Using the method of dynamic–mechanical analysis, the structuralisation kinetics of condensed starch solutions, cooled down to the temperature of 20 °C, was investigated. A close correlation of spatial crosslinking with local processes of macromolecule associations was discovered. It was found that depending on the concentration intervals of starch solutions, equilibrium nodes of the spatial network assume the form of either single or double hexagonal structures made up of bispiral chain associates. The increase of gel crosslinking, together with the passage of time, is the result of increasing the node functionality of the spatial network. Starch is one of the most common natural polymers. That is why the main objective of basic research on starch as well as of technologically oriented papers is the elaboration of methods of optimal utilisation of this raw material and establishment of foundations allowing prognostication and control of the course of technological processes aimed at obtaining products of desirable, planned-in-advanced properties. One of the major objectives of the basic research conducted so far on starch has been to recognise its structure in its native state. In this condition, starch is found in the form of spherulitic granules distinguishing themselves by radial anisotropy. The high degree of orientation in starch spherulites of its polymolecular constituents, linear amylose and branched amylopectin, is the source of the optical double refraction manifesting itself when observing starch grains in polarised light ,2. SinceCrystalline forms of the second starch constituent—amylopectin—can be attributed to the identical bihelical organisation of branches to that described above  packed iThe second branch of the physicochemical direction of research of starch systems, which is currently developing very strongly, is the problem of structure changes of the discussed systems in conditions of the hydrothermal processing frequently accompanying culinary processes. During the heating of water starch suspensions to temperatures of over 50 °C, starch spherulites swell and lose their optical anisotropy, which is identified with the melting of the polymer crystalline phase. Complicated structural changes taking place during the melting of crystalline starch areas are subjected to interpretations from the thermodynamic point of view ,11,12 baStarch solutions obtained in normal cooking conditions of suspensions show tendencies of constituent aggregation following their cooling and, at sufficiently high concentration, the capability to develop a permanent spatial structure ,15.The aggregation process of starch structural elements taking place in the described conditions, which is frequently accompanied by a liquid phase as a result of synthesis, is traditionally referred to as retrogradation. Due to the fact that retrogradation affects many quality parameters of almost all products of starch processing, investigations of a complex of phenomena accompanying starch retrogradation constitutes the subject of many analyses and monographic studies, in particular, those that aim at elucidating the mechanism of bread staling ,16,17.Already, the results of the first roentgenographic investigations  have revIn order to characterise the molecular preconditioning of the starch retrogradation process, measurements were performed of the structuring kinetics of starch solutions cooled down to the temperature of 20 °C in an atmosphere of saturated water vapour, using for this purpose the DMA method. This allowed the determination of components of the complex modulus of elasticity of the developing spatial network and assessment of the density of the segments of macromolecules making up the spatial network.G′(t) and G″(t) presented in The character of the dynamic–mechanical changes of the starch solution properties taking place with the passage of time, counted from the moment of reaching ambient temperature at various concentrations, illustrates the model courses of the dependences G′) and loss modulus (G″), at different concentrations, increase, tending towards asymptotic values.With the passage of time, both storage modulus  and loss modulus (G″) values increases together with the increase in the starch concentration. This is due to the fact that the retrogradation process is faster at more concentrated systems of starch dispersion than in a dilute dispersion [The growth increment of the storage modulus  dependent on starch concentration. The approximate relationship between the storage modulus of highly elastic polymer networks and the segment concentration in these networks is determined by the following dependence [In addition, the attainment of plateau values for both t starch . This ispendence :(1)n≅G′Rn∞(c) are approached. These changes can be described by the Avrami-type [on and ∞n designate initial and final network segment concentrations, k is the Avrami crystallisation rate constant, and m is the Avrami exponent of time.The observed increase of the hydrogel storage modulus together with the passage of crosslinking time at different concentrations indicates that the concentration of effective segments in this network increases as asymptotic values of ami-type  Equationm and k of the Avrami equation characterised the mechanism of nucleation, geometry of the crystallisation, and growth of crystallites. Constant k characterised a crystal growth rate dependent on the temperature and molecular weight [The parameters r weight ,32,33.m) according to the theory is an integer value ranging from 1 to 4  [The Avrami exponent (chanism) .G′ and G″ components in the course of the observed crosslinking can be described by the equation of the same type:oG and ∞G designate, respectively, initial and final values of storage modulus.In crosslinked systems, the bond density and durability exert a decisive influence on their mechanical and rheological properties; hence, the change kinetics of the It was found that the Avrami exponent m assumes the value of 1 throughout the examined interval of starch concentrations.This appears to suggest that the node formation processes of the spatial network of the examined gels attain only the nucleation phase of crystalline forms.k changes  constant relationship was obtained as a result of the relaxation of velocity measurements of identical wheat starch gels, using for this purpose the NMR technique [3. This indicates a close correlation of spatial structuring with local association processes.A very similar course of echnique ,38 in a μο is the chemical potential of a pure diluent, v1 is a volumetric share of the diluent, v2 is a volumetric share of the polymer in the system, χ is a parameter of the polymer–diluent interaction, n is the number of segments of a volume unit of swelled gel, and f is the node functionality of the spatial network of the gel.In the case of polymer systems of an established spatial network containing no individual polymer molecules, according to the statistical–thermodynamic theory , the cheWhen the process takes place in the presence of diluent vapours, then the chemical potential of this phase is determined by the following expression:o.p = p Therefore, the condition for the stabilisation of the chemical potential equilibrium requires evolution of the constituent values of Equation (4), leading to neutralisation of the left side of this equation:When a gel spatial structure is formed in the atmosphere of the diluent saturated vapour, then its pressure over the solution This can take place not only following changes in the spatial network segment concentration and their functionality but also as a result of the increased effectiveness of polymer concentration combined with decreasing the volume taken up by this polymer. The phenomenon of the release of pure diluents accompanying the process, known as syneresis, is a frequent manifestation of the starch retrogradation process . Therefo∞ in the polymer concentration function in the solution calculated on the basis of Equation (2) as well as curves obtained in the result of additional fitting of these courses to Equation (7).3 and over 0.07 g/cm3 show distinctly greater diversity. The initial gel structure at low starch concentrations is determined by nodes of spatial networks that occur, primarily, in the form of single hexagonal structures made up of bispiral chains of associated molecules, i.e., nodes of functionalities of the order of 30. In the case of aggregate organisation in characters corresponding to the polymorphic type A form, their functionality should assume the value f = 28. Then, it follows that the structures of the re-organised regions in retrograded starch differ from those in native starch. B-type crystals are found in retrograde cereal starch [3 is exceeded, a discrete change of network node functionality of the examined gels takes place, indicating the possibility of the development of hexagonal binary structures. This was also corroborated in the research results of earlier publications [It is evident from the data analysis presented in l starch ,40 that ications ,38 in whf:of and ∞f designate, respectively, initial and equilibrium functionalities of nodes; k is the kinetics constant; and m is the Avrami constant.In conditions of unstable gel spatial structure when, as a result of crosslinking, network segment concentration and the functionality of its nodes can change, the analysis of the state of the starch–water system cannot be carried out according to the method proposed above. It is only possible to perform an approximate analysis on the basis of the interdependence between network segments n, node concentration w, and their functionality kinetics , accordiThen, Equation (2) can be presented in the following form:n~c2 (c-polymer concentration). On the other hand, when the node concentration is assumed as a constant, the increase of polymer concentration will cause an increase of the average functionality proportional to c. The diagrams shown in ∞n) and initial (on) network segment concentrations are subject to linearisation in function c2, adopting, however, slopes that depend on the system condition and starch solution concentration interval below and above c≈ 0.06 g/cm3.Using the method of computer fitting, it can be demonstrated that assuming constant node functionality, the increase of polymer concentration in a crosslinked gel should lead to the increase of network node concentration in accordance with the dependence: c ≈ 0.06 g/cm3 i.e., respectively, 3.7 and 26.5. This means that within the range of low starch concentrations, the initial gel structure is determined by tetrafunctional nodes that are bispiral chain associates, whereas within the range of concentrations above 0.06 g/cm3, hexagonal associates of bispiral structures are involved. The discrete change of node functionality observed at starch concentration of 0.07 g/cm3 is probably connected with the occurrence of areas of spatial arrangement that are characteristic for solutions of liquid crystal polymers [These data suggest that with gel aging, fragments of starch macromolecules, gradually incorporated into the network, undergo adsorption on these nodes, increasing their functionality. It is evident from the obtained values of node functionality of the equilibrium network and from the ratio of directional coefficients of anamorphoses presented in polymers . The occpolymers ,42.It is evident from earlier considerations that the formation of a spatial network in starch gels is the result of random linkages between different fragments of macromolecules neighbouring with one another in the solution, leading to spiralisation and their association to forms of bihelical associates. Therefore, individual nodes of the gel network are characterised by varying functionalities assuming values from about 4 to several dozen.iΔU segment linkages in individual nodes and, consequently, leads to the distribution of mechanical relaxation times resulting from the following dependence:This is associated with the energy differentiation of The mechanical–rheological properties of polymers are conditioned by the function course of the relaxation times distribution . TherefoDτ and top Gτ spectral limits, while outside this interval, H(lnτ)/δG = 0.Hence, the standard function of the distribution of relaxation times can be approximated by the rectangular distribution for which:ω ≅ 1/averageτ, it can be assumed [At sufficiently broad distributions and frequencies  assumed  that. into consideration, Equation (13) assumes the following form:GU − ΔDU) = δU is the difference of the activation energy of the relaxation processes accompanying the deformation of the gel network segments connected with the strongest and weakest nodes of this network.Using the empirical c ≤ 0.06 g/cm3 concentrations and values from approximately 30 to 60 kJ/mol for starch concentrations exceeding 0.06 g/cm3.It is evident from the data shown in the drawings in The author applied successfully the same procedure in the study  in orderTriticum durum wheat . The chemical composition of wheat starch was as follows: moisture, 12.2%; protein, 0.44%; lipid, 0.39%; ash, 0.21%; and 90% amylopectin. The starch used in the studies was dried to a constant mass at the temperature of 100 °C prior to the preparation of solutions. Starch dispersion samples weighing 100 g were subjected to thermal treatment at a constant temperature of 90 °C, in an LWC2M water bath  for a period of 1 hour and stirred continuously with the assistance of an IKA EUROSTAR agitator power control-visc 6000 of IKA WERKE GmbH (Staufen Germany). Solutions of 0.04 to 0.13 g/cm3 starch concentration obtained in this way were used to fill the measuring system of the rheological analyser and cooled to the required temperature with the velocity of 4 °C/min.Investigations were carried out on starch hydrogels obtained from starch solutions of G’) and the loss modulus (G″). The storage modulus is associated with the part of potential deformation energy that is maintained in the course of periodical deformations. Loss modulus (G″) is associated with the part of energy that undergoes dissipation in the form of heat. The analyses were performed considering the linear viscoelastic region in each sample. The frequency of the vibrations of the systems amounted to 1.2 Hz. Measurements of the starch structuralisation kinetics were carried out at a set temperature of 20 °C (with up to 0.2 °C accuracy).A dynamic mechanical thermal analyser (DMWT)  was used for measurements. A parallel plate with a 50 mm diameter and 1.0 mm gap measuring system was used. The sample perimeter was covered with a thin layer of high-temperature-resistant silicone grease  to prevent dehydration of the sample edge and moisture evaporation from the sample. The following components of the complex modulus of elasticity were calculated: storage modulus (SD) were calculated with the assistance of the Statistica 10.0 PL software. The values of changes of m, k, f, and n parameters were obtained by the method of computer fitting .The mean and standard deviations were calculated from the rheological measurements on three newly prepared samples in accordance with the procedure described earlier. The data presented on Figures are the result of averaging of the results obtained in individual replications. Mean values as well as standard deviations (In order to characterise rheological attributes of the examined starch–water systems, the analysis of kinetic changes of basic parameters determining their mechanical–rheological properties was carried out. The research results of starch gel systems obtained using the DMA method indicate that with the passage of time counted from the moment they reach ambient temperature, a spatial network with segment density dependent on starch concentration develops in the examined systems already during the process of cooling down. It is worth mentioning that the kinetics of the run of crosslinking processes conforms to the description of the Avrami-type equation. The kinetic parameters of crosslinking that determine the extent and rate of spatial network node development in gel systems, similar to the initial and final densities of network segments, are preconditioned by the concentration of starch in the system.It is evident from the dependence between segment concentration in the gel network and node concentration as well as the functionality of these nodes that at a given starch concentration in the system and at constant temperature, the increase of gel stiffness in time function occurs as a result of node expansion involving the binding of new macromolecule segments. This leads to the increase of node mean functionality stabilising the spatial network.


