Phlogiellus bundokalbo venomagainst neurodegenerative diseases. Spider venom is a rich cocktail of neuroactive compounds designed to preycapture and defense against predators that act on neuronal membraneproteins, in particular, acetylcholinesterases (AChE) that regulate synaptictransmission through acetylcholine (ACh) hydrolysis - an excitatoryneurotransmitter - and beta-secretases (BACE) that primarily cleave amyloidprecursor proteins (APP), which are, in turn, relevant in the structuralintegrity of neurons. The present study provides preliminary evidence on thetherapeutic potential of Terebriomolitor larvae in vivo and fractions werescreened for their inhibitory activities against AChE and BACE invitro. Spider venom was extracted by electrostimulation and fractionated byreverse-phase high-performance liquid chromatography (RP-HPLC) andcharacterized by matrix-assisted laser desorption ionization-time flightmass spectrometry (MALDI-TOF-MS). Neuroactivity of the whole venom wasobserved by a neurobehavioral response from P. bundokalbo demonstratedneuroactivity by inducing excitatory movements from T.molitor for 15 min. Sixteen fractions collected produceddiverse mass fragments from MALDI-TOF-MS ranging from 900-4500 Da. Eleven ofsixteen fractions demonstrated AChE inhibitory activities with 14.34% (±2.60e-4) to 62.05% (± 6.40e-5) compared with donepezil which has 86.34% (±3.90e-5) inhibition (p > 0.05), while none of the fractions were observedto exhibit BACE inhibition. Furthermore, three potent fractions againstAChE, F1, F3, and F16 displayed competitive and uncompetitive inhibitionscompared to donepezil as the positive control.  The whole venom from P. bundokalbo contains compounds thatdemonstrate neuroactivity and anti-AChE activities invitro, which could comprise possible therapeutic leads for thedevelopment of cholinergic compounds against neurological diseases. The venom of  Helodema suspectum), captopril from Brazilian pit vipers(Bothrops jararaca), bivalirudin from medicinal leechs, ziconitide from the cone snails(Conus magus), fibrin sealant from South American rattlesnakes, and apilic antivenom from melittin andPLA2 from Africanized honeybees (Apis mellifera)+. P. bundokalbo venom was plotted in a 3D landscape as shownThe diversity of compounds from the whole and fractionated venom from The whole and fractionated venom samples were screened for BACE inhibitoryactivities in comparison with the negative control or the untreated BACEreaction solution. However, most of the venom-treated samples demonstratednegative percentage inhibition which suggests that the fractional constituentsof the spider venom did not exhibit BACE inhibition as indicated in invitro compared to the whole venom, Donepezil (positive control),and untreated AChE reaction (negative control). Out of the 16 fractions, 11 ofthem exhibited anti-AChE activities, as demonstrated in  The venom fractions were screened for AChE inhibitory activities M and Vmaxvalues derived from the Michaelis-Menten and Lineaweaver-Burke kinetics comparedto the untreated AChE reactions.Moreover, F1, F3, and F16 exhibited potent anti-AChE activity which was alsoselected for substrate competition experiments to characterize the possible modeof inhibition and binding affinity to AChE against Donepezil and whole venom.Graphical results reported in M) measures the affinityof substrate concentrations at ½ of the maximum velocity (Vmax) of anenzyme. The corresponding values are shown in The kinetic parameters Michaelis constant  for 15 min as represented in Similarly, F1, F3, F16, whole venom, and Donepezil were administered between thesutures of the third metathoracic segment of P. bundokalbo showed that allthe peptide masses were not similar to masses of 3 isoforms of peptides fromPhlogiellus sp. recorded on Arachnoserver which areµ-theraphotoxin-Phlo1a (4103.79 Da), µ-theraphotoxin-Pho1b (4137.78 Da), andµ-theraphotoxin-Phlo2a  (3276.33 Da) [P.bundokalbo by family and order as shown in Database search of the monoisotopic oxidized peptide masses of the whole andfractionated spider venom from 6.33 Da) ,55,56. Oinvitro and in vivo [Current therapies for treating neurological diseases are ineffective and have sideeffects associated with their use; therefore, there is a need to develop noveltherapies. In this regard, previous studies have shown that neuroactive compoundsobtained from the spider venom with neuroprotective effects  in vivo .N- and O- linked glycosylations,hydroxylations, carboxylations, and bromination of specific amino acids. Thesetypes of PTMs alongside structurally related isoforms, variations, and molecularscaffolds, contribute to different mechanisms of pharmacologically based toxindiversifications in spider venom [The spider venom constitutes diverse mixture molecules composed of 16% of smallcompounds, 11% of acylpolyamines, 6% of linear peptides, 60% cysteine-knottedmini-proteins, 1% of neurotoxic proteins and 6% enzymes ,74-76. Ter venom -82. Phlogiellus bundokalbo venom, the chromatographic profilecontains peaks detected at 215 nm and 280 nm which generally detect peptides asindicated in In In a similar way, analysis of the chromatogram suggests that the compoundspresent on peaks 1 and 2 are characterized to be polar in nature, peak 3 to 14to be semi-polar in nature, while peaks 15 and 16 are characterized to benon-polar due to their affinity with the solvent system. Likewise, peaks 2-16exhibited broad peaks which can be attributed to the solvent conditionsestablished in the RP-HPLC or to the nature of the fractions themselves, whichmay contain different compounds in a single peak. Moreover, multiple peaks from5-14 may indicate similar characteristics of each compound present in thechromatogram with respect to their amphiphilicity. The elution of each peak isinfluence by the nature of the solvent system wherein TFA and ACN increases theresolution of the peaks in the chromatogram by acting as counter ions to thesilanol groups in the stationary phase and increase the hydrophobicity of themobile phase, respectively, to prevent the adsorption of peptides in the column,85. P.bundokalbo fractions indicates that early eluting fractions, F1 andF2, contain peptidic compounds that are less than 2000 Da. Moreover, semipolareluting fractions, F3 - F14, have peptidic constituents with mass ranges of 2000Da-4500 Da, and late eluting fractions, F15-F16 contain peptide masses rangingfrom 1000-2000 Da. These can be attributed to the nature of the sequence,folding, functional groups, and PTMs, which may confer different interactionsbetween constituents in the whole spider venom and their corresponding retentiontime to the stationary phase [However, these wavelength ranges are not specific only for peptide bonds andaromatic amino acids as they can be detected for small molecules such asacylpolyamines, organic acids, nucleotides, and free amino acids which are alsopresent in the whole spider venom ,83. Furtry phase .P. bundokalbo in theArachnoserver, shows that most of the peptide masses from the venom nosimilarity to the available Phlogiellus sp. toxins in thedatabase. Moreover, it is interesting to note that some of the fractions whichdemonstrated either anti-AChE or anti-BACE activities have shown similarity ofpeptide masses to other spider toxins that are related to P.bundokalbo by family Theraphosidae and orderAraneae suggesting that the peptides from P.bundokalbo have not yet recorded in the database [Finally, manual curation of peptides from database .BACE is a dimeric and transmembrane aspartic endoprotease localized in the Golgiapparatus of neurons that cleaves several neuronal peptide substrates, includingamyloid-precursor protein (APP) . Itattain vitro. However, most fractional constituents of the spider venom did not exhibitinhibition towards BACE, possibly because the enzyme has numerous peptidesubstrates where BACE cleavage may occur depending on the degree of activity. Since t3COLQ super complex [AChE (E.C. 3.1.1.7) is a 550 amino acid residue carboxylesterase monomer thathydrolyzes acetylcholine into its choline and acetate derivatives . It loca complex ,97. The  complex . However, according to the cholinergic hypothesis in AD, excessive alterations inAChE alongside reduced cortical innervations, corticocortical glutamatergicneurotransmission, coupling of muscarinic M1 receptors to secondary messengersinside the post-synaptic neuron are contributory factors towards the aggravationof tau protein formation, amyloidogenesis, and amyloidosis which are symptomatichallmarks of AD . Thus, cinvitro against AChE, which are the diversity of compounds andconcentrations of each fraction, the molecular size of the compound present ineach fraction, and the nature of the spider venom fractions with respect totheir amino acid compositions, functional groups and PTMs towards AChEinhibition.Based on the screening results for AChE inhibitory activities, the spider venomgenerally exhibited weak inhibition in comparison with Donepezil, as indicatedin invitro. Since spider venom contains different sizes ofpost-translationally modified peptides and polypeptides with molecular weight(MW) ranges greater than 2,000 Da, and are more significant than Donepezil, asmall molecule with M2 of 379.42 Da, it is unlikely that the binding sites forAChE inhibition are the same for the spider venom and donepezil, thus,contributing to weak inhibition [First, the presence of various compounds in each fraction influences the weakinhibition of AChE which is manifested in the broad and multiple peaks in thechromatogram and the mass distribution profiles in MALDI-TOF-MS. Most notably,this is evident in peaks 6-11 of the chromatogram in hibition ,82. Smalhibition . in vitrocan be attributed to the binding interactions of the compounds at the peripheralanionic sites nearby the choline-binding pocket of the active site, whichcontains positively charged groups such as amino groups and other N-containingcompounds [Trittame loki,contains AChE as part of its arsenal. These may also explain the negativeinhibition because the additional AChE from P. bundokalbo venommay promote ACh catalysis than inhibiting it [Furthermore, this can be supported by the kinetic inhibition characterization asindicated in ompounds . Aside fompounds ,101. On ompounds ,103. On ompounds  thattheiting it . Nonetheiting it .T. molitor principallycontains high levels of neurotransmitters for chemo- and mechanosensoryreceptors neurons in the peripheral nervous system (PNS) and central nervoussystem (CNS) of insects. Interestingly, the nervous system of the T.molitor is comprised of several receptors such as acetylcholine,GABA, and glutamate receptors to mediate the regulation of synaptic transmission[T. molitor is comparable with that of a human because theseinsects have functional equivalents of neurotransmitters and neuronal membraneproteins in the body, which are relevant to study for neurodegenerative diseases[T. molitor as an organism to study theneuroactivity effects upon administration of Donepezil and spider venomin vivo. The scoring was based on the rate of motility andconvulsive/paralytic-like effects of the venom and Donepezil towards the larvae,wherein 4 is the highest while 1 is the lowest. This is highly attributed to thein vivo action of the venom fractions, whole venom, andDonepezil on T. molitor, wherein spider venom toxins may causea build-up of neurotransmitters at the synapse which may interact at thepre-synaptic neuronal receptors for a prolonged amount of time in the nervoussystem of the T. molitor, and phenotypically results toexcitations and increase motor activities [T. molitor and AChEinhibition studies suggest that the fractional constituents from P.bundokalbo venom are insightful compound ds to focus on cholinergictherapeutics with respect to neurological diseases [Aside from this, behavioral results from smission. Roughlydiseases. Thus, ttivities ,51,55. Btivities ,107,108.diseases . Throughdiseases ,15,110. P. bundokalbo spider venom fractionsfrom Mindanao, Philippines, contain compounds that showed notable behavioralactivities in vivo and AChE inhibitory activities but not in BACEin vitro. Hence, further research on P.bundokalbo venom should focus on developing it for cholinergic studiessuch as investigating cholinergic-related membrane proteins  aside from AChE that contributes towards plaque andtangle degradation, modulation of neuroinflammation, and neuroprotective effectsagainst Alzheimer’s disease and other neurological diseases. Similarly,dose-dependent evaluation, purification, identification, and structural elucidationof putative cholinergic compounds from the spider venom are essential to developinginto its more potent, stable, and target-specific forms against neurodegenerativediseases. In this preliminary study, the


