . The twoWimshurst and colleagues conducted a study of 24 male county cricket players divided into four “training” groups who participated in six weeks of training . One groBrenton and colleagues performed a study in which twelve “emerging” cricket batsmen from an Australian state cricket squad were included. Six of the subjects received visual-perceptual training, and six subjects served as controls . The traBrenton and colleagues also completed a study of 39 district club level specialist male batters aged 18–36 . Particit-tests were performed, but the results of statistical tests are not given to support their statements that eye/hand coordination, eye/foot coordination, visual reaction time, depth perception, dynamic acuity, or batting performance are “significantly” improved.Kumar and Kadhiravan completed a study that assessed changes in visual skills and batting performance of male cricket players who underwent sports vision training for 12 weeks . Thirty The criteria for evaluating the quality of clinical trials in sports vision were described recently and can be applied in assessing the studies in this manuscript . These cVisual skills that have been discussed in this paper can be divided into high-level and low-level skills based on the classification scheme provided by Hodges and colleagues . Some, bIn terms of higher-level visual skills, there is evidence from one study that simple reaction time may be shorter in cricket players , but in A question of interest is whether the just-described findings on the significance of various visual skills can inform the vision training methods employed in cricket. In a recent paper on sports vision training, Poltavski and colleagues  reviewedAn interesting point to consider going forward is that while studies on the advantages of hardware skills in cricket are equivocal thus far, in many of the training studies, the vision training includes or is focused on hardware skills such as accommodation and vergence eye movements. If training these visual skills improves performance in cricket, then it may be that the training results in supra-normal lower-level visual skills, or that the training results in improvements in related skills such as attention in addition to changes in the visual skills that are trained and tested.In spite of the diverse levels of the cricket players in the studies discussed here, the conclusions were often similar. However, it is possible that the visual skills or visual behaviors of the most elite cricket players vary from those of even near-elite players ,59. Simi


The rapid development of the cassava industry has increased our demand for dried cassava, but the supply of raw materials and the production of cassava starch have declined due to the reduction in the planting area. Moreover, the anti-nutritional factors contained in natural cassava limit the application of cassava. Therefore, it is necessary to study processing methods to improve the utilization value of cassava. We used three processing methods  and three conditioning temperatures  to process cassava and cassava feed so as to improve the utilization of cassava. The results found that high-temperature conditioning reduced broilers’ growth performance and nutrient utilization, and steam conditioning and puffing conditioning increased the in vitro starch digestibility and nutrient utilization of broilers. Specifically, cassava starch had lower amylose content and amylose/amylopectin at a conditioning temperature of 60 °C in combination with puffing conditioning or steam conditioning, and puffing conditioning had the highest crude protein digestibility (in the starter period) and apparent metabolizable energy (in the grower period) at 60 °C.p < 0.01) were higher at conditioned 90 °C than that at 60 or 75 °C, and PU was higher than SC and MC (p < 0.01) (0.25–2 h). The amylose content and amylose/amylopectin at conditioned 60 °C or PU were lower (p < 0.01) than that of 75 or 90 °C or SC, whereas the opposite was true for amylopectin content (p < 0.01). The resistant starch content of SC or PU was lower (p < 0.01) than MC. In the in vivo study, broilers fed diets conditioned at 60 °C or SC had a lower (p < 0.05) feed-to-gain ratio than those fed diets conditioned at 90 °C or PU diets. The ileum apparent digestibility of starch and AME were higher (p < 0.05) for broilers fed SC diets than for those fed MC diets. These results indicate that cassava starch promoted starch digestion rate by reducing amylose content and amylose/amylose under PU combined with a conditioning temperature of 60 °C, ileum digestibility of starch in broilers fed SC diets was higher than MC diets regardless of conditioning temperature, and SC diets increased AME and decreased F/G to promote growth performance of broilers.As an important food crop, cassava is rich in nutrients and high in starch content and is widely used in the production of industrial raw materials. However, the utilization value of cassava is limited due to the reduction of planting area and the existence of anti-nutritional factors. Therefore, we evaluated in vitro cassava starch digestibility and in vivo growth performance of broilers in a 3 × 3 factorial arrangement of treatments using three processing methods , steam conditioning (SC), and puffing conditioning (PU)) and three conditioning temperatures  to screen for the optimal processing method and conditioning temperature to improve the utilization of cassava. In the in vitro cassava starch digestion study, the digestibility and digestion rate ( Euphorbiaceae family), sweet potato, and potato are known as the world’s three major potato crops, of which cassava is highly adaptable, resistant to drought and acidic soil, and can be grown on poor land. At the same time, cassava is a potentially high biomass-producing crop [Cassava . The animal procedures used in this study strictly abide by the Administrative Measures of Gansu Province on Experimental Animals (2021–2025). Glucose and starch standard samples were purchased from Shanghai Zhanyun Chemical Co., Ltd. . All other reagents used in this study were analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. .