Obstructive left ventricular assist device (LVAD) lesions are uncommon but are being increasingly recognized, particularly with the increased use of advanced imaging modalities. While heart failure symptoms and LVAD power fluctuations have a broad differential, obstructive lesions in the LVAD circuit should be considered. We present a unique case of a patient supported on HeartWare HVAD  therapy, who experienced postural dizziness with objective orthostatic hypotension and occasional ventricular tachycardia. With fluctuations in LVAD flow and power, a CT scan with three-dimensional reconstruction was obtained showing outflow graft kinking. The patient was brought to the cardiac catheterization laboratory for investigation and consideration of outflow graft intervention. However, intracardiac echocardiography revealed the presence of an inflow cannula obstruction with position changes and catheter interrogation involving the outflow cannula suggestive of a gradient across the kinked area as an unlikely cause for the presentation. This case highlights the importance of a thorough interrogation for obstructive lesions in the setting of heart failure symptoms, particularly postural symptoms, in a patient on LVAD therapy, even when not identified on routine echocardiography. Among hemodynamic complications related to left ventricular assist device (LVAD) therapy, obstructive lesions can be some of the most difficult to manage. Kinking in the outflow cannula is increasingly being recognized, courtesy of advanced imaging modalities, including three-dimensional 3D) cardiac CT reconstructions ,2. TreatD cardiacA 69-year-old gentleman with ischemic cardiomyopathy was implanted with a HeartWare HVAD , as destination therapy. The outflow cannula graft was anastomosed to the descending aorta due to a porcelain ascending aorta. His postoperative period had been uneventful. Due to poor hemodynamics on pulmonary artery catheter monitoring, including mild biventricular filling pressure elevation and a cardiac index below 2.0 L/min/m2, his LVAD speed was set to 3000 revolutions per minute (RPM). However, in the recovery phase after implant, the patient had recurrent dizziness and ventricular tachycardia, preventing rehabilitation.Given the debilitating symptoms, examination and echocardiography were undertaken to understand the pathophysiologic alterations. At 3000 RPM, with the patient lying down, blood pressure was 106/79 mmHg (mean 89 mmHg). Upon sitting, blood pressure dropped to 78/68. HVAD waveforms demonstrated a sawtooth pulsatility pattern in concert with a drop in power (5 W to 4.7 W) and estimated flow (3.7 L/min to 3.0 L/min). LVAD parameters and blood pressure normalized when he returned to a laying position. By echocardiography, the left ventricular end-diastolic dimension (LVEDD) decreased from 64 mm at baseline to 44 mm with sitting. The outflow cannula was poorly visualized.While dehydration or low preload could have explained the apparent orthostatic findings, his examination was consistent with euvolemia. Furthermore, his right ventricular function was preserved on echocardiography. At this juncture, the pathogenesis of the intermittent symptoms was felt most likely due to inflow cannula obstruction.However, given the atypical anastomosis and postural symptoms, evaluation of the outflow graft was felt to be warranted. Therefore, a CT angiogram with 3D reconstruction was obtained, wherein a site of outflow graft kinking was observed Figure . The infIntracardiac echocardiography (ICE) was utilized given limited transthoracic images. At 3000 RPM, there was no aortic valve opening. The interatrial septum was midline and the interventricular septum was deviated to the right Figures , 2B. TheSimultaneously, to assess the outflow graft, a multipurpose guide catheter was threaded into the aorta, while a high-fidelity micromanometer wire was advanced into the outflow graft beyond the kink, shown fluoroscopically in Figure The present case demonstrates the imperative to fully evaluate hemodynamics in symptomatic patients supported on LVAD therapy. Dynamic inflow cannula obstruction can be difficult to appreciate, particularly if position-dependent and in patients with challenging echocardiographic images. The most important clues to this etiology of paroxysmal symptoms were the prominent decrease in left ventricular dimensions with position change and on echocardiography a decrease in inflow cannula velocities ,7.With dynamic symptomatology, inflow obstruction, and 3D reconstruction of the outflow graft concerning hemodynamically significant obstruction with poor baseline hemodynamics, further evaluation was required to delineate the significance of each pathology. The present case conjured a unique pathophysiologic mechanism for suction: that unkinking of the outflow graft may have resulted in significantly improved flow due to the afterload sensitivity of the pump, contributing to suction events. This diagnostic challenge prompted an invasive investigation, which confirmed that dynamic inflow obstruction was solely responsible for the clinical findings.Outflow kinking is a difficult complication to manage effectively, as even if identified invasively, there is a lack of data about criteria for intervention based on outflow graft hemodynamics . FurtherThe importance of thorough hemodynamic evaluation is critical for evaluating symptoms, particularly when dynamic and multiple possible etiologies exist. Given the known limitations of surface echocardiography, consideration should be given to intracardiac echocardiography and invasive hemodynamic assessment may be warranted.


L* a* b*). Total dietary fiber content was measured according to the AOAC 2009.01 method. Structural and morphological changes were determined by means of SEM, XRD, DSC, and GC-MS analyses. Microwave-assisted dextrinization of potato starch led to light yellow to brownish products with increased solubility in water and diminished crystallinity and gelatinization enthalpy. Dextrinization products contained glycosidic linkages and branched residues not present in native starch, indicative of its conversion into dietary fiber. Thus, microwave-assisted heating can induce structural changes in potato starch, originating products with a high level of dietary fiber content.Dietary fiber can be obtained by dextrinization, which occurs while heating starch in the presence of acids. During dextrinization, depolymerization, transglycosylation, and repolymerization occur, leading to structural changes responsible for increasing resistance to starch enzymatic digestion. The conventional dextrinization time can be decreased by using microwave-assisted heating. The main objective of this study was to obtain dietary fiber from acidified potato starch using continuous and discontinuous microwave-assisted heating and to investigate the structure and physicochemical properties of the resulting dextrins. Dextrins were characterized by water solubility, dextrose equivalent, and color parameters ( Nowadays, there is growing interest in the correlation of nutrition with human health. Several studies have been conducted to develop functional foods that provide caloric intake and contain bioactive compounds . ConsideDF preparations were previously obtained by dextrinization, which occurs while heating starch in the presence of acids . During Microwave irradiation is referred to as a type of electromagnetic irradiation in the frequency range from 0.3 to 300 GHz and wavelength from 1 mm to 1 m . MicrowaMicrowave irradiation has already been used in starch modification ,21,22, iTo the best of our knowledge, this article presents, for the first time, a comprehensive and comparative study on dietary fiber obtained from potato starch under continuous and discontinuous microwave heating conditions. It was hypothesized that the discontinuous process would be more effective than the continuous one. In order to confirm this hypothesis, the dextrins obtained by both methods were subjected to structural and physicochemical tests and their properties were compared.The solubility of dextrins at 20 °C was affected by the microwave-assisted heating conditions, including the microwave power intensity and the processing time . When coAlthough the dextrose equivalents (DE) values  were lowa* and b* confirm share of red and yellow colors of dextrins The reduction in whiteness and predominance of beige/brown can be related to the progress of the caramelization reaction [L* parameter and higher values of the ∆E parameter. This is in line with previous observations, which showed that harsher conditions  resulted in a darker color of dextrins [After exposure to microwave irradiation, the white color of native potato starch changed . All dexreaction ,32,33. Idextrins ,12,13,34Total dietary fiber content for dextrins obtained by continuous microwave-assisted heating ranged from 14.5% to 21.5% , which wThe XRD pattern for potato starch contained diffraction peaks (2θ) at 5.59°, 15.12°, 17.21°, 19.54°, 22.40°, 23.90°, and 26.27° , which cFor potato starch continuously heated a, a 2-foo, Tp, and Tc were significantly higher than the ones obtained for the native potato starch -linked Glcp -Glcp were observed. The results are in line with observations of Nunes et al. [p. In the context