Desulfovibrio, Faecalibaculum, and Lactobacillus and lower levels of Muribaculum compared to ND mice. However, GWD and SGW mice showed a significantly different gut microbial composition, such as a lower proportion of Lactobacillus and Desulfovibrio, and higher proportion of Faecalibaculum and Muribaculum compared to WD mice. High starch diet-induced dysbiosis caused increase of lipid accumulation and inflammation-related proteins’ expression, thereby leading to non-alcoholic fatty liver disease. However, GWD and SGW showed lower levels than that, and it might be due to the difference in the gut microbial composition compared to WD. Taken together, diets high in gelatinized starch and high in gelatinized starch supplemented with salt induced mild metabolic disorders compared to native starch.Diets high in gelatinized starch and high in gelatinized starch supplemented with salt-induced metabolic disorders and changes in gut microbiota have scarcely been studied. In this study, mice on wheat starch diets (WD) exhibited significantly higher body weight, white adipose tissue (WAT), and gut permeability compared to those on normal diet (ND). However, gelatinized wheat starch diet (GWD) and NaCl-supplemented gelatinized wheat starch diet (SGW) mice did not increase body and WAT weights or dyslipidemia, and maintained consistent colon pH at ND levels. WD mice showed higher levels of  Asian diets are characterized by high carbohydrate and salt contents, which is reported to be an important cause of metabolic diseases. High-carbohydrate diet (HCD) is known to induce hepatic steatosis and cholesterol deposition in the same manner as a high-fat diet . Starch,High salt intake is also known to cause various metabolic diseases. It is the major cause of high blood pressure, leading to cardiovascular diseases  and is rIt is well known that gut and liver are connected by portal vein and bidirectionally communicate. Changes in the gut microbial composition can cause disruption of the gut barrier function, thereby causing translocation of microbial products, such as endotoxins and microbial-associated molecular patterns . These pIt has been reported that high-carbohydrate or high-salt diets are known to alter gut microbiota. However, changes in gut microbiota caused by diets high in gelatinized wheat starch and high in salt supplemented with gelatinized wheat starch have not yet been reported. Therefore, we investigated the effect of these diets on mice in this study.® O.C.T.™ Compound was purchased from Sakura Wheat starch, 4 kDa fluorescein isothiocyanate (FITC)-dextran, and NaCl were obtained from Sigma-Aldrich . Primary antibodies to western blot, such as peroxisome proliferator-activated receptor-γ (PPAR-γ), peroxisome proliferator-activated receptor-α (PPAR-α), sterol regulatory element-binding protein 1 (SREBP-1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), interleukin-6 (IL-6), TLR-4, tumor necrosis factor-α (TNF-α), and β-actin, were purchased from Abcam . Mouse and rabbit secondary antibodies were purchased from Thermo Fisher Scientific . Tissue-TekTo prepare gelatinized wheat starch, 10 kg of wheat starch powder was heated and stirred in 100 L of water for 60 min at 70 °C, quickly cooled to −70 °C, and lyophilized. Lyophilized gelatinized starch was used for mouse diets.n = 9) and fed a normal diet (ND), high-wheat starch diet (WD), gelatinized wheat starch diet (GWD), and NaCl-supplemented gelatinized wheat starch diet (SGW) for 8 weeks. Mice were housed three per cage and the body weight was recorded every week and food intake was measured two times a week until the end of the study.Six-week-old male C57BL/6J mice were purchased from Central Lab Animal Inc.  and the animals were maintained in a humidity-controlled animal facility with a 12-h light/dark cycle at 23 °C and provided with food and water ad libitum. All animal studies were approved by the Animal Welfare Committee of the Korea Food Research Institute (KFRI-M-18025). After acclimation, mice were divided into 4 groups  was used for the ND diet and its composition was 18.0% of calories in fat, 24.0% of calories in protein, and 58.0% of calories in carbohydrate. WD contained 17.1% of calories as fat (8.8% from soybean oil and 91.2% from lard), 17.3% of calories as protein (97.5% from casein and 2.5% from L-cysteine), and 65.6% of calories as carbohydrates (85% from wheat starch and 15% from sucrose). The composition of GWD was the same as that of WD except that wheat starch was replaced by gelatinized wheat starch. SGW was composed of 4% NaCl in addition to GWD .After 8 weeks, the mice were fasted for 12 h and euthanized by anesthesia. Blood was drawn into microfuge tubes, and serum was collected thereafter. Total cholesterol and LDL cholesterol levels were quantified using a Cholesterol Assay Kit , and triglyceride levels were analyzed using a Triglyceride Assay Kit , according to the manufacturers’ instructions.Total protein was extracted from the liver with PRO-PREP™ . Proteins were separated using a polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. The membranes were incubated overnight with the primary antibodies against PPAR-γ, PPAR-α, SREBP-1, ACC, FAS, MCP-1, IL-1β, IL-6, TLR-4, TNF-α, and β-actin at 4 °C and then incubated with secondary antibodies at 23 °C. Then, bands were detected using a ChemiDoc™ XRS+ imaging system .At week 4 and 8, fresh fecal samples were collected, and fecal DNA was extracted for the analysis of microbial composition. Hypervariable V3–V4 region of 16S rRNA amplicons were generated using a MiSeq  at Macrogen  according to the manufacturer’s instructions. Microbial diversity was assessed using the QIIME software and operational taxonomic units (OTUs) were defined at ≥97% sequence homology . TaxonomIntestinal permeability was analyzed at 8 weeks as previously described . Mice weHematoxylin and eosin (H&E) staining and oil red O (ORO) staining were performed for histological analysis. For H&E staining, liver and WAT were fixed with 4% formaldehyde and processed with paraffin. Then, the tissues were sliced and stained with H&E. For ORO staining, liver tissues were frozen with O.C.T. Compound and sliced. Then, sections were fixed in 4% paraformaldehyde and then stained with ORO. All sections were scanned and analyzed using a Pannoramic 250 Flash III slide scanner  and CaseViewer software (3DHISTECH Ltd).p-value <0.05 was considered statistically significant.The data are presented as the mean ± standard error of the mean. One-way ANOVA analysis and Tukey’s analysis were used for the statistical significance of the differences among groups. All data were obtained using GraphPad Prism software . A Over the course of 8 weeks, WD mice showed a significant increase in body weight a and theDesulfovibrio, Faecalibaculum, Romboutsia, and Lactobacillus genera and lower levels of Alistipes and Muribaculum genera compared to ND mice. Compared to WD mice, GWD mice have a lower proportion of Lactobacillus and Desulfovibrio and higher proportion of Faecalibaculum, Muribaculum, and Bacteroides. Interestingly, the gut microbial composition of SGW was similar to GWD mice. Moreover, GWD and SGW showed a significantly higher proportion of Akkermansia, which has been reported to combat obesity. In summary, high-starch diet reduced the gut microbial diversity and markedly changed the gut microbial composition; however, diets high in gelatinized starch and high salt supplementation shifted the gut microbial composition.All HCD groups showed fewer OTUs and lower Shannon and Simpson indices compared to the ND group a. PrinciIn gut permeability analysis, the levels of plasma FITC-dextran and area under the curve (AUC) significantly increased in every HCD group. Among these, SGW mice exhibited the highest increase in permeability a,b. ThesTo evaluate diet-induced changes in lipid metabolism, protein expression of lipid metabolism regulatory factors, such as SREBP-1, PPAR-α, and PPAR-γ, was analyzed. SREBP-1 and PPAR-γ protein expression increased two-fold while PPAR-α protein expression decreased in the WD mice liver . MoreoveThe correlation of changes in the gut microbiota with diet-induced metabolic disease was assessed by comparing inflammatory cytokine expression. As shown in Development of NAFLD in the liver was detected using H&E staining and ORO staining. As shown in High carbohydrate and/or high salt diets are known to be one of the major causes of metabolic diseases. Gelatinized starch is also considered to be a potent inducer of obesity than native starch. Starch gelatinization occurs by granule swelling and crystalline structure loss in the presence of heat and water. It involves breaking of intermolecular hydrogen bonds into amylose and amylopectin, thus increasing starch digestibility. Starch gelatinization is one of the most important factors in the determination of digestibility and nutritional properties. It is well known that native starch is degraded slowly by amylases but gelatinization of starch increases sensitivity to amylases . TherefoMuribaculum genus, previously classified as S24-7, is known to decrease in mice on high-fat diet and is associated with the regulation of body weight [Lactobacillus spp. treatment has an anti-obesity and probiotic effect in diet-induced obesity [Lactobacillus in WD, which is expected to be strongly associated with obesity or metabolic disease. In support of our results, several studies have reported a significant increase in the proportion of Lactobacillus in NAFLD [Faecalibaculum, one of lactic acid producers, has not been studied as much in this context yet, but its role in metabolic disease has been studied to some extent [Muribaculum compared to WD mice, and the proportion of Lactobacillus was almost similar to ND mice. Interestingly, the SGW group had a similar gut microbial composition to GWD and it also showed a markedly higher composition of Muribaculum and lower composition of Lactobacillus than those of WD. It can be concluded that the change in gut microbiota by diets high in gelatinized starch and high in salt supplemented with gelatinized starch cause weight and lipid metabolism to be similar to ND mice. In addition, the increased proportion of Akkermansia, known to be an intestinal microorganism with anti-obesity effects, was confirmed. This also seems to have played a critical role in maintaining normal weight in GWD and SGW mice, despite having a lower proportion of Muribaculum compared to ND mice. Therefore, both groups showed a higher proportion of Faecalibaculum than WD mice, which is expected to have a negative impact on other tissues.To determine whether the changes in body weight and lipid metabolism were caused by changes in gut microbial composition, 16s rRNA analysis was performed. The high-starch diet exhibited significantly increased Fermicutes to Bacteroidetes ratio and increased proportion of Proteobacteria compared to ND. This is consistent