However, their mechanical performance is still uncertain. The aim of this study was to evaluate the mechanical behavior of a combination of a zirconia abutment and monolithic all-ceramic zirconia and lithium disilicate crown manufactured with a chairside workflow, connected to titanium implants with two types of internal connection—tube in tube connection and conical connection with platform switching. They were thermally cycled from 5 °C to 55 °C and were subjected to a static and fatigue test following ISO 14801. The fractured specimens from the fatigue test were examined by SEM (scanning electron microscopy). Simulations of the stress distribution over the different parts of the restorative complex during the mechanical tests were evaluated by means of  FEA. The mechanical performance of the zirconia abutment with an internal conical connection was higher than that of the tube in tube connection. Additionally, the use of disilicate or zirconia all-ceramic chairside CAD/CAM monolithic restorations has similar results in terms of mechanical fracture and fatigue resistance. Stress distribution affects the implant/restoration complex depending on the connection design. Zirconia abutments and monolithic restorations seem to be highly reliable in terms of mechanical resistance. Nowadays, osseointegrated implants are becoming the most common treatment alternative among clinicians in order to restore missing teeth as they have been proven to have high survival rates in the scientific literature. Conventionally, these implants were restored using titanium abutments and porcelain fused to metal crowns. However, using a metallic component has numerous drawbacks. The aesthetic result of the restoration could be dimmed by the absence of naturalness, and not only the white aesthetics could be jeopardized, but the pink aesthetics could also be affected if a grey shadow appears in the gingival margin. The use of materials with improved biomimetic characteristics such as dental ceramics has allowed us to achieve better aesthetic results regarding the appearance of our restorations ,5,6,7,8.Developments in ceramic materials, equipment, and technology have made it possible to manufacture an indirect aesthetic restoration in a single appointment. CAD/CAM was created to give the clinician the possibility of designing and manufacturing a restoration without the need for laboratory assistance, provisional restorations, or conventional impressions . The comThe implant connection should also be considered as is an important variable of the restorative complex; traditionally, hexagonal external connections were used. However, in recent years, internal connections have become popular as they preserve more crestal bone. In spite of this, there is a wide range of internal connections, and their biological and mechanical properties are not the same. Conical connections have gained the upper hand in recent years due to their internal fit and less screw loosening. These conical connections incorporate the concept of platform switching. This consists of the placement of abutments or other prosthetic elements with a narrower diameter than that of the implant platform. In the last decade, many clinical and histomorphometric studies have been published to demonstrate the efficacy of this procedure in preserving peri-implant bone ,18,19,20Therefore, zirconia abutments with a titanium base are promising candidates to substitute for titanium abutments from an aesthetic and biological point of view. However, information on the mechanical performance of zirconia abutments supporting ceramic crowns and the influence of the implant connection type is limited. Because of this scarcity of mechanical in-vitro studies and lack of significant clinical studies, the mechanical performance of chairside CAD/CAM zirconia implant abutments supporting translucent lithium disilicate and zirconia monolithic crowns should be analyzed. The aim of this study is to evaluate the mechanical behavior of chairside customized zirconia abutments with two different implant connections under static and fatigue conditions. Moreover, in vitro tests were reproduced using engineering tools like von Mises and finite element analysis, as they can consider all the different variables  and calculate the stress distribution of a system in which dental implant components are involved ,25.The first null hypothesis is that no differences in mechanical performance would be seen between the systems with the two types of connection . The second null hypothesis is that no differences in mechanical behavior would be registered between the systems with the two different crown materials (lithium disilicate and zirconia).n = 15) were included depending on the material of the crown: group zirconia (Group Z) and group lithium disilicate (Group LD):Subgroup Tube ZZ: 15 specimens, using a tube in tube connection implant and a customized zirconia abutment with a monolithic zirconia CAD/CAM crown with standardized anatomy of a maxillary premolar.Subgroup Tube ZLD: 15 specimens, using a tube in tube connection implant and a customized zirconia abutment with a monolithic lithium disilicate CAD/CAM crown with standardized anatomy of a maxillary premolar.Subgroup Conical ZZ: 15 specimens, using a conical connection implant and a customized zirconia abutment with a monolithic zirconia CAD/CAM crown with standardized anatomy of a maxillary premolar.Subgroup Conical ZLD: 15 specimens, using a conical connection implant and a customized zirconia abutment with a monolithic lithium disilicate CAD/CAM crown with standardized anatomy of a maxillary premolar.This in vitro study  included 30 Camlog Screw Line implants on a Promote plus surface (4.3 mm diameter and 11 mm height) with a transmucosal part of 0.4 mm, with a tube in tube trilobular connection, and 30 Conelog Screw Line implants (4.3 mm diameter and 11 mm in height) with a conical connection. For both implants, a customized zirconia abutment with a titanium base was used. Two subgroups  and the other with a replica of Conelog Screw Line implants (diameter 4.3 mm in the same position) were used.For the digitization of the casts, the digitalization unit Cerec Onmicam Intraoral Scan (Sirona) was used. A prior wax up of the restoration was performed with the aim of standardizing the anatomy of all of the crowns. After the digitalization of both models, we proceeded to design the restorations choosing the biogeneric copy option with the software CEREC 4.4 . A special scanbody  was used to locate the replicas directly on the titanium bases for the zirconia abutments. Once the models were digitalized, we proceeded to design an anatomical zirconia abutment for the Camlog Titanium base CAD/CAM and for Conelog Titanium base CAD/CAM with the same shape and anatomy.Once the abutments were designed, they were milled using the MCXL milling unit of the Cerec system. Pre-sintered blocks of Incoris Zi (Dentsply Sirona), as well as the following milling burs were used: Step Bur 20 and Cylinder Pointed Bur 20. For complete sintering of the zirconia, the In-Fire Htc Speed  oven, which is specific for this material, was used in order to obtain a correct shrinkage of the material. All-ceramic monolithic crowns were milled from pre-sintered blocks of Emax-CAD  lithium disilicate and Incoris Tzi (Sirona) zircona. For the crystallization of the lithium disilicate, the Programat CS2  oven was used. For the complete sintering of the zirconia, the In Fire Htc Speed (Sirona) oven, which is specific for this material, was used in order to obtain the correct shrinkage of the material.®, Danville Materials) with 50 microns aluminium oxide particles at a pressure of 1 atm, and they were cleaned in an ultrasonic cleaning unit. The lithium disilicate crowns were treated with 4.5% hydrofluoric acid for 20 s, washed up with water and cleaned in an ultrasonic unit, then silanized with monobond Plus (Ivoclar Vivadent). The zirconia abutments were luted to the titanium CAD/CAM base with a dual polymerization resin cement speed Cem Plus (Ivoclar Vivadent), positioning it with hand pressure. Before luting the crowns, a randomization table was performed in which the implant, abutment, and crown were involved. Once the randomization was completed, each abutment was screwed to the corresponding implant with an insertion torque of 20 N·cm, following the manufacturer’s recommendations, using a torque wrench and sealing the screw access with polytetrafluoroethylene (PTFE).Before cementation, the internal and external surfaces of the zirconia abutments and the internal surfaces of the zirconia crowns were sand-blasted with a sandblasting unit for clinical use  was used for a time between 2 and 4 s for each surface of the crowns and the abutment. Once the excess was removed, the photopolymerization was completed by 40 s per surface. Representative pictures for each group are presented in Dual polymerization resin  was used to cement the crowns. To standardize the cementation phase, all restorations were cemented using a clamping jaw and a torque wrench with a pressure of 40 N/cmThe aging of the specimens was carried out using an automated thermocycling device for dental materials . The speAll of the specimens were mounted perpendicular to the horizontal plane in the center of epoxy resin holder  with a Young’s modulus Conelog Screw Line implants comparable to that of the human mandible , followihttps://www.shimadzu.eu/trapezium-x-software, accessed on 18 May 2021). The test machine was programmed to stop the force test process for a greater displacement of 1.5 mm. The maximum force (Fm) was considered as the load–bearing capacity (p) at which the load–displacement curve first deviated by 10% from the regression line in the elastic interval was recorded as an indicator for the initiation of plastic deformation . The load was applied to the specimens’ occlusal surface of the palatal cup at a crosshead speed of 0.5 mm/min with an angle of 30° . Load .The mechanical fatigue tests were carried out using an electromagnetic testing machine  in dry conditions operated under load control and with a sinusoidal wave form at 2 Hz. The load ratio (minimum to maximum loading ratio) was equal to 10 with a unidirectional and tilted (30°) pulsating load. A fatigue life test was carried out until the specimens showed failure, or it was interrupted at Ny system ) for they system . Six samBased on previous studies ,30,32, t® software . Descriptive statistics are expressed as means and standard deviations (SD) for the quantitative variables. A comparative analysis was performed by comparing fracture resistance and plastic deformation data among the four groups using Analysis of Variance (ANOVA). The statistical significance was set at p < 0.05.Statistical analysis of all of the variables was carried out using SPSSAll of the tested specimens were photographed with a Canon 700 D with a 100 mm macro