O-glucoside, a single-glucose-containing flavanone glycoside, significantly inhibited PTP1B with an IC50 value of 37.14 ± 0.07 µM. Hesperetin, which lacks a sugar molecule, was the weakest inhibitor compared to the reference compound, ursolic acid (IC50 = 9.65 ± 0.01 µM). The most active flavanone hesperetin 5-O-glucoside suggested that the position of a sugar moiety at the C-5-position influences the PTP1B inhibition. It was observed that the ability to inhibit PTP1B is dependent on the nature, position, and number of sugar moieties in the flavonoid structure, as well as conjugation. In the kinetic study of PTP1B enzyme inhibition, hesperetin 5-O-glucoside led to mixed-type inhibition. Molecular docking studies revealed that hesperetin 5-O-glucoside had a higher binding affinity with key amino residues, suggesting that this molecule best fits the PTP1B allosteric site cavity. The data reported here support hesperetin 5-O-glucoside as a hit for the design of more potent and selective inhibitors against PTP1B in the search for a new anti-diabetic treatment.In the present study, we investigated the structure-activity relationship of naturally occurring hesperetin derivatives, as well as the effects of their glycosylation on the inhibition of diabetes-related enzyme systems, protein tyrosine phosphatase 1B (PTP1B) and α-glycosidase. Among the tested hesperetin derivatives, hesperetin 5- Diabetes mellitus (DM) has emerged as a major threat to human health, and the expected number of diabetic patients will exceed 642 million by 2045 globally ,2. DM isRecent studies on the pathological mechanism revealed that DM has a close relation to the protein tyrosine phosphatase (PTP) family, which plays a pivotal role in the regulation of insulin function by dephosphorylating the tyrosine residues of proteins . Among tO-glycoside or C-glycoside forms in plants. There are several reports that flavonoids can regulate or modulate blood glucose levels after food intake -1-naphthyl}-L-alanyl)amino]pentyl}oxy)-2-naphthoic acid (compound 23) and 3--2-ethyl-benzofuran-6-sulfonic acid (4-sulfamoyl-phenyl)-amide (compound 2), with structures defined in Molecular docking is one of the most frequently used approaches in structure-based drug design because of its ability to predict, with a substantial degree of accuracy, the conformation of small-molecule ligands within the appropriate target binding site. To further understand the mechanisms of action of the flavonoids with inhibitory activity against PTP1B, we performed molecular docking simulations. Specifically, we used AutoDock vina to predict and investigate the interactions between five flavonoids  drastically abolished inhibitory activity against PTP1B. In addition, aglycone hesperetin, which lacks a sugar moiety, showed weak inhibitory activity against PTP1B. Based on these observations, it was clear that the presence of a sugar moiety at the C-5 position is very important for PTP1B inhibitory activity. On the other hand, both hesperidin (hesperetin 7-O-rutinoside) and neohesperidin (hesperetin 7-O-neohesperidoside) contain two disaccharides but exhibited different levels of potency against PTP1B, due to the different positions of the glycosidic linkage. Hesperidin sugars consist of the α-1,6 interglycosidic linkage and showed significant inhibitory activity against PTP1B (IC50 = 58.15 ± 4.18 μM), whereas neohesperidin sugar moieties consist of α-1,2 interglycosidic bonds and display weak inhibitory activity against PTP1B (IC50 = 143.63 ± 3.04 μM). Thus, based on these results, it was clear that the position of the sugar moiety to be appears important for PTP1B inhibition. Interestingly, all flavonoids were inactive as observed in the α-glucosidase inhibitory assay, hinting that this pathway may be less important for the bioactivity of flavonoids. Thus, the main approach of the present study was to analyze the selective inhibition of PTP1B by naturally occurring flavonoids. Research on PTP1B inhibitors as potential therapeutic options for the treatment of DM and obesity has reached a peak, as approximately 300 PTP1B inhibitors have been developed from a variety of natural sources, with the largest representation from flavonoids -1-naphthyl}-L-alanyl)amino]pentyl}oxy)-2-naphthoic acid (compound 23) were obtained from the RCSB Protein Data Bank (PDB ID: 1NNY) [O-glucoside, hesperetin 5-O-glucoside, and hesperetin were obtained from the PubChem Compound (NCBI), with compound CIDs of 10621, 442439, 147394, 18625123, and 72281, respectively, and were protonated (pH 7.0) using MarvinSketch program . The automated docking simulation was performed using AutoDock Tool (ADT) to assess the appropriate binding orientations and conformations of PTP1B with the different compounds. A Lamarckian genetic algorithm method implemented in AutoDock vina was employed. For docking calculations, Gasteiger charges were added by default, the rotatable bonds were set by the ADTs, and all torsions were allowed to rotate. The grid maps were generated by the Autogrid program where the grid box size of 80 × 80 × 80 had a default spacing of 0.375 Å. The respective X, Y, Z coordinates of the center were 56.019, 31.367 and 22.486, respectively. The docking protocol for a rigid and flexible ligand docking consisted of 10 independent genetic algorithms, while the other parameters used were default parameters of the ADT. The binding aspects of the PTP1B residues and their corresponding binding affinity scores were regarded as the best molecular interaction. The results were analyzed using UCSF Chimera [The X-ray crystallographic structure of PTP1B, with its selective inhibitor 3--2-ethyl-benzofuran-6-sulfonic acid (4-sulfamoyl-phenyl)-amide (compound D: 1NNY) ,53 websi Chimera , while tt-test , and were noted at p < 0.05.All results are expressed as the mean ± SEM of triplicate samples. The statistical significances were analyzed using the one-way ANOVA and Student’s O-glucoside is a potent PTP1B inhibitor with a mixed inhibitory mechanism. Hesperetin 5-O-glucoside, with its single sugar moiety, displayed reasonable binding energy and the highest binding affinity for PTP1B inhibition. The structure-activity relationship studies indicated that the single sugar moiety at the C-5 position was crucial for the activity against PTP1B inhibition. Our results indicated that flavanones containing one sugar moiety at the 5-position, such as hesperetin 5-O-glucoside, best fit the PTP1B allosteric site cavity for PTP1B inhibition. Further in vivo and cellular-based studies are needed to help clarify the detailed mechanism of action of these flavonoids in the brain membrane and other organs.In this work, a promising flavonoid scaffold was found as a potential effective PTP1B inhibitor for the treatment of DM. Hesperetin 5-


In vitro experiments revealed competition of Ca2+ and Mg2+ in the activation of ryanodine receptors (RyRs) as well as inhibition of RyRs by Mg2+. The impact of RyR modulation by Mg2+ on calcium release is not well understood due to the technical limitations of in situ experiments. We turned instead to an in silico model of a calcium release site (CRS), based on a homotetrameric model of RyR gating with kinetic parameters determined from in vitro measurements. We inspected changes in the activity of the CRS model in response to a random opening of one of 20 realistically distributed RyRs, arising from Ca2+/Mg2+ interactions at RyR channels. Calcium release events (CREs) were simulated at a range of Mg2+-binding parameters at near-physiological Mg2+ and ATP concentrations. Facilitation of Mg2+ binding to the RyR activation site inhibited the formation of sparks and slowed down their activation. Impeding Mg-binding to the RyR activation site enhanced spark formation and speeded up their activation. Varying Mg2+ binding to the RyR inhibition site also dramatically affected calcium release events. Facilitation of Mg2+ binding to the RyR inhibition site reduced the amplitude, relative occurrence, and the time-to-end of sparks, and vice versa. The characteristics of CREs correlated dose-dependently with the effective coupling strength between RyRs, defined as a function of RyR vicinity, single-channel calcium current, and Mg-binding parameters of the RyR channels. These findings postulate the role of Mg2+ in calcium release as a negative modulator of the coupling strength among RyRs in a CRS, translating to damping of the positive feedback of the calcium-induced calcium-release mechanism.Ryanodine receptor channels at calcium release sites of cardiac myocytes operate on the principle of calcium-induced calcium release.  Since the cytosolic concentration of free Mg2+ is by several orders of magnitude over that of Ca2+, magnesium ions compete effectively with calcium ions for binding to the RyR calcium-activation site despite their relatively low affinity  based on RyRs with the same, realistic calcium dependence of the steady-state open probability in the presence of Mg2+ but with different kinetic assumptions. Postulating binding of Ca2+ ions to the RyR activation site as instantaneous and unbinding of Mg2+ from the calcium activation site as slow predicted 2-3 simultaneously open RyR channels at the peak of a spark  showed a decreased inhibition of RyR activity by Mg2+ ions , increased non-spark leak, increased spark size, and decreased spark frequency were observed ; in atris effect , was shos effect . Thus, t2+ binding. The explored parametric space of the CRS model included the description of stochastic RyR gating that allowed binding of Mg2+ to the RyR magnesium inhibitory site, Mg2+/Ca2+ competitive binding to the RyR calcium-activation site, and allosteric regulation of RyR opening by Ca2+ and Mg2+ binding to the activation site. The simulated CRS activity in response to a random opening of a single RyR was analyzed for the fractions of generated quarks, blips, and sparks, as well as for the amplitudes and time courses of generated CREs. Inclusion of the kinetics of Ca- and Mg-binding to the RyR gating model allowed us to redefine the role of magnesium ions in the CRS function.In this study, we used a model of the calcium release site  based on1. Parallel generation of random numbers was performed using OMPRNG , 32 GB RAM. Individual calcium release events were simulated in parallel using the OpenMP APIg OMPRNG . The prog OMPRNG . Simulatg OMPRNG , pandas g OMPRNG , SciPy . These experiments were performed on RyRs in isolated cardiac microsomal vesicles incorporated into lipid bilayers. RyR single-channel activity was recorded using Cs+ or Cs2+ ions as charge carriers in the presence of variable concentrations of total ATP (0 – 3 mM), free Mg2+ (0 – 1.3 mM), and free Ca2+ (0.1 – 100 μM) at the cytosolic side and 1 mM free Ca2+ at the luminal side  is the vector of probabilities of individual RyR states, as the sum of the time-dependent probabilities of all open states. The initial vector P(0) was calculated from the Q-matrix at [Ca2+]b as the solution of the system of equations -th part of the interval between open probabilities at [Ca2+]b and [Ca2+]e.where 2+ diffusion in the CRS assumed open RyRs as a source of constant single-channel calcium current iCa, flowing into calcium buffers that do not saturate .The description of Casaturate . It alsoRS model  correspodel  at basal concentrations of free Ca2+ (100 nM) and free Mg2+ (1 mM), i.e., probabilities of the states given that the channel is open:The above assumptions