with the gut microbial composition in metabolic diseases revealed by many researchers . The Mury weight ,26. Morey weight . In this obesity . Howeverin NAFLD . Faecalie extent . These ce extent ,32. HoweMuribaculum and Akkermansia and lower levels of Lactobacillus may induce a decrease in gut–liver axis alteration and lipid metabolism, resulting in mild NAFLD.HCD is known to cause NAFLD by inducing inflammation and altering lipid metabolism in the liver . The chaAkkermansia [Faecalibaculum and decreased proportion of Muribaculum, GWD did not showed an increase of inflammatory cytokines. These results indicated that an increased proportion of Akkermansia may play a positive role in liver inflammation. Although the SGW group showed lower expression of inflammatory cytokines than WD, on the other hand, they exhibited a significant increase in the expression of inflammatory cytokines compared to GWD mice. However, high salt supplementation did not increase TLR-4 expression, so it may not be due to changes in gut microbiota-induced alteration of the gut–liver axis. Increased inflammatory cytokines without elevation of TLR-4 suggest that high salt supplementation has a direct negative effect on the liver. These results may be related to higher gut permeability in SGW mice. Further research is needed, however, to determine the effects of high-salt intake on the gut–liver axis and liver inflammation.It is well known that endotoxins, produced by gut microbiota, induce TLR-4 activation through alteration of the gut–liver axis, thereby inducing a pro-inflammatory response by TNF-α and IL-6 activation in the liver . Therefoermansia . HoweverMuribaculum and increased Faecalibaculum compared to ND. For these reasons, we carefully suggest that diets high in gelatinized starch and high in gelatinized starch supplemented with salt cause mild metabolic disorders compared to native starch. However, further studies on the increased expression of inflammatory cytokines in liver and gut permeability in high salt-supplemented diets are needed.GWD and SGW altered WD-induced change of gut microbiota, such as an increase of Bacteroidetes and decrease of Firmicutes and Proteobacteria. Hence, it can be concluded that gelatinized starch did not increase body and WAT weight. Moreover, high salt supplementation did not affect gelatinized starch-induced change in gut microbiota. Both GWD and SGW groups had milder NAFLD compared to WD. These results may be due to decreased


No ambiente de grande competição entre periódicos científicos, em que a batalha pelo melhor artigo científico, maior número de citações e maior fator de impacto (FI) parece não ter limites, os Arquivos Brasileiros de Cardiologia (ABC Cardiol) tem tomado ações no intuito de contribuir para o crescimento de outros periódicos com foco similar na comunidade científica nacional. O objetivo principal é permitir que a nossa melhor ciência seja veiculada por periódicos científicos de qualidade cada vez mais alta, obtenha a visibilidade que merece, e possa contribuir de forma mais impactante na comunidade científica mundial.Journal of Citation Reports (JCR), os ABC Cardiol têm tido um aumento crescente de submissões nacionais e principalmente internacionais.2Nossa taxa de aceitação não passa de 15% e com tendência a diminuir ainda mais. Nesse cenário, muitos artigos de qualidade significativa não podem ser aceitos para publicação nos ABC Cardiol, incluindo artigos oriundos dos nossos profícuos programas de pós-graduação. Infelizmente, tal fato tem como efeito indesejável a diminuição da exposição da nossa melhor ciência em fontes de indexação como o PubMEd, Scielo e outros.Com o atual FI próximo a 1,7 pela Com essa situação em mente, o corpo editorial dos ABC Cardiol tem comunicado aos autores desses artigos não aceitos para publicação a possibilidade de indicação ou transferência da submissão desses manuscritos para periódicos nacionais em ascensão, que poderiam ser adequados para a divulgação daquela ciência específica. É importante ressaltar que a decisão de transferência ou submissão a periódicos sugeridos é uma decisão única e exclusiva dos autores. Essa é uma logística do tipo “win-win situation”, pois ganham tanto os autores que podem ter seus artigos publicados de forma ágil em periódicos de prestígio e em crescimento, como os periódicos que terão probabilidade de maior número de citação e poderão melhorar e ampliar sua indexação em bases científicas mundiais.International Journal of Cardiovascular Sciences (IJCS). Ambas são mantidas pela SBC e indexadas na Scielo, o que permite que ambas utilizem o mesmo sistema de submissão, o ScholarOne, e possam realizar transferências diretas, com as revisões incluídas, desde que autorizadas pelos autores. Os ABC Cardiol têm um foco mais intenso em doença cardiovascular, e o IJCS na multidisciplinaridade, incluindo aspectos da nutrição, fisioterapia entre outros, o que torna a relação entre esses periódicos sinérgica e não competitiva. Esta parceria com o editor-chefe do IJCS, Dr. Claudio Tinoco Mesquita, tem impulsionado ambos os periódicos, de forma muito efetiva, à maior visibilidade e impacto.Como exemplos de colaboração, temos a mais íntima entre os ABC Cardiol e o Journal of Transcatheter Interventions (JOTCI) formam, com os ABC Cardiol, uma grande família de periódicos científicos na área da ciência cardiovascular. Contudo, esses periódicos não fazem parte da Scielo, não permitindo, assim, sua integração ao sistema do ScholarOne sem custo associado. Por isso, quando um artigo é rejeitado para publicação pelos ABC Cardiol, o modelo inclui o envio de e-mail aos autores, contendo um link para o contato direto com os periódicos que, na visão do corpo editorial dos ABC Cardiol, seriam adequados para veiculação daquele material científico. Isso facilita e agiliza uma possível submissão pelos autores.Em modo de colaboração intermediária, a ABC Imagem Cardiovascular e a 3Também agradeço ao editor-chefe Dr. Silvio Henrique Barberato da ABC Imagem Cardiovascular que tem mantido uma parceria muito próxima com os ABC Cardiol.Neste ponto, gostaria de fazer um agradecimento pessoal ao editor-chefe da JOTCI, Dr. Pedro Beraldo de Andrade, pela divulgação dessa colaboração em um editorial publicado em sua revista, que comentou também sobre a primeira publicação internacional gerada por esse modelo na JOTCI.Brazilian Journal of Cardiovascular Surgery. Fica aqui a ideia para discutirmos com os respectivos editores chefes Dr. Bruno Guedes Baldi e Dr. Domingos M. Braile.Ainda, julgamos importante implementar uma colaboração com outras sociedades de especialidade mantenedoras de periódicos de grande importância para nossa ciência nacional, como o Jornal Brasileiro de Pneumologia e o Journal of the America College of Cardiology (JACC), ligada ao American College of Cardiology dos Estados Unidos, e da família de periódicos da European Heart Journal (EHJ), ligada a European Society of Cardiology (ESC) na Europa. Apenas como exemplo, entre 2011 e 2018, sete curtos anos em tempo editorial, o EHJ saiu de um FI de 10,4 para 24,8, o European Journal of Heart Failure de 4,8 para 13,9, o European Journal of Preventive Cardiology de 2,6 para 5,6, EHJ Cardiovascular Imaging de 2,3 para 5,2, e o Europace de 1,9 para 6,1. Esses números são realmente impressionantes em termos de aumento de FI em um período muito curto. Este modelo parece ser muito eficaz em melhorar a divulgação da ciência e permitir ao mesmo tempo o crescimento do FI dos periódicos membros da família.A proximidade dos periódicos em modelo de família permite maior sucesso de cada um dos periódicos individualmente. Essa tem sido a rotina na ciência mundial, em especial na área de cardiologia, a exemplo do crescimento impressionante da família de periódicos do Como família ou como “amigos”, creio que a colaboração efetiva entre periódicos nacionais de grande reputação permite um crescimento do grupo de periódicos que não seria possível isoladamente. Espero que essa colaboração inicial e preliminar possa estimular uma maior discussão e interação futura mais intensa entre periódicos com objetivos comuns e focos científicos próximos. Convido a todos a pensarmos juntos em modelos que permitam real crescimento da nossa comunidade científica dentro da ciência mundial. A qualidade da nossa produção científica já é fato; precisamos minimizar as dificuldades que ainda temos de divulgar efetivamente nossa melhor ciência. Este deve ser um trabalho conjunto dos nossos periódicos científicos nacionais.Entre as ações do ABC Cardiol nesta direção estão a modernização do portal digital do periódico e utilização de ferramentas digitais modernas. Entre elas ferramentas que sugiram artigos semelhantes em outros periódicos quando uma busca é realizada em seu portal digital, com foco nos periódicos nacionais da família cardiovascular. As oportunidades são muitas e temos que trabalhar para aproveitá-las integralmente. Arquivos Brasileiros de Cardiologia (ABC Cardiol) has taken actions to foster the growth of other journals with similar scope in the national scientific community. The main objective is to promote dissemination of our best science by journals of increasingly higher quality, so that it can attract deserving attention and make a stronger contribution to the world scientific community.In the environment of intense, and apparently unlimited competition among scientific journals for the best article, and the highest number of citations and impact factor (IF), the 2 Our acceptance rate is no more than 15% and tends to decrease even more. In this scenario, many papers of significant quality cannot be accepted for publication in the ABC Cardiol, including articles from studies developed in our successful postgraduate programs. This has an undesirable effect – the reduced exposure of our best science in indexation sources such as PubMED, Scielo, among others.With the current IF of nearly 1.7 according to the Journal of Citation Reports (JCR), the ABC Cardiol has attracted more and more submissions from national and international authors.With this in mind, the editorial board of the ABC Cardiol has suggested to the authors of manuscripts not accepted for publication, either the possibility of recommendation of these papers for publication, or the transfer of these papers to other potential national journals that would be adequate for dissemination of that specific scientific information. It is important to point out that the decision to transfer or submit the manuscript to one of the journals recommended by the ABC Cardiol is solely and exclusively made by the authors. This is a “win-win situation” where both authors and journals win, the first for having their manuscript rapidly published by high quality journals, and the second for increasing the likelihood of citations and indexation in international scientific databases.One example of such collaboration is the up-close relationship between the