lens with extensor rings and an annular flash following a photographic protocol.For the microstructural analysis of the deformed specimens after the static mechanical test, the samples of each group were embedded in a transparent resin . The samples were cut on the longitudinal axis using a cutting machine with a diamond saw . The final slice of each specimen was polished with silicon carbide papers and finally with a silica suspension.The microstructural study of the deformed specimens after the static mechanical test and the fatigue fracture surfaces of the broken samples were examined using SEM  at different magnifications.A finite element analysis was conducted in which different simulations were carried out for the different restorative options. It was intended to obtain the values in terms of the stress for each of the specimens, and to compare them with each other in static and cyclic fatigue conditions according to the ISO 14801. The CAD models of each piece were modelled in Solidworks 2019  and were imported into Ansys Workbench 2019 , where the corresponding finite element models were created, and different properties were applied to each piece depending on the material, boundary conditions, contacts, existing loads, stereolithography (STL) mesh, etc. The element was defined by 10 nodes with three degrees of freedom at each node: translations in the nodal x, y, and z directions. The element had plasticity, hyperelasticity, creep, stress stiffening, large deflection, and large strain capabilities. It also had a mixed formulation capability for simulating deformations of nearly incompressible elastoplastic materials, and fully incompressible hyperelastic materials. The material properties were assumed to be homogeneous, isotropic, and linearly elastic, and the applied state equation was the Hooke Law through Young and Poisson Modulus specification . The totThe crowns that were cemented to both the abutment or Ti base, as well as the implant to the mold, are considered to have a “bonded” type contact. For the rest of the pieces, a “friction” type contact was considered between the materials. It was verified that there were no contact opening problems. The three contact surfaces between the testing machine and the epoxy resin were simulated as a fixed support. The preload (first torque) of the screw was previously adjusted in order to ensure the reality in the application and the distribution of the Von misses forces, and then the external load was applied according to the ISO norm. Therefore, the state of the contacts and the accuracy of the created mesh were analyzed before considering the created models to be correct. Finally, von Mises stress maps and safety factor maps were performed in FEA.m) of 1050 ± 92 N and the Conical ZLD subgroup obtained a Fm = 1102 ± 112 N. The Tube ZZ subgroup obtained a Fm = 1015 ± 73 N and the Tube ZLD subgroup obtained a Fm = 975 ± 64 N. There were no statistical differences (p > 0.05) between the subgroups. However, the highest values were registered in the conical connection group. No abutment or crown was fractured during the static test. On the other hand, significant differences (p < 0.05) were observed in the load at plastic deformation (Fp) among the different connection configurations . The highest values were registered in the conical system.The Conical ZZ subgroup obtained a maximum load . On the other hand, no differences in mechanical behavior were registered between the systems with the two different ceramic restoration materials (zirconia and lithium disilicate). Therefore, the first null hypothesis was rejected and the second null hypothesis was accepted.A challenging aspect in treatments with dental implants is the placement and subsequent restoration in high aesthetics areas, mainly due to the fact that the level of peri-implant bone support and soft tissue dimensions are critical factors in the final result. It goes without saying that from a biological and histological point of view, the platform switching is, without a doubt, indicated. However, the effect of placing narrower CAD/CAM zirconia implant abutments with conical implant−abutment connections for platform switching supporting single all-ceramic crowns made with a chairside workflow on the transmission of forces along the implant−abutment complex and its corresponding mechanical behavior is unknown. In this research, one of the study variables was the implant design. Two types of configurations have been studied. On the one hand, a cylindrical connection with a regular platform and without platform switching using an abutment of the same diameter, on the other hand, a conical and the concept of platform switching incorporated into its design, forcing the use of a narrow abutment for the restoration. Logic would suggest that placing a narrower abutment of a smaller diameter would increase the concentration of the stresses at the level of the abutment neck and affect its mechanical performance. However, the results of this investigation indicate the opposite.In the case of the static test, the mean fracture resistance and plastic deformation point of the zirconia abutment with an internal conical connection group were higher than that of the zirconia abutment with the tube in tube connection. However, in both cases, these were still greater than the typical masticatory forces (148–354 N) ,37, and 6 at a load as high as 400 N. As we have already mentioned, the weakest component of the whole restorative complex was the screw.After performing the fatigue tests, a radically different behavior was observed between both implant configurations. While the study groups with tube implants largely failed during the tests, conical implants survived 2 × 10The fact that there was so much difference between both implants from a mechanical fatigue point of view lies in the fact that the transmission of forces to the clamping screw was higher in the tube implant than in the conical implant. This may be due to the difference existing in terms of the design of its internal connection, a cylinder versus a cone, although a narrower abutment was used. The failure pattern of the tube implants during the fatigue tests was the same in most specimens. With the microstructural analysis, it has been observed that, in addition to the implant, in all of the specimens, the screw was fractured. With the scanning electron microscope, it was possible to observe the areas already mentioned previously that showed the screw fatigue fracture. This suggests that the component that failed in the first place was the screw, perhaps fracturing before the implant. On the other hand, the tube connection and its indexing system with three edges on the implant platform mean that not only tensions were concentrated at this level, but also that the implant walls were thinner. The consequence, as it has been observed in the macro-structural analysis, was a vertical fracture that began at the level of these notches in a crown-apical direction.Therefore, the sensitivity of tube connection groups to cyclic stresses was higher than for the conical groups. In a fatigue loading regime (in vivo mastication), a monolithic all-ceramic zirconia or lithium disilicate crown with a zirconia abutment manufactured with a chairside CAD/CAM connected to a titanium implant with an internal tube in tube connection may fail at a typical force generated in the oral cavity ≈400 N) in a very short period of time . This critical maximum load value is close to the average occlusal force applied at the cusp during chewing and swallowing for humans in normal conditions ,39. On t0 N in a In this study, both the customized zirconia abutment and the crowns were manufactured in ceramic materials following a chairside workflow. In a study conducted by Joda et al. , in whicIt can be summarized that with a digital workflow, it is possible to optimize the treatment in order to achieve a reasonable cost−benefit ratio for the unit implant restorations. Keeping this in mind, it is more justified to follow a digital workflow. As the laboratory costs represent the greatest expense, using a chairside system to manufacture the restorations can greatly increase the benefit to the clinic. However, these systems require a learning curve that covers different phases, beginning with the impression taking using an intraoral unit, and continuing with the design of the restoration, which requires certain familiarity, and the handling of the software and the manufacture and sintering of the restoration for which some technical skills are needed. In addition, corresponding ovens for the correct sintering of the different materials are required. In this study, we decided to follow a chairside workflow using the CEREC system, which has its own intraoral, design, and manufacturing unit. Likewise, different materials have been used: zirconium oxide for the manufacture of a customized abutment, and translucent zirconia and disilicate for the manufacture of the crowns. In our study, the results are more than promising, leaving this work system in a very good position, despite all of the pertinent precautions having been taken regarding the design and manufacture of the restorations. Among the results obtained and assessing the type of catastrophic failure, it should be noted that neither the abutment nor crown fractured during any of the tests. It should be remembered that they were subjected to loads of up to 400 N.The findings of this study suggest that the fracture resistance of the zirconia abutment with an internal conical connection was higher than that of the zirconia abutment with a tube in tube connection. On the other hand, the risk of long-term mechanical and biological complications is expected to decrease using zirconia abutment with an internal conical connection versus tube in tube connection. Consequently, implant platform switching, which has been proven to have biological advantages over platform matching, and helps to minimize the marginal bone loss, does not seem to be a critical parameter for the mechanical integrity of the implants. It seems that the configuration and design of the connection is more important than the diameter of the abutment. Moreover, there are no significant differences between the two all-ceramic crowns. Both monolithic zirconia and monolithic lithium disilicate have the same mechanical behavior.Additional research is needed to confirm these results to other systems and to evaluate the differences after the simulated clinical function. It is difficult to predict the clinical results on the basis of in vitro studies, as many factors affect the oral cavity.CAD/CAM zirconia abutments with conical implant−abutment connections for platform switching supporting single all-ceramic crowns made with a chairside workflow are highly reliable in terms of fracture resistance, both in static and in fatigue loads.The stress distribution affects the implant/restoration complex depending on the connection design. The component more susceptible to fracture is the screw. In the conical connection group, the screw is subjected to a lower stress level.The mechanical behavior of the restorative complex does not seem to be affected by the different crown materials (zirconia or lithium disilicate) used in this study.Within the limitations of this in vitro study, the following can be concluded:


Porcine epidemic diarrhea virus (PEDV) is the causative agent of PED, an enteric disease that causes high mortality rates in piglets. PEDV is an alphacoronavirus that has high genetic diversity. Insights into neutralizing B-cell epitopes of all genetically diverse PEDV strains are of importance, particularly for designing a vaccine that can provide broad protection against PEDV. In this work, we aimed to explore the landscape of linear B-cell epitopes on the spike (S) and membrane (M) proteins of global PEDV strains. All amino acid sequences of the PEDV S and M proteins were retrieved from the NCBI database and grouped. Immunoinformatics-based methods were next developed and used to identify putative linear B-cell epitopes from 14 and 5 consensus sequences generated from distinct groups of the S and M proteins, respectively. ELISA testing predicted peptides with PEDV-positive sera revealed nine novel immunodominant epitopes on the S protein. Importantly, seven of these novel immunodominant epitopes and other subdominant epitopes were demonstrated to be neutralizing epitopes by neutralization–inhibition assay. Our findings unveil important roles of the PEDV S2 subunit in both immune stimulation and virus neutralization. Additionally, our study shows the first time that the M protein is also the target of PEDV neutralization with seven neutralizing epitopes identified. Conservancy profiles of the epitopes are also provided. In this study, we offer immunoinformatics-based methods for linear B-cell epitope identification and a more complete profile of linear B-cell epitopes across the PEDV S and M proteins, which may contribute to the development of a greater next-generation PEDV vaccine as well as peptide-based immunoassays. Alphacoronavirus genus and it is an enveloped, positive-sense, single-stranded RNA virus (A swine disease named porcine epidemic diarrhea (PED) is one of the main diseases that causes huge economic losses in the swine industry worldwide, including Asia, America, and Europe , 2. PorcStudies on the genetic profile of PEDV have demonstrated that the PEDV genome is highly diverse . OutbreaWhile the M protein is the most abundant in the PEDV particle and plays an important role in the viral assembly process , 10, theThe COE domain has been identified as an immunogenic domain containing B-cell epitopes recognized by neutralizing antibodies , 15, 18;in-silico evaluation  database were retrieved and a phylogenetic tree was generated. Consensus sequences obtained from each group were then subject to linear B-cell epitope prediction using multiple immunoinformatics tools. We developed our own prediction methods for identifying potential B-cell epitopes based on four known epitopes . The predicted peptides were then experimentally validated using ELISA by testing synthetic peptides with PEDV-positive sera. Their potential as a neutralizing epitope was then investigated using neutralization–inhibition assay. Based on this approach, the whole set of neutralizing linear B-cell epitopes as well as immunodominant epitopes across the PEDV S and M protein was identified.). The consensus sequence of each group of both proteins was next generated and aligned as shown in ). For the S protein, only the consensus sequences of groups S2 and S14 were clustered in genotype G1, while all the other sequences were clustered in genotype G2 ; we, thus, created three new prediction methods, designated methods A, B, and C, for identifying and selecting linear B-cell epitopes as described in To search for a prediction tool/method that gives the best performance in terms of accuracy and epitope coverage, we aligned and compared all predicted peptides obtained from each prediction tool with the reference epitopes. Four well-known B-cell epitopes of the PEDV S protein, namely, i) peptide M (MQYVYEPTYYML) , ii) S1DThe consensus sequences from all 14 groups of the S protein and 5 groups of the M protein were subject to linear B-cell epitope prediction. Putative B-cell epitopes were identified and selected based on prediction methods A, B, and C, and prediction of the M protein group 3 (M3) is shown as a representative Table 1.We further analyzed the strain coverage of each predicted epitope as information toward epitope conservancy could be beneficial to the design and development of a vaccine, particularly a universal vaccine. For the M protein, six out of seven predicted epitopes cover higher than 70% of 560 accession numbers with four epitopes  having strain coverage greater than 90%  were further used in ELISA analysis. A total of 12 F1 pigs and 11 F3 pigs showed neutralizing activity that passes the criteria Figure 6p < 0.05). All seven peptides from the M protein showed reactivity with sera from at least one pig strain . Two subdominant epitopes, S1B-15 and S1B-16, are located within the COE domain. Furthermore, we demonstrated that antibody response in the sera with high neutralizing titer (NT ≥ 64) and low neutralizing titer (NT = 32) against some epitopes was significantly different (p < 0.05) as indicated in Three well known epitopes, SS2, SS6, and 2C10 , 25, werg strain Table 2TLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVT-636)  is a part of the S1D domain (aa. 636–789) containing neutralizing epitopes . The S1BGVT-636)  as underGVT-636) . Besides50 of PEDV. Neutralization inhibition mediated by a peptide was determined at the endpoint neutralizing antibody titer, at which the amount of antibodies is only sufficient to neutralize the virus and depletion of neutralizing antibodies will result in the loss of the neutralization potential of the serum. Four sera including F1 1-3, F1 1-4, F1 1-5, and F3 1-10 were used in the assay. Peptides corresponding to the known epitopes SS2, SS6, and 2C10 were used as references, and irrelevant peptides N(MHC) and E(CTL) were included as negative controls. All three reference peptides exhibited their ability to inhibit the neutralizing activity of the sera, while irrelevant peptides did not . A combined result from both sera suggested that all seven epitopes of the M protein had the potential to be neutralizing epitopes. However, the three M peptides, namely, M-1, M-2, and M-6, could inhibit the neutralizing activity of both sera tested, suggesting them to be the most promising targets in the M protein for virus neutralization.Neutralization ability of the antibodies against each epitope were further tested using neutralization–inhibition assay as shown in the schematic representation in In the presence of the S peptides, four pig sera showed different patterns of neutralization inhibition as summarized in ). Close-ups and the secondary structure of all the other epitopes are shown in Surface representation of all epitopes is depicted on the prefusion structure of the trimeric S protein of the PEDV strain USA/Colorado/2013 (PDB: 6VV5)  using PyThe genetic diversity of PEDV, particularly in the gene encoding S protein , has limAfter prediction, the predicted B-cell epitopes of the PEDV S and M proteins were verified with PEDV-positive sera. Pig sera with PEDV-neutralizing activity were screened by neutralization assay with CV777 lineage virus (ACC_PEDV). Both PEDV genotypes (G1 and G2) were found and reported to be the cause of several PEDV outbreaks in Thailand in the past recent years ; therefoStatistical analysis of antibody responses in both F1 and F3 pig sera defined 11 immunodominant within the PEDV S proteins. Interestingly, although PEDV  and SARS-CoV-2 (betacoronavirus) belong to different genus, the landscape of immunodominant epitopes on the S protein of these two coronaviruses remarkably resembles. The following epitopes including i) S1B-1, ii) S1B-19 (19.1 and 19.3), iii) S2B-22, and iv) S2B-29 and S2B-30.2 are located at the following regions: i) the N-terminus next to the signal sequence; ii) CTD, upstream region of the S1/S2 cleavage site; iii) FP region; and iv) upstream region of the HR2, respectively, which have also been indicated to be the main sites of immunodominant epitopes on the SARS-CoV-2 S protein recently reported by our group  and Li eThe neutralizing activity of the antibodies targeting these novel epitopes was addressed using neutralization–inhibition assay, by which the target antibodies are depleted/blocked with their cognate peptides. This technique has been previously used to identify neutralizing epitopes from SARS-CoV-2  and coxsThe neutralizing epitopes we identified are located at the regions that are functionally important during virus infection. Epitopes S1B-1 to S1B-9 are located at the NTD of the S1 subunit, the region that is involved in binding to sialoglycoconjugate receptor on the host cell , 44. TheWhile most of the known neutralizing B-cell epitopes are located in the S1 subunit, information of B-cell epitopes in the S2 subunit is limited with only one neutralizing epitope identified. The epitope 2C10 located at the C-terminus of the S2 domain has been identified as a neutralizing epitope , 25. In In addition to the S protein, we showed that M protein is also a target for virus neutralization. Based on neutralization–inhibition assay, epitopes M-1, M-2, and M-6 are the most promising as evidenced by the loss of neutralizing ability of both tested sera in the presence of these three peptides. The neutralization potential of antibodies against coronavirus M protein has been previously reported in the study of transmissible gastroenteritis virus (TGEV), a PEDV closely related alphacoronavirus, that the antibodies against the TGEV membrane protein had neutralizing activity in the presence of complement . TogetheAs our B-cell epitopes are predicted based on consensus sequence of each group of the S and M proteins, the epitope sequence may not match 100% with all PEDV strains/variants. However, some epitopes are highly conserved as evidenced by a high percentage of strain coverage among global PEDV strains. Epitopes in the M protein were found to be more conserved compared with those in the S protein. Within the S protein, epitopes in the S2 subunit are more conserved than those in the S1 subunit. These conserved epitopes may serve as candidates for the development of a multi-epitope universal vaccine.Taken together, the method combining immunoinformatics with immunoassays enabled the identification of novel neutralizing linear B-cell epitopes on the PEDV S and M proteins. Even though these B-cell epitopes are derived from a consensus sequence, some are highly