of dextrins’ resistance to enzymatic digestion, in addition to the presence of such bonds, the presence of branched molecules, i.e., with more than two groups -OH involved in the formation of glycosidic bonds, is also beneficial. In each sample low amounts of  and -Glcp were presented, and in some dextrin samples it was also possibly to quantify small amounts of -Glcp. Increasing microwave power intensity and heating time seemed to favor the molecules branching. For dextrins prepared under continuous and discontinuous microwave-assisted heating, the presence of molecules other than (1 → 4)-Glcp, terminal, and -Glcp differed depending on the modification conditions used. The values of the relative percentage of non-starch glycosidic linkages ranged from 4.6% to 7.2% for samples heated once for 75 s; 8.0% to 9.2% for samples heated once for 90 s; 10.6% to 12.3% for samples heated 10 times for 15 s; and 13.4% to 17.3% for samples heated 10 times for 30 s. These results were in line with the 17.8% and 5.8% of linkages other than (α1 → 4) reported by Bai and Shi [According to GC-MS analysis, native potato starch contained more than 90% of (1 → 4)-linked Glcked Glcp . For alls et al. , who use and Shi  for pyroPotato starch and analytical grade reagents were purchased from Sigma-Aldrich, Poznan, Poland; enzymatic kits were purchased from Megazyme, Wicklow, Ireland.v/v) solutions of hydrochloric and citric acids to a final concentration of both acids amounting to 0.1% w/w related to dry starch basis. Acidified starch was mixed and distributed on the surface of the tray. The prepared material was dried at 110 °C for 2 h in order to obtain a final water content of less than 5%. Afterwards, it was weighed (5 g) into 35 mL glass vessels and heated in a Discover SP microwave reactor  at 40 W, 45 W or 50 W for 75 s or 90 s, for the continuous process, and at 100 W, 110 W or 120 W for 15 s or 30 s, for the discontinuous process. During the continuous process, the samples inside the vessels were heated once, while during the discontinuous process each of the samples inside the vessels was heated 10 times under the selected conditions (the vessels’ content was mixed after each heating cycle to increase uniformity of microwave heating and modification of starch level). The different microwave irradiation conditions used in the study and operational advantages and disadvantages are presented in Dextrins were prepared by weighing 80 g of potato starch, spreading it onto a glass tray, and spraying it with 0.5%  in water was calculated using Equation (1):Solubility in water at 20 °C was measured according to Richter’s method . DextrinDextrose equivalent of dextrins was determined using Schoorl–Regenbogen’s method . DextrinL* (luminosity), a* (red/green color), b* (yellow/blue color) components were determined with the CIELab color profile. Color difference was calculated from Equation (2):L*, a*, b* between native starch and dextrins, respectively. Measurements were performed 10 times for each sample.The color of dextrins was measured using a Chroma Meter CR-400 . High molecular weight dietary fiber (HMWDF), comprising insoluble dietary fiber (IDF) and dietary fiber soluble in water but precipitated in 78% aqueous ethanol (SDFP), and dietary fiber soluble in water and not precipitated in 78% aqueous ethanol (SDFS) were determined following the AOAC 2009.01 method . BrieflyThe granular shape and surface morphology of native potato starch and the prepared dextrins were observed using a Tescan VEGA 3SBU scanning electron microscope . The accelerating voltage was 3 kV. Adhesive tape was attached to circular stubs, then all samples were sprinkled onto tape without coating with any conductive material. All samples were observed, and micrographs were taken at magnification of ×2000.cX) for all samples were obtained using Equation (3):cH and aH are the intensities for the crystalline and amorphous profiles with typical diffraction reflex (121) at a value of 2θ between 17° and 18°, as shown in Phase analysis of dextrins was carried out using powder X-ray diffraction (XRD) Rigaku Miniflex D 600 diffractometer  with D/teX Ultra silicon strip detector, Cu-Kα radiation. To assess the crystallinity, the method described by Hulleman et al.  was usedw/w). Samples were heated from 10 °C to 120 °C at 3 °C min−1. The onset (To), peak (Tp), and conclusion (Tc) temperatures; gelatinization temperaturę range ΔTr = (Tc − To); and enthalpy change (ΔH) expressed in J g−1 dry starch were calculated from thermograms.Gelatinization properties of native starch and dextrins were determined by differential scanning calorimetry (DSC), following a previously described method, but with some modifications ,57. For 3I was added and allowed to react in room temperature under vigorous stirring. After 20 min, 2 mL of distilled water was added, and the solutions were neutralized with 1 M HCl. Subsequently 3 mL of dichloromethane was added, and the solutions were manually shaken and further centrifuged at 11,600× g. The water phase was removed, and the precipitate was washed twice with distilled water. The samples were evaporated to dryness. To ensure a complete methylation, this step was repeated. Afterwards, the samples were hydrolyzed with trifluoroacetic acid (TFA) at 121 °C for 1 h, with subsequent evaporation of the acid. For the carbonyl reduction, 300 μL of 2 M NH3 and 20 mg of sodium borodeuteride were added. The mixtures were incubated at 30 °C for 1 h and then excess of borodeuteride was removed by addition of glacial acetic acid. The partially methylated alditols were acetylated by adding 1-methylimidazole and acetic anhydride. After 30 min at 30 °C, the excess of acetic anhydride was removed and the partially O-methylated alditol acetates (PMAA) were extracted with dichloromethane in two steps. Then, the dichloromethane was evaporated, and the samples were dissolved in anhydrous acetone, which was evaporated prior the GC-MS analysis.For determination of glycosidic linkages composition, dextrins were converted to partially O-methylated alditol acetylates . BrieflyPMAA obtained in the p < 0.05.The results were subjected to statistical analysis using Statistica 13.3 software . A completely randomized design was applied for all the experiments. Analysis of variance was performed. Mean comparison was done using Duncan’s new multiple range test. All assays, except for color measurement (where the tests were repeated 10 times), were performed in triplicate, and their results were averaged if the difference was not statistically significant at p linkages, absent in the native starch, thus highlighting the higher total dietary fiber content. Moreover, the discontinuous heating decreased the starch crystallinity, changing the granules surface morphology and originating samples with higher dextrose equivalent values and darkest coloration, thus revealing a more pronounced degree of starch modification. The applied dextrinization processes allowed products with a high total dietary fiber content to be obtained.Dextrinization of starch acidified with hydrochloric and citric acids using microwave radiation in a single-mode reactor was successfully carried out. The use of a single-mode microwave reactor allowed high repeatability of conducted processes to be created. The use of discontinuous process (10-fold heating with mixing between cycles) proved to be more effective than the continuous one (single heating). The dextrins obtained by discontinuous heating showed higher solubility and higher content of, among others, (1 ⟶ 2), (1 ⟶ 3), , , and  Glc


Journal of the American Academy of Orthopaedic Surgeons published the inaugural edition of the “Blue Journal”—JAAOS Global Research & Reviews . The mission of this JAAOS expansion was to improve the availability of quality orthopaedic knowledge through an open access, peer-reviewed, international journal. After 5 years of steady growth, I am proud to announce that we are achieving this lofty goal.In 2017, the JAAOS Global has reached many critical milestones:(1) Formation of an International Advisory Board, representing eight countries and five continents;(2) Ten Deputy Editors from eight different countries;(3) Indexed in MEDLINE, Scopus, PubMed Central, Emerging Sources Citation Index, and Directory of Open Access Journals;(4) Over 90,000 unique visitors to the website;(5) 366 submissions in 2021, an increase of 23% from 2020;(6) Over 140 articles published in 2021 (30 published in 2017);JAAOS Global partnerships with international orthopaedic associations.(7) Five Since the publication of our first issue in March 2017, https://links.lww.com/JG9/A211) from around the world. These volunteers provide an invaluable service to the Journal by using their expertise to provide constructive feedback to our authors. This has led to the early successes of JAAOS Global and positions the JAAOS portfolio for continued growth in the future.These accomplishments would not be possible without the assistance of our talented Deputy Editors and hundreds of reviewers (see list at Over the next few years, we will continue to expand our global partnerships. Our goal is to solicit more articles from international authors. This will help us to accomplish our mission of disseminating high-quality orthopaedic knowledge around the world.