ABC Cardiol and the International Journal of Cardiovascular Sciences (IJCS), both held by the SBC and indexed in Scielo. This allows that articles are submitted through the same unique system, the ScholarOne, and can be directly transferred, including the revisions, once authorized by the authors. While the ABC Cardiol focuses more on cardiovascular disease, the IJCS focuses more on multidisciplinarity, including other areas such as Nutrition and Physiotherapy, which makes the relationship between these journals synergistic rather than competitive. Thus, the partnership with the Editor-in-Chief of the IJCS, Dr. Claudio Tinoco Mesquita, has effectively boosted the recognition and the impact of both journals.ABC Imagem Cardiovascular and the Journal of Transcatheter Interventions (JOTCI), together with the ABC Cardiol, constitute a large family of scientific journals in the cardiovascular field. However, these journals are not members of Scielo, and hence cannot be integrated to the ScholarOne system without additional cost. For this reason, when a manuscript is rejected for publication by the ABC Cardio, an e-mail is sent to the authors, containing a link to journals that, according to the ABC Cardiol’s editorial board, may be adequate to publish that scientific material. This model facilitates and speeds up the (re)submission of the manuscript.In an intermediate level of collaboration, the 3 I would also like to thank the Editor-in-Chief Dr. Silvio Henrique Barberato of the ABC Imagem Cardiovascular for the close relationship with the ABC Cardiol.At this point, I would like to thank the Editor-in-Chief of the JOTCI, Dr. Pedro Beraldo de Andrade, for reporting about this collaboration in an editorial published in the JOTCI, and commenting about the first publication of an article resulting from this model in an international journal. Jornal Brasileiro de Pneumologia and the Brazilian Journal of Cardiovascular Surgery. This is my suggestion for consideration by the respective Editors-in-Chief Dr. Bruno Guedes Baldi and Dr. Domingos M. Braile.Also, we find it important to implement a collaboration to other societies that support other journals relevant to the national science, such as theThe proximity of the journals in a “family” model allows a greater success of each journal individually. This has been a tendency of the world science, particularly in Cardiology, as exampled by the impressive growth of the American College of Cardiology (JACC)’s family of journals, associated with the USA American of Cardiology, and the European Heart Journal’s (EHJ) family, associated with the European Society of Cardiology (ESC) in Europe. As example, in the period from 2011 and 2018 , the IF of the EHJ increased from 10.4 to 24.8, the European Journal of Heart Failure had its IF increase from 4.8 to 13.9, the European Journal of Preventive Cardiology from 2.6 to 5.6, the EHJ Cardiovascular Imaging from 2.3 to 5.2, and the Europace from 1.9 to 6.1. These increases in IF, in such a short period of time, are impressive. Therefore, this model seems very effective in improving the dissemination of science while enabling the increase of the IF of each member of the family.Either as a family or as “friends”, I believe that an effective collaboration between national renowned journals allows the growth of a group of journals that would not be seen by the journals alone. Hopefully, this initial and preliminary collaboration will lead to a broader discussion and closer interaction between journals with similar aims and scope. I invite everyone to think together about models that would increase the visibility of our scientific papers in the world science. Although the high quality of our scientific production is already recognized, we still need to minimize the barriers to effectively disseminate our science. This must be a joint effort of the Brazilian scientific journals.Among the actions of ABC Cardiol in this direction are the reshaping of the journal’s digital portal and the use of modern digital tools. Among them are tools that suggest similar articles in other journals when a search is carried out on its digital portal, focusing on national journals in the cardiovascular family. The opportunities are many and we have to work to take full advantage of them.


Nevertheless, statistically comparisons resulted in no significant effect of the differently sized microslits on SBS (p > 0.05). Additionally, MS  demonstrated greater SBS and bonding durability than that of CON , confirming that the micromechanical retention with laser-milled microslits was effective at enhancing the bonding strength and durability of the acrylic resin and zirconia. Polycrystalline zirconia-based ceramics are a newly accessible material for improving removable prosthodontic treatment, as the bond strength with acrylic resin can be greatly enhanced by laser milling.Heightened aesthetic considerations in modern dentistry have generated increased interest in metal-free “zirconia-supported dentures.” The lifespan of the denture is largely determined by the strength of adhesion between zirconia and the acrylic resin. Thus, the effect on shear bond strength (SBS) was investigated by using an acrylic resin on two types of zirconia ceramics with differently sized microslits. Micromechanical reticular retention was created on the zirconia surface as the novel treatment (microslits (MS)), and air-abrasion was used as the control (CON). All samples were primed prior to acrylic resin polymerization. After the resin was cured, the SBS was tested. The obtained data were analyzed by using multivariate analysis of variance(α = 0.05). After the SBS test, the interface failure modes were observed by scanning electron microscopy. The MS exhibited significantly higher bond strength after thermal cycles ( Metal alloys, such as cobalt-chromium, titanium, and gold, are widely used in removable prosthodontic treatment as materials for the frameworks of removable particle dentures (RPDs). These metal alloys possess favorable characteristics, such as excellent mechanical strength and thermal conductivity. However, the demand for metal-free treatments has increased due to various factors, such as escalating precious metal prices , concernEnhanced technology, such as computer-aided design and computer-aided manufacturing (CAD/CAM), has been successfully integrated into dentistry from the engineering sciences. The polycrystalline zirconia-based ceramics with superior mechanical properties, which can only be processed by CAD/CAM, have been widely utilized in clinical dentistry . ZirconiPrevious literature has reported that zirconia ceramics are potentially alternative materials for connectors, clasps, and other components used in the framework of RPDs ,15 due tIn the last decade, with the advancement of laser techniques, the use of lasers as an alternative pretreatment method for dental procedures has become prevalent, such as by applying laser treatment to dental materials to improve the bonding properties between materials and resin-based luting agents  or materHerein, we aim to investigate the effects of micromechanical retention (microslits of different sizes) created by lasers on durability and bond strength regarding two different zirconia ceramics and an acrylic resin. The theory is that (1) laser pretreatment should increase the shear bond strength (SBS) and durability of the zirconia ceramics and acrylic resin; (2) the different sizes of microslits (laser grooves) will alter the SBS; and (3) the SBS would be unchanged after artificial aging via thermal cycling.The details of the materials used in this study are provided in Two types of zirconia ceramics (Y-TZP and Ce-TZP/A) were processed with the CAD/CAM system and then sintered in a high-temperature furnace. The temperature was raised to 1450 °C and maintained for 2 h. The zirconia ceramics were then fabricated into disk-shaped specimens with a diameter of 10 mm and a thickness of 2.5 mm a. Eighty disk-shaped specimens were prepared for each zirconia ceramic. All specimens were then ground flat with 600-grit silicon carbide abrasive paper, followed by steam cleaning and air-drying. Subsequently, specimens were randomly distributed into four groups according to the surface treatments (n = 20 per group for each zirconia ceramic).2O3) particles of 50 μm, and jet pressures of 0.3 MPa, for 10 s. The distance from the orifice to the zirconia ceramic’s surface was approximately 10 mm (group CON), as shown in 4) laser . The laser irradiation conditions were 70 kHz frequency, 1065 nm pulse-width, 60 mm irradiation distance, and 5–6 min radiation time. The microslits (laser grooves) followed an isometric grid pattern with groove dimensions of 50 μm (group 50MS), 75 μm (group 75MS), and 100 μm (group 100MS), as shown in Specimens of air-abrasion pretreatment (group control (CON)) were treated by air-abrasion with grain-sized alumina  was positioned on the surface of each zirconia ceramic specimen to define the bonding area a and wasOne hour after the bonding procedure, the specimens were immersed in water at 37 °C for 24 h. This state was defined as a “zero-thermal cycle.” The 24 h shear bond strength of half the number of specimens in each group (n = 10) was tested at the zero-thermal cycle. The remaining specimens (n = 10) were placed in a thermal cycling apparatus  and cycled between a cold bath (4 °C) and a hot bath (60 °C) with a one-minute dwell time per bath for 20,000 cycles. The specimens immersed in water for 24 h  and those after 20,000 thermal cycles were subjected to SBS testing. The specimens were attached to a shearing jig  and the After the SBS test, the fractured interfaces were observed with an optical microscope  at 8× magnification to determine the mode of failure. The failure modes were classified into (1) adhesive failure , (2) cohesive failure , and (3) mixed failure (combination of cohesive and adhesive failure). The fractured surfaces were also observed by scanning electron microscopy (SEM) at 500× magnification , at a high vacuum level and an operating voltage of 2 kV.The data collected for all tests were calculated, and the homogeneity of variance was primarily analyzed using Levene’s test. Data comparisons were conducted using one-way analysis of variance (ANOVA) with post hoc Scheffé test and post hoc Tukey’s HSD (honestly significant difference) tests. All statistical analyses were performed using IBM SPSS statistical software , and the level of statistical significance was set at 5%.p < 0.05). However, in MS there was no dramatic reduction (<25%) observed in the SBS values irrespective of the artificial aging process or the different conditions of laser treatment . p < 0.05). However, no dramatic reduction (< 9%) in SBS values occurred in MS regardless of the different conditions of laser treatment , or whether or not artificial aging was performed.The results of SBS testing of Ce-TZP/A