allowed the use of the Naraghi-Neher approximation for calculations of free calcium concentrations during Ca flux in the cytosolic half-space delimited by the plane of the CRS  and the Si = 1 if the state i corresponds to an open state and 0 otherwise. The initial state of the closed channels was chosen based on the calculated probabilities from Eq. 6 but using (1 – Si) instead of Si. Calcium influx upon the first channel opening changed the calcium distribution in the CRS and, correspondingly, the Q-matrix of individual RyRs. The index of the RyR that would change its state was sampled from the distribution of probabilities of state transitions of individual RyRs, which was for the thj RyR equal to the ratio of its transition rate of leaving the microstate M(j), equal to –QM(j)M(j), to the sum of leaving rates over all the RyRs, QM(j)M(j). The time of the next transition was sampled from the exponential distribution of waiting times with the rate QM(j)M(j). If the transition changed the macrostate of the channel, i.e., opened a closed channel or closed an open channel, the transition would lead to a new distribution of [Ca2+] in the CRS and a new corresponding set of RyR transition rate constants. The algorithm was repeated until the end of the simulation. From each RyR in the CRS, a dataset of 500 CREs was collected at each set of tested parameters.where NO(t) ⋅ iCa) as if emanating symmetrically from a virtual spherical source of 100 nm radius and binding to cytosolic buffers, including the calcium selective fluorophore Fluo-4 and the non-diffusible buffers troponin, sarcolemma, and SR membrane  and  ms–1 , resulting in 81 combinations that had KAMg in the range  μM. The allosteric coefficient of Mg2+ at the RyR activation site was varied from 0.5 to 500, i.e., from 0.15× to 154× the reference value  and between  ms–1 , resulting in 81 combinations that had KIMg in the range  μM.To study how changes in Mg-RyR interactions affect the characteristics of CREs, the parameters of Mge values . All valce value  in 17 stCalcium release sites containing 20 RyRs with the geometric arrangement corresponding to a typical RyR cluster in cardiac dyads  were genvi and the group vicinity v were determined according to For quantitative characterization of the geometrical arrangement of RyRs, we used the concept of vicinity , an exteNRyR is the number of RyRs in the CRS, and Cij is the reciprocal value of the distance between the thi and the thj RyR (v = 0.058 and the RyR vicinities spanned the range 0.034 - 0.072.where  jth RyR . Out of  jth RyR . The gro2+ released during the event (nCa). Calcium release events were classified as quarks, blips, and sparks as previously described , iCa is defined in the model, so the respective coupling strength can be directly evaluated for each CRS model if the weight factor α can be found. As shown in vi or iCa and the measured characteristics of the corresponding simulated calcium release events was suboptimal, which could be misinterpreted as if the CREs were not fully controlled by the determinants of CRS, at odds with current understanding of local calcium release. Optimization of the weight factor allowed to find coupling strength values that correlated with the measured CRE characteristics according to Hill function for the whole parameter range of inspected CRS determinants (where the index rminants .fMg), activation , and inhibition  and defined the effective coupling strength asTo account for the effect of Mg-RyR binding parameters on the coupling strength among RyRs, we included terms for allosteric coupling . Arel) and frequency of quarks and sparks (Fq and Fs) on the other hand by a proper weight factor. In the present case, however, it would not be appropriate to optimize the functional relationships between the effective coupling strength and CRE characteristics Fq, and Fs using the Hill function, since the Mg-binding parameters do not characterize the state of the CRS but kinetic processes in the RyR molecule. Defining an a priori relational function could lead to a mutual dependence of the fitted parameters and a potential bias in weight factor estimates. To avoid this, we approached this problem using information theory as follows:Finding the effective coupling strength function (Eqs. 8-10) requires evaluation of the weight factors of individual components, namely, the RyR vicinity, single-channel current, and kinetic parameters of the activation and inhibition processes. The values of weight factors can be found by optimizing the effective coupling strength function to the distribution of measured CRE characteristics (X) equal to the effective coupling strength, there is a dependent variable (Y) representing the CRE characteristics. The information content of a random variable X is quantified by its entropy H(X) (P(X) of the variable. For a discrete variable,For a random variable (opy H(X)  that is n is the number of possible values of X. The entropy H(X) defines the uncertainty of the random variable. A large value of H indicates a large number of states with low probabilities and the measurement of such a variable provides a large amount of information, while H = 0 means a deterministic outcome and the measurement of such a variable will not provide any information because its value is exactly known.where X and Y, I is the amount of information obtained when X is known and vice versa  between random variables ce versa , definedX and Y are independent variables. This definition indicates that when the maximal MI between the effective coupling strength and the CRE characteristics is reached, the effective coupling strength provides the most certain prediction for CRE characteristics.where Fq, and Fs . MI was calculated using a non-parametric method based on entropy estimation from k-nearest neighbor distances b and (tC)b are the values of the RyR open and closed time at basal [Ca2+].where  based on a quantitative description of RyR gating that accounted for Ca2+ and Mg2+ binding kinetics, RyR distribution in a typical CRS, and the cytosolic Ca2+ buffer near RyRs and around the CRS. The reference parameter set of RyR gating was obtained by fitting the available experimental data of RyR open probability, open time, and activation time at a range of cytosolic concentrations of free Ca2+ and free Mg2+ that encompassed the physiological values (section “Kinetic Parameters of the Ryanodine Receptor Gating Model”). Then we analyzed the characteristics of simulated calcium release events generated at the physiological free [Mg2+] concentration (1 mM) with the reference RyR model . The consequences of variation in parameters related to Mg2+ binding on the activity of RyR channels and the ensuing CREs were determined in sections “Effect of Mg-Binding Parameters on Calcium Dependence of Ryanodine Receptor Activation” and “Mg-Binding Parameters Affect Calcium Release Events.” Finally, we solved the relationship between RyR activity and the characteristics of CREs on the grounds of the effective coupling strength that, in addition to RyR vicinity and calcium current, accounts also for magnesium binding to RyR, and determined the effect of Mg-binding parameters on the characteristics of CREs .To study the role of Mg2+ activation and Mg2+ inhibition (2+- and Mg2+-binding (MWC-Ca/Mg) RyR model has 60 states . In comparison with CRSs based on the two-state RyR model of iCa (7% at 0.15 pA and 15% at 0.6 pA) on account of quarks, while in the two-state model it decreased with iCa since in the two-state model the fraction of sparks increased more steeply with iCa than in the MWC-Ca/Mg RyR model (0.06% of sparks at 0.15 pA and 13% of sparks at 0.6 pA in the present model). The time to peak of blips and sparks was curtailed at higher iCa, similar to the two-state model. However, the time to peak of sparks decreased with iCa in the present model while it increased in the two-state model. The time to end of quarks and blips shortened with iCa, as it did in the two-state model. In contrast to the two-state model, the time to end of sparks did not depend significantly on iCa. The amount of calcium released by CREs increased proportionally with iCa, less steeply than in the two-state model.As shown in 2+ concentration of 1 mM. The calculated dependences of RyR characteristics on [Ca2+] for the allosteric coefficient fMg at the RyR activation site and the on/off rate constants of Mg2+ at both the activation and the inhibition sites are summarized in Before characterization of calcium release events in the CRS, we inspected the single-channel RyR behavior of the MWC-Ca/Mg RyR model for a range of Mg-binding parameters at the physiological cytosolic free Mg2+ binding to the RyR activation site, that is, a decrease of KAMg by either an increase of kAonMg (purple), or a decrease of kAoffMg (light green), led to decreased calcium sensitivity (right shift) of the RyR open probability, open time, and closed time. At the basal calcium concentration, these changes decreased PO and tO but increased tC. Vice versa, changes that impeded Mg2+ binding [a lower kAonMg (pink) or a higher kAoffMg (dark green)] had the opposite effect. A change of KAMg led to a change of the apparent KCa in inverse proportion. Changing both on- and off-rate constants by the same fraction, that is, without changing KAMg (light and dark blue), did not affect the calcium dependence of PO, tO, and tC.As shown in fMg relative to the reference value had a non-linear effect on the calcium dependence of RyR characteristics , while it shifted the calcium dependence of open time in this [Ca2+] range but had no effect at low and high [Ca2+] range. Notably, at the lowest calcium concentration, a larger fMg decreased PO and substantially increased tC, while a smaller fMg increased PO and decreased tC. Variation of fMg exerted no effect on the maximal PO, tO, or minimal tC, in agreement with Eqs. 2 and 14.Variation of the allosteric coefficient 2+] was speeded up the most by an increased rate constant of Mg2+ unbinding from the RyR activation site, kAoffMg (dark green), somewhat less by decreasing kAonMg (pink), and even less when both rate constants were increased in proportion (dark blue). The RyR activation rate was slowed down the most by decreased kAoffMg (light green) or increased kAonMg (purple), but less when both on- and off-rate constants of Mg2+ binding decreased (light blue). Changes of fMg had negligible effects on the rate of RyR activation.The rate of RyR activation by a step change of ), but they had a profound effect on the maximum open probability  or decreasing kIoffMg (light green) decreased PO, while increasing kIoffMg (dark green) or decreasing kIonMg (pink) increased PO. In effect, a change of KIMg led to an almost proportional change of the maximum PO. Changing both kIonMg and kIoffMg proportionally  did not affect PO at any [Ca2+]. On the other hand, the values of tO were insensitive to changes of kIoffMg. However, a change of kIonMg or a proportional change of both kIonMg and kIoffMg affected the calcium sensitivity of tO in the direction of the change but changed the maximum tO in the opposite direction.By definition, changes of the on- and off-rate constants for Mg2+], the open time was not affected by rate constants of Mg2+ binding to the inhibition site. However, increasing kIonMg or decreasing kIoffMg increased the closed time, and decreasing kIonMg or increasing kIoffMg decreased it. Simultaneous equivalent changes in both kIonMg and kIoffMg did not affect tC. At high [Ca2+], change of kIoffMg or both kIonMg and kIoffMg in one direction changed the mean closed time in the opposite direction; however, changes of kIonMg in either direction did not change it. The parameters of Mg2+ binding to the inhibition site did not affect the rate constant of RyR activation, as expected.At the basal  tC >> tO, this effect, summarized in tC. The event frequency was changed approximately in proportion  or inverse proportion  to the change of rate parameters. All parameters of Mg2+ binding affect the event frequency and the fraction of sparks in the same direction, and therefore they have a pronounced