conserved among the global PEDV strains, which represent a promising vaccine target for the development of a universal epitope-based vaccine as well as for antibody detection. Importantly, the immunoinformatics method developed in this study can serve as a useful tool for the prediction of linear B-cell epitopes from proteins of interest.https://www.ncbi.nlm.nih.gov/protein/). The retrieved protein sequences were analyzed individually based on a defined name and length using Sublime Text 3 program. Based on sequence similarity, the amino acid sequences of the S and M proteins were grouped using alignment and phylogenetic tree tools in the SeaView program (version 4). In the SeaView program, these amino acid sequences were aligned using MUSCLE and grouped using a tree based on distance analysis, NJ method, ignore gap, and bootstrap 1,000 replication. The consensus sequence of each group was generated using the MUSCLE method in Unipro UGENE program   , 50. Thehttp://www.cbs.dtu.dk/services/BepiPred/), which predicts linear B-cell epitopes from a protein sequence, using a random forest algorithm trained on epitopes and non-epitope amino acids determined from crystal structures, followed by performing sequential prediction smoothing (http://tools.iedb.org/bcell/), except IUPred. IUPred (https://iupred.elte.hu/) was used to predict intrinsically unstructured proteins which infers to coil probability  and M (5 groups) proteins, we further analyzed the conservancy of our predicted epitopes. Epitope conservancy was analyzed by calculating the percent strain coverage of the predicted epitopes in comparison to all of the sequences in the group using the formula below.Peptides corresponding to the predicted epitopes were chemically synthesized . All synthetic peptides include i) 57 predicted epitopes; ii) 3 known reference B-cell epitopes, SS2 (YSNIGVCK) , SS6 LQ, and 2C1All procedures of sample collection from pigs raised in the farms were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Institute for Animals for Scientific Purpose Development and carried out under the regulation and permission of King Mongkut’s University of Technology Thonburi IACUC protocol no. KMUTT-IACUC-2019/018. For the control group, pigs were raised in the animal research facility of Mahidol University. All procedures were carried out under the regulation and permission of the Faculty of Veterinary Science, Mahidol University IACUC protocol No. MUVS-KA-2021-01-01.g for 10 min in a refrigerated centrifuge to separate the serum. Serum was aliquoted and separated to new tubes and stored at −20°C until used.Blood samples (10 ml) were collected from pigs naturally infected with PEDV. Infected pigs were observed and diagnosed by a veterinarian based on signs and symptoms of PED. Pigs showing PED signs or had been diagnosed with PED within the past 4 weeks were used as subjects. Bloods were collected from two different pig strains: F1 sow  and F3 fattening pigs . Blood was collected from 13 female F1 pigs and 14 either male or female pigs raised in three different farms. Control sera were prepared from 10 uninfected F3 pigs (age 7 weeks old) raised in the animal research facility of Mahidol University. Bloods were left to clot at room temperature for approximately 2 h, followed by centrifugation at 1,500×). Vero cells were cultured in complete DMEM medium supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin , and then incubated at 37°C with 5% CO2. The seed virus was sequentially propagated in Vero cell with PEDV medium  . To prepare the PEDV stock, Vero cells were cultured in 10 T-175 flasks. When the cell confluency was over 90%, the old medium was removed and the cells were infected with PEDV resuspended in 20 ml of PEDV medium. When the cytopathic effect (CPE) reached 70%, the medium was harvested and pooled. Sodium chloride (NaCl) was added to the medium to a final concentration of 0.5 M and polyethylene glycol  was then added to a final concentration of 8%. After an overnight incubation, PEDV was harvested by centrifugation at 8,000 rpm for 20 min. The viral pellet was next resuspended in 5 ml of endotoxin-free PBS, aliquoted in a small volume to new tubes, and stored at −80°C. Virus titer was determined using 50% tissue culture infectious dose (TCID50) method. Virus suspension was diluted in 10-fold serial dilutions and pipetted into 10 wells of confluent Vero cells. After 3 days post-infection, PEDV-infected cells were observed and counted using immunofluorescence staining assay. TCID50 was next calculated.PEDV used in this study is a lineage of PEDV CV777, designated ACC_PEDV (classification is shown in ® 594 (Abcam) diluted 1:1,000 in PBS, was added to the cell and incubated for 1 h at RT. The cells were washed twice with PBS and then the nucleus was stained with DAPI (Sigma) diluted 1:1,000 in PBS and incubated at RT for 30 min, followed by washing once with PBS. Fluorescence images were directly examined under an inverted fluorescence microscope (Olympus DP74).Three days post-infection, Vero cells in 96-well plate were fixed with 1% formaldehyde at RT for 15 min and then washed with PBS once. Cells were permeabilized with cold 90% methanol and then incubated at 4°C for 5 min. Cells were washed once with PBS and blocked with 2% FBS/PBS for 1 h at RT. The blocking solution in each well was replaced with 50 µl of mouse anti-PEDV (Median Diagnostic) diluted 1:1,000 in PBS. Following an incubation for 2 h at RT, the cells were washed twice with PBS. Secondary antibody, donkey anti-mouse IgG conjugated with Alexa Fluor50/ml was prepared using PEDV medium. PEDV (10 TCID50 in 50 μl) was mixed with 50 μl of diluted serum or medium (control) and incubated at 37°C with 5% CO2. After, a 1-h incubation, the mixture of 100 μl was transferred into 96-well plates containing approximately 90% confluent monolayer of Vero cells. Each condition was performed in duplicate. After a 3-h incubation, the medium was removed and replaced with new PEDV medium. At day 3 post-infection, immunofluorescence staining assay was performed and the number of infected cells was counted. Endpoint neutralizing antibody titer was determined based on the serum dilution that completely inhibited virus infection. As all of the sera from the control pig did not neutralize PEDV at dilution 1:32 and higher, we thus set the cutoff of neutralizing antibody at neutralization titer 32 . Only sera with neutralization titer ≥32 were subject to ELISA and neutralization and inhibition assay.All pig serum samples were 2-fold serially diluted with DMEM containing 1% Pen/Strep to the dilution ranging from 1:32 to 1:4,096. PEDV at the concentration of 200 TCID2SO4), and the optical density was measured at a wavelength of 450 (OD450) .Synthetic peptides (0.75 nmol in 50 μl PBS/per well) were added into 96-well ELISA microplates (Greiner Bio-One). After an overnight incubation at 4°C, plates were washed three times using PBS containing 0.05% Tween 20 (PBST). Plates were blocked using 100 μl of PBST containing 5% FBS and incubated with agitation at room temperature (RT) for 1 h. Pig sera diluted 1:50 in PBST containing 1% FBS were added to the plates. After a 2-h incubation at RT, the plates were washed three times with PBST, and goat anti-pig IgG HRP antibody (Abcam) diluted 1:10,000 in PBST containing 1% FBS was added to the well (60 μl/well), followed by an incubation at RT for 90 min. After a three-time wash with PBST, the TMB substrate (BioLegend) was added (70 μl/well) and the plate was incubated in the dark at RT for 30 min, allowing the color to develop. The reaction was stopped by adding 35 μl of 1 M sulfuric acid  was added to the peptide–serum mixture. After an incubation at 37°C for 1 h, the mixture (100 μl) was transferred to the Vero cells grown in 96-well plates. Following an incubation at 37°C with 5% CO2.for 3 h, the virus suspension was removed from the well and a new medium was added into the wells. In addition, the conditions that the Vero cells were incubated with i) medium alone, ii) serum alone, iii) PEDV alone, and iv) the mixture of PEDV and serum were also included in the assay as control conditions. All conditions were performed in duplicate. Following a 3-day (F1 1-4 and F1 1-5) or 5-day (F1 1-3 and F3 1-10) incubation, PEDV infection in Vero cell was investigated using immunofluorescence staining assay. Neutralization inhibition mediated by a peptide was determined at the endpoint neutralizing antibody titer.Neutralization–inhibition assay was conducted as previously described by Shi et al.  with somPutative neutralizing B-cell epitopes were aligned and compared with full-length S protein of PEDV USA/Colorado/2013 strain for the identification of epitope position. To localize each epitope within the trimeric PEDV S protein, the prefusion structure of the PEDV spike named 6VV5  was usedU test. The difference between the two groups was determined based on p <0.05. Immunodominant and subdominant epitopes were defined with the following criteria. In response to a particular peptide, if all sera from both F1 and F3 pigs exhibited OD450 value higher than OD450 mean of the control group + 2SD, such peptide was considered an immunodominant. On the other hand, if all sera from both F1 and F3 pigs exhibited OD450 value higher than OD450 mean of the control group + 1SD, such peptide was considered a subdominant epitope.The statistical significance of the samples in different groups was analyzed using SPSS 22 for Windows software . Data analyses were performed using non-parametric Mann–Whitney The original contributions presented in the study are included in the article/The animal study was reviewed and approved by King Mongkut’s University of Technology Thonburi Institutional Animal Care and Use Committee (IACUC). Written informed consent for participation was not obtained from the owners because informed consent was obtained through verbal agreement between the veterinarian and farm owners.KP designed and performed the experiments, analyzed and interpreted the data, wrote the original draft, and revised the manuscript. MR supervised the immunoinformatics methods and experimental design. PM provided the materials and reagent. KK prepared the uninfected pig sera. TH provided financial support, materials, and reagents. PJ collected the blood samples from PEDV-infected pigs. YR designed the experiments, analyzed and interpreted the data, provided supervision to KP, and reviewed and edited the manuscript. All authors read the manuscript and gave comments.This study received funding from B.F. Feed Company Limited. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. The PhD study of KP is funded by the Development and Promotion of Science and Technology Talents Project (DPST).Author TH is academic director of B.F. Feed Company Limited. PJ is employed by B.F. Feed Company Limited.The remaining 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.


We present the technique of biologic tuberoplasty, where an acellular dermal allograft is used to cover the tuberosity in patients with massive irreparable cuff tears to prevent bone-to-bone contact between the tuberosity and acromion when active elevation is attempted. This technique can be performed in patients with massive rotator cuff tears who are low-demand and have significant medical comorbidities, poor bone quality, or who are not candidates for a reverse shoulder arthroplasty or who cannot tolerate a lengthy rehabilitation protocol. It is less time-consuming than a superior capsule reconstruction and more bone-sparing than traditional tuberoplasty. Patients with massive rotator cuff tears involving the supraspinatus and a portion of the infraspinatus lose their force couple, leading to superior humeral head migration with abutment of the tuberosity against the acromion upon deltoid activation. The center of rotation moves superiorly with deltoid contraction, developing an acromiohumeral articulation. This results in bone-to-bone contact between the acromion and humerus, leading to pain and acetabularization of the acromion over time. Coverage of the tuberosity with the acellular dermal allograft acts as an interpositional tissue and prevents bone-to-bone contact between the tuberosity and acromion. The procedure is demonstrated on a right shoulder with the patient in the lateral decubitus position. A burr is used to remove any osteophytes or hypertrophy of the tuberosity until a bleeding cancellous surface is achieved. The length of the tuberosity is measured at 30 mm and the width at 20 mm. An ArthroFlex that is 3 mm thick is then measured to these dimensions and cut to size. Three puncture holes are made and also punched to widen them in the medial and the lateral edges of the graft for easier passage of FiberTape sutures. Knotless SwiveLocks are then placed in the anteromedial, posteromedial, and central aspect of the tuberosity footprint. A 10-cc syringe with the end cut off is used to allow for easier passage of the graft. The FiberTapes, repair stitch, and loop stitch are then retrieved through the syringe from each anchor, leaving behind the pull stitch from each anchor. The sutures from each anchor are kept separated and clamped to the syringe for suture management. The repair stitch from each anchor is then passed in a mattress fashion from the edge of the graft and is then passed through the loop of the loop stitch and folded over. The pull stitch from that anchor is then pulled, bringing the repair stitch into the knotless mechanism and locking it in like a finger trap so it cannot back out. This is performed for each anchor. The mattress stitches will be used to pull the graft into the shoulder. The FiberTapes from each anchor are then passed through the pre-punched holes. The FiberTapes are then passed back through the lateral edge of the graft in an expanded speedbridge configuration. This will allow better control of the graft when lateral row anchors are placed for fixation and will eliminate dog ears in the graft. The pull sutures from each anchor are then sequentially pulled, allowing the mattress passes to shorten and thus reducing the graft into the shoulder. The graft can be seen entering the subacromial space. The pull stitches are pulled until the graft is secured to the medial edge of the footprint. Although there appears to be disorganization of sutures, when the FiberTape limbs are pulled individually, the slack is taken out of them and the graft and sutures become more manageable. The posteromedial corner of the graft is grasped and reduced to that corner and the pull stitch is pulled one last time to tighten it and secure the graft to that corner. The same step is repeated for the anteromedial corner followed by the central pull stitch. The posteromedial FiberTapes are then retrieved from a posterolateral cannula and passed into the SwiveLock anchor to fixate the graft. The steps are then repeated for the anterolateral FiberTapes and then followed by the central FiberTapes. The graft will prevent bone to bone contact between the acromion and tuberosity. Patients with massive rotator cuff tears involving the supraspinatus and a portion of the infraspinatus lose their force couple, leading to superior humeral head migration with abutment of the tuberosity against the acromion upon deltoid activation.For patients with significant comorbidities who are unable to undergo an extensive operation, the options are limited. Many of these patients have poor bone quality and are at high risk for rotator cuff retear, graft tear, or anchor pull-out.This article describes the technique of biologic tuberoplasty using an acellular dermal allograft, providing a quicker, bone-sparing, less time-consuming option for patients with massive rotator cuff tears who are low-demand and have significant medical comorbidities .Table 1AThe tips and pearls of this technique are listed in Once the graft site has been prepared, measurements of the graft dimensions (length and width) are achieved with the use of an arthroscopic measurement probe . On the The repair stitch and the looped end of the shuttle stitch from each anchor are then retrieved through the syringe, leaving behind the pull end of the shuttle stitch . The sutThe described technique of biologic tuberoplasty provides a viable option for patients with massive irreparable rotator cuff tears who cannot tolerate a long, extensive operation or lengthy rehabilitation, who may have poor bone quality, or who may not be a candidate for shoulder arthroplasty. This procedure has several advantages especially for elderly, low-demand patients with significant medical comorbidities but with functional mobility, whose primary goal is pain relief .Patients with massive rotator cuff tears involving the supraspinatus and a portion of the infraspinatus lose their force couple leading to superior humeral head migration with abutment of the tuberosity against the acromion upon deltoid activation.,Fenlin et al.The interest in combining the placement of an acellular dermal matrix with a minimal tuberoplasty stemmed from the findings of Mirzayan et al.,,,,The use of autologous graft or acellular dermal allograft as spacers in patients with degenerative joint disease has been explored in various orthopaedic procedures in the hand, wrist, elbow and ankle.


Correction to: European Journal of Clinical Pharmacology10.1007/s00228-020-03021-9The correct affiliation 1 is presented in this paper.The original article has been corrected.