The current dynamics of the COVID-19 pandemic have become a serious concern with the emergence of a series of mutant variants of the SARS-CoV-2 virus. Unlike the previous strain, it is reported that the descendants are associated with increased risk of transmission yet causing less impact in terms of hospital admission, the severity of illness, or mortality. Moreover, the vaccine efficacy is also not believed to vary among the population depending on the variants of the virus and ethnicity. It has been determined that the mutations recorded in the spike gene and protein of the newly evolved viruses are specificallyresponsible for this transformation in the behavior of the virus and its disease condition. Hence, this study aimed to compare the immunogenic profiles of the spike protein from the latest variants of the SARS-CoV-2 virus concerning the probability of COVID-19 severity. Genome sequences of the latest SARS-CoV-2 variants were obtained from GISAID and NCBI repositories. The translated protein sequences were run against T-cell and B-cell epitope prediction tools. Subsequently, antigenicity, immunogenicity, allergenicity, toxicity, and conservancy of the identified epitopes were ascertained using various prediction servers. Only the non-allergic and non-toxic potential epitopes were matched for population relevance by using the Human Leucocyte Antigen population registry in IEDB. Finally, the selected epitopes were validated by docking and simulation studies. The evaluated immunological parameters would concurrently reveal the severity of COVID-19, determining the infection rate of the pathogen. Our immunoinformatics approach disclosed that spike protein of the five variants was capable of forming potential T and B-cell epitopes with varying immune responses. Although the Wuhan strain showed a high number of epitope/HLA combinations, relatively less antigenicity and higher immunogenicity results in poor neutralizing capacity, which could be associated with increased disease severity. Our data demonstrate that increased viral antigenicity with moderate to high immunogenicity, and several potential epitope/HLA combinations in England strain, the USA, India, and South Africa variants, could possess a high neutralizing ability. Therefore, our findings reinforce that the newly circulating variants of SARS-CoV-2 might be associated with more infectiousness and less severe disease condition despite their greater viremia, as reported in the recent COVID-19 cases, whichconsequently determine their increased epidemiological fitness. The recently emerged SARS-Cov-2 is an RNA virus with high mutating efficiency, and the infection is transmissible between humans . It enteAlthough reverse vaccinology techniques have been conventionally used to design peptide-based vaccines , advanceSince the so-called UK variant with mutations like N501Y, D614G, etc., has been widely reported in many countries from October 2020, four representative sequences of the S protein with the characteristic mutations deposited from England, Karnataka in India, Georgia in the United States, and Western Capein South Africa were retrieved. Among the sequences retrieved, three were genome sequences obtained from GISAID submitted by England, India, and South Africa. The protein sequence of the USA variant was instantly obtained from the NCBI-Protein database. To compare these latest variants, the primary Wuhan SARS-CoV-2 genome and the sequence of S glycoprotein were separately retrieved from the NCBI-Protein database. The spike gene of England, India, and South African variants were translated into protein sequences using the BioEdit-translate program.All five sequences chosen for the study were 1273 amino acids long. Protparam results for physicochemical properties revealed that the composition of the acidic and basic amino acids in the S protein of England, the USA, India, and South Africa showed a slight variation when compared to the Wuhan reference strain.Due to the high number of basic amino acids in all the isolates, they were found to be acidic. Although the instability index value of all five SARS-CoV-2 isolates was found to be within the stable range, it was slightly increased in the spike protein of the recently evolved variants. Interestingly, the negative GRAVY index values of the spike protein in all the five isolates indicated their hydrophilic nature .The consTo gain insight into their phylogenetic relationships, a maximum likelihood phylogenetic tree was generated for the genome sequences of all the SARS-CoV-2 variants isolated from England (EPI_ISL_655762), India (EPI_ISL_1708422), South Africa (EPI_ISL_1706561), USA (MW494124.1), and China (NC_045512.2) in comparison with Bat Coronavirus (MW251308.1), SARS-CoV-1 (MK062180.1) and MERS virus (MW086530.1). The resulting cladogram revealed that all the SARS-CoV-2 isolates were in clade I while the SARS-CoV-1 strain remained in clade II, indicating close evolutionary relationships shared by the SARS-CoV-2 isolates with each other . It was + unique T-cell epitopes from the S protein sequence of the Wuhan isolate.After evaluating their antigenicity based on the VaxiJen scores, only 77 epitopes were found to be above the threshold value of 0.5. When the qualified 77 antigenic epitopes were further evaluated, only 35 of them possessed immunogenicity scores greater than 0.00. Besides this, the results revealed that 28 of the 77 epitopes were non-allergic and non-toxic peptides in the Wuhan isolate. All the identified peptides were 9mers. The final set of immunogenic, non-allergic, and non-toxic peptides was evaluated for their MHC I binding affinity using the IEDB recommended NetMHCpanEL 4.1 prediction method. With a reference panel of 27 alleles, this prediction step facilitated the selection of good-quality cytotoxic T-cell epitopes. About 20 CTL epitopes with the percentile rank less than or equal to 1.5 for the MHC I allele binding were filtered out, which were further narrowed down to the 11 best CTL epitopes.The identification of cytotoxic and helper T-cell epitopes in the target antigens is one of the key steps not only in the epitope-driven vaccine design process but also crucial for understanding the immunologic profiles of each SARS-CoV-2 variant. Undoubtedly, it is important to identify and compare the immuno-dominant epitopes that are capable of eliciting an immune response in the host and are responsible for the clinical outcome.Thus, T-cell epitopes were computationally identified on the S protein sequences of the SARS-CoV-2 isolates collected from China, England, the United States, India, and South Africa.With the selected threshold value of 1.00, NetCTL 1.2 server initially predicted 167 CD8Similarly, from the 86 unique CTL epitopes that were identified on the spike protein of the England isolate, we selected 36 epitopes that exhibited increasedantigenicity, 21 of them with positive immunogenicity scores. Based on the binding affinity of the epitopes with the highest number of MHC-I alleles, seven immuno-dominant epitopes of the England variant wereselected as highly antigenic epitopes. They could potentially interact with a set of the class I MHC alleles with high affinity and elicit the desired humoral immune response in the host system. When the latest variant isolated from the USA was analyzed, 159 unique CTL epitopes were initially recognized, which were further narrowed down to 75 qualified antigens from the VaxiJen scores. Out of the 75 epitopes, 38 peptides were either allergic or toxic. Those antigens with positive immunogenicity scores were selected to be highly antigenic peptides that could interact with the MHC I alleles with high binding affinity. Herein, for the 39 peptides that were predicted to be immunogenic, MHCI binding ability was evaluated. The sevenbest epitopes were identified, and their associated alleles were cataloged. Similarly, 67 of the 146 unique CTL epitopes that were initially identified from the Indian isolate were antigenic, and 41 were immunogenic candidates. Twenty-eight out of the 40 epitopes were discovered to be non-allergic and non-toxic peptides. In the end, 14 were qualified to be the best epitopes based on their binding affinity to the MHC I molecules. Lastly, when we analyzed the spike protein of the South African SARS-CoV-2 isolate, it possessed 162 unique CTL epitopes. Of all the variants, the S protein of the South African isolate showed the highest number of 70 antigenic and 37 immunogenic peptides. Finally, 17 out of 28 non-allergic and non-toxic peptides qualified as potential CTL epitope candidates . AlthougWe used the IEDB server to determine the binding affinity of the class II MHC human leukocyte antigen alleles with the helper T lymphocytes using human HLA-DR, HLA-DP, and HLA-DQ combinations. Witha reference panel of 29 HLA alleles, we predicted the binding affinity of MHC II allele-associated 15mers for the spike glycoprotein of the three different SARS-CoV-2 variants with the help of the IEDB recommended method. The epitopes identified were ranked based on their percentile rank scores given in the output. The lower the percentile ranked score, the higher the binding affinity of the epitopes for HTL receptors. Only those peptides with the percentile score threshold ≤ 1.5 were considered as having higher binding affinity for MHC II alleles and thus were selected as the best helper T-cell epitopes . The IEDSince the spike glycoprotein of SARS-CoV-2 is structurally oriented on its outer surface, it optimally enhances the specific binding of the pathogen to the ACE II host receptor. For this reason, it was considered an ideal target for B-cell epitope screening. Potential B-cell linear and discontinuous epitopes were identified within the regions they exist in the S protein structure models of China, England, the USA, India, and South Africa using the IEDB-Bepipred linear epitope prediction tool. According to the Bepipred results, out of 35 epitopes predicted, we identified six linear antigenic, non-allergic, and non-toxic epitopes in the S protein of the Wuhan isolate , which wUsing IEDB—ElliPro, we obtained a total of 88 linear B-cell epitopes with 12, 15, 17, 22, and 22 epitopes identified in China, England, the USA, Indian, and South African variants, respectively. When compared, we noticed that some of the sequential B-cell epitopes predicted in all the five SARS-CoV-2 isolates were similar in terms of length and amino acid composition. The length of the epitopes predicted ranged from 6 to 104. However, certain peptides such as ‘STEKSNIIRGWI’ and ‘TNSPRRARSVA’ were identified in England and USA isolates alone. The conformational epitopes predicted in the Indian and South African variants were noticeably the same. Peptides such as ‘PREGVFVSNGTHWFV’, ‘IAIPTNFTISVTTEI’, ‘SGWTAGAAAYYVG’, ‘VVVLSFELLHAPA’, and ‘SPRRARSVA’ were recognized as overlapping helper T-cell epitopes, which shows the comprehensive immunogenic potential of the epitope candidates against SARS-CoV-2 infection.As far as the B-cell epitopes were concerned, peptides identified as the best epitopes within Wuhan isolate were unique when compared to the recently evolved England and the USA variants, as expected. The peptides predicted in England and the USA variants were the same with the peptide ‘TESNKKFLPFQQF’ unique to the USA isolate only. Interestingly, the B-cell epitopes identified in the Indian and South African isolates exhibited the same patterns, which were different from the England and USA variants .147–154 (‘KNNKSWME’), spike496–501(’GFQPTN’/‘GFQPTY’),and spike1142–1149(‘QPELDSFK’) exhibited excellent surface accessibility in the spike trimer. Spike72–75(‘GTNG’) was found to be commonly predicted in both England and USA variants, while spike181–183(‘GKQ’) was noticed to be unique to the Wuhan isolate only. In parallel, spike460–462(‘NLK’) was observed to be unique to the Indian isolate only.However, spike1158–1161 (‘NHTS’) peptide was recognized to be a common segment between the Indian and the South African variants. The conformation of these residues was visualized in the Pymol graphics system and highlighted as a sphere  value determined that the complexes of the England and Indian variants showed the least deviation, unlike those of the other two variants A. AlthouSARS-CoV-2 undergoes rapid evolution leading to the emergence of different lineages and many variants of the pathogen, which have been identified from different parts of the world since its outbreak. Certain mutations reported have been associated with higher transmission rates, complex clinical presentation, and severity of the disease condition, while others didnot. D614G substitution caused by the SNV (A23403G) in the spike protein of a particular SARS-CoV-2 lineage was identified in early February 