follow a similar trend to those of Y-TZP, as seen in From the representative SEM images of the debonded zirconia-resin interface , it was 2O3 particles (CON) [2O3  was approximately 0.23 μm for Y-TZP and 0.24 μm for Ce-TZP/A [The shear bond strengths of the acrylic resin and two types of zirconia ceramic (Y-TZP and Ce-TZP/A) were evaluated after traditional surface pretreatment, air-abrasion with 50 μm and 0.3 MPa Ales (CON) , and a nes (CON) ,33 postues (CON) , reporte4) to make microslits on the surface of zirconia ceramics [Compared with the traditional mechanical surface treatment methods, laser treatment is suggested to be more controllable and stable. Thus, lasers have been widely used in medicine over the past decade. In dentistry, the erbium-doped yttrium-aluminum-garnet (Er: YAG) laser is the most frequently used laser system. Based on prior related experiments, it was determined that although Er: YAG lasers can improve the surface roughness of zirconia ceramics , they receramics . The repceramics . Three iceramics . TherefoPrevious studies have reported that when acrylic resin bonds to other materials, the insufficient bonding strength at the adhesive surface causes microbreakage due to differences in the thermal expansion coefficient between the two substrates . During The minimum acceptable SBS value is 5 MPa at the interface of resin-based materials and substrate, as specified in ISO 10477 . Additio4 laser also needs to be assessed in future studies. CNC lasers are not popularly used in dentistry due to their size, so a reduction in size, such as a desktop CNC laser machine, is also an essential issue that requires further investigation. Furthermore, when applying laser as a surface treatment method, the high temperature and heat of laser might cause microstructure change in zirconia ceramics and might further affect the material properties; thus, the internal structure changing mechanism of materials should also implement in future studies. Regardless, the present study provides substantial evidence to support the application of Nd: YVO4 lasers in dentistry, in order to substantially improve the adhesive properties of acrylic resin bond to zirconia ceramics.Additional research is required to determine the thickness of the RPD framework made by zirconia ceramics and combine it with microslits. Furthermore, the processing time and the strength of the Nd: YVO4 laser effectively improve the bonding strength and durability of acrylic resin bond to zirconia ceramics. After artificial aging, the bond strength of the air-abrasion treated zirconia ceramics (CON) decreased, but that of the laser etched materials (MS) remained high. Yet, there was no difference in the bond strength between the different sizes of microslits diameter .The limited results of this in vitro study prompt that micromechanical microslits etched with a Nd: YVO


European Journal of Midwifery would like to thank all our reviewers who have contributed to the journal in Volume 1 (2017).The editors of Carrie BonsackUnited StatesSigne EgenbergNorwayRamón EscurietSpainJennifer FenwickAustriaBelén FerrerSpainSusan FlemingUnited StatesPeter GallagherNew ZealandJane HendersonUnited KingdomBengt KayserSwitzerlandJulia LeinweberAustraliaColin MartinUnited KingdomRui Filipe Oliveira MiguelotePortugalPolona MivšekSloveniaJane MorrowAustraliaJaana NevalainenFinlandRhona O’ConnellIrelandVictor PulgarUnited StatesMirjam SnellenAustraliaChuenjitwongsa SupachaiThailandLinda SweetUnited StatesStefania TriunfoSpain


Essential for the human use of a radiotracer is its production and quality control under GMP-compliance. For this purpose, all analytical methods have to be validated. [18F]PSMA-1007 is easily radiosynthesized in a one-step procedure and isolated using solid phase extraction (SPE) cartridges followed by formulation of a buffered injection solution and for the determination of its chemical and radiochemical purity a robust, fast and reliable quality control method using radio-HPLC is necessary. After development and optimizations overcoming problems in reproducibility, the here described radio-HPLC method fulfills all acceptance criteria—for e.g., specificity, linearity, and accuracy—and is therefore well suited for the routine quality control of [18F]PSMA-1007 before release of the radiopharmaceutical. Recently a European Pharmacopeia monograph for [18F]PSMA-1007 was published suggesting a different radio-HPLC method for the determination of its chemical and radiochemical purity. Since the here described method has certain advantages, not least of all easier technical implementation, it can be an attractive alternative to the monograph method. The here described method was successfully validated on several radio-HPLC systems in our lab and used for the analysis of more than 60 batches of [18F]PSMA-1007. Using this method, the chemical and radiochemical purity of [18F]PSMA-1007 can routinely be evaluated assuring patient safety.For the PET imaging of prostate cancer, radiotracers targeting the prostate-specific membrane antigen (PSMA) are nowadays used in clinical practice. [ This method has certain advantages over the recently published monograph method and can be an attractive alternative. The successfully validated radio-HPLC method was routinely used for the analysis and subsequent release of more than 60 batches of [18F]PSMA-1007 over more than one year assuring patient safety.In order to use a radiotracer in human applications, this tracer has to be produced and its quality ascertained under full GMP-compliance. The production should take place fully automated in a cleanroom environment. The fully automated production of PSMA-1007 this is the reference compound PSMA-1007. Additionally, other impurities may be formed during the labelling reaction which may be known or unknown impurities. The chemical purity of [18F]PSMA-1007 can be accessed by HPLC using UV detection. The radiochemical purity can be evaluated using the same analytical method by having a radioactivity detector in series. Typical methods for the quality control of radiopharmaceuticals include radio-HPLC, radio-TLC, and gas chromatography for determination of residual solvents. For some commonly used radiopharmaceuticals—e.g., [18F]FDG, [18F]FLT, and [18F]FET—these acceptance criteria as well as suitable analytical methods are described in monographs of the current European Pharmacopeia [18F]PSMA-1007 a monograph (“PSMA-1007 (18F) injection”; 07/2021:3116) detailing its acceptance criteria has recently been published [18F]PSMA-1007 established in our lab as already published PSMA-1007 injection solution is recommended as 10 µg/mL (0.1 mg/Vmax) which has recently also been defined in the monograph of [18F]PSMA-1007. The peak area of this reference solution has to be determined in calibration measurements and routinely checked before analyzing the quality control sample of [18F]PSMA-1007 injection solution since this area is the basis for the comparison of peak areas of other impurities: the peak area of any impurity has to be less than or equal to the peak area of a reference solution of PSMA-1007 (10 µg/mL). The sum of the peak areas of all impurities including PSMA-1007 has to be less than or equal to 5 × the peak area of the reference solution. Peak areas ≤ 0.3 x the peak area of the reference solution can be disregarded.The limit value for the unlabeled compound PSMA-1007 (reference standard) in the [18F]PSMA-1007 is recommended to be ≥95%, also in accordance with other established radiopharmaceuticals. Radiochemical impurities have to be ≤5%. Recently a radiochemical purity of ≥91 % for [18F]PSMA-1007 has been suggested in the monograph 3116 where the impurity caused by [18F]fluoride has to be ≤5%. This is fulfilled by the here recommended acceptance criterion. The radiochemical purity of [18F]PSMA-1007, both guidelines were taken into consideration. Using this analytical method, the following specification parameters of [18F]PSMA-1007 injection solution were determined: chemical identity of PSMA-1007, radiochemical identity of [18F]PSMA-1007, amount of PSMA-1007 as well as chemical and radiochemical impurities of the injection solution. For GMP-compliance all these parameters have to be validated. The here successfully validated radio-HPLC method for the determination of the chemical and radiochemical purity of [18F]PSMA-1007 has the following parameters  guideline “Validation of Analytical Procedures” . For therameters :This method is different to the recently suggested one in the Eur. Ph. monograph “PSMA-1007 (18F) Injection”  whose pa18F]PSMA-1007 may use this method, too, when explaining its advantages to their authorities and showing that it is suitable.The most severe difference between the two methods is the mobile phase: while the in this work validated method uses the addition of 0.1% TFA to the aqueous phase, the monograph method uses a phosphate buffer. A salt buffer has the huge disadvantage that it may crystallize in the HPLC system resulting in blockages. To reduce this risk the system has to be cleaned very thoroughly after the analysis which takes some time. Moreover, the preparation of a salt buffer is more elaborate, error-prone and time consuming in comparison to the addition of TFA to water. Another difference is the run time of the method which has to be at least double the retention time of the analyte. In the here validated method it takes 19 min to again reach the starting conditions of the method in comparison to approximately 25 min for the monograph method. Since the HPLC run has to be completed before release of the radiopharmaceutical the shorter the run time the better. Still another difference between the two methods is the UV wavelength chosen for the measurements (254 nm vs. 225 nm): generally, the adsorption of solvents is higher at lower wavelengths which may result in an increase of baseline noise. A parameter which makes the monograph method technically more difficult to implement, is the slightly elevated temperature of 30 °C versus room temperature in the here presented method. This necessitates a column oven which may not be present in every HPLC system representing a knock out criterium for these systems. Regarding the column stationary phase, the monolithic as well as the core-shell phase technology show high resolution, productivity and sensitivity. Comparing both methods regarding their differences, the here developed and validated method has certain advantages over the monograph method which may make it attractive to users worldwide. European users which have to consider the monograph for [18F]PSMA-1007 in our lab. The following parameters, whose acceptance criteria are based on monographs for radiopharmaceuticals as well as guidelines for method validation PSMA-1007.After successful validation, the results for chemical and radiochemical purity on both systems have been compared for several batches of [18F]PSMA-1007 injection solution the chemical identity of PSMA-1007 as well as known impurities have to be validated. Furthermore, unknown impurities may occur in the product solution. With an external reference standard, the chemical identity of PSMA-1007 can be verified using its retention time. The retention time of the product should be at least 3 times the systems dead volume. For the validation of the suitability of the method to determine the chemical identity of PSMA-1007, a reference standard solution of PSMA-1007 was injected. Possible known impurities which may occur in the injection solution of [18F]PSMA-1007 can be the labeling precursor of PSMA-1007 (trimethyl-ammonium-precursor for direct labeling) and defluorohydroxy-PSMA-1007. These compounds were also separately injected and identified by means of their retention times. 