−1.The study was a completely randomized design with 3 processing methods , steam conditioning (SC), and puffing conditioning (PU)) × 3 conditioning temperatures . The cassava roots were cut into small pieces using a slicer machine  after removing peels, stems, leaves, purified and impurities, then sun-dried on a concrete floor for about 2–3 days until the moisture content dropped below 14% , then grv/w = 10:1) overnight. The mixture was ground in a mortar and filtered through 200 mesh sieves. The filtrate was then centrifuged at 3000× g for 15 min. The upper layer containing impurities was removed. The precipitate was suspended in distilled water and then centrifuged again. This process was repeated several times until the supernatant was clear. The sediment was dehydrated with absolute ethanol twice, dried at 40 °C for 6 h, then ground into powder, screened through 100 mesh sieves, and stored at −20 °C. The determination of starch content refers to GB 5009.9-2016, and the content of amylose refers to GB/T 15683-2008.The extraction of cassava starch was conducted as described by El Halal et al. (2019) . Cassava2h and DC4h is starch digestion coefficient at incubation time 2 h and 4 h, respectively. Moreover, 0.9 is the coefficient to convert molecular mass from glucose to starch monomer units.The in vitro digestibility property of cassava starch was determined based on a previously published method that simulated the digestive behavior of the digestive tract of the broiler  with modt (%) is the starch digestibility at incubation time t, and DST (%) is the content of digestible starch that will digest at a fractional rate of KDS . The slope of each curve was calculated by Excel software, giving the KDS value.Since in vitro starch digestion follows first-order kinetics, the following curve equation was used to estimate in vitro starch digestibility .DCt = D2) as an indigestible marker to determine nutrient digestibility. The composition of the starter (1–21 d of age) and grower (22–42 d of age) periods was formulated with reference to the recommended according to the NRC (1994) [The study utilized a 3 × 3 design, using processing methods , steam conditioning (SC), and puffing conditioning (PU)) and conditioning temperatures  as main factors. A total of 1008 one-day-old male Cobb broilers (47.5 ± 2 g) purchased from a commercial hatchery  were randomly divided into 9 treatments with 8 replicates (cages) (14 broilers per replicate). The broilers were fed a cassava–soybean basal diet with 0.4% titanium dioxide (TiOC (1994)  to meet C (1994) . Feed an2 by the marker ratio technique [The apparent digestibility of the ileum and total tract were determined utilizing TiOechnique . On days2SO4 (v/w = 1:10). The excreta and feed samples were dried at 65 °C, ground into 1 mm screen with a hammer mill  for the determination of DM, CP, and apparent metabolizable energy (AME). The contents of DM (method 930.15) and CP (method 954.01) in feed, excreta, and ileum digesta samples were measured using standard procedures of AOAC (1995) [2 was determined according to the method of Short et al. (1996) [At 18 to 21 d of age and 38 to 41 d of age, excreta was collected twice daily from each replicate (cage). Daily excreta from each cage was collected and mixed well before subsampling. Feather and dander were removed from the excreta and were acidified with 10% HC (1995) . TiO2 wa. (1996) . Gross e. (1996) . The folp ≤ 0.05 and highly significant if p < 0.01 (**).The data were analyzed by two-way factorial arrangement (ANOVA) to determine the main effects (conditioning temperature and processing methods) and their interaction using the General Linear Models procedure of SPSS statistical software . A cage served as an individual experimental unit. The Duncan method was used for multiple comparisons when the difference was significant. All results were presented as mean values and the standard error of the mean (SEM). The difference between means is significant (*) if p < 0.01) than at 60 °C or 75 °C, and PU demonstrated the highest (p < 0.01) digestibility, followed by SC and MC (0.25–2 h). The interactions (p < 0.05) were observed between processing methods and conditioning temperatures on starch digestibility (0.25–2 h) and starch digestion rate. The starch digestion rate varied from 2.7% (combination of 60 °C and MC) to 5.8% (combination of 75 °C and PU), with a difference of 3.1% between maximum and minimum values. The digestibility of cassava starch was highest at 60 °C or 75 °C in combination with PU.In vitro digestibility of cassava starch increased gradually with increased incubation time (0.25–3 h) until reaching an asymptotic level . The stap < 0.01) than at 75 °C or 90 °C, whereas the opposite was true for