2020 ,19. AnotOur study methodology was based on immunological ‘lock and key theory’, wherein our immuno-genetic makeup is the lock and the SARS-CoV-2 variants are the key . Our appPhysicochemical properties predicted for the protein sequences revealed that the mutations in the spike protein of the recently evolved variants have enhanced its structural stability, which in turn promote its efficient binding with the ACE-II receptors as reported previously . AlthougIn the present study, immunoinformatics-based approaches that we exploited predicted potential T-cell epitopes, revealing that certain T-cell epitopes among them were dominantly antigenic, immunogenic, highly conserved, and common in all fiveSARS-CoV-2 isolates studied. However, each variant displayed a few unique epitope candidates as well. In congruence with the previous research findings, a few of the potential epitopes we predicted in our study have been reported in the spike protein of the Wuhan strain ,32. SincOverall, the combination of a high number of the best CTL epitopes with several HLA combinations, relatively low antigenicity, and high immunogenicity in the Wuhan spike protein demonstrated its poor neutralization ability. We believe that this reduced neutralization ability could be responsible for increased severity with prolonged symptoms, which in turn might have an impact on the cytokine storms that were previously observed in several COVID-19 cases ,35. In chttps://www.gisaid.org/, accessed on 3 December 2020) deposited from England (accession no EPI_ISL_655762), India (accession no EPI_ISL_1708422), and South Africa (accession no EPI_ISL_1706561) during November and December 2020. The Spike gene sequence was extracted at positions from 21,563 to 25,384 in the genome sequences and translated into S glycoprotein in one of the frames using BioEdit. Since the S glycoprotein of the isolates from Wuhan, China, and the United States deposited in January 2021 were available in NCBI, the protein sequences were directly retrieved from the NCBI-Protein database  in FastA format using the accession numbers NC_045512.2 and MW494124.1. Since each isolate that was chosen showed 97 to 100% sequence identity with the sequence of other isolates reported to GISAID during the same timeframe from the same geographical area, this study used a single representative sequence of each variant.We collected the Spike glycoprotein of five different SARS-CoV-2 isolates from five different countries based on the mutations recorded in various geographical regions from the start of the pandemic until January 2021. To begin with, we collected the whole genome sequences of SARS-CoV-2 isolates with N501Y, A570D, D614G, S982A, A262T, E484K, and K417Nmutations from the GISAID repository. Among the different variability metrics that were employed by PVS, we identified the conservative fragments in the multiple sequence alignment by plotting the variability. Additionally, the Expasy-Protparam tool  allowed us to qualitatively determine the protein by computing several physicochemical parameters such as molecular weight, theoretical PI, instability index, half-life, aliphatic index, and grand average of hydropathicity (GRAVY) for the selected B-cell epitopes.To determine the level of the conservancy, all the directly retrieved and translated spike glycoprotein sequences were subjected to variability analysis. A multiple sequence alignment (MSA) was performed for the sequences using the BioEdit-ClustalW multiple alignment program. The absolute site variability in the MSA created was ascertained using Protein Variability Server (PVS)  with the bootstrap value set to 1000 and Kimura-2 chosen as the tree drawing method.The evolutionary relationship of five different SARS-CoV-2 isolates from China, England, the United States, India, and South Africa was analyzed using Molecular Evolutionary Genetics Analysis X (MEGA X). Whole genome sequences of the selected isolates were aligned initially via the MEGA-MUSCLE program using the default parameters, and the alignment was exported in MEGA and FastA format. The genome sequences of SARS-CoV-1 (MK062180.1) and Middle-East Respiratory Syndrome virus (MW086530.1) retrieved from the NCBI-Nucleotide database were used as a reference for comparing with the above-mentioned SARS-CoV-2 virus genomes.Whole-genome sequence of Bat coronavirus (MW251308.1) was used as an outgroup for the tree construction . A maximhttps://services.healthtech.dtu.dk/, accessed on 11 January 2021). When the protein sequence was given as input, 9mer CTL epitopes were predicted from the query sequences for all the MHC-I supertypes. This server works based on the training dataset combining the prediction of MHC-I binding peptides, proteasomal C-terminal cleavage, and transporter associated with antigen processing aspects. Only those epitopes with a combined score greater than 1.00 were selected as CTL epitopes. To be stringent in the filtering process, this threshold was adjusted to 1.00, unlike in the previously reported experiments [http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html, accessed on 13 January 2020) with 0.5 threshold value, followed by MHC-I binding immunogenicity in Immune Epitope Design Database (IEDB) . The calculated immunogenicity scores indicates the probability of eliciting an immune response in the host. Epitopes with immunogenicity scores greater than zero were selected as positive epitopes.To understand the CTL and HTL-mediated host immune responses, T-cell epitopes with MHC-I and MHC-II binding affinities were predicted for the highly antigenic S glycoprotein of each variant using NetCTL 1.2 server . The cut-off value of IC50 was set to less than 200nM [http://tools.iedb.org/mhcii/, accessed on 20 January 2021), and those alleles with a percentile rank less than 1.5 were selected for further evaluation, as they were considered to be promising alleles with high binding affinity with their corresponding epitopes.MHC-I alleles interacting with each of the unique immunogenic peptides were predicted by the IEDB-MHCI prediction server , which individually accepted the sequence of the S protein of each variant, was chosen for the prediction of linear epitopes. With 0.5 as the specificity threshold value [http://tools.iedb.org/ellipro/, accessed on 25 January 2021) to identify conformational B cell epitopes using the homology-modeled three-dimensional structure of the surface glycoprotein of each variant with a minimum score value of 0.7 and a maximum distance of 6 Å. Ellipro also predicted discontinuous epitopes on the protein sequences submitted. Those epitopes predicted as potential linear epitopes with a high VaxiJen score were selected as the best B-cell linear epitopes. Discontinuous epitopes are increasingly explicit and have more dominant attributes over linear epitopes [With the help of online prediction servers, we forecasted the B cell epitopes on the S glycoprotein sequence of the SARS-CoV-2 variants under study. IEDB-Bepipred Linear Epitope Prediction 2.0 method .ToxinPred web server was utilized to examine the toxicity of the identified T-cell epitopes . ToxinPred works based on the Support Vector Machine (SVM) algorithm for classifying the toxic and non-toxic peptides depending on their parameters such as mutations, hydropathicity, hydrophilicity, hydrophobicity, and charge.For determining the allergenicity of the identified B and T-cell epitopes, AllerTop 1.0 server was used  with a sequence identity threshold of 90 percent. The degree of conservancy was determined based on the protein sequence set of SARS-CoV-1 Urbani strain; Wuhan, England, and USA isolates of SARS-CoV-2; and Bat-Coronavirus and reference comprising the epitopes at a specific sequence identity level.The conservation of the identified epitopes across diverse antigens was checked for their conservancy levels using the IEDB-Epitope Conservancy Analysis tool .Population coverage score was individually calculated against the population of the whole world, South East Asia, United Kingdom, Italy, France, the United States, and South Africa to compare the similarities and differences among those promising T-cell and B-cell epitope candidates across various countries. Due to the difference in the MHC restriction of T-cell response, identifying highly immunogenic peptides from different isolates with diverse HLA binding specificities represents more population coverage in the defined geographical regions. Population coverage for individually qualified T-cell epitope candidates and their binding HLA alleles was assessed by the IEDB Population Coverage Analysis tool . Similarly, the selected epitopes were modelled using PEP-FOLD server . Protein–protein docking was performed for the peptide structures and the HLA alleles using PatchDock server .To evaluate the binding affinity between the predicted epitopes selected from each SARS-CoV-2 variant and their corresponding MHC alleles, a molecular docking and simulation study was performed using the in silico tools. For this purpose, the crystal structure of the HLA protein molecules, namely HLA-A*02:06 (3OXR), HLA-B*51:01 (1E27), HLA-B*08:01 (1M05), HLA-A*32:01 (6AT5), HLA-B*57:01 (5VUF), HLA-B*15:01 (6VB3), HLA-A*02:03 (3OX8), HLA-A*01:01(4NQX), HLA-B*35:01 (4LNR), HLA-C*06:02 (5W6A), HLA-A*30:01 (6J1W), HLA-B*07:02 (6AT5), and HLA-A*68:02 (4I48), were retrieved from the RSCB Protein Data Bank (PDB) and prepared for further analysis. For those HLA alleles, namely HLA-A*30:02 and HLA-A*23:01, whose crystal three-dimensional structures were not available in PDB, their molecular sequences were obtained from IMGT/HLA database, and eventually their tertiary structures were modeled using SWISS-MODEL  for 15,000 ps using OPLS force field. We selected TIP3P water model for solvating complexes followed by the addition of ions to neutralize the solvent system. Periodic boundary conditions were set up for orienting the protein–peptide complex. Energy minimization of each system was performed by the steepest descent approach with a tolerance of 1000 kJ/mol/nm. Equilibration of the system was done employingNVT and NPT ensembles with the snapshot interval set to 100 ps. The trajectories were set to be generated every 2 fs and save every 2 ps. All of the trajectories were concatenated to calculate and plot root mean square deviation (RMSD), root mean square fluctuation (RMSF) data, and the protein–protein complexes were analyzed using xmgrace.Finally, based on their binding energy, for top-scoring protein–protein complexes, molecular dynamics simulation was carried out with GROMACS-2020 (We have attempted to address the raising concerns about the impact of the accumulating mutations in the spike protein of the SARS-CoV-2 variants on their immunogenic potential in driving host–pathogen interactions, the severity of the disease conditions, and the epidemiological fitness by applying the fundamental immunology principles while carrying out in silico simulations.Taken together, there exists a considerable difference between the antigenicity, immunogenicity, and the number of potential epitope/HLA combinations between the newly emerged SARS-CoV-2 variants. Our results corroboratively suggestthat the Wuhan spike protein possesseda higher number of T-cell epitopes with reduced antigenicity and increased immunogenicity, which might lead to increased severity of the disease condition. Nevertheless, the predicted T-cell epitopes disclosed increased antigenicity and moderate to high immunogenic potential with best epitope/HLA combinations in the British, the USA, Indian, and South African strains, making them less virulent and dominantly circulating variant during the current pandemic trends. Therefore, we suggest that the emerging virus strains could be weaker than the original COVID-19 strain, having more transmitting capacity. Alternatively, the emergence of variants could diminish the host immune response by evolving HLA-specific escape mutations, becoming more lethal at a population level. These immunogenic differences that arise among the succeeding variants of SARS-CoV-2 might interfere with the neutralizing activity of the current generation vaccines.Further, this study highlights that multiple peptides predicted from new variants may be useful to tailor the current vaccines against the latest variants as they have shown worldwide coverage. Nonetheless, further experimental validations are required to confirm the implications of the immunomodulatory effects of the predicted T- and B-cell epitopes among the newly emerged variants before they could be used for controlling the COVID-19 infection caused by the latest descendants of SARS-CoV-2 through the generation of effective immune response and long-term memory with the help ofproperly tailored vaccines.