18F]PSMA-1007, PSMA-1007 as well as possible known impurities.For the evaluation of the chemical purity of the PSMA-1007 injection solution. Since their identity is not known, they cannot be injected separately. As recommended, impurities may be disregarded if they are below the limit of 0.3 × the peak area of the reference solution of PSMA-1007 (10 µg/mL). Nonetheless unknown as well as known impurities have to be adequately separated from PSMA-1007 in order to not interfere with the determination of its amount.Furthermore unknown impurities may occur in the [18F]FET [18F]PSMA-1007 [18F]PSMA-1007 was analyzed. The UV-chromatogram showed a resolution of 4.0 and 3.8 to the neighboring peaks of PSMA-1007 on the UltiMate 3000 system and a resolution of 3.9 and 5.2 on the ICS5000 system. The acceptance criterion of at least 2.0 for the resolution to unknown impurities was therefore also fulfilled on both systems. The specificity of the method for known as well as unknown impurities to PSMA-1007 was proven.The specificity which ensures that unknown as well as known impurities are suitably separated from PSMA-1007, can be validated by injection of a solution containing all known impurities and determination of the resolution between the compounds. In reference to the Eur. Ph. monograph of [18F]FET , the resSMA-1007 , is not A very important parameter for the suitability of a method for routine measurements is its precision or repeatability. The precision expresses the closeness of the results when repeatedly measuring a homogenous sample with the same analytical method. For validating the precision of the method, the samples used for the determination of the linearity were injected 4–5 times for each concentration and the relative standard deviations (RSD) of the peak areas of each level were calculated which should be less than 2%.18F]PSMA-1007 production, the injection solution contained 90% physiological saline (0.9% NaCl) and 10% ethanol as well as sodium ascorbate for stabilization. For the validation the reference standard PSMA-1007 was solved in physiological saline/ethanol (90:10 v/v) without sodium ascorbate because its peak eluted at 0.6 min (dead volume) and was huge in comparison to the PSMA-1007 peak. Measurements of PSMA-1007 with or without sodium ascorbate were otherwise completely comparable. Since solubility of PSMA-1007 in aqueous solution is poor, the reference standard was first solved in 50 µL DMSO and then diluted accordingly. Using normal plastic tubes and pipette tips for dilution resulted in completely unreproducible results which could be expected since droplet formation in the tips was seen during dilution. This problem was solved by using so called “protein low bind” tubes and tips for preparation of the standard solutions.When first starting the [18F]PSMA-1007 in our lab was the UltiMate 3000 system. The first system used for validation of the HPLC method for [18F]PSMA-1007 batches and comparing them to the results of the same batches on the UltiMate 3000 system huge discrepancies occurred. Peak areas during validation measurements and measurements of the reference standard solution were approximately half the size for the ICS5000 system used compared to those of the UltiMate 3000 system. This was not the case for measurements of [18F]PSMA-1007 batches which were almost comparable in size. Therefore, when measuring a reference standard solution of PSMA-1007 before the batch analysis on both systems, the results were comparable  vs. 10.24 µg/mL (UltiMate 3000)). However, the following batch analysis resulted in apparently much higher amounts of PSMA-1007 when measuring with the ICS5000  leading to a very poor intermediate precision between the two systems. Normally an ICS5000 system is used for ion chromatography and also other detectors, e.g., electrochemical detectors, are used. Since production of [18F]FDG was cancelled in our lab, we reused the ICS5000 as a HPLC system by bypassing the electrochemical detector and using only the UV and radioactivity detectors. A huge difference between the ICS5000 and the UltiMate 3000 system is the mode of injection by the respective autosampler: for the injection of a sample in the ICS5000 system the sample is first drawn into a buffer line and then pushed through the sample loop onto the column (push mode) [18F]PSMA-1007 batches , no sample remained in the buffer line leading to higher peak areas and seemingly higher amounts of PSMA-1007. Due to direct injection on the UltiMate 3000 system such effects were not observed, leading to comparable peak areas between reference standard samples and [18F]PSMA-1007 samples. But when using this system for the analysis of [sh mode) . In the sh mode) . This di18F]PSMA-1007 was changed from 90% physiological saline (0.9% NaCl) and 10% ethanol as well as sodium ascorbate for stabilization to 90% PBS  and 10% ethanol necessitating a new validation of the method concerning certain parameters using reference standard solutions in the new matrix. Again, five different concentration levels of PSMA-1007 were analyzed and the precision calculated as the RSD between the determined peak areas  were 0.9989 (UltiMate 3000) and 0.9968 (ICS5000), fulfilling the acceptance criterion of R2 > 0.99. The second validation was performed with standard solutions in PBS containing 10% ethanol  or five (ICS5000) times each. For the linearity determination of the method, the average values of these peak areas were plotted against the known concentration and a linear regression was performed. Again, the first validation was performed with standard solutions in physiological saline (0.9%) containing 10% ethanol a. The ca ethanol b. Using Analyzing the chromatograms of the lowest concentration level of the range of PSMA-1007 (8 µg/mL) in order to confirm that it is not below the limit of quantitation (LOQ), the signal-to-noise-ratio (S/N) was determined for both systems and both injection matrices. For the standard solution in physiological saline the S/Ns were 239 (UltiMate 3000) and 64 (ICS5000), respectively. For the standard solution in PBS the S/Ns were 565 (UltiMate 3000) and 248 (ICS5000), respectively. Therefore, for both systems and both matrices the acceptance criterion of S/N > 10 was easily fulfilled.To determine the limit of quantitation of the analytical method, the standard solutions of PSMA-1007 were further diluted until the LOQ was reached. These measurements were also conducted for both systems and both injection matrices. The S/N ratios were calculated by the Chromeleon 7.2 software. With the exception of the solutions in physiological saline on the ICS5000 system, the LOQ for PSMA-1007 using the validated method was 0.75 µg/mL which is well below the lowest concentration level of the range (8 µg/mL). Given the problems of the slight insolubility of the PSMA-1007 standards in physiological saline when injecting on the ICS5000 system as discussed above, the lower sensitivity (LOQ 1.5 µg/mL) of this method could be expected. Concerning the disregard limit for impurities (0.3 x area of reference peak PSMA-1007 (10 µg/mL) ≙ 3 µg/mL), it is essential that this peak area is detectable which was indeed feasible. The accuracy of a method is a measure for the conformity of a determined value with the assumed or accepted true value. The accuracy can be either surmised from the parameters specificity, linearity and precision when the reference matrix has no influence on the measurement or can be calculated as the average bias (positive or negative deviation from 100%). For the reference standards in physiological saline and the UltiMate 3000 system the average bias for the different concentrations were between −0.05% and 0.97% while they were between 0.03% and 2.53% for the ICS5000 system. As can be seen, the accuracy of the method with reference standards in physiological saline was significantly less for the ISC5000 system in comparison to the UltiMate 3000 system. When evaluating the reference standards in PBS solution, the average bias for the UltiMate 3000 system was between −0.03% and 0.27% whereas values between −0.01% and 0.28% were achieved for the ICS5000 system. For both systems the accuracy of the method using reference standard solutions in PBS was excellent, comparable to each other and higher in comparison to the standard solutions in physiological saline. The acceptance criterion of an average bias < 5% was easily fulfilled validating the accuracy of the method.18F]PSMA-1007 injection solution were successfully validated. While for the UltiMate 3000 system the matrix of the PSMA-1007 standard solutions made no difference, this was not the case for the ICS5000 system. Using this system, it was essential to use the standard solutions in PBS.All parameters for establishing the HPLC method concerning the chemical purity of [18F]PSMA-1007, both guidelines were taken into consideration. The following parameters were evaluated: radiochemical identity of [18F]PSMA-1007, specificity, recovery, linearity, limit of quantitation, and intermediate precision.Another very important specification parameter of a radiopharmaceutical is its radiochemical purity which can be determined using radio-HPLC and radio-TLC. For validation of a radio-HPLC method, some modifications of the ICH guideline concerning radiochemical parameters are necessary. To this point, a guideline for the validation of analytical methods for radiopharmaceuticals was published by the EANM . While d18F]PSMA-1007, its retention time was compared to that of co-injected reference standard PSMA-1007. For the UltiMate 3000 system the retention time of [18F]PSMA-1007 was 7.82 min whereas it was 7.73 min for PSMA-1007. The deviation between the two times is therefore 0.09 min or 1.15% in relation to the retention time of the standard compound. Considering the arrangement of both detectors (1. UV and 2. radio) a shift to higher retention times for the radiochemical compound is to be expected. For the ICS5000 system the retention time of [18F]PSMA-1007 was 8.39 min whereas it was 8.30 min for PSMA-1007. The deviation