amylopectin content (p < 0.01) (p < 0.01) than at 60 °C or 75 °C, whereas the opposite was true for SDS content (p < 0.01). The amylose (24.8% vs. 26.7%), amylose/amylopectin (0.54 vs. 0.62), and SDS (2.4% vs. 6.5%) content of PU were lower (p < 0.01) than SC, whereas the opposite was true for amylopectin and RDS content (p < 0.01). The RS content of SC or PU was lower (p < 0.01) than MC. Significant interactions (p < 0.05) were observed between conditioning temperatures and processing methods on the content of amylose, amylopectin, amylose/amylopectin, RDS, SDS, and RS.The amylose content and amylose/amylopectin at conditioned 60 °C were lower ( < 0.01) . The RDSp < 0.05) ADFI than those fed diets conditioned at 75°C or 60 °C in the starter period (p < 0.05) for broilers fed MC diets in the starter period. In the grower period, broilers fed diets conditioned at 60 °C had higher (p < 0.05) ADG than those fed diets conditioned at 90 °C. Broilers fed PU diets had higher (p < 0.05) ADG and ADFI than those fed MC and SC diets. Broilers fed diets conditioned at 90 °C or PU diets had higher (p < 0.05) F/G in both the starter and grower periods. No interactions (p > 0.05) between processing methods and conditioning temperatures were observed for ADG, ADFI, and F/G in both the starter and grower periods.Broilers fed diets conditioned at 90 °C had higher (r period . ADG andp < 0.05) ileum apparent digestibility of CP than those fed diets conditioned at 90 °C in both the starter and grower periods (p < 0.01) ileum apparent digestibility of CP than those fed MC diets (76.0% vs. 72.3%) in the starter period. The ileum apparent digestibility of starch was higher (p < 0.05) for broilers fed SC diets than for those fed MC diets in both the starter and grower periods. Significant interactions (p < 0.05) were observed between processing methods and conditioning temperatures on CP apparent digestibility in the starter period and starch apparent digestibility in both the starter and grower periods. The combination of 60 °C and PU diets generated the highest ileum apparent digestibility of CP (78.1%) in the starter period (p < 0.05).Broilers fed diets conditioned at 60 °C had higher ( periods . Broilerp > 0.05) by processing methods and conditioning temperatures in both the starter and grower periods (p < 0.05) AME than those fed diets conditioned at 75 °C or 60 °C in both the starter and grower periods. In the starter period, broilers fed PU diets had higher (p < 0.05) nitrogen retention than those fed MC or SC diets. However, broilers fed SC diets had higher (p < 0.05) AME than those fed MC diets in both the starter and grower periods. Significant interactions (p < 0.05) were observed between processing methods and conditioning temperatures on AME in the grower period.The DM apparent digestibility of the total tract was not affected  ,43. The The temperature required to promote starch digestion is not uniform but related to the characteristics of the starch varieties and processing methods. In vitro digestibility and digestion rate of cassava starch increase with increasing conditioning temperature, which is consistent with the findings obtained by Ali et al. 2020) . Interes20 . InteBroilers fed PU diets had higher ADFI than those fed MC diets. Broilers fed diets conditioned at 90 °C or PU diets had higher F/G in both the starter and grower periods. These results agree with those of Liermann et al. (2019) , who demBroilers fed diets conditioned at 90 °C had higher ADFI than those fed diets conditioned at 75 or 60 °C in the starter period. In the grower period, broilers fed diets conditioned at 60 °C had higher ADG than those fed diets conditioned at 90 °C, which is consistent with the findings of Abdollahi et al. (2011) , who fouIt is worth noting that the effective utilization of starch is not only related to the digestion rate but also depends on the passage time of starch in the digestive tract; the longer the starch stays in the small intestine, the more complete the starch digestion . BroilerThe AME was lower for MC diets than for SC and PU diets during both the starter and grower periods. Rodriguez et al. (2020)  reportedHigh temperature during the puffing process leads to the denaturation of proteins or inhibits the action of digestive enzymes. Meanwhile, an increased conditioning temperature increases water-soluble non-starch polysaccharides in the intestine, resulting in an increased viscosity of the digesta and influencing the digestion of starch . TherefoThe conditioning temperature of SC diets at either 60 °C or 90 °C decreased the ileum digestibility of DM, compared with those fed nonconditioned MC diets. Moreover, broilers fed diets conditioned at 60 °C had higher ileum digestibility of DM and starch compared to those conditioned at 90 °C . SimilarIn conclusion, cassava starch promoted starch digestion rate by reducing amylose content and amylose/amylose under PU combined with a conditioning temperature of 60 °C. The ileum digestibility of starch in broilers fed SC diets was higher than MC diets regardless of conditioning temperatures. And SC diets increased AME and decreased F/G to promote growth performance of broilers. In addition, high conditioning temperature is not conducive to poultry feed manufacture. The current findings may suggest that broiler diets can be conditioned at lower temperatures if combined with puffing or steam conditioning and also benefit from improved feed utilization.