Escherichia coli expression system. The immunoreactivity of recombinant substitutes was preliminarily confirmed by western blot analysis using epitope-specific antibodies. The sandwich enzyme-linked immunosorbent assay demonstrated that either FN3-epitopes-polyN or FN3-epitopes-4GS was highly sensitive, and FN3-epitopes-polyN exhibited better kinetics to specific antibodies than FN3-epitopes-4GS, showing a linear dose-response relationship in the concentration range of 0.06–12.85 ng/ml, which suggest that the polyN linker was more suitable for constructing the FN3-based substitute antigens compared to the 4 GS linker. Furthermore, the serum stability test and differential scanning calorimetry analysis showed that the recombinant FN3-epitopes-polyN maintained the original stability of FN3. Therefore, it was confirmed that FN3 could be engineered to construct a stable biomacromolecular substitute for displaying double epitopes of antigen proteins, such as NT-proBNP. In summary, a cost-effective strategy to produce NT-proBNP substitute antigens with good immunoreactivity and physicochemical stability was established in this work, which may provide potential uses for the production of other substitute antigens in the future.Construction of substitute antigens based on alternative scaffold proteins is a promising strategy in bioassay technology. In this study, we proposed a strategy for constructing substitute antigens derived from 10th human fibronectin type III (FN3) using two peptide epitopes of terminal pro-brain natriuretic peptide (NT-proBNP) as an example. The base sequences encoding the two antigenic epitopes of NT-proBNP were recombined into the FG loop region and the C-terminus of FN3, fused by 4 GS or polyN linker. The fusion proteins  were expressed and purified cost-effectively using an  Escherichia coli. In previous studies, we efficiently expressed monobodies and receptor proteins based on FN3 as a protein scaffold in E. coli (The 10th human fibronectin type III (FN3) domain has become one of the most widely used non-antibody scaffolds for engineering novel binding proteins . Several E. coli .in vitro. Studies have shown that linear epitopes are determined by the amino acid sequences rather than the 3D shapes, unlike the discontinuous conformation epitopes  linker or 4 GS (GGGGS) linker was introduced to ensure a better connection between the two epitopes; thus, two different fusion proteins, FN3-epitopes-4GS and FN3-epitopes-polyN, were synthesized. The results demonstrated that the FN3-based biomacromolecular substitutes with the same immunoreactivity of NT-proBNP could be rapidly and efficiently produced in the E. coli expression system, avoiding the complexity and high cost of expression in eukaryotic cell lines or synthesis by chemical methods. In summary, this study demonstrated that FN3 could be used as a scaffold protein to display the two specific antigenic epitopes to produce the corresponding stable substitute antigens of NT-proBNP cost-effectively via the E. coli system, with good biophysical characteristics of high stability and immunoreactivity.Using NT-proBNP as an example, we developed a strategy for producing stable recombinant antigen proteins by employing the FN3 scaffold protein to display two epitopes via the E. coli BL21 (DE3) used for protein production experiments was obtained from Sangon Biotech . Kanamycin, IPTG, TMB, and HEPES were obtained from Sigma-Aldrich (United States). The monoclonal antibody, HRP-conjugated mouse anti-human NT-proBNP 4NT1C-13G12, and mouse anti-human NT-proBNP 4NT1-15C4 were purchased from Hy-Test (Finland). HisTrap™HP (1 ml) for His-tagged protein purification was obtained from GE Healthcare (United States). PVDF membrane and 6x-His tag monoclonal antibody were purchased from Thermo Fisher Scientific (United States). All other chemicals and solvents were purchased from Sangon Biotech .NcoI and HindIII sites. Moreover, the genes encoding 4 GS linker or polyN linker were introduced upstream of epitope 62–73 generating two similar but different plasmids [named pET28a (+)-FN3-epitopes-4GS and pET28a (+)-FN3-epitopes-polyN]. The successful insertion of linker and epitope DNA into the plasmids was confirmed by DNA sequencing.The pET28a (+) vector and restriction enzymes were obtained from TaKaRa Biotechnology . Plasmids were constructed using standard molecular techniques. The genes encoding scaffold protein FN3, whose FG loop and C-terminus were replaced with two epitopes of NT-proBNP, were chemically synthesized by Nanjing GenScrip Tech Ltd. , and cloned into the pET28a (+) vector by http://raptorx.uchicago.edu/) for the protein secondary structure prediction and template-based tertiary structure modeling. The amino acid sequence of FN3 was retrieved from the RCSB PDB database (PDB:1FNA) and used as a target for homology modeling. The alignment of two protein structures was performed in PyMOL .To obtain the 3D structures of terminal linear-epitopes, homology modeling was performed using RaptorX (E. coli BL21 (DE3) cells. A single colony was picked and grown overnight at 37°C in 3 ml Luria-Bertani (LB) medium containing 50 μg/ml kanamycin (Kan). This culture was inoculated at a ratio of 1/100 into 50 ml LB media and further grown at 37°C with shaking until the OD600 reached 0.4–0.6, followed by addition of 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce the expression of FN3-epitopes at 25°C with shaking. Expression was allowed to continue for 6 h. After harvesting the cells by centrifugation, the crude product was extracted by ultrasonication and centrifugation at 12,000 g, and the proteins of interest were purified using HisTrap™HP (1 ml). In addition, to optimize protein purification, the supernatant of the cell lysate containing target proteins was first heated at 40°C for 10 min. Thereafter, the precipitate was removed at high speed to obtain the supernatant for importing to the HisTrap™HP. The expression and purification of target proteins were monitored by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) or western blot analysis. Protein concentrations were determined by bicinchoninic acid (BCA) assay kit .To produce recombinant FN3 proteins  that display the two epitopes of NT-proBNP, the plasmid pET28a (+)-FN3-epitopes-4GS or pET28a (+)-FN3-epitopes-polyN was transformed into After ultrasonication and centrifugation, the supernatants from bacterial extracts containing the proteins of interest were used for the thermal denaturation experiments. In brief, 80 μL of the supernatant was heat-denatured separately at different temperatures  for 10 min. The supernatant and pellet were separated by centrifugation at 12,000 g. Finally, the results were analyzed using SDS-PAGE gels.13-20) or mouse anti-human NT-proBNP 4NT1-15C4 . HRP-conjugated goat anti-mouse IgG was used as the secondary antibody. All blots were visualized using a Chemidoc™ XRS + system with Image Lab™ image capture software .The pellets corresponding to 1 OD unit of cells were resuspended in 0.1 ml PBS with loading buffer and boiled for 10 min. After cooling to room temperature, 0.01 ml samples were separated by 15% SDS-PAGE gels and transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane-bound to proteins was first incubated with primary antibody HRP-conjugated mouse anti-human NT-proBNP 4NT1C-13G12  as capture antibodies and incubated for 3 h at 37°C. Bovine serum albumin  was added to each well and incubated at 4°C overnight. Serially diluted purified protein was added to each well and incubated for 3 h at 37°C. HRP-conjugated mouse anti-human NT-proBNP 4NT1C-13G12 (0.35 μg/ml) served as a detection antibody and was incubated for 1.5 h at 37°C. The colored reaction product was developed by addition of 3,3′,5,5′-Tetramethylbenzidine (TMB) and the reaction was terminated after 30 min by 1 M sulphuric acid. The absorbance of each well was measured at a UV wavelength of 450 nm. Data were analyzed using Prism GraphPad software. Curves were adjusted using a sigmoid dose-response (variable slope) equation, Y=Bottom + (X^Hillslope)*(Top-Bottom)/(X^HillSlope + EC50^HillSlope)), where X is the logarithm of concentration and Y is the response. Y starts at Bottom and goes to Top with a sigmoid shape.DSC detection was performed using a standard procedure . The purin vitro stability of the recombinant proteins in human plasma, the purified FN3 and FN3-epitopes-polyN protein were spiked into the pooled plasma to a final concentration of 50 μg/ml. The plasma sample was incubated at 37°C for 80 h. Equal volumes of samples were collected at different time points and then detected by western blotting using 6x-His tag monoclonal antibody.To evaluate the To produce a biomacromolecule for displaying double antigenic epitopes of NT-proBNP, we chose the two positions (FG loop and C-terminus) of FN3 that are relatively far from each other in spatial structure as implantation sites to avoid possible interaction between the two epitopes. The amino acid sequence RGDSPASSK of the FG loop region of FN3 was replaced by epitope 12–21 of NT-proBNP. Furthermore, the epitope 62–73 of NT-proBNP was recombined with the C-terminus of FN3, and the linker KKGKGKKGK was added to facilitate coupling with other molecules, such as Biotin and peroxidase. Specifically, we introduced polyN or 4 GS linkers to fuse the above two epitopes in the FN3 domain to ensure the normal functions of the target proteins, resulting in the construct named FN3-epitopes-4GS or FN3-epitopes-polyN .E. coli BL21 (DE3). The recombinant FN3-epitopes could be recognized by commercial monoclonal antibodies specific to different regions of NT-proBNP, corresponding to amino acid residues 13–27 and 61–76. The recombinant proteins FN3-epitopes-polyN (16.7 kDa) and FN3-epitopes-4GS (14.5 kDa) were expressed under the control of the IPTG-inducible T7 promoter   . However21 (DE3)  under thFurthermore, we performed a thermal denaturation experiment to detect the level of degradation of FN3-epitopes-polyN protein under different temperature conditions (37–70°C) to facilitate the purification process by affinity chromatography. The results revealed that the FN3-epitopes-polyN protein transferred gradually from the supernatant to precipitate as the temperature increased. Approximately 30% of the heteroprotein precipitated at 40°C, whereas 60% of the target protein still existed in the supernatant at this temperature . Taking r2 values of standard curves were greater than 0.99  cells, reaching a yield of at least 40 mg/L in a 2 L shake-flask cultivation.Another important consideration is the low cost-benefit ratio to expand the clinical applications of using FN3-epitopes-polyN for NT-proBNP detection. As the results presented here, through the optimized chromatography purification process, the level of contaminating proteins was significantly reduced by up to 40% after the whole-cell lysates were heated. It is not easy to purify native NT-proBNP as a standard antigen as the concentrations of NT-proBNP or BNP in human serum are rather low and often contaminated with high concentrations of other proteins. It is expensive to produce antigen protein NT-proBNP from eukaryotic cell lines owing to high-cost cell culture and the easy degradation of NT-proBNP . In contE. coli. This work makes it possible to produce a large-scale stable detection calibrator in E. coli cost-effectively. This method may have general implications on producing other artificial recombined proteins, particularly for synthesizing unstable complex proteins in humans.In conclusion, using the two selected epitopes of NT-proBNP as an example, our proposed approach provides a simple and efficient strategy to produce biomacromolecular substitutes by engineering the FN3 scaffold in


It has been well known that electric pulse can be utilized to enhance the plasticity of metals, which is attributed to the change of dislocation dynamics, e.g., localized planar slip to homogeneous wavy slip. Here, we show another effect of pulse current, which facilitates texture weakening through room-temperature dynamic recrystallization and additionally improve the plasticity of a polycrystalline Mg-3Al-1Sn-1Zn alloy. By conducting a tensile test under electrical pulse, we found that the peak flow stress and fracture strain depend strongly on current density. As peak current densities increases, the flow stress drops and the fracture strain increases. Our Electron Backscatter Diffraction results suggest that dynamic recrystallization occurs at room temperature, which develops a weakened texture. Our work provides a new insight into electroplasticity mechanism in Mg alloys. Wrought magnesium alloys attract increasing attentions in lightweight structural applications because of their low density, high specific strength and stiffness, and good damping capacity ,2,3. HowHowever, the physical origin of electroplasticity is still not clear in the literature, due to the variety of different metallic materials. For example, Zhao et al.  recentlyIn this work, electropulsing with different current densities was imposed to an Mg-3 Al-1 Sn-1 Zn (ATZ311) alloy during uniaxial tension at room temperature. The microstructure evolution and the recrystallization mechanism of ATZ311 alloy during electropulsing treatment were investigated in detail based on EBSD analysis. We show that dynamic recrystallization under pulsed current not only lowers the flow stress and accelerates recrystallization, but also refines grain and weakens texture. This work may provide a new insight to understand the dynamic recrystallization assisted by electropulsing in magnesium alloy.The extruded Mg-3 Al-1 Sn-1 Zn (wt. %), denoted as ATZ311 in this work, a sheet with a thickness of 1.2 mm was selected as raw material. Subsequently, the sheet was annealed at 200 °C for 1 h to relieve the residual stress, which acts as the initial alloy. The initial alloy has a near-equiaxed grain structure with an average grain size of ~43 μm .−3 s−1. The uniaxial tensile experiment under pulse current along extruded direction (ED) is performed on a WDW-200 tensile machine , with an initial strain rate of 10P). It is clear that the flow stress drops but the fracture strain increases with an increase of JP, which is in complete agreement with the case of other metallic systems under pulse current [2, respectively, which indicates that increased peak current densities lead to upward initial temperatures. Specifically, under 20 A/mm2, the temperature change with increasing strain is very small, with a maximum temperature of only 41 °C, close to room temperature. Under 30 A/mm2, the temperature remains near constant before the strain of ~24%, but the temperature gradually increases until failure, with a maximum temperature of ~239 °C. current ,11,13. T2. When the strain is 24%, the material has fractured at 20 A/mm2, while necking occurs at 30 A/mm2. So, the tensile tests are interrupted at a strain of 8% and 24% to investigate microstructure evolution in this work.At a strain of 8%, the material reaches ultimate tensile strength at 30 A/mm2. As noted earlier, this current density almost did not produce high temperature. Accordingly, this tensile deformation can be regarded as room temperature. As it is well known, both basal slip and {10–12} twinning is firstly activated during deformation. On the whole, from At the initial stage, the formation of twins can hinder the dislocation slip because the twin boundary originating from the crystal reorientation between the twinned zones and surrounding zone within grain can effectively hinder dislocation slips, thereby resulting in a strong dislocation pile-up at the twin boundary ,19. As fSuch a process offers a possibility for the nucleation of multiple twinning dislocations owing to the interaction of slipped dislocation with the twin boundary . An aval2. In the initial sample, there are a few {10–12} twins in the parent grains and still no recrystallized structure  existed within the parent grains. This indicates the occurrence of dynamic recrystallization (DRX) in this strain.tructure a,c, alth2. At 100 °C, it is not enough to drive the occurrence of DRX, implying that electrical effect can significantly promote DRX, besides the thermal effects of the pulse current. Note that the DRX degree in different parent grains is different possibly due to orientation discrepancy [From crepancy .According to an investigation on the influence of initial texture on the dynamic recrystallization in AZ31 alloy, it was shown that DRX takes place preferentially in those of grains favorable to the dislocation slips . In addiWe counted the misorientation distribution for more than 60 grains in 2.c-axis rotates from being parallel to the ED to ND, which strengthens ND component. The start of tension deformation is accompanied by basal <a> slip, even for those grains with low Schmid factor of basal slips [During tension, those grains with ED component are in a favorable orientation for {10–12} twinning see in a due to al slips . For theal slips . Consequal slips ,28.2, the reduction of the texture intensity can be attributed to detwinning because of no recrystallization in this case. Under, 30 A/mm2, apparent recrystallization occurred, which gives rise to an obvious decrease of the texture intensity. As mentioned earlier, twin boundaries provide nucleation sites, promoting DRX. It must be pointed out that further DRX at twin boundaries should weaken the twin texture caused by {10–12} twinning [With further strain to 24%, the texture intensity decreases in both conditions. Under 20 A/mmtwinning . Meanwhi2, which could be attributed to gliding dislocations across twin boundaries under electron wind force, while significant DRX formed under 30 A/mm2. We proposed that electrical effect can significantly promote DRX except for the thermal effects of the pulse current because pulsed current only causes a low temperature of ~100 °C that is difficult to promote DRX. In this case, further DRX at twin boundaries and continue DRX should develop random texture, therefore, weakening initial texture. By investigating the room temperature dynamic recrystallization, our work may provide a new insight into electroplasticity mechanism in Mg alloys.In this work, we systematically investigated the microstructure evolution of Mg-3Al-1Zn-1Sn alloy during uniaxial tension under pulsed current, during a room temperature dynamic recrystallization process. Our results show that the flow stress drops but the fracture strain increases with an increase of peak current densities. In the strain of ~8%, {10–12} twins are observed at both conditions, but no DRX. Due to {10–12} twins and non-basal slips, basal texture are strengthened and {11–20} is transformed into {10-10} being parallel to ED. Further strain to ~24%, detwinning occurred under 20 A/mm


In this study, we evaluated the fracture resistance of three commercially available prefabricated primary zirconia crowns and their correlation with dimensional variance. Methods: a total of 42 zirconia crowns were selected from three companies, (1) NuSmile primary zirconia crowns, (2) Cheng Crowns zirconia, and (3) Sprig EZ crowns. The crowns were divided into two groups based on their location in the oral cavity and further divided into subgroups based on the brand. All of the samples were subjected to fracture tests using a universal testing machine. Results: the mean load observed was highest with Cheng Crowns zirconia anterior crowns (1355 ± 484) and the least load was seen with Sprig EZ anterior crowns with a mean load of 339 ± 94. The mean load observed was highest with Cheng Crowns zirconia posterior crowns (1990 ± 485) followed by NuSmile posterior crowns and the least load was seen with Sprig EZ posterior crowns with a mean load of 661 ± 184. Conclusion: the Cheng crowns showed the highest fracture resistance amongst all three groups. Overall, the zirconia crowns (anterior and posterior) tested showed optimum mechanical properties to withstand the masticatory forces. Early childhood caries (ECC) is the most common childhood disease and is often accompanied by serious comorbidities affecting the quality of life of children, their families, the community, and the health care system . VariousIn recent years, prefabricated primary zirconia crowns for both anterior and posterior primary teeth have been developed ; howeverThe purpose of this study was to evaluate the fracture resistance of three commercially available prefabricated zirconia crowns and their correlation with dimensional variance.X-axis, height—Y-axis, and width—Z-axis). Modeling wax (GEO Crowax and Perfect Wax) was used to create a negative replica of the crowns, which was later invested and turned into metal dies. The crowns were then tried out onto the corresponding metal dies to ensure a passive fit between each crown and metal die, which were then embedded into cylinders filled with orthodontic resin material for placement in the metallic holders. The crowns were cemented onto the dies with glass ionomer luting cement  following the manufacturer’s instructions and allowed to set for 24 h. A pressure of 30 Newtons was applied onto the crowns during cementation using a customized surveyor. The fracture resistance (N) was tested using a measured universal testing machine  with a 90° angle at a ramp rate of 1mm/min. The samples were placed into an appropriate metallic holder and the forces were applied onto the crowns with two different tips (chisel for anterior and flat rounded for posterior) until the crown fractured. The values were recorded at the time of fracture, and the Instron was reset after each value was recorded. Furthermore, the load was balanced, and the tip attached was returned to its starting position to ensure standardization.The study was carried out following institutional review board approval at the College of Dentistry at Prince Sattam bin Abdulaziz University (ID: PSAU2020025). Forty-two zirconia crowns were used from three companies: NuSmile primary zirconia crowns , Cheng Crowns Zirconia , and Sprig EZ Crowns . The crowns were divided into 2 groups based on their location in the oral cavity (18 anterior/24 posterior) and then into subgroups based on the brand. The sizes used were (A4R) for NuSmile anterior (NA) and (E5L) for NuSmile posterior (NP), (CR4) for Cheng Crowns anterior (CA) and (ELL5) for Cheng Crowns Posterior (CP), (E4) for Sprig anterior (SA), and (K5) for Sprig Posterior (SP). The maximum dimensions of the samples were measured using an electronic dental caliper . Data were compared using 1-way analysis of variance at 95% confidence intervals (CI) and a significance level of 0.05 was used. Tukey’s post hoc test was used for inter-group comparisons. ANOVA was considered as there were more than two groups and the mean value for each group was compared. Data were explored for normality by checking the data distribution and using Kolmogorov-Smirnov and Shapiro-Wilk tests.x-axis, y-axis, and z-axis); the total load was applied for the three different crowns. The mean load observed with the anterior crowns was highest with Cheng crowns (1355 ± 484), followed by NuSmile crowns (1192 ± 120). The least load was seen with Sprig crowns with a mean load of 339 ± 94. The mean load observed was statistically significant with an ANOVA value of 20.76 and a p-value of <0.001. The mean load observed with the posterior crowns was highest with Cheng crowns (1990.63 ± 485.39) followed by NuSmile crowns (1013 ± 240). The least load was seen with Sprig crowns with a mean load of 661 ± 184. The mean load observed was statistically significant with an ANOVA value of 34.77 and a p-value of < 0.001. p-values among anterior crowns and posterior crowns along different axes. p < 0.05 was considered as statistically significant. The following A total of 42 crowns were tested . Failure was not noted in any of the samples; therefore, no samples were excluded. Zirconia has emerged as an ideal material to use in fabricating crowns since it meets clinical requirements and has the ability to withstand masticatory forces ,13,14,15In the present study, Cheng’s zirconia crowns demonstrated the highest fracture resistance followed by NuSmile; the least fracture resistance was demonstrated by Sprig.Townsend et al. (2014) measured the fracture resistance of three commercially available pediatric zirconia crown companies , and Kinder Krowns) and its correlation with crown thickness and used pre-veneered stainless-steel crowns as a control group. The findings concluded that the pre-veneered group was stronger than any of the zirconia groups and that there was a directly proportional relationship between an increase in both thickness and resistance to fracture. However, in this study, EZpedo (Sprig) zirconia crowns were more resistant to fracture than Cheng zirconia crowns . MoreoveA similar study conducted by Al Shobber et al. 2017) compared the fracture resistance of two commercially available primary esthetic crown companies (NuSmile and Cheng Crowns) and two types of esthetic crowns (zirconia and pre-veneered stainless-steel crowns). Similarly to this study, the samples were tested for their fracture resistance using an Instron model 5965, with a 90° angle and ramp rate of 1mm/min, after the samples were cemented and mounted onto negative replicas. The results showed that Nusmile zirconia measured the highest fracture resistance (937 ± 131 N) followed by Cheng Crowns zirconia (751 ± 102 N); NuSmile pre-veneered (482 ± 76 N) and Cheng Crowns pre-veneered (415 ± 12 N) did not demonstrate a major difference. Overall, the study concluded that zirconia crowns were higher in fracture resistance compared to pre-veneered crowns, especially NuSmile crowns [ comparedStefan Kist et al. (2019) conducted a study in which prefabricated esthetic crowns were compared with conventional restorative crowns. They compared commercially available zirconia crowns, CAD/CAM-manufactured zirconia crowns, pre-veneered stainless-steel crowns, and conventional stainless-steel crowns under two variables which were fracture loaded under three conditions . Pre-veneered stainless-steel crowns exhibited the highest fracture resistance (6251 N) within the no-treatment group, while CAD/CAM zirconia crowns had a fracture resistance of 2444 N and prefabricated zirconia crowns had a maximum fracture resistance of 1582 N. However, in the saliva aging and chewing simulation groups, the fracture resistance of pre-veneered stainless-steel crowns was negatively affected (5348 N for the aging group and 3778 N for the chewing simulation group) while the fracture resistance of the zirconia crowns remained similar. Furthermore, when gauging the survival rate, zirconia crowns and pre-veneered stainless-steel crowns had a 100% survival rate during chewing simulation, but only 41.7% in the conventional stainless-steel group [Another study conducted by Beattie et al. (2011) evaluated the fracture resistance of three brands of primary esthetic stainless-steel crowns. The crowns were cemented onto idealized epoxy dies with GIC and the samples were then tested using a universal testing machine. Moreover, in order to replicate a cusp contact environment, a ball fixture was used to deliver the forces on the samples with a ramp rate of 1mm/min. Data collected from the study were analyzed using a one-way analysis of variance. The study concluded that forces needed to fracture any of the three brands of SSCs were quite similar to each other . In addition, the recorded forces also exceeded the natural forces produced by biting [Furthermore, Singh et al. (2020) evaluated the effects of full-mouth rehabilitation on bite forces in dental pediatric patients in both primary and mixed dentition. Thirty subjects—comprising an almost even number of male and female participants—were included in this study, and the maximum bite force was voluntarily recorded for each participant before the start of dental treatment and one month after completion. The collected data were analyzed, considering variables that might affect the maximum bite force generated by a participant such as gender, age, height, weight, and extent of caries. The total mean maximum force of all of the participants before treatment was 167 N, in which the male subjects recorded a mean biting force of 175 N and the female subjects recorded a mean biting force of 166 N. Prior to the completion of dental treatment, the bite forces of the participants were once again measured, and a mean maximum bite force of 182 N was recorded [When considering the above-mentioned results obtained by Singh et al. (2020), it is clear that all three commercially available crowns have optimum mechanical properties which can tolerate excessive masticatory forces.From our study, there is a direct correlation between the increase in dimensions and the zirconia crowns’ ability to withstand a higher load/force. The overall dimensions of the crowns were shown to be somewhat correlated to the crown’s strength. Importantly, a potential reason for Sprig zirconia crowns demonstrating the lowest fracture resistance scores was because of a retentive feature within the crowns. Cheng’s zirconia crowns showed the highest fracture resistance (Newton), followed by Nu Smile and, lastly, Sprig. In general, the zirconia crowns (anterior and posterior) tested showed optimum mechanical properties to withstand the masticatory forces.


Grain quality is one of the key targets to be improved for rice breeders and covers cooking, eating, nutritional, appearance, milling, and sensory properties. Cooking and eating quality are mostly of concern to consumers and mainly determined by starch structure and composition. Although many starch synthesis enzymes have been identified and starch synthesis system has been established for a long time, novel functions of some starch synthesis genes have continually been found, and many important regulatory factors for seed development and grain quality control have recently been identified. Here, we summarize the progress in this field as comprehensively as possible and hopefully reveal some underlying molecular mechanisms controlling eating quality in rice. The regulatory network of amylose content (AC) determination is emphasized, as AC is the most important index for rice eating quality (REQ). Moreover, the regulatory mechanism of REQ, especially AC influenced by high temperature which is concerned as a most harmful environmental factor during grain filling is highlighted in this review. Rice is one of the most important staple foods, feeding more than half of the population in the world. Developing varieties with high quality is a major aim for rice breeders . Starch Wx on chromosome 6 is the major locus for rice AC and has been detected in almost all studies (Wx gene encodes granule-bound starch synthesis I (GBSSI), which is the key enzyme for amylose synthesis in rice  × Oryza nivara (Oryza sativa indica (Caiapo) × Oryza glaberrima  under different environments, most CSSLs showed varied D-value with their parents lines. Such results suggested that most QTLs responsible for AC determination are not stable under varying environments, and we deduced that these loci might be involved in genetic-environment interactions of AC control. Thus, AC is genetically controlled by the major locus Wx and several minor loci, such as qSAC3, qAC8-1, and qAC8-2, which could stably affect AC under multiple conditions. Fine mapping and characterization of the candidate genes of qSAC3, qAC8-1, and qAC8-2 will help us to understand their relationship with Wx and establish the exact genetic basis of AC control.The genetic control of rice AC is relatively complex. Genetic studies using different populations, such as doubled haploid (DH), recombinant inbred lines (RILs), BCmFn, and chromosome segment substitution lines (CSSLs), have been performed , and a s studies . The Wx  in rice . Two allectively . Subsequisolated . Variatijaponica , Oryza sa nivara , and Oryaberrima . Interesus study . Compareh low AC . In addi studies . MoreoveMany key enzymes, such as ADP glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble starch synthase (SS), starch branching enzyme (SBE), and starch debranching enzyme (DBE), are involved in starch synthesis in rice seeds. Most of the enzymes that have isozymes and isoforms preferentially expressed in endosperm are responsible for starch synthesis in rice seeds, such as GBSS1 , SS1, SS2a , SS3a , SBE1 , and SBE2 . Previous studies proposed that AM and AP were synthesized by different enzymes in rice. AM is mainly synthesized by GBSS1, while AP is synergistically regulated by multiple enzymes, such as SSs, SBEs, and DBEs . Howeverss1 mutant, chains of DP 8–12 are decreased, whereas DP 6–7 chains are increased, which indicates that SS1 elongates DP 6–7 chains to DP 8–12 chains of AP (SS3a is mainly responsible for the generation of long chains (DP ≥ 30) in AP  to the L-type (mostly in indica cultivars) and increase GT significantly as well  in AP . The ss3ndosperm . This res1 seeds . However sterile . These d sterile . The actactivity . SS2a is quality , 2011. I as well .SS1 would cause a great reduction in AP. However, the appearance of seeds and starch granules remained normal, and the AC remained unchanged in the ss1 mutant , which are mainly produced by SS1, might be important substrates for AM synthesis.It was generally believed that SSs  were only involved in AP synthesis. Recent studies noted that these SSs might also affect AM synthesis. The short chains of AP produced by SSs could supply substrates for the synthesis of AM . SS1 is 1 mutant . Moreovepe seeds , and knoensation . We deduSS2a might be another SS gene involved in AM synthesis. Introducing the high activity allele IndSS2a  into rice plants could raise the AC whereas the effects are ecotype dependent  in AP and no obvious alteration in AM synthesis  of AP might be important substrates for AM synthesis and both SS1and SS2a play important roles in this process. More evidence for this conception should be collected in the future by using other technologies, such as radio isotope tracer.ependent . In the atically , while ict on AC . The totd by Wxb . Therefoynthesis , althougsbe1 seeds, both intermediate chains of DP 12–21 and long chains of DP ≥ 37 were reduced which resulted in a GT decrease in sbe1 seeds  seeds  double mutant, similar to ss1/ss3a, normal starch granules could not be formed, and very few seeds could be produced  seeds . This inproduced . These rWx gene, and many allelic Wxs, such as aWx, bWx, inWx, mqWx, lvWx, and wx, explain a major AC variation in rice germplasm. With the development of biotechnology, more novel Wx alleles were generated and many important rice materials with different AC were produced by genetic modification or gene editing recently, and most of them occurred at coding and promoter region of Wx gene , which is responsible for AM synthesis in Arabidopsis leaves, was newly found to help the GBSS protein localize to starch , another homolog of PTST1 in rice, is the most likely such gene. FLO6 contains the CBM48 domain at the C-terminus and can interact with GBSSI, GBSSII, and ISA1 to help them target starch in endosperm. Total starch and AC decreased significantly, and starch granules were abnormal in the flo6 mutant  proteins involved in RNA binding and metabolism in plant mitochondria. The processing of mitochondrial NAD genes, such as NAD1 and NAD5, was defective in flo10, flo14, or flo18 mutants  domain-containing protein  mutants , which catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3), is essential for the early development of endosperm . Fertilie genome . Seed de mutants . Some MA quality . ChIP-PCsilenced . Other t OsMADS1 , OsMADS3OsMADS34 , and OsMOsMADS29 , also co altered . Moreoveelopment . OsMADS7h OsFIE1 . All thejaponica cultivars under high temperature  at the splicing site of the first intron in bWx causes low splicing efficiency (bWx under cool temperature conditions (18°C) were much higher than those under optimal temperature conditions (25°C) and HT conditions (33°C). Two major mature transcripts could be generated from the bWx allele under optimal temperature conditions. The large one is spliced after CT repeats (site 2), and the small one is spliced near the donor site of aWx  under HT was much smaller than that in Nipponbare (japonica). Using the CSSLs between 9311 and Nipponbare, several QTLs, qHAC8a, qHAC8b, and qHAC4, responsible for AC stabilization under HT were characterized. Introducing the indica allele of these loci into Nipponbare could enhance the splicing efficiency of bWx, which suggested that increasing the pre-mRNA processing efficiency of the Wx gene might be an important regulatory mechanism for maintaining AC stability at HT  whose activity was affected by Ca2+ sensor calmodulin (CaM) in plants  and the National Natural Science Foundation of China (31401031).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.