between the two times is therefore 0.09 min or 1.08% in relation to the retention time of the standard compound. Moreover, for this system considering the arrangement of both detectors (1. UV and 2. radio) a shift to higher retention times for the radiochemical compound is to be expected. The acceptance criterion that the deviation between retention times has to be less than 10% is fulfilled for both systems and the method is suitable for the determination of the radiochemical identity of [18F]PSMA-1007. An overlay of the respective UV and radiochromatogram of the co-injection of PSMA-1007 reference standard and a batch of [18F]PSMA-1007 on the ICS5000 system is shown in The radiochemical identity of a compound can be validated by co-injection of an external standard and the comparison of their retention times. The retention time of the radiolabeled compound can be either larger or smaller than that of the external standard, depending on the arrangement of the UV and radiochemical detectors. For the validation that the method is suitable for determining the radiochemical identity of [18F]PSMA-1007 has to be determined. Therefore the method has to be specific for known and unknown radiochemical impurities of [18F]PSMA-1007. A known impurity of [18F]PSMA-1007 is unreacted [18F]fluoride. In order to confirm a suitable resolution between [18F]PSMA-1007 and [18F]fluoride, a sample of [18F]PSMA-1007 injection solution was spiked with [18F]fluoride. For the UltiMate 3000 system the retention time for [18F]fluoride was 0.65 min resulting in a resolution of 48.8 to [18F]PSMA-1007. For the ICS5000 system the retention time for [18F]fluoride was 0.66 min resulting in a resolution 49.2 to [18F]PSMA-1007. Additionally, to known impurities also unknown impurities may occur in the injection solution. For validating a suitable specificity of the method for unknown impurities, the same batch samples of [18F]PSMA-1007 injection solution were analyzed and the resolution to neighboring peaks was determined. In case of the UltiMate 3000 system, the resolution to the neighboring peak was 2.8 and for the ICS5000 system it was 5.47. Therefore the acceptance criterion of a resolution of ≥1.5 was fulfilled for known as well as unknown impurities on both systems, validating a suitable specificity of the method for the determination of the radiochemical purity of [18F]PSMA-1007 injection solution.Both the chemical purity and the radiochemical purity for each batch of [18F]PSMA-1007 was only tested on the UltiMate 3000 system and was very good with 98.2% fulfilling the acceptance criterion of a recovery of >95%. The deviation from 100% may be due to an error in measurement of the radioactivity which can be expected since the measurement geometry of the sample solution and the recovered HPLC run differs.In radiochemical HPLC analysis it is a well-known problem that some radiochemical compounds may be partially retained on the HPLC column therefore falsifying the results of the radiochemical purity of the injection solution. To confirm that this is not the case for a given method, the recovery of a sample can be determined. For this purpose, a known amount of radioactivity is injected, the whole HPLC run is collected and its amount of radioactivity is measured. After half-life correction the percentage of recovered radioactivity can be calculated. The recovery of [x)/area(t0)) × 100) and compared to the expected value. From these values the linearity of the detector can be determined. For the determination of the linearity of the radiodetector of each HPLC system three independent measurement series were evaluated and the correlation coefficients of the linear regressions were determined. In case of the UltiMate 3000 system R2 for the radiodetector was 0.998 and for the ICS5000 system it was 0.9999. The acceptance criterion of R2 ≥ 0.99 was fulfilled for both systems.A very important parameter of an analytical method is the linearity of the used detector over the range of the method. The linearity was not only validated for the UV detector see  but alsoThe linearity of the radiodetector can also be validated during the determination of the limit of quantitation see  of the r18F]PSMA-1007 in our lab, the specified radioactivity range is 100–2000 MBq/mL. With the achieved LOQ on both systems, a change in radiochemical purity of ±0.5% should be measurable.As the expected amounts of radioactivity for radiochemical impurities of a radiopharmaceutical are low, another important parameter of the method is the limit of quantitation of the systems radiodetector. This can be evaluated by repeated injection of the same radiopharmaceutical solution at specified time intervals. Due to radioactive decay the amount of radioactivity declines and the sample can be measured until the limit of quantitation (S/N > 10) is reached. The LOQ was determined for both systems and the S/N ratio was calculated by Chromeleon 7.2 software in each radiochromatogram. The LOQ for the UltiMate 3000 system was 6.45 kBq while it was 4.6 kBq for the ICS5000 system. It is desirable to determine the radiochemical purity of a radiopharmaceutical within ±0.5% and this should be achievable also at the lowest radioactivity concentration allowed in the specification of the product. In the case of [18F]PSMA-1007 were independently integrated by three different persons who routinely perform quality control of this radiotracer. The intermediate precision for the determination of the radiochemical purity of [18F]PSMA-1007 by three different analysts was very good with RSDs between 0.22% and 0.57% fulfilling the acceptance criterion of < 2%. The method is well suited for use by different operators.Another parameter to be validated in an analytical method is the intermediate precision which can take the analyst into account: different operators may achieve different results. Since injection onto the HPLC systems is automatic in our lab, differences in injection due to the analyst will not occur. However, in contrast to the automatic integration of the software in the UV chromatograms, the radiochromatograms often need to be re-integrated. To validate the intermediate precision for this re-integration by different analysts, radiochromatograms of 10 batches of [18F]PSMA-1007 were successfully validated on both systems. The radio-HPLC method for the determination of the chemical and radiochemical purity of [18F]PSMA-1007 injection solution was successfully validated, enabling its use for a GMP-compliant routine quality control of the radiopharmaceutical. For the ICS5000 system it was essential that the PSMA-1007 standard solutions were prepared in PBS. Generally, the method using standard solutions in PBS gave better results for both HPLC systems.All parameters concerning the radiochemical purity of [18F]PSMA-1007, more than 60 batches were analyzed in our lab. Sometimes both systems, the UltiMate 3000 as well as the ICS5000, were used in parallel for the analysis. 18F]PSMA-1007 injection solution analyzed on both systems demonstrating the excellent comparability of both systems.Using the successfully validated radio-HPLC method for the determination of the chemical and radiochemical purity of [18F]PSMA-1007 on both systems is summarized in A comparison of the results for the chemical as well as radiochemical purity for 5 different batches of [18F]PSMA-1007 on both systems is excellent, validating the methods suitability for the routine quality control of this radiotracer.The conformance of the batch results for the chemical as well as the radiochemical purity of PSMA-1007 was synthesized on a Nuclear Interface Tracerlab FX FN system as described earlier PSMA-1007 injection solution. Reference standard PSMA-1007 (1 mg) was dissolved in 50 µL DMSO and diluted with one of the matrices to achieve a concentration of 0.58 µmol/mL. This stock solution was further diluted to the concentrations of 12 µg/mL to 8 µg/mL PSMA-1007. It was essential that all dilutions were carried out using “protein low bind” tips as well as tubes. For the determination of the specificity of the method, standard solutions of PSMA-1007 labeling precursor and hydroxy-PSMA-1007 in a concentration of 12 µg/mL were prepared accordingly.As solvent for the reference standard solutions either a physiological saline based mixture or a PBS based mixture was applied. These matrices were achieved by mixing of 30% EtOH (8 mL) with either 0.9% NaCl (22 mL) or PBS (22 mL) representing the composition of the [18F]PSMA-1007.For the validation of the chemical identity, the reference standard PSMA-1007 was injected and its retention time determined. The retention times of known impurities like PSMA-1007 labeling precursor and hydroxy-PSMA-1007 were also determined by single injection. The specificity of the method was validated by analyzing a mixture of the reference standard PSMA-1007 and known impurities. Resolution between the peaks was calculated by the Chromeleon 7.2 software and had to be >2.0. Specificity of the method regarding unknown impurities was determined by analyzing the resolution of neighboring peaks to PSMA-1007 in the UV chromatogram of a typical batch of PSMA-1007, a sample of a batch of [18F]PSMA-1007 injection solution was spiked with non-radioactive reference standard PSMA-1007 and analyzed. The retention time of PSMA-1007 in the UV chromatogram was compared with that of [18F]PSMA-1007 in the radiochromatogram. Both peaks have to co-elute with a deviation of < 10% between both retention times. Depending on the arrangement of the UV and radiodetector in the HPLC system the retention time of the radioactive compound is either shifted to higher (1. UV and 2. radio) or lower (1. radio and 2. UV) retention times.For validation of the radiochemical identity of [18F]PSMA-1007 peak to known as well as unknown radioactive impurities has to be validated. A known impurity of [18F]PSMA-1007 injection solution is unreacted [18F]fluoride. In order to analyze the resolution between the peak of [18F]fluoride and [18F]PSMA-1007, a sample of [18F]PSMA-1007 was spiked with [18F]fluoride solution under the addition of citrate buffer for reducing tailing of the [18F]fluoride peak. The resolution between the resulting peaks in the radiochromatogram was calculated using the Chromeleon 7.2 software and has to be > 1.5. For validation of the specificity to unknown impurities, the resolution of [18F]PSMA-1007 to neighboring peaks in a given batch was also calculated using the software.As for validation of the chemical purity, the method also has to be specific for the determination of the radiochemical purity. For this purpose the resolution of the [18F-radiopharmaceuticals may be that using the given analytical method unreacted [18F]fluoride may stick to the HPLC column falsifying the results of the analysis. To rule that out, the recovery of a sample of the radiopharmaceutical has to be determined. For this purpose a known