R2 = 0.64). The observed algal bloom was detected one month before the algae reached its major extent, being the reflectiveness of the green band a good indicator of the onset of the algal bloom event. Favorable conditions for the algal bloom were also recorded two months before its major extend through a chain of thermistors.The condition of Lake Zirahuén was evaluated before and after a significant algal bloom took place. The state of the lake was tracked using measurements of chlorophyll‐a concentration from a fluorometer attached to a conductivity, temperature, and depth (CTD) profiler and complemented with the use of satellite images from Sentinel‐2. It was found that the current state of the lake is in transition from an oligotrophic to a mesotrophic level with some evidence of deterioration, but still being considered as a clear lake. With the use of empirical algorithms to retrieve chlorophyll‐a from satellite data it was found that the images presented an acceptable feasibility (The trophic state of Lake Zirahuén was evaluated before and after a significant algal bloom took place.The lake is classified as being in transition from oligotrophic to mesotrophic. Nonetheless, it continues to have good clarity.Satellite images improve in the description of the spatial‐temporal variability of the lake. In particular, the green band reflectiveness. The presence and detection of an algal bloom event.  Analyzing lakes based on the amount of nutrients reaching their basin is of great importance because the proper management and conservation is vital due to the high impact this has on the lake's ecological and social environment Carlson, . Also, bIn lakes, the concentration of chlorophyll‐a (Chl‐a) is widely used to measure the biomass of phytoplankton, and hence, the concentration of nutrients, that is, the Chl‐a is a common biological indicator of eutrophication  according to the Köppen classification  November 23, 2018, (ii) March 19, 2019, and (iii) June 19, 2019. That is, before and after an algal bloom took place at the beginning of the year 2019. Also, a thermistors chain and a series of weekly satellite images were used with approximately one year of data.The water quality and thermal structure of Lake Zirahuén was measured using a CTD  profiler XR‐620 , using additional sensors for Chl‐a. The CTD was dropped manually from a boat acquiring a vertical speed of about 1 m/s and sampling at a rate of 1 Hz. All of the variables were linearly interpolated to smooth values around the thermocline by removing spikes due to sensor issues.To continuously measure the temperature in the lake, a water thermistors chain was placed with 2 Hobo temperature loggers  at depths of 26 and 31 m, sampling at a rate of 12 min near the northern part of the lake at a depth of ~32 m. The sample period was from November 22, 2018, to November 18, 2019.Because monitoring a lake is an intricate task that demands time and resources, an excellent alternative procedure for lake monitoring is the remote sensing techniques that have been developed and improved in recent years, particularly the use of satellite sensors  is a Copernicus program of the European Space Agency (ESA) consisting of two polar‐orbiting satellites: Sentinel‐2A and Sentinel‐2B. The multi‐spectral instrument onboard each satellite is able to recover data in 13 spectral bands at a variable and high resolution of 10, 20, and 60 m within 290 km of swath and with a revisit time of 5 days (European Space Agency (ESA), λB4=665nm, λB5=705nm, and λB6=740nm central wavelengths, respectively, , For relatively deeper lakes in which a thermal structure forms, the development of a thermocline is vital for the dynamic control of Chl‐a and other biogeochemical substances  are satellite products that have the function of presenting the target area as it is seen with the naked eye based on the B2, B3, and B4 spectral bands , respectively. In particular, the TCI for March and June present a lake with a green tone Figure , which iThis anomalous behavior on the lake's color can be used as a proxy for tracking throughout the year the growth of vegetation within the lake , as shown in Figure These aspects are discussed in the following section in the context of the algal bloom event that took place at the beginning of the year and apparently was detect clearly by the satellite imagery.Before going further into the algal bloom event, this section begins with some considerable differences between the observed Chl‐a values obtained during the campaigns of this study compared with the values recorded in previous studies of the lake Table .These differences may be due to both the measurement method and the equipment used. Rendón et al.,  and VergHowever, it cannot be ruled out that the higher concentration of this pigment found compared to those recorded in previous studies is due to a true increase in the degree of eutrophication of the lake, that is, a deterioration of its waters due to both natural and anthropogenic processes.As already mentioned, it was not possible to perform transparency measurements. However, as a way to validate the trophic classification found above, it is possible to use the transparency values of the lake from previous studies and calculate an average to obtain a classification of the trophic state of the lake. Then, according to the average values of transparency from several studies and the OECD criteria, Lake Zirahuén is classified as mesotrophic Table , which cThe observations taken during March were for most the more remarkable campaign given that the lake is considered in the range of oligotrophic‐mesotrophic level. In the previous and after campaigns, no significant spatial variations on the trophic state were found, so it is thought that the higher variability was due to the layer of suspended organic matter covering the lake. During this campaign, the lake was covered by a dense layer of organic matter because of the excessive proliferation of algae Figure , which, Although this might seem inconsistent  since eutrophication means excess of nutrient and hence higher values of Chl‐a, the results are showing the opposite, that is, lower values of Chl‐a during a clearly visual confirmation that the lake was undergoing an algal bloom Figures  and 1c. It can be said that the algal bloom started around early February and remained until around mid‐June ; Funding acquisition (lead); Investigation ; Project administration (lead); Supervision ; Writing‐original draft . Néstor Alberto Vega‐Álvarez: Conceptualization ; Investigation ; Writing‐original draft . Tzitlali Gasca‐Ortiz: Conceptualization ; Formal analysis ; Investigation ; Methodology ; Writing‐review & editing .Appendix S1Click here for additional data file.


Heat treatment is used as a pre-processing step to beneficially change the starch properties of wheat flour to enhance its utilisation in the food industry. Heat-treated wheat flour may provide improved eating qualities in final wheat-based products since flour properties predominantly determine the texture and mouthfeel. Dry heat treatment of wheat kernels or milled wheat products involves heat transfer through means of air, a fluidising medium, or radiation—often resulting in moisture loss. Heat treatment leads to changes in the chemical, structural and functional properties of starch in wheat flour by inducing starch damage, altering its molecular order , pasting properties as well as its retrogradation and staling behaviour. Heat treatment also induces changes in gluten proteins, which may alter the rheological properties of wheat flour. Understanding the relationship between heat transfer, the thermal properties of wheat and the functionality of the resultant flour is of critical importance to obtain the desired extent of alteration of wheat starch properties and enhanced utilisation of the flour. This review paper introduces dry heat treatment methods followed by a critical review of the latest published research on heat-induced changes observed in wheat flour starch chemistry, structure and functionality. Triticum aestivum L.) is a staple crop that provides energy, proteins, dietary fibre, vitamins, minerals and phytochemicals that are essential dietary components for many individuals daily [Wheat , resulting in the occurrence of the Maillard reaction. In addition, caramelisation takes place which allows for the development of improved colour, aroma and flavour as well as increased antioxidant activity ,22. At hDry heat treatment of whole wheat kernels or flour involves heat transfer through convection, conduction or radiation. Roasting is achieved by using sand, a fluidising bed roaster, a conventional oven or a forced convection continuous tumble roaster (FCCT). Radiation methods include microwave heating and infrared radiation whereas gun puffing, vacuum steam and superheated steam thermal methods are based on a change in pressure. The principle of forced convection has been known for a long time, but the use of forced convection continuous tumble (FCCT) roasting in cereal products has only emerged in recent years. It is based on the principle of using forced airflow at atmospheric pressure to roast a sample. Hot air is continuously circulated throughout the chamber, allowing for even heat transfer and increased efficiency . The rotSand roasting is a traditional method used in India for grain processing . It invoThe fluidised bed roaster (FBR) provides a roasting technique in which a solid sample is exposed to a fluidising medium such as flue gas, allowing the solid sample to act as a liquid . A vibraDry heat treatment by means of microwave heating is advantageous, as it has the ability to achieve high heating rates, the start-up time is short, the energy is efficiently used and little space is required . MicrowaDhua et al.  noticed Infrared (IR) radiation provides an efficient heat treatment—the processing time is short, as the temperature of the sample rapidly increases during IR heating . IR radiUsing IR radiation as a dry heat treatment increased the digestibility of the starch and appeared to be effective in decontaminating low moisture foods from bacteria, yeasts and moulds . Wheat kRadiation penetrates the grain, giving rise to heating that begins at the centre of the grain . Heat isIn recent years, the use of superheated steam (SS) on wheat has emerged as a viable pre-processing method to enhance product quality. It uses sensible heat to increase the temperature of the product above its saturation temperature at a specified pressure . Steam wSS drying is associated with higher temperatures and shorter time duration compared to conventional drying. This can mitigate the losses of heat-sensitive compounds . PreviouVacuum steam treatment is a promising pre-processing technique for low-moisture food products such as wheat to inactivate the microbial load, owing to its high level of heat transfer . Vacuum Gun puffing is based on instantaneous evaporation and expansion when the pressure of the wheat grains containing superheated steam is reduced to a low pressure ,34. The Starch is comprised of two glucose polymers, namely amylose and amylopectin, that influence its physicochemical properties . Heat trα-Amylase and β-amylase digest amylose and amylopectin to produce maltodextrins consisting of D-glucose units with different chain lengths . Dry heaThe morphology of wheat starch granules greatly contributes to the functional properties of wheat flour. Schoeman and Manley  comparedSand roasting and microwave heating resulted in a lowered bulk density after the heat treatment . The desMariotti et al.  noticed The crystallinity of wheat starch is determined by the ratio of amylose and amylopectin, the number of double helices, the extent of amylopectin destruction as well as the crystal size ,39. HighThe proportion of A- and B-type granules in the bimodal distribution of wheat starch affects the structural and functional properties of wheat  and, eviV-type polymorphs are divided into Type I and Type II complexes depending on the thermal transitions and the degree of crystallinity of the produced complex. High-temperature treatment (>90 °C) of low moisture foods resulted in the formation of Type II amylose–lipid complexes forming clear semi-crystalline structures . Oven anMilling of wheat affects the structural and functional properties of flour , such asThe extent of the starch damage content is not only related to the milling technique used but also to the hardness of the wheat. Wheat is classified as soft, medium or hard, depending on the endosperm texture and the protein content . MillingThe type and severity of the heat treatment determine the extent of the starch damage. Snelling et al.  noticed The change in pressure and the remarkably high processing temperatures used during gun puffing resulted in kernels with nearly complete disintegration of the starch granules. Values of up to 80% of damaged starch or gelatinised starch were observed as a result . ExcessiStarch gelatinisation, requiring excess water and heat, involves the absorption of water, resulting in several molecular transitions, i.e., granule swelling, the loss of birefringence, the reduction of crystallinity and the disruption of the granule structure . In a liMoisture plays an essential role during the gelatinisation of starch molecules. Pores and cavities on the starch surface allow for enhanced moisture migration into the granule . Ma et aGelatinisation enthalpy, i.e., the energy required for a sample to gelatinise, indicates the melting of double helices and the disruption of the molecular order . Thus, gGranule size also influences the degree of gelatinisation. Larger granules demonstrated higher gelatinisation enthalpy, indicating a more ordered arrangement of polysaccharide chains, thus a greater crystallinity . This isWater absorption is dependent on the presence of hydrophilic groups to bind water molecules ,28. PartPadalino et al.  indicateSimilar outcomes occur in wheat kernels exposed to gun puffing. The highly expanded structure due to cavity formation, the porosity of the internal structure and the structural changes of the outer layers of the puffed grains led to a much greater ability to absorb water. The puffed wheat kernels can hydrate four to five times more water than raw grains within the same period . In a limited moisture environment at high temperatures, starch and protein will compete for moisture absorbance. Starch has the ability to gelatinise, and the protein will form a gluten network . ThermalIt is known that amylose may retard the swelling of starch granules by stabilising the granule structure. Therefore, amylopectin (and even more so for long-chain amylopectin molecules) greatly contributes to the water absorption, swelling ability and pasting behaviour of starch, which explains why waxy starches (amylose-free granules) have great swelling power . HoweverMariotti et al.  showed tPasting profiles are influenced by changes in the functional properties of wheat flour, such as the degree of gelatinisation, complex formation with other compounds and α-amylase activity. The changes in viscosity, measured by a Rapid Visco Analyser (RVA), are recorded as a function of time and temperature to determine the pasting behaviour of a starch sample . ThermalPeak viscosity correlates with the resistance of swollen granules to shear, which relates to the swelling power of starch . It greaExtensive SS heat treatment promoted the melting and gelatinisation of starch, which decreased the viscosity of the flour due to the inability of the starch granules to absorb water and swell ,6. This During microwave cooking, the vibrational motion of the water molecules and the re-orientation of the other polar molecules to the oscillating microwave field greatly affected the pasting properties of the wheat flour ,29. As mThe hardness of the wheat, which relates to the protein content, once again leads to changes in the functional properties. The pasting profile of the flour produced from the wheat kernels with a higher protein content was shown to be different from those containing less protein . A signiUpon the cooling of the non-crystalline starch structures, amylose and amylopectin molecules reassociate to form a new crystalline order. Retrogradation, therefore, involves the transition of a rubbery state starch solution to an ordered crystalline structure in the form of a viscoelastic gel during cooling ,39. The Breakdown viscosity indicates the heating stability of a cooked gel, where its value relates to the rate of rupture of swollen starch granules when maintained at a constant high temperature . FCCT anWith regards to end-product quality, moisture greatly contributes to bread staling, as the moisture migration from the gluten and starch matrix within the crumb to crust are responsible for crumb firmness . Hu et aVacuum steam treatment at 85 °C for 4 and 8 min showed a significant increase in the firmness of the crumb texture . During baking, higher temperatures result in more retrogradation . This coThermal treatment disrupts the starch granule structure and induces protein aggregation. As a result, protein–starch crosslinking, amylose–lipid complexes and inter- and intramolecular hydrogen bonding occur, which evidently forms an uninterrupted structure increasing the diameter of the granule . ProteinSchoeman and Manley  noted a 2 for 3 min at a 0.2 m emitter–sample distance was the optimal condition to stabilise the wheat germ without significantly reducing the antioxidant content. Non-polar lipids are found within the germ and aleurone layer. However, during milling, approximately half of these lipids are transferred to the produced flour. Upon storage, lipase hydrolyses triacylglycerols into free fatty acids, monoacylglycerol and diacylglycerol, which result in acid rancidity. Acid rancidity negatively affects the baking quality of whole wheat flour by reducing its functional properties . FortunaAmylose–lipid complexes are naturally occurring structures often formed during heat treatment. As lipids are hydrophobic, these complexes have an inability to absorb water, consequently affecting starch functional properties, including pasting, swelling power, the leaching of amylose, viscosity and the retrogradation of starch ,65. FurtDry heat treatment of wheat generally results in the partial gelatinisation of starch due to modifications in its structural properties. In addition, a reduction in damaged starch occurs after the milling of wheat kernels previously exposed to intermediate heat treatment conditions. However, mild and severe treatments generate an increase in starch damage content. Thermal treatment of wheat and/or flour also increases the amorphous content, resulting in changes in the functional behaviours of starch, e.g., increasing its viscosity and lowering the pasting temperature. The decrease in α-amylase activity observed during thermal treatment may also contribute to an increase in viscosity. In contrast, FCCT and oven roasting also increased the α-amylase activity but increased (insignificantly) viscosity was observed. Similar trends in the changes in the functional and structural properties of starch occurred due to dry heat treatment. New interactions, due to the heat treatment, of starch with proteins, lipids or other starch molecules may result in a lowered solubility index but an enhanced ability to retain water. Heat treatment, at optimal conditions, is a viable solution for the pre-processing of wheat flour to enhance its utilisation. Temperature, time and tempering conditions appear to be very important variables in influencing the quality of the resulting products from heat pre-processed wheat kernels. Future studies are therefore required to optimise these conditions to produce flour with better functionality and improved utilisation.


Pazala, G. (P.) eurous asakurae and G. (P.) mullah chungianus from Taiwan Island, are reported. Both mitogenomes are circular, 15,228 bp and 15,240 bp in length respectively, and consist of 37 genes, including 13 PCGs, 22 tRNAs, and two rRNAs. The Bayesian phylogenetic tree containing the focal taxa and 31 other Papilioninae members clustered them with G. (P.) mullah  and then G. (P.) parus  inside tribe Leptocircini, which agrees with their taxonomic positions. The findings of this study would benefit future understanding of phylogeography and conservation of subgenus Pazala.The mitogenomes of two insular subspecies of  Pazala Moore, 1888 (Lepidoptera: Papilionidae), represented by two endemic subspecies, Graphium  eurous asakurae  and G. (P.) mullah chungianus   eurous asakurae came from Taroko, Hualien , and G. (P.) mullah chungianus was collected at Fushan, Yilan . The specimens were deposited in the zoological museum (insect collection) of Yunnan University, Kunming, China . Genomic DNA was extracted from the thoracic muscle tissue of the adult using phenol-chloroform and isopropanol protocol  with the previously reported mitogenomes of G. (P.) mullah  (KJ472924)  parus  (MT198821)  .The samples used in this study were collected from Taiwan Island. G. (P.) eurous asakurae (MW549198) and G. (P.) mullah chungianus (MW549197) are circular, 15,228 bp and 15,240 bp in length respectively. The base composition of G. (P.) eurous asakurae is 39.81% for A, 40.58% for T, 7.85% for G, and 11.76% for C; while that of G. (P.) mullah chungianus is 40.11% for A, 40.85% for T, 7.60% for G, and 11.43% for C. Both mitogenomes contain 37 genes, including 13 PCGs, 22 tRNAs, and two rRNAs, plus a non-coding control region. The plus (+) strands of both mitogenomes encode nine PCGs , while the minus (–) strands encode four PCGs . The gene arrangement and character of both genomes agree with recently reported ditrysian Lepidoptera mitogenomes   mullah, while G. (P.) parus is placed in the basal position of Pazala. All Pazala taxa are related to other Graphium species within Leptocircini, forming a monophyletic clade, supported by the maximal support values (To validate the two mitogenomes, a Bayesian phylogenetic tree was reconstructed using PhyloSuite 1.2.2 (Zhang et al. t values .


The tooth dies were printed with a resin material  using a 3D printing software (3D Sprint® Client Version 3.0.0.2494) from a 3D printer . The printing layer thickness was 50 µm. Then, a total of twenty-eight (N = 28) stylized crowns were milled out of AmannGirrbach  (n = 7), Cercon HT  (n = 7), Cercon XT  (n = 7), and Vita YZ XT  (n = 7). Following sintering the crowns, sandblasting was performed and they were bonded to the tooth dies with the resin cement  and permitted to self-cure under finger pressure for 6 min. The crowns were loaded on the occlusal surface in a universal testing machine (MTS Centurion) with a stainless-steel ball indenter (7 mm radius) with a loading rate of 1 mm/min to contact the stylized crowns on each of the four cusps until failure. A rubber sheet (1.5 mm thickness) was positioned between the crown and indenter, which helped with the load distribution. Statistical analysis was done using SPSS version 20 . The fracture loads were analyzed using Dunnett’s T3 test, and the number of cracks was analyzed using the Mann–Whitney U test among the groups. The significant level was set at p value = 0.05. The mean fracture loads were 3086.54 ± 441.74 N, 4804.94 ± 70.12 N, 3317.76 ± 199.80 N, and 2921.87 ± 349.67 N for AmannGirrbac, Cercon HT, Cercon XT, and Vita YZ XT, respectively. The mean fracture loads for the surfaces with the greatest number of cracks  were on the lingual surface for AmannGirrbach and Cercon HT, on the distal and mesial for Cercon XT, and on the buccal for Vita YZ XT. We found that the AmannGirrbach had the most overall cracks. Cercon XT had the greatest number of occlusal cracks and appeared to be the most shattered. Cercon HT had the least number of cracks. In conclusion, Cercon HT presented the best strength properties, the highest fracture load, and no visible cracks. AmannGirrbach presented the lowest strength properties.Recently, translucent zirconia has become the most prevalent material used as a restorative material. This study aimed to compare the crown fracture load of the four most common different translucent zirconia brands available in the market at 1.5 mm thickness. Standardized tooth preparations for a full ceramic crown were designed digitally with software (AutoCAD) by placing a 1.0 mm chamfer margin and 1.5 mm occluso-cervical curvature for the crown sample manufacturing. Stylized crowns were chosen to control the thickness of the crown. The axial and occlusal thickness were standardized to 1.5 mm thickness except at the central pit, which was 1.3 mm thick. The STL file for the tooth dies was prepared using software (3Shape TRIOS In recent decades, there has been considerable development in the materials and technologies in prosthetic and restorative dentistry ,2,3,4. EThe CAD/CAM technique has superior results with more time efficiency, an improvement in cost/effectiveness, and higher-precision prostheses compared to conventional manufacturing techniques . With thAt present, there are various types of zirconia restorations based on strength and opacity due to advances in zirconia manufacturing. Both the opaque zirconia crown and the translucent lithium disilicate crown require thickness (minimum 1.5 mm) for sufficient strength. One important factor for the longevity of zirconia restorations is the mechanical and fracture load properties of the restoration apart from the marginal fit, fracture resistance, and esthetics ,10,11. SThe proper manipulation of restorations with adequate thickness presents increased fracture strength when bonded to tooth tissue ,15. Axia® ZirCad LT and MO, Ivoclar Vivadent; Lava™ Plus, 3M; BruxZir®, Glidewell Laboratories; and KATANA™ HT, Kuraray Noritake), (b) 4 mole % Y-TZP  , and (c) 5 mole % Y-TZP  .Currently, zirconia can be classified into three groups based on the yttria content as follows : (a) 3 mThis study aims to compare the crown fracture load of the four most common different translucent zirconia brands  available in the market with 1.5 mm thickness. The hypothesis of this study was there is no difference in the fracture load of the four most common different translucent zirconia brands .Tooth preparations for the full ceramic crown were done digitally with software (AutoCAD) by placing a 1.0 mm chamfer margin and 1.5 mm occluso-cervical curvature for the crown sample manufacturing without periodontal ligaments and roots, since the rigidity of the specimen is taken into account . Stylize® Patient Monitoring, Copenhagen, Denmark). Tooth dies were printed with a resin material  using a 3D printing software (3D Sprint® Client Version 3.0.0.2494) from a 3D printer . The printing layer thickness was 50 µm. Then, a total of twenty-eight (N = 28) stylized crowns were milled out of AmannGirrbach  (n = 7), Cercon HT  (n = 7), Cercon XT  (n = 7), and Vita YZ XT  (n = 7). The compositions of each material used in this study with their translucency values are shown in The STL file for the tooth dies was prepared using computer software  and allowed to self-cure under finger pressure for 6 min. The crowns were loaded on the occlusal surface in a universal testing machine (MTS Centurion) with a stainless-steel ball indenter (7 mm radius) with a loading rate of 1 mm/min to contact the stylized crowns on each of the four cusps until failure ,29,30. Ap value = 0.05. The sample size was calculated using G*Power 3.1.9.7, which obtained a minimum of three per group, and the power was 99% [The descriptive statistics were calculated from the SPSS version 20 . The fracture loads were analyzed using Dunnett’s T3 test, and the number of cracks was analyzed using the Mann–Whitney U test among the groups. The significant level was set at  was 99% . p value < 0.0001).The fracture load results are shown in p value < 0.0001) and Cercon HT vs. Cercon XT , while there was no statistical significant difference between Cercon HT vs. Cercon XT  and Cercon HT vs. Cercon XT .The cracks on the surfaces of zirconia crowns are shown in The advancement in zirconia manufacturing technology has developed various new zirconia compositions with esthetic enhancements. Zirconia is polymorphic and can be present in three crystallographic structures with the same chemical composition: monoclinic, tetragonal, and cubic ,33. At rThe aging process results from the progressive spontaneous transformation of the metastable tetragonal phase to the monoclinic phase (t–m transformation) in the presence of water or water vapor at relatively low temperatures (approximately 30 °C up to 400 °C), which is a phenomenon known as hydrothermal or low-temperature degradation. Regarding the aging and degradation of zirconia, some theories have described the aging mechanism. Yttria, which is used as a dopant in zirconia to achieve phase stability, is exhausted through a reaction with water in the presence of moisture, resulting in a tetragonal to monoclinic (t–m) phase transformation ,37,38. − and forming lattice defects that cause the t–m phase transformation [2 derived from water likely fills the oxygen vacancies of the matrix and induces a t–m phase transformation [The zirconia bond of Zr–O is disturbed by water, thereby, creating stress due to the diffusion of OHormation . O2 deriormation . This trormation . Sato anormation  proposedThis study aimed to compare the crown fracture load of the four most common different translucent zirconia brands available on the market; AmannGirrbach , Cercon HT , Cercon XT , and Vita YZ XT . Based on the results, the hypothesis that there is no difference in the fracture load of the four most common different translucent zirconia brands was approved. We found that the Cercon HT presented the best strength properties, the highest fracture load, and no visible cracks. AmannGirrbach presented the lowest strength properties. The reason that Cercon HT showed more fracture load/resistance may be due to the lower yttria content compared with the other groups. Cercon XT and Vita YZ XT had higher yttria contents and had less phase transformations and less fracture resistance.The yttria contents of AmannGirrbach and Cercon HT are similar; however, the fracture toughness of AmannGirrbach was lower than Cercon HT. The reason could be from the process of the manufacturing process for making translucent zirconia, such as by adding more cubic phases to increase the translucency without changing the phase transformation. Similarly, although the yttria contents of various crown materials are close to each other with similar load applications, the Cercon HT could receive more loads, which could be due to the lowe yttria content being converted to the monoclinic phase.There are several methods for making translucent zirconia, such as by increasing the stabilized yttrium, increasing the cubic phase ratio without reducing the toughness, and reducing its grain size . Hence, Lawson et al.  performeBoth zirconia crowns luted with resin cement showed a greater fracture load than the resin-modified glass-ionomer cement. In addition, the 3Y-PSZ had a greater fracture load than the 5Y-Z and lithium disilicate for the resin cement. Another study found that the lithium disilicate restorations showed significantly greater load-bearing properties compared wtih 5Y-PSZ but was similar to 4Y-PSZ . In our Similarly, Kim et al.  comparedIn our study, the thickness had a 1.0 mm chamfer margin and 1.5 mm incisal-cervical curvature. Hence, the fracture load was higher in our study compared to their study. However, a study by Nakamura et al.  mentioneLopez-Suarez et al.  comparedSimilarly, Alshiddi et al.  studied Some limitations of this study include that we could not create more samples due to financial restrictions. Longer fatigue would be helpful for better prediction of the performance of the crowns, and the differences in the material composition can affect the fracture load results. Further studies can be done to study the effects of the cement on the fracture load of these ceramic crowns. In addition, the fracture load can be studied by comparing the studied zirconia crowns of varying thicknesses.The crown fracture loads of the studied four most-common different translucent zirconia brands  were in the acceptable range. Compared to the maximum bite force, all materials presented an acceptable fracture load value. Cercon HT presented the best strength properties, the highest fracture load, and no visible cracks compared to the other three zircona brands. AmannGirrbach presented the lowest strength properties.