effect on the spontaneous spark frequency at the examined iCa of 0.6 pA. Since the fraction of blips is only weakly affected by Mg-binding parameters, the frequency of blips was changed only modestly.The last problem we addressed in this study was the impact of Mg-binding parameters of the RyR on the frequency of spontaneous occurrence of individual calcium release types at basal [Cain silico model of the calcium release site was based on a novel model of RyR gating that incorporated Mg2+ binding kinetics  of the RyR inhibition site on the RyR open and closed times and their relationships to the spark termination, CRE frequency, and relative occurrence of visible and invisible CREs (see below).The kinetic description confirmed previous results of the equilibrium model of the RyR inhibition site  that the2+ ions influence the single-channel activity of RyRs considerably. Magnesium ions act through their allosteric competition with Ca2+ at the RyR activation site as well as through their binding at the RyR inhibition site. This means that modification of the Mg-binding properties of RyR channels by pharmacological, metabolic, or structural changes in RyR monomers may substantially affect the calcium release function of cardiac myocytes.To sum up, the MWC-Ca/Mg RyR model predicts that at the physiological cytosolic concentration of 1 mM, Mg2+ unbinding from the calcium activation site is much slower than the rate of Ca2+ binding, in line with experiments , CRS models produced mostly quarks or blips. Individual records of blips were formed by 2 to 6 RyRs open at their peaks (at around 3 ms), and terminated fast, within 5 to 6 ms, by simple stochastic attrition (iCa was higher (0.4 or 0.6 pA), CRS models produced similar quarks and blips as at lower iCa amplitudes but also a significant fraction of large sparks. With the reference parameter set, these sparks were formed by 7 to 13 RyRs (out of 20 in the CRS) open at their peaks (30 to 40 ms) and terminated very slowly (within 60 to 70 ms) by the attrition mechanism. However, the average rate of spark termination was very sensitive to the Mg-binding rate constants of the RyR inhibitory site (see below).Simulations of the reference CRS model predict that a large fraction of spontaneous local calcium release events (quarks and blips) result from the activation of a subset of RyR channels in a CRS . When thttrition . When thF/F0 > 3) and prolonged. If real, such large calcium release flux would cause a rapid dissipation of the calcium gradient across SR membrane and fast termination of calcium release by a mechanism dubbed “induction decay” , it was not possible to use fitting for the determination of the weight factors, since it was not a priori known whether they follow the same Hill equations, in contrast to our previous study  would have no effects on the calcium dependence of the steady-state PO, tO and tC  and mean open time (0.2 ms) at 100 nM Ca2+ then corresponds to 36 - 54 RyR openings per second in this volume. Of these, there would be 2.7 - 8.4 blips per second and 0.02 - 7 sparks per second at a calcium current of 0.15 - 0.6 pA (see iCa and a high predominance of blips or with a higher iCa and a comparable occurrence of both blips and sparks. However, our simplified calculations predict that blips cannot be detected when iCa is lower than 0.4 pA (2+-ATP binding kinetics are necessary to decide this point.The presented model provided a high fraction of quarks, i.e., initiating RyR openings that activated no other RyRs. In real experiments, quarks are not observable for their small calcium yield. To determine whether this finding is consistent with experimental data, we compared the predicted fraction of CREs of different types with the observed number of calcium release events in cardiac myocytes. The number of ryanodine receptors per volume unit of a myocyte has been estimated as 60 - 90 RyRs per μm3 . Assumin6 pA see . These c6 pA see  in intac6 pA see . The valn 0.4 pA ; thus a 2+ binding on spontaneous sparks and waves in a simplified kinetic model of the CRS (The Mg-binding parameters substantially affect the frequency of RyR openings by modulating the RyR closed time at the basal calcium concentration . A simil the CRS . Additio the CRS . Such ch the CRS .2+ to both the activation and the inhibition site negatively modulated the effective coupling strength in a qualitatively similar way. In other words, these findings reveal the role of Mg2+ ions as a damping agent that protects calcium release sites from uncontrolled activation at threshold calcium stimuli. This finding is of principal physiological importance since it helps to explain the graded behavior of calcium release at the level of ryanodine receptors, which impedes the positive feedback inherent to the calcium-induced calcium release mechanism.The binding of MgThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.BI, IZ, and AZ planned the research and wrote the manuscript. BI and AZ designed the simulations. BI wrote the software, derived mathematical expressions, and performed simulations. BI, JB, IZ, and AZ analyzed and interpreted the experimental and simulated data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


VS,avg) and for the top 30 m ; a method based on the fundamental site period (T0); and a hybrid method based on the combination of VS,30, T0 and VS,avg. The study specifically aimed to evaluate the importance of the site classification parameters on the resulting microzonation maps. VS,30 is capable to present the geological and geotechnical site conditions, however, the results may be further improved by considering Vs,avg in shallow and T0 in thick layers of soil sediments as secondary parameters. The T0 method gives also satisfactory results with T0 showing a better correlation to Vs,30 than to Vs,avg. The versatile hybrid method may be challenging to apply in certain cases with its nine different site categories and parameters.A seismic microzonation study was conducted to refine the seismic hazard model for the city of Saguenay, Canada. The Quaternary geology underlying Saguenay shows complex glacial and post-glacial stratigraphy with a number of buried valleys filled with fluvioglacial and glaciomarine sediments. High impedance contrast between rock formations and surficial sediments is prone to seismic amplification. To evaluate their applicability, advantages and limitations in capturing the geological specificity of the study area, four site classification methods were applied: the current National Building Code of Canada (NBCC) and Eurocode 8, both mainly based on the average shear-wave velocity for the surficial sediments ( An important aspect of geotechnical earthquake engineering is related to the evaluation of the expected intensity and the dominant period of the seismic ground motion at a given location. Knowledge of the geological and geotechnical properties of the surficial sediments is important in this respect since they tend to modify the amplitude and frequency content of the incoming seismic waves, a phenomenon known as the site effect  until their energy is dissipated  and the soil undrained shear strength (Su). Other classification schemes utilize the fundamental site period T0  requiring site-specific geotechnical investigations includes liquefiable soils, sensitive or highly organic clays > 3 m in thickness, or plastic clays > 8 m thick.The NBCC seismic provisions, basically a replica of NEHRP BSSC , recogniVS,30 ranges  can be a useful site parameter complementary to VS,30, as it decreases the standard deviation of residuals in the modern ground motion prediction equations . The seismic activity of this region was reassessed following the 1988 M6.0 Saguenay earthquake. This intraplate earthquake, with a mid-crustal depth of 29 km and a moderate magnitude, occurred 35 km south of downtown Saguenay  and 122 cone penetration tests (CPTs) for the Saguenay region were acquired from the Quebec Ministry of Transport  with an average vibration period less than 0.1 s . The areas covered with these soil types produce the main site classification difference between the NBCC and T0 methods on one side and Eurocode and the hybrid method on the other.Figure (i)T0 site classification methods identify rock and stiff soil conditions. On the other hand, for Eurocode 8 and the hybrid method these site conditions are soft soils since they both take into account VS,avg of the surficial sediments instead of VS,30 and T0 by the former two methods.Medium thickness sediments, 5 < H < 30 m: The site evaluations are different in this stratigraphic column. NBCC and (ii)Thick sediments, H > 30 m: the observed evaluations of site conditions are in fair agreement, soft soils, by all four classification methods.(iii)VS,30 based site classifications, NBCC and Eurocode 8, evaluate this soil column with medium stiffness, whereas T0 and the hybrid method identify rather soft soil conditions. This is because, in the two former schemes, the soil thickness has more weight in the final results than the stiffness (Vs). A typical example is the differences in site classification of stratigraphic columns ii and iii due to the presence of thick stiff sandy soils.Thick sediments, H > 30 m: the (iv)T0 < 0.2 s).Shallow sediments, H < 5 m: this geological setting is probably the least challenging and all site classification methods are in agreement evaluating rock or shallow rock site conditions with a short fundamental period , average shear-wave velocity for the total thickness of the surficial sediments  and the fundamental site period (T0). Four different classification schemes were applied and compared: NBCC, Eurocode 8, fundamental site period and the hybrid approach based on all main site parameters. All of the classification methods have their own advantages and limitations with respect to the local geological and geotechnical conditions. The following are the major conclusions drawn from this study:VS,30, NBCC and Eurocode 8, appear the most consistent with the local conditions. The results could be further improved considering secondary parameters, e.g., VS,avg in shallow (H < 30 m) and T0 in deeper soil sediments (H > 30 m). In such a case, the impact of the stiffness and thickness of the surficial sediments will be better accounted for.The site classifications based on VS of these soils is combined with VSrock yielding VS,30 considerably higher than VS. Since such site conditions cover a significant portion of the study area, their dynamic response should be analysed more in detail.Site class E in Eurocode 8 and the hybrid method refers to medium to soft soils, 5 < H < 20 m, on top of bedrock. In NBCC, the Eurocode 8 doesn’t include the hard-rock site condition as is the case with the site class A in NBCC. Classification of rock sites into two categories helps distinguish the site effect in crystalline hard rocks and more fractured sedimentary rocks formations.VS,30 and T0 was observed in shallow sediments (H < 30 m) and a relatively weaker correlation in deeper sediments (H > 30 m), whereas the correlation between Vs,avg and T0 is practically inexistent. This suggests that the addition of Vs,rock to the Vs soil in the top 30 m improves the correlation between Vs,30 and T0. Due to this strong correlation, NBCC site classification yields similar patterns as the T0 scheme.A strong correlation between T0 is affected considerably more by the thickness of the overlying sediments than by VS,avg. Therefore, most of the shallow deposit conditions have a resonance period < 0.4 s, which highlights the potential for seismic amplification in the short period range.The site classification based on The hybrid site classification proposes a multitude of classification parameters, which, in certain cases, may lead to confusion in selecting the appropriate site class. However, the results arrange the site conditions mainly into two major groups: rock and soft soils. Stiff and medium stiffness soils share only a limited part of the study area as opposed to the NBCC site classification.A seismic microzonation study was conducted in the complex geologic environment underlying the city of Saguenay. The main seismic site classification parameters considered in the analyses were: average shear-wave velocity of the top 30 m  and T0 in deeper soil sediments (H > 30 m) as secondary parameters. In such a case, the impact of the stiffness and thickness of the surficial sediments will be better accounted for.As an overall conclusion, this study demonstrates that site classification based on


The aim of this study was to explore the effects of an emerging and efficient heating technology, superheated steam (SS), on the allergenicity and molecular structure of ovomucoid (OVM). OVM was treated with 120–200 °C of SS for 2 to 10 min. The allergenicity (IgG/IgE binding abilities and cell degranulation assay) and molecular structure  changes were investigated. The IgG-binding ability of OVM decreased and the releases of β-hex and TNF-γ were inhibited after SS treatment, indicating that the protein allergenicity was reduced. Significant increases in oxidation degree, free SH content and surface hydrophobicity were observed in SS-treated OVM. The protein dimer and trimer appeared after SS treatment. Meanwhile, obvious changes occurred in the primary structure. Specifically, serine can be readily modified by obtaining functional groups from other modification sites during SS treatment. Moreover, the natural OVM structure which showed resistance to trypsin digestion was disrupted, leading to increased protein digestibility. In conclusion, SS-induced OVM aggregation, functional groups and amino acids modifications as well as protein structure alteration led to reduced allergenicity and increased digestibility. Egg is the main daily protein source for human beings, and also one of the major ingredients in the food industry . HoweverPrevious studies have shown that heating, high pressure, glycation and hydrolysis can decrease the protein allergenicity ,4,5. HeaSuperheated steam (SS) treatment is an emerging heating method and has attracted more and more attention for its advantages, including higher heat transfer efficiency, low-oxygen and pollution-free environment as well as good quality of processed products . During The objective of the study is to find a new, easily operated, efficient and safe technology to reduce the allergenicity of proteins. Our preliminary experiments showed that SS is an effective approach to modifying egg proteins and reducing the allergenicity. It is necessary to perform experiments to prove those effects.Previous research demonstrated that egg proteins allergies are mainly originated from immunoglobulin G (IgG) and immunoglobulin E (IgE)-mediated rapid allergic reaction. In addition, macrophages are also important effector cells that mediate immune response . The medThe protein allergenicity changes are reported related to the alteration in molecular structure in terms of primary structure, main functional groups and degree of protein aggregation ,2,3,4,5.OVM, D-glucose, goat antirabbit IgG-HRP conjugate, goat antihuman IgE-HRP conjugate and trypsin were purchased from Sigma-Aldrich . Dithiothreitol (DTT) was purchased from Thermo Fisher Scientific Inc. . The KU812 cells were obtained from iCell Bioscience . All other reagents used were of analytical reagent grade. Ultrapure water from a water purification system  was used throughout this study.The sera of six egg white allergy (EWA) patients were purchased from Plasma Lab International. The specific IgE levels of six groups of sera were 10.8, 26.3, 24.9, 13.5, 16.5 and 12.8 kU/L. The six groups of sera were mixed and served as human anti-OVM IgE serum and stored at −80 °C until used.v/v, 1:1). The booster immunization was taken after one, two and three weeks at a dose of 0.4 mL, respectively. After the rabbits were anesthetized, the rabbit anti-OVM IgG plasma was collected. After being centrifuged at 4 °C, the rabbit anti-OVM IgG serum was separated and stored at −80 °C until used.Preparation of rabbit anti-OVM IgG serum: Male Japanese rabbits (100 days)  2014–0005) were purchased from Longping . After acclimatizing in a breeding room for 10 days, the rabbits were intravenously injected with 1 mL OVM (0.8 mg/mL) emulsified with the Freund’s complete adjuvant  value of a well with the control and SS-treated OVM, respectively.The IgG and IgE-binding abilities of the OVM samples were estimated with indirect competitive ELISA according to our previous report . Rabbit 5 cells was in each well. The culture condition was 37 °C with CO2 (5%). The cells were then activated with human anti-OVM IgE serum for 24 h, and followed by, stimulated by 50 μg/well of samples for 4 h. Cells treated with PBS buffer were served as the negative control. The release of β-hex, TNF-γ, histamine and IL-6 were analyzed by ELISA kits, following the manufacturer’s instructions [The basophil histamine release test was carried out according to the method of Wang et al. . KU812 cructions ,17.−1·cm−1, and the result was expressed as nmol/mg protein.The carbonyl content of the OVM samples was determined according to the method of Zhang et al.  with sliThe surface hydrophobicity of the OVM samples was determined by an 8-aniline-1-naphthalene sulfonic acid (ANS) fluorescence probe method . The sam2HPO4 (pH 7.0); then, the volume was adjusted to 100 mL; the reagent was stored at 4 °C in the dark. The protein samples were diluted to 2 mg/mL with 0.1 mol/L phosphate buffer (pH 8.0) containing 0.086 mol/L Tris-HCl, 0.09 mol/L Gly, 4 mmol/L EDTA and 8 mol/L urea. The diluted protein solution (1 mL) was mixed with buffer solution (8 mL), and centrifuged at 8000 r/min for 20 min. Following this, 4.5 mL of supernatant was added to 0.5 mL of Ellman’s reagent. The mixed solution was reacted at room temperature for 15 min after evenly oscillated. The absorption at 412 nm was measured. The free SH content was calculated according to the following formula:6/(1.36 × 104); 1.36 × 104 is the molar absorption coefficient of DTNB; C is the protein concentration (mg/mL).The free SH content of OVM samples was determined according to the method of Ellman  with sliThe molecule weight of OVM was determined by MALDI-TOF MS  according to the method of Liu et al. . OVM samThe OVM samples were digested by trypsin at 37 °C for 24 h. The digested sample solution was separated via a nanoliter flow HPLC system . Solution A was an aqueous solution with 0.1% formic acid. Solution B was acetonitrile binary solution with 0.1% formic acid and 84% acetonitrile. The digested sample solution was injected into a RP-C18 column to remove insoluble or impure substances and then separated by another RP-C18 column at a flow speed of 300 μL/min. Gradient elution was then carried out.2 scans) showing the mass-to-charge ratio of the polypeptide and polypeptide fragments were collected at each full scan. Raw files were obtained from the corresponding database using Proteome Discoverer 1.4. Some parameters were set as follows: Enzymes, non-specified; Missed cleavage, 2; Modification, carbamidomethyl, oxidation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, nitro.After separation, the eluant was injected into an LTQ-Orbitrap Fusion Velos mass spectrometer  for analysis by tandem mass spectrometry (MS/MS) to identify protein modification forms and sites with a positive ion detection mode. The precursor ions were subjected to high-energy collisional dissociation (HCD) fragmentation to detect fragment ions. Twenty fragment maps . Statistical data were determined based on a two-tailed The IgG and IgE binding abilities of OVM were estimated through indirect competitive ELISA with sera from rabbit and EWA patients, respectively. As shown in However, there were no obvious changes in the IgE binding rate of OVM samples treated by SS, except that at 200 °C for 8–10 min B. The re450) of cells increased after SS treatment, suggesting that SS heating can promote KU812 cells proliferation. Generally, the absorbance (OD450) of cells increased with the increase of OVM concentration and SS processing period. These suggested that a higher OVM concentration and longer SS processing period promoted the KU812 cells proliferation.To analyze the potential cytotoxic activities of the tested substances, KU812 cells were incubated for 24 h with increasing concentrations of OVM treated by SS at different temperatures and periods. As shown in The degranulation rate of basophils and mast cells are key factors that determine the immunoreactivity of allergens. In this work, the effects of SS treatment on the release of β-hex, TNF-γ, histamine and IL-6 in KU812 cells are shown in Histamine is another key mediator found in KU812 cell granules which is released during antigen-specific IgE binding and plays an important role in the induction of adverse physiological symptoms of an allergic reaction . InteresIL-6 is an important mediator produced in the process of mast cell degranulation. As shown in The above results indicated that the degranulation ability of the basophils was decreased, although there was little change in the IgE binding ability. In conclusion, SS treatment on OVM inhibited the anaphylactic reaction and reduced the release of β-hex and TNF-γ in the process of basophilic granulocyte degranulation. The allergenicity of SS-treated OVM decreased in the KU812 cell.Generally, the content of carbonyl is an oxidative indicator of the protein . In thisSurface hydrophobicity is an important index that indicates the binding ability between antibody and antigen. As one type of molecular force involved in immune reactions, surface hydrophobicity plays an important role in assisting in epitope recognition . As showOVM consists of three structurally independent tandem homologous domains and possesses nine intramolecular disulfide bridges but lacks interdomain disulfide bonds. This is the reason that OVM is resistant to heat denaturation and structural change. The surface hydrophobicity analysis suggested that an unfolding of OVM conformation could occur after SS treatment, which was related to the reduction of disulfide bridges. As shown in OVM has a molecular mass of 28.0 kDa, comprising 186 amino acids and 20–25% of carbohydrate . Heat trIn order to identify the specific protein sites susceptible to SS treatment and explore the mechanism of the decreasing in OVM allergenicity, it is necessary to find out the precise modification sites of OVM. Previous studies have suggested that the antigenic epitopes in protein sequence mainly originated from hydrophobic amino acids and amino acids containing sulfhydryl groups ,31. TherOVM has a signal sequence of 24 amino acids and a protein sequence of 186 amino acids. Therefore, in this work, the SS-induced changes in 210 amino acids of OVM were explored and mainly concentrated in oxidation, nitro, phospho, carboxymethyl, glygly and sulfo modifications. Oxidative modification is typically associated with lower protein solubility, which can significantly hinder and confound the identification . TherefoInterestingly, it was observed that the phospho modification of OVM occurred after treatment at 120, 140 and 200 °C for 10 min. There were two phospho sites S6 and T38 in native OVM, and three new phospho sites were observed in OVM treated by SS at 120, 140 and 200 °C for 10 min. It is worth noting that no phosphate groups participated in this reaction. Therefore, this result indicated that the phospho groups have shifted from phospho sites to non-phospho sites, which was similar to the observation in our previous research . This fiThere was also an important finding in regard to glygly modification, which plays an important role in life activities. Similarly, three new glygly sites S71, S72 and S78 were observed in OVM treated at 120, 140 and 200 °C for 10 min, without the participation of the glygly group. The internal transfer might be the reason for the generation of new glygly modification sites in the OVM molecule. Finally, the sulfo modification sites among native and SS-treated OVM at 120, 140 and 200 °C for 10 min were also compared. There were four new sulfo sites  observed in OVM treated at 200 °C for 10 min. The sulfuric groups may be originated from cysteine or other sulfo sites. Interestingly, serine could be readily modified with the acquisition of functional groups from other modification sites during SS treatment.OVM molecule is organized into three well-separated domains which play an important role in inducing allergic reactions. It is important to explore the relationship between the sensitive modification sites and the three-dimensional (3D) structure of SS-treated OVM. The modification sites of OVM treated at 120 °C, 140 °C and 200 °C for 10 min are shown in SS treatment, particularly at high temperatures and long processing times, could induce the OVM aggregation, increase surface hydrophobicity, modify the functional groups and amino acids in OVM. These phenomena disrupted and buried some allergen epitopes and made it difficult for the combination of antibodies and antigens ,15. TherTo our knowledge, this is the first study investigating the allergenicity and structure changes of OVM under SS treatment. A decrease in the IgG-binding ability was observed for SS-treated OVM with no significant changes in the IgE binding ability. SS treatment reduced the release of β-hex and TNF-γ, promoted the release of histamine, while having no significant effect on IL-6 release in KU812 cells. An obvious increase in the oxidation degree, free SH content and surface hydrophobicity were observed. Trimer and tetramer aggregations generated after SS treatment. In addition, there were obvious changes in the primary structure. Furthermore, some amino acids could be readily modified by obtaining functional groups from other modification sites during SS treatment. It can be inferred that SS-induced OVM aggregation, functional groups and amino acids modifications as well as protein structure alteration led to the reduction in allergenicity and increase in digestibility.


Ptyonoprogne rupestris (ECM) roosts at the ‘Gorham’s Cave Complex’ UNESCO World Heritage site in Gibraltar, which is best known for its occupation by Neanderthals at times when ECMs were also present. Its complex geomorphology allows the study of use of different micro-sites (caves) within the roost. We used mark-recapture to test whether birds showed fidelity to micro-sites for roosting, and for differences in condition of birds across micro-sites. ECM showed very high fidelity towards micro-sites, within and between years, with > 90% chance of recapture at caves where they were first caught. Condition of birds differed between micro-sites, suggesting differences in roost quality between caves; birds were more likely to be recaptured at the micro-site where birds were in best condition, indicating higher survivorship. Our results demonstrate extremely fine-scale fidelity at the largest roosting site documented for ECM globally. Implications for conservation are discussed. The study provides current knowledge of a bird that has been using these caves since the Pleistocene and more generally on these caves as refuges.Species present in the fossil record may continue to exist at an archaeological site, allowing study that fine-tunes our picture of the ecological past. A large wintering population of Eurasian Crag Martins  Though used less often, the study of current processes at such sites can provide information on the ecology of species present in archaeological sites2, enriching our understanding beyond the information provided by taphonomy. Here we argue that the neontological perspective to ecology is a separate tool to the traditional palaeoecological approach, and is a powerful weapon in support of taphonomic analyses and interpretations. We use the Eurasian Crag Martin Ptyonoprogne rupestris (ECM) at the Gorham’s Cave Complex, UNESCO World Heritage Site3, as a case study to illustrate the potential of the method.An advantage of archaeological sites is that they offer opportunities to reconstruct past ecological communities4. Although Asian populations are migratory, those around the Mediterranean Basin—which includes a large proportion of the European range—are largely sedentary, with birds from further north (such as the Alps and southern Carpathians) moving mainly to the Mediterranean region4 and Iberian birds from the peninsular interior moving south and towards the coasts5. Thus, the species is the only aerial insectivore for which much of the European Population winters around the Mediterranean6. It nests solitarily or in small colonies7 but during the winter it can form large roosts4, which presumably increases the likelihood of discovery of ECM fossil material.ECM are distributed from Morocco and Portugal in the west to the Pacific coast of China in the east10). The ECM has had a relationship with the archaeological site known today as the Gorham’s Cave Complex in Gibraltar for tens of millennia, at least since the Late Pleistocene11, suggesting particularly stable and optimal conditions for this species despite severe climatic fluctuations. The species is currently present at the site during the autumn and winter13, providing opportunities for research on the existing population that could shed light on past events and aid in the conservation of potentially vulnerable sites.Gibraltar’s fossil avifauna is well studied , particularly as some winter roosts are known to hold a large proportion of a species or population (e.g.22) and are therefore exceptionally vulnerable to contingent events.The winter is a potentially limiting period for the survival of populations of migratory birds wintering in temperate zones21). They open an avenue for future research and also illustrate the value of interdisciplinary research. Viewed from the millennial perspective, ECM conservation at the Gorham’s Cave Complex is not just about the protection of a living species but is also about the conservation of ecological and eco-cultural processes that are very much a part of our universal patrimony.Here we report, for the first time, detailed evidence that birds are not only faithful to winter roosts (both within- and between-years) but that they are also faithful to particular locations (micro-sites) within these roosts. The Gorham’s Cave Complex is estimated to harbour ~ 1–2% of the European population of ECM (more below). Our results are important to our understanding of the ecology of a species present at the site for millenia, as well as the conservation needs of communally roosting migratory bird species, which have typically received much less attention at their wintering quarters (e.g.Weekly counts of ECM returning to their roosting site (see description of The Site below) during evenings showed considerable fluctuation between weeks Fig. . ECM werZ = 8.16, P < 0.00000001), with the great majority of recaptures occurring at the cave where the birds were originally ringed (92 of 101 birds recaptured: a 91.1% chance of recapture at the cave where each bird was ringed). Furthermore, 37 birds were recaptured that had been ringed during separate seasons  and all of these were captured at the caves where they had originally been ringed . Micro-site fidelity therefore held constant between years. There was also a significant difference in the strength of fidelity between caves within the roost during the 2019-2020 wintering period (Fisher’s exact: P 0.02), with birds initially ringed at Gorham’s Cave being captured less frequently in other caves than vice-versa . There was a significant difference between mean residuals generated for each cave, showing that recapture rates were highest at Gorham’s Cave, then Cave F, and lowest at Vanguard Cave  that had been ringed there during previous seasons strongly suggest that, not only do ECM return to roost in Gibraltar between years as had already been reported24. The latter is an unlikely factor for differences observed among ECM in Gibraltar because they roost at a single site, departing for their foraging grounds and arriving to roost together. Peregrine Falcons Falco peregrinus hunt ECM on their approach to the roosting site and other predators such as Feral Cats Felis catus may take birds that roost closer to the ground9, but thermoregulatory demands seem the most likely factor separating the micro-sites. This could have important implications on energy conservation during a period when aerial insects are scarce. Certainly, differences in temperature between roosting sites of Cave Swallows Petrochelidon fulva have been suggested as an explanation for differences in condition of birds at different sites25.The condition of birds differed between the micro-sites, with birds in best condition at Gorham’s Cave and worst at Vanguard Cave. This suggests a difference in the quality of each cave as a refuge, which could be the driver of micro-site fidelity. Benefits that are thought to underlie communal roosting in birds include a reduction in thermoregulatory demands, a decrease in predation risk, and an increase in foraging efficiencyThe very high level of fidelity observed within and between years suggests that these patterns in use of micro-sites remain quite stable. As well as showing strong ties to particular micro-sites, the rate of recapture and proportion of birds recaptured more than once was highest at Gorham’s Cave and lowest at Vanguard Cave, the same pattern as that of body condition. The finding therefore reinforces the evidence that Gorham’s Cave provides better conditions as a micro-site for roosting. If winter mortality is an important driver in ECM population dynamics, micro-site selection could be an important factor in fitness of ECM. The finding helps to elucidate the value of these caves as winter refuges for this and other taxa.Riparia riparia regularly visit breeding colonies other than their own26 and it is possible that birds captured at a different micro-site were merely visitors that would nevertheless have returned to their own micro-sites to roost, especially when considering the proximity of these to each other within a single roosting site. However, a certain level of interchange can also be expected if micro-sites differ in their quality and birds in better condition can compete favorably for better sites. Under such a scenario, the population roosting in the micro-site with the higher proportion of birds in a good condition can be expected to be more stable than others. This is indicated by our results, which suggest that birds first captured in Gorham’s Cave showed even less mobility between micro-sites than those first captured at the other two caves. A third possibility is that fidelity to micro-sites is lower among immature birds27. Our incomplete ageing data could support this idea, but in any case, age of birds and competition for better micro-sites may not be independent factors, because adult ECM wintering at Gibraltar are larger and heavier than juveniles13. Incidental observations of birds chasing each other off ledges (pers. obs.) reinforces the likely role of competition in assortment of birds across micro-sites.A few birds were subsequently captured at different micro-sites from those where they had been first caught. The number was very small and this does not necessarily indicate that they were roosting at the new micro-site. For example, Sand Martins 5, but our results suggest that numbers of ECM wintering at the Gorham’s Cave Complex remain comparable to those recorded during the 1970s, or perhaps exceed them. Some 1500 ECM have been recorded at a winter roost in Benicàssim on the Valencian coast of Spain28 but at its peak, the Gibraltar roost is much the largest on record and could be the largest globally, holding perhaps ~ 1–2% of the European post-breeding population of the species (based on29). Although the species’ conservation status is currently listed as ‘Least Concern’30, the scale of the aggregation illustrates the vulnerability that even such species may have to contingent events. The status of the Gorham’s Cave Complex as a UNESCO World Heritage Site, located within the Gibraltar Nature Reserve, helps to ensure the conservation of this important wintering population, which is as much a part of the site’s Pleistocene past as its present.The wintering population of ECM at Gibraltar was assumed to have decreased markedly in recent times31. The size of the roost is large and was estimated at 1500–3100 in the 1970s13. Wintering ECM arrive in Gibraltar in early October and most leave in early March12. They leave the roost each day to forage to the north in nearby Spain, returning to the caves in the evening en masse, along a single trajectory13. The site is geomorphologically complex, with a series of eight caves and numerous cavities that lie side by side at sea level. They were formed by a combination of tectonic and eustatic processes3 and span some 300m along a north-south axis. All of these caves and cavities are used by roosting ECM, allowing the identification of micro-sites (caves and cavities) within the roost that are in close proximity to each other. Aerial insects are a scarce resource in temperate environments during the winter months32 and any factor influencing body condition could impact survivorship of an aerial insectivore wintering in the Western Palaearctic, including selection of wintering and roosting sites. This, combined with the site’s morphology, makes the Gorham’s Cave Complex an ideal site at which to study micro-site selection ecology in ECM.The Rock of Gibraltar holds a winter roosting site of ECM at the Gorham’s Cave Complex Fig. , on its We carried out monitoring of ECM wintering at a roost in Gibraltar that consists of a series of caves alongside each other at sea level, primarily during the autumn–winter period of 2019-2020. The monitoring consisted of weekly counts of birds returning to roost, and of regular ringing and measuring sessions. The ringing data for 2019–2020 were augmented with data collected at the site between 2016 and 2018.The eight caves at the site are all micro-sites within a single ECM roost and three of them, which lie just above the current sea level and are the only ones accessible from land, were studied: Gorham’s Cave (36° 07′ 13.86″ N 5° 20′ 32.57″ W UTM 30 N 289190.5 3999856.5), Vanguard Cave  and Cave F  . The approach of the birds as they return to roost is described elsewhere33 to estimate the total roosting population size of ECM at the site during the 2019–2020 period. The latter was achieved by estimating the number of birds roosting at each cave and then combining these for a total population size, although we recognize that birds also use other micro-sites7; (pers. obs.). Due to differences in sample sizes of birds recaptured, 95% confidence limits for the estimated roosting population size at each cave were drawn from the t-distribution for Gorham’s Cave and the Poisson distribution for Vanguard Cave and Cave F33.We used a combination of the results of the counts and the Schnabel Index for mark-release-recapture data from a series of dates34. Routinely, birds are released without ringing when their condition is poor. One bird was captured in a condition that was too poor for it to be ringed. The reporting recommendations of the ARRIVE guidelines35 were followed.Trapping and ringing were carried out at the three caves at least once a week. All licences required under the laws of Gibraltar were obtained and protocols were approved by the Ethics Committee of the University of Gibraltar. Ringing and handling of birds was carried out under the auspices of the Gibraltar Ornithological & Natural History Society (GONHS), which carries out its bird ringing under licence from the Ministry for the Environment, HM Government of Gibraltar, under the 1991 Nature Protection Act. Gibraltar-based ringers are licensed by the British Trust for Ornithology (BTO), and we adhered closely to the technical and ethical standards of the BTO for handling and ringing birds34. Due to the different dimensions of the caves, we used different mist net sizes at each one. A 6m-length net was used at Vanguard Cave, 12 m and 3 m nets at Cave F, and 3 × 6 m nets mounted vertically on triple high poles at Gorham’s Cave.The majority of the data used in this study were collected between October 29th 2019 and March 4th 2020. In addition, trapping and ringing had taken place intermittently at Vanguard and Cave F during the winter since 2016, and trapping took place at the site throughout autumn 2020. We used the BTO A-sized rings, in accordance with guidelines for other European hirundinesThe number of trapping sessions, and the range of dates of these at each cave during the 2019–2020 autumn-winter season, was: 10 Gorham’s Cave , 11 Vanguard Cave , 11 Cave F . Seven extra trapping sessions took place at Vanguard Cave and Cave F before the 2019-2020 autumn-winter season, on: 01/28/2016, 02/16/2016, 02/13/2018, 02/21/2018, 12/04/2018, 01/08/2019 and 02/21/2019. There were eight additional trapping sessions during the autumn of 2020, on: 10/29/2020, 11/02/2020, 11/12/2020, 11/15/2020, 11/19/2020, 11/24/2020, 12/02/2020 and 12/03/2020. 1511 different birds were processed between 2016–2020, of which 156 were captured at least twice. 796 individuals were processed during the 2019–2020 autumn-winter season, the period for which most of our analyses are based: 369 at Gorham’s Cave, 221 at Vanguard Cave and 206 at Cave F. Of the birds recaptured that had been ringed at the site during previous seasons, eighteen were from the 2019–2020 season , fifteen were from the 2018–2019 season , four were from the 2017–2018 season , and one was from the 2015–2016 season . A bird was recaptured that had been ringed elsewhere in Gibraltar (the GONHS Jews’ Gate Field Centre) on the 14/01/2014, 2233 days before it was captured again on the 25/02/2020.34, which includes recording the weight of birds in grams (g) to 0.1 g and length of wing in millimeters (mm) to 0.5 mm. Birds were aged whenever this was possible but ageing of ECM became increasingly difficult towards the end of the winter period, increasing the possibility of confusion with adults9. For this reason, age was excluded from most of the analyses. Birds could not be sexed because sexes are similar in appearance, including size36. We captured birds only during the evening, to ensure that condition of birds was not a factor of weight-loss whilst roosting, since ECM at the site are known to weigh less during mornings than the evenings13. Birds captured were roosted in boxes and released at the site the following morning.Biometric measurement of all birds was carried out by a single person (CP) in order to maximize consistency. We followed the standard processing procedure of the BTO12 assumed that movement of birds between different parts of the roost at Gibraltar is considerable, this was never tested. The proximity of different parts of the roost from each other means that all micro-sites are potentially equally accessible to ECM using the site. It is expected that they should be able to use micro-sites interchangeably, given especially their approach during evenings along a fixed narrow route. Any fidelity to micro-sites must thus be explained by factors other than distance between individual micro-sites. The multiple cavities at the roosting site, and the ease with which we were able to access these, allowed a unique opportunity to test whether individual birds repeatedly used the same micro-sites within the roost, both within and between winters. We used the data gathered to test the following hypotheses: (1) that a degree of fidelity to different spaces within the roost (‘micro-sites’) exists among ECM, with individuals more likely to be recaptured at the same cave than in a different cave, (2) that any fidelity observed will translate to a difference in quality of roosting sites, as indicated by differences in condition of birds according to micro-site, and (3) that the incidence of recapture should be highest at the cave at which birds are in the best condition.Although Elkins & Etheridge37 and were carried out on SPSS statistical software (IBM). We used a binomial Z test to analyze whether recaptured birds that were initially ringed during the 2019–2020 season were returning to the cave where they were first trapped/ringed, (1) within the 2019–2020 season and (2) between this and separate seasons. We also used a 3 × 2 Fisher’s exact test to test for differences, between caves, in the frequency with which birds ringed at one cave were captured at another. Multiple recaptures of birds were excluded from all of these analyses on fidelity in order to avoid bias.Statistical analyses followed Sokal & Rohlf38—using only data collected during the 2019–2020 season. For individual birds that were trapped more than once, we used wing length and weight on the date of first capture. We then grouped, by cave, the residuals of the regression and used a one-way ANOVA to explore differences in mean condition of birds between caves, with condition expressed as the relationship between wing length and weight. We also used linear regression to explore the relationship between daily recapture rate at all caves and the number of days from the first day of trapping at each cave, with the latter as the explanatory factor. Again, we segregated the residuals of the regression by cave and used a one-way ANOVA to explore differences in recapture rates between caves. We used Pearson’s chi-squared test with Yates’s correction for small sample sizes39 to explore differences in the likelihood of recapture of birds on more than one occasion at each cave. Because differences in weight and wing length have been recorded between adult and juvenile ECM in Gibraltar13, we used Pearson’s chi-squared test to explore the relationship between age and use of the different micro-sites for all the birds that we were able to age (n = 395 of 796 birds processed), to see whether this was consistent with our other findings.We explored the relationship between wing length and weight using linear regression analysis, to control for the possible effect of body size on weight—on the basis that wing length provides a good measure of body size in passerines


In this paper, we propose a novel information criteria-based approach to select the dimensionality of the word2vec Skip-gram (SG). From the perspective of the probability theory, SG is considered as an implicit probability distribution estimation under the assumption that there exists a true contextual distribution among words. Therefore, we apply information criteria with the aim of selecting the best dimensionality so that the corresponding model can be as close as possible to the true distribution. We examine the following information criteria for the dimensionality selection problem: the Akaike’s Information Criterion (AIC), Bayesian Information Criterion (BIC), and Sequential Normalized Maximum Likelihood (SNML) criterion. SNML is the total codelength required for the sequential encoding of a data sequence on the basis of the minimum description length. The proposed approach is applied to both the original SG model and the SG Negative Sampling model to clarify the idea of using information criteria. Additionally, as the original SNML suffers from computational disadvantages, we introduce novel heuristics for its efficient computation. Moreover, we empirically demonstrate that SNML outperforms both BIC and AIC. In comparison with other evaluation methods for word embedding, the dimensionality selected by SNML is significantly closer to the optimal dimensionality obtained by word analogy or word similarity tasks. In recent years, word2vec has been widely applied to many aspects of Natural Language Processing (NLP) and information retrieval such as machine translation ,2, text The selection of the dimensionality for word2vec is important with regard to two aspects: model accuracy and computing resources. It is crucial to have a model of dimensionality high enough to learn the regularity of the data, but too high a dimensionality tends to cause overfitting. For instance, the experiment results in  demonstrHowever, few studies have focused on the dimensionality selection problem. Most research evaluating the effectiveness of word embedding focuses on word analogy and word similarity tasks . These eOur contributions are two-fold. First, we introduce an effective dimensionality selection method for word2vec based on information criteria. Moreover, our proposed approach does not require handcrafted evaluation datasets, and therefore, can be applied to any type of data not limited to English or verbal-data. This is important and necessary to be able to choose a reasonable dimensionality of the word2vec model when applying it to various fields in information retrieval. Second, from the perspective of information theory, we propose the Sequential Normalized Maximum Likelihood (SNML) criterion in a novel combination with some heuristics for the dimensionality selection problem. The application of our proposed criterion enjoys valuable theoretical guarantees from information theory as well as experimentally ensures that the selected dimensionality is able to capture regularity from the data as well as meet the preferences of models with relatively low but sufficient dimensionality. To the best of our knowledge, this study presents the first application of information criteria in the field of embedding methods as well as the first heuristic comparison of the SNML codelength. These positive results not only encourage wide use of the proposed method in other embedding methods but also suggest a promising solution to evaluate hyper-parameters of a machine learning model.Representations of words in a vector space have been studied exhaustively in the NLP literature. Beginning with a one-hot vector (the very first representation of words), other word representation methods such as latent semantic analysis  and lateAs the SG model often uses the negative sampling technique, in this study, we work with both original SG (oSG) and Skip-gram with Negative Sampling (SGNS) to clarify the idea behind our approach. In order to apply information criteria on the SG, we summarize it and introduce our notations for both oSG and SGNS.E and F are the parameter matrices of the shapes d is the dimensionality of the embedding vector space.SG normally takes text data as input to the whole training process. This text data is then processed into pairs of word and context  network . The preUnlike oSG, SGNS learns the probability that a particular context occurred around a word or not: function . In the Unlike our approach, Yin and Shen  consider129, and the best 5% dimensionalities range from 67 to 218, while the best dimensionalities in the WordSim353 (WS), MTurk771 (MTurk), and Google word analogy (WA) datasets are 56, 102, and 220, respectively [However, the selected number of dimensions does not agree with the optimal dimensionality performance based on the other evaluation tasks. For example, the best dimensionality of SG chosen by PIP loss is ectively . MoreoveWord2vec is classified as a self-supervised machine learning model. Therefore, the number of dimensions can be selected by comparing the value of the loss function on the validation dataset. An alternative approach to dimensionality selection involves using information criteria such as the Akaike Information Criterion (AIC) , BayesiaSince AIC, BIC, and MDL have different backgrounds with regard to the estimation of expected log-likelihood and approximation of the log marginal likelihood, we need to carefully choose the criteria to be used in specific cases. In fact, AIC and BIC rely heavily on the asymptotic theory, which states that as the data size grows to infinity, the estimated parameters converge in probability to the true values of the parameters. However, the asymptotic theory does not apply to word2vec, i.e., as the number of data increases to infinity, we can obtain different optimal parameters set . Therefore, AIC and BIC are not guaranteed to work theoretically. Nonetheless, several empirical studies have applied them successfully.Unlike AIC and BIC, MDL with Normalized Maximum Likelihood (NML) codelength is an accurate model selection criterion for real-world data analysis based on limited samples. NML is also known as the best codelength in the context of the minimax optimality property .However, choosing the best method for dimensionality selection is still an experimental task in word2vec. In the next section, we describe in detail the application of MDL to the dimension selection problem and the reason for choosing this method. We then provide empirical comparisons between the methods listed in this section.In order to apply these information criteria to the dimensionality selection problem, we introduce our notations for the AIC and BIC first.Word2vec was derived based on the distributional hypothesis of Harris , which srinciple  is a powThe MDL principle states that the best hypothesis  for a given set of data is the one that leads to the best compression of the data, namely the minimum codelength . SpecifiAssuming that we are able to encode a data series n.Given a model class, there are many methods to estimate the shortest codelength of a given dataset such as: two-part codelength, Bayesian codelength , NML or However, the PC term involves extensive computations and is not realistic to implement. Instead, we apply the SNML codelength  in this delength . Since tIn addition, the SNML codelength function Heuristic comparisonThe computation of the SNML codelength still costs n records of data, we can use the codelength of a small set of data. In fact, the results of our experiments show that focusing on the last several thousand records of data is sufficient to compare model classes.A simple observation reveals that if the codelength of data obtained with model class d1 vs. d2 dim); specifically, it is calculated by To facilitate comparisons among dimensionalities that are markedly different from one another  increases, and distribution Q is similar to function Q, and the sampling size is chosen to be 1/10 the size of the context set to balance the computation time and sampling error.This estimation asymptotes to the true value as Synthetic dataWe compared the above-mentioned model selection criteria using SG trained on three datasets: synthetic data, text8, and Wikipedia.Synthetic data were generated based on several random questions from the WA dataset. Assuming a numeric context set, we generated categorical distributions on this set for all words for which the parameter vectors of the corresponding distributions satisfy the constraints in the questions. For example, corresponding to question: https://github.com/truythu169/snml-skip-gram).The implementation for generation of such categorical distributions is also available on GitHub  with the oracle criterion. The experimental results are shown in The results for oSG show that the BIC exhibits a monotonous decrease, while the optimal dimensionality chosen by the oracle and SNML is 16 and 17, respectively. On the one hand, AIC and CV choose a more distinct dimensionality: 10 and 13, respectively. For SGNS, the oracle chooses 20 dimensions, SNML and CV choose 15, and WA achieves the highest score at 25 dimensions. On the other hand, the BIC chooses 10 dimensions, while the AIC chooses 30.In both oSG and SGNS, SNML chooses the dimensionality closest to the oracle criterion. Thus, SNML outperforms both AIC and BIC. Note that the synthetic data are designed to achieve a 100% WA score using contextual distribution; however, the scores achieved by using embedded vectors are sensitive to noises and change significantly according to the dimensionality.We compared the SNML criterion with the NLP word analogy task (using WA) and word similarity tasks ). As knowledge regarding the underlying true distribution of the data is lacking, it is difficult to determine the best dimensionality selection method. However, assuming the existence of the true contextual distribution, NLP tasks will roughly prioritize models closest to the true distribution. Therefore, the dimensionality selected by a good method is expected to be close to the optimal dimensionalities for NLP tasks. Note that the evaluation method using scores of NLP tasks is available only in the case of English text data; therefore, it is reasonable to apply a method that achieves the same results as the NLP tasks-based method to any other type of data.We experimented with at least three runs for each dataset, and the average results are shown in The main results of the study are shown in Furthermore, the SNML criterion tends to favor smaller dimensions, although it is sufficient to ensure good performance on other NLP tasks while heavily penalizing model classes that tend to overfitting. This characteristic of SNML helps us avoid choosing large models, and therefore, the available resources should be fully utilized. On the other hand, although AIC favors bigger dimensions and the performance of the model is slightly reduced, this approach is computationally advantageous over SNML. This advantage makes AIC useful in some situations.To the best of our knowledge, this is the first study that applies information criteria to dimensionality selection for embedding methods.P in Equations . Our proposed framework can be applied to other embedding methods as well as other neural network-based models once a likelihood function corresponding to any embedding method is defined. For example, in the case of BERT , the likquations –9) to o to oP inThe optimal dimensionality selected by SNML is low; however, it is sufficient to ensure good performance in terms of significant closeness of optimal dimensionality in NLP tasks. However, deep learning models (DL) usually benefit from over-parameterization properties, i.e, the performance of models is not significantly reduced due to the increasing number of parameters. Furthermore, there exist other approaches to the overfitting problem, such as regularization, early-stopping, randomly drop-out, etc., or strategies to adopt large DL models to small data, such as transfer learning, semi-supervised learning, etc. In the paper, we introduce an alternative method to the same problem from an information-theoretic view. The optimal dimensionality selected by the proposed framework can benefit from the over-parameterization property of DL by adopting alternatives such as applying other codelength methods or considering other parameterization methods for the likelihood function. Future challenges in this field include determining which modification results in the most improvement for the dimensionality selection strategy.When considering word2vec SG as a probability distribution estimation problem, the optimal dimensionality can provide an estimation of contextual distribution as close as possible to the true distribution-generated data. We tested information criteria (AIC and BIC) and SNML with some heuristics to select such a dimensionality. The experimental results on synthetic data showed that the SNML could choose a dimensionality such that the corresponding probability model is able to learn the contextual distribution closest to the true distribution-generated data. The experiments on text datasets showed that SNML has the ability to choose a desirable dimensionality with regard to two aspects, although low dimensionality is sufficient to ensure good performance in terms of significant closeness of optimal dimensionality in NLP tasks without a hold-out test dataset. Furthermore, SNML typically outperforms AIC, BIC, CV, and PIP in the selection of good dimensionality for NLP tasks in our experiments. Our method therefore holds promise for choosing the most appropriate dimensionality in word2vec when training with data not limited to English or non-verbal.To the best of our knowledge, this is the first study that applies information criteria to dimensionality selection for word embedding. In fact, the limitations associated with computation or asymptotic estimation of NML or SNML codelength make it difficult to apply such criteria in these areas. By introducing some heuristics in the SNML codelength calculation, we have discovered a new and useful approach, namely MDL-based knowledge embedding. Our proposed approach can be applied to other embedding methods once a likelihood function corresponding to any embedding method is defined. A more detailed evaluation will be left for future study.