radioactive amount of [18F]PSMA-1007 injection solution was injected, the whole run was collected and then its radioactive amount was determined using an ionization chamber. After half-life correction of the obtained value both values can be compared and the percentage of recovery can be calculated. The recovery should be > 95%.A common problem while determining the radiochemical purity of 0, t1 = 56 min, t2 = 110 min, and t3 = 178 min. The percentage of the integrated peak area was calculated (=(area(tx)/area(t0)) × 100) and compared to the expected value. From these values the linearity of the detector can be determined by plotting the measured percentage values against the expected percentage values. For the determination of the linearity of the radiodetector of each HPLC system three independent measurement series were evaluated and the correlation coefficients of the linear regressions of the average values were determined.Linearity of the radiodetectors was validated using two different methods. For the first method an equal volume from the same radiopharmaceutical solution was injected four times at specified time intervals. The time intervals depend on the half-life of the radionuclide so that an expected amount of radioactivity remains in the sample and is injected . For fluorine-18 the respective time intervals were: tAnother method for determination of the linearity of a radiodetector is the repeated measurement of an equal volume of the same radiopharmaceutical solution at fixed timepoints as realized for the determination of the limit of quantitation. The obtained integrated peak areas are plotted against the expected radioactive concentration of the sample at the timepoint of injection  and the correlation coefficient of the linear regression can be determined.The determination of the limit of quantitation of the radiodetectors was realized by consecutive measurements of the same radiopharmaceutical solution. Due to the radioactive decay the measured integrated peak areas decrease over time resulting in smaller S/N ratios with every measurement. The S/N ratio was determined using Chromeleon 7.2 software and the limit of quantitation was the last radioactive concentration which fulfilled the criterion of a S/N > 10. The radioactive concentration at a given timepoint was calculated using the decay equation.18F]PSMA-1007 injection solution were integrated independently by 3 different analysts. For all batches the relative standard deviation of the results regarding the radiochemical purity of [18F]PSMA-1007 by the different operators was calculated using Microsoft Excel 2013. For the determination of the intermediate precision of the method concerning re-integration of the radiochromatograms, 10 batches of [18F]PSMA-1007, a tracer for imaging of prostate cancer, by radio-HPLC. The described radio-HPLC method fulfilled all acceptance criteria—for e.g., specificity, linearity, and accuracy—and is therefore well suited for the routine quality control of [18F]PSMA-1007 before release of the radiopharmaceutical. Albeit it is different to the method recently published in a European Pharmacopeia monograph, it has certain advantages, not least of all an easier technical implementation. Therefore, this method is an attractive alternative for users all over the world. Even though European users have to consider the monograph for [18F]PSMA-1007, they may use this method when explaining its advantages to their authorities and showing that it is suitable. The here described method was successfully validated on several radio-HPLC systems in our lab and used for the analysis of more than 60 batches of [18F]PSMA-1007. Preparation of the PSMA-1007 standard solutions in PBS proved essential for the ICS5000 system and beneficial for the UltiMate 3000 system. Using this method, the chemical and radiochemical purity of [18F]PSMA-1007 can routinely be evaluated assuring patient safety.For the use of a radiotracer in human applications its production as well as its quality control have to be GMP-compliant. In this study we developed, optimized and validated a fast and reliable method for the routine determination of the chemical and radiochemical purity of [


Betacoronavirus (SARS-CoV-2). A computational approach was adopted to identify multiepitope vaccine candidates against SARS-CoV-2 based on S, N and M proteins being able to elicit both humoral and cellular immune responses. In this study, the sequence of the virus was obtained from NCBI database and analyzed with in silico tools such as NetMHCpan, IEDB, BepiPred, NetCTL, Tap transport/proteasomal cleavage, Pa3P, GalexyPepDock, I-TASSER, Ellipro and ClusPro. To identify the most immunodominant regions, after analysis of population coverage and epitope conservancy, we proposed three different constructs based on linear B-cell, CTL and HTL epitopes. The 3D structure of constructs was assessed to find discontinuous B-cell epitopes. Among CTL predicted epitopes, S257-265, S603-611 and S360-368, and among HTL predicted epitopes, N167-181, S313-330 and S1110-1126 had better MHC binding rank. We found one putative CTL epitope, S360-368 related to receptor-binding domain (RBD) region for S protein. The predicted epitopes were non-allergen and showed a high quality of proteasomal cleavage and Tap transport efficiency and 100% conservancy within four different clades of SARS-CoV-2. For CTL and HTL epitopes, the highest population coverage of the world’s population was calculated for S27-37 with 86.27% and for S196-231, S303-323, S313-330, S1009-1030 and N328-349 with 90.33%, respectively. We identified overall 10 discontinuous B-cell epitopes for three multiepitope constructs. All three constructs showed strong interactions with TLRs 2, 3 and 4 supporting the hypothesis of SARS-CoV-2 susceptibility to TLRs 2, 3 and 4 like other Coronaviridae families. These data demonstrated that the novel designed multiepitope constructs can contribute to develop SARS-CoV-2 peptide vaccine candidates. The in vivo studies are underway using several vaccination strategies.The causative agent of severe acute respiratory syndrome (SARS) reported by the Chinese Center for Disease Control (China CDC) has been identified as a novel  Betacoronavirus (SARS-CoV-2) [The causative agent of severe acute respiratory syndrome (SARS) reported by the Chinese Center for Disease Control (China CDC) has been identified as a novel S-CoV-2) . The genS-CoV-2)  mainly eS-CoV-2) . EnhanceS-CoV-2) . The triS-CoV-2) . S1 and S-CoV-2) . Being tAnti-viral drugs, broad-spectrum antibiotics such as Remdesivir, Chloroquine, Ribavirin, Favipiravir or Baricitinib are potential therapeutic strategies used to reduce the viral load  by blockHowever, each type of vaccine has a number of advantages and disadvantages. Although platforms based on DNA or mRNA are flexible and effective for antigen manipulation, peptide-based vaccines are customizable multipurpose therapeutics which does not have the implication of stability or translation  and by tThe reference sequences of the targeted proteins including S, N and M proteins of SARS-CoV-2 were obtained from the NCBI database and used as an input for more bioinformatics analyses.http://www.cbs.dtu.dk/services/BepiPred-2.0/) for linear B-cell prediction of the conserved regions in S, N and M proteins of SARS-CoV-2 to produce the B-cell mediated immunity. In this study, epitope threshold value was set as 0.5  [A successful vaccine must elicit strong cellular and humoral immune responses. Thus, it is important to show that the constructed immunogens are able to induce protective immunity. It should be considered that optimal peptide-based vaccines must be presented in a desired secondary structure of peptides in order to induce a specific humoral response. In this subsection, we used BepiPred-2.0 prediction module (ctively) .The initial step on applying bioinformatics to design synthetic peptide vaccines is to determine whether epitopes are potentially immunoprotective. T-cell epitopes presented by MHC are linear form containing 12 to 20 amino acids. This fact facilitates modeling for the interaction of ligands and T-cells with accuracy . Bindinghttp://www.cbs.dtu.dk/services/NetMHCpan/) to predict high-potential T-cell epitopes. This server is meant to predict MHC I binding with accuracy of 90–95% [For MHC class I, we adapted Artificial Neural Networks  [For MHC class II, we used NetMHCIIpan 4.0 server (CIIpan/)  to predihttp://www.cbs.dtu.dk/services/NetCTL) to assess the prediction of antigen processing through the MHC-I antigen presentation pathway. In this method, weight on C-terminal cleavage set on 0.15, and tap transport efficacy and epitope identification were set on 0.05 and 0.75, respectively.Best ranked peptides extracted from NetMHCpan database were used in transporter associated with antigen presentation (TAP) transport efficacy and proteasomal cleavage analysis. In MHC class I presentation pathway, this section is as essential as binding affinity prediction. We employed NetCTL 1.2 server combined with Tap transport/proteasomal cleavage tools (http://tools.immuneepitope.org/tools/conservancy/) was employed [Up to now, more than 16667 full-sequences of SARS-CoV-2 have been registered globally in GISAID database classified into four clades of V, G, S and O. To calculate the degree of conservancy of each epitope, IEDB epitope conservancy tool (employed . This tohttp://tools.iedb.org/population/) to assess the coverage rate of population for each epitope.Due to a phenomenon known as denominated MHC restriction of T-cell responses, selecting multiple epitopes with different HLA binding specificities will afford more increases in population coverage. Prediction based on HLA binding at population level in defined geographical regions where the peptide-based vaccine might be employed is essential. Since MHC polymorphisms are dramatically at different frequencies in different ethnicities, without careful consideration, a vaccine with ethnically biased population coverage will result. In this study, we used IEDB population coverage tool  (http://https://webs.iiitd.edu.in/raghava/igpred/index.html) was developed for predicting different types of B-cell epitopes inducing different classes of antibodies. We used this server to identify epitope tendency for inducing IgG and IgA antibodies.IgPred module  (https:/http://crdd.osdd.net/raghava/IL-10pred/) and IFNepitope [http://crdd.osdd.net/raghava/ifnepitope/index.php) to predict Il-10 and Interferon-gamma inducing peptides, respectively. We used Support Vector Machine (SVM)-based model as prediction model in both servers. Other features including SVM threshold left at the default value. Through using these servers, we improved insight into the future in vivo studies.It is important to understand that all MHC class II binders will not induce the same type of cytokines. Thus, we used IL-10 Pred  (http://Nepitope  webserve3P server (http://lpa.saogabriel.unipampa.edu.br:8080/pa3p/pa3p/pa3p.jsp) using AFDS-motif, Allergen online (6aa and 80-word match) algorithms. The specificity of these methods is 95.43% (6aa), 92.88% (80aa), and 88.1% (ADFS) [The prediction of potential allergenicity is an important step in safety assessment as proteins and polypeptides have significant roles in inducing allergenic reactions. The allergenicity of the selected epitopes was calculated by PA% (ADFS) .http://galaxy.seoklab.org/cgi-bin/submit.cgi?type=PEPDOCK). This study presents an example of GalexyPepDock performed by each epitope and available PDB file of HLA alleles, separately.To estimate the formation of MHC-peptide complex, we used GalexyPepDock peptide-protein flexible docking server  , cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) were designed.http://us.expasy.org/tools/protparam.html) [The physicochemical properties of the designed LBL, CTL and HTL epitopes including molecular weight, theoretical PI, positive and negative charge residue, solubility and stability were evaluated by ProtParam online server (am.html) .https://zhanglab.ccmb.med.umich.edu/I-TASSER/) was used for modeling the 3D structure of designed constructs. This server is in active development with the goal to provide the most accurate protein structure and function predictions using state-of-the-art algorithms. After analysis, the models with the highest confidence score (C-score) were selected for refinement analysis.I-TASSER server   was used to refine predicted tertiary structures. GalaxyRefine2 performs iterative optimization with several geometric operators to increase the accuracy of the initial models. Final Refined models were analyzed by SAVE5.0 (https://servicesn.mbi.ucla.edu/SAVES/) server to validated tertiary structures. SAVE server gives Ramachandran plot of the whole structure, determines the overall quality of tertiary structure, and calculates buried protein atoms, stereochemical quality and atomic interaction of predicted 3D structure.GalaxyRefine 2 Server  (http://https://tools.iedb.org/ellipro/help/) to find discontinuous B-cell epitopes. ElliPro web-based server uses modified Thornton’s method along with residue clustering algorithms. In this study, epitope prediction parameters (minimum score and maximum distance) were set to default values (0.5 and 6).Prediction of discontinuous B-cell epitope needs tertiary structure of a protein or polypeptide since the interaction between antigen epitopes and antibodies is very important. As regards, after refinement and validation analysis, the 3D structure of constructs were assessed by the Ellipro server  (https:/http://www.rcsb.org/) and then protein-protein docking between three vaccine constructs and TLRs were performed by ClusPro server [https://cluspro.bu.edu/). ClusPro uses three steps algorithms containing 1) Rigid-body docking, 2) Cluster retained conformations, and 3) Refine by CHARMM minimization.TLRs are sensors recognizing molecular patterns of pathogens to initiate innate immune system. It was demonstrated that TLRs 2, 3 and 4 are more susceptible to Coronaviridae family including SARS-CoV and MERS-CoV –35. Thuso server   were obtained from NCBI. The sequence was downloaded in a FASTA format to carry out further analyses.i.e., S, N and M), and the ability of epitopes in inducing different classes of antibody in IgPred server were analyzed. In S protein, S1133-1172 (VNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGI), S440-501 (NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTN), S59-81(FSNVTWFHAIHVSGTNGTKRFDN) and S304-322 (KSFTVEKGIYQTSNFRVQP), and in N protein, N232-269(SKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYN), N164-216(GTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGD), N1-51(MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTAS) and N361-390(KTFPPTEPKKDKKKKADETQALPQRQKKQQ) were chosen as they had the ability to induce antibody  epitopes is a crucial step in epitope-driven vaccine design as MHC class I restricted CTL plays a critical role in controlling viral infections. In this study, we employed NetMHCpan and NetMHCIIpan as mentioned procedure in below.Identification of CD8The SARS-CoV-2 protein sequences were analyzed by NetMHCpan 4.1 server to identify the most immunodominant regions. In each protein, peptides with the highest binding affinity scores were determined as high-potential CTL epitope candidates. In each protein, the best epitopes with higher binding affinity were selected as the putative CTL epitope based on calculated average immunogenicity scores. Chosen MHC-I epitopes were listed in The SARS-CoV-2 protein sequences were analyzed by NetMHCIIpan 4.0 server to identify MHC-II epitope. Epitopes with the maximum number of binding HLA-DR alleles were selected as putative HTL epitope candidate. Chosen MHC-II epitopes were listed in 257-265 and S603-611 have the highest epitope identification score of 3.14 and 3.07, respectively.Tap transport and proteasomal cleavage are as important as binding affinity in antigen presentation pathway to CTLs. In this case, NetCTL1.2 server was used. All of the epitopes shown in 27-37 with 86.27%. For helper T-cell epitopes, the highest population coverage of the world’s population was calculated for S196-231, S303-323, S313-330, S1009-1030 and N328-349 with 90.33%.As mentioned above, MHC polymorphisms are dramatically at different frequencies in various ethnicities. Thus, careful consideration should be given to the way of effective vaccine development. In this study, population coverage was estimated separately for each putative epitope in different geographical regions Tables  and 6. Fhttps://www.rcsb.org/). All potential epitopes and MHC PDB files were submitted to the server separately. Then, top models with the highest interaction similarity score  were selected for each peptide and its MHC. For CTL epitopes, N103-113, S868-876, M36-46, S1094-1102, M102-111, S1051-1061, S360-368, S191-199 and S686-696 had the highest average of interaction similarity score, respectively  [The SARS-CoV-2 has become a major global public health issue and scientists are struggling to find the best way to treat the disease and develop a vaccine against the virus. Numerous immune-bioinformatics methods have been developed in vaccine researches which can potentially save time and resources. These tools could help us to identify antigenic domains to design a multi-epitope vaccine. Since now we know enough information about SARS-CoV-2’s genomics and proteomics, we can design peptide vaccines based on a neutralizing epitope. These immunoinformatics methods have made a significant impact on the immunology researches and we can see many examples of us (HIV) , human pus (HIV) , 39, SARus (HIV) , rhinovius (HIV) .in-silico approaches were intended to design a universal SARS-CoV-2 vaccine for induction of B- and T-cell immunity with efficient reactions to the virus and long-term immune responses based on the S protein of the virus and also two other structural proteins including N and M.SARS-CoV-2 is an RNA virus tending to mutate more frequently . These met al. suggested certain peptides in E protein of SARS-CoV-2 as promising epitope vaccine candidates against T-cell [et al. proposed one construct including E, S, N and M proteins as an epitope vaccine candidate [et al. suggested some putative B- and T-cell epitopes based on S, M and E proteins of SARS-CoV-2 [et al. designed a multiepitope vaccine candidate based on N, M and open reading frame (ORF) 3a of SARS-CoV-2 [et al. tried to predict B- and T-cell epitopes of SARS-CoV-2 in comparison with SARS-CoV [There are some efforts based on designing epitope-based vaccines against SARS-CoV-2 since the outbreak. In one study, Abdelmageed t T-cell . In anotandidate . On the RS-CoV-2 . Also, ERS-CoV-2 , and TeiSARS-CoV . These pAccording to the aforementioned finding, we tried to use computational and bioinformatics methods on the formulation of new SARS-CoV-2 vaccine against its structural proteins including S, N and M proteins in a more comprehensive way. In the beginning, the whole genome of SARS-CoV-2 was analyzed. Then, three major structural proteins including S, N and M were chosen for further analyses. We identified epitopes corresponding to B-cells and T-cells to design constructs being able to elicit both humoral and cellular immunity. We used BepiPred tool to predict putative B-cell epitopes and chose 8 putative epitopes of S and N protein being able to induce antibodies like IgG. In contrast, M protein of the virus could not induce any class of antibodies.+ and CD4+ T-cells play a major role in antiviral immunity, we tried to evaluate the binding affinity to MHC class I and II molecules. Choosing S, N and M proteins of the virus as the antigenic site, we used NetMHCpan and NetMHCIIpan prediction tools to identify the most immunodominant regions. From all peptides predicted, we chose 20 putative epitopes for MHC class I and 20 putative epitopes for MHC class II. Since S protein is currently the most promising antigen formulation, we put the focus on the S protein epitopes and chose 10 epitopes of S protein and 10 epitopes of other proteins for each MHC class I and II. The IFN-γ and IL-10 cytokines were also measured as candidates in MHC class II epitopes as they promote the development of T-helper cells being required for B-cell, macrophage and cytotoxic T-cell activation. Among the CTL predicted epitopes, S257-265, S603-611 and S360-368 and among HTL predicted epitopes, N167-181, S313-330 and S1110-1126 had better MHC binding rank.As CD8To predict antigen processing through the MHC class I antigen presentation pathway, we used NetCTL1.2 server. All of the predicted epitopes had upper cut off identification scores (> 0.75) showing a high quality of proteasomal cleavage and Tap transport efficiency. We also measured the epitopes for conservancy analysis. All predicted epitopes were 100% conserved within four different clades of SARS-CoV-2. In general, the selected epitopes had the potency to produce an immune response against S, V, G and O clades of SARS-CoV-2.27-37 with 86.27%, and for S196-231, S303-323, S313-330, S1009-1030 and N328-349 with 90.33%, respectively. Overall, these results suggest a specific binding of CTL epitopes and HTL epitopes to the prevalent HLA molecules in the targeted populations. Another prominent obstacle in vaccine development is the probability of allergenicity since many vaccines stimulate the immune system into an allergenic reaction. In this study, we used PA3P to predict potential allergenicity and all of the epitopes were analyzed as non-allergen.Population coverage is another important factor in vaccine design. We measured population coverage rate for CTL and HTL epitopes in 16 specified geographical regions. For CTL epitopes, and helper T-cell epitopes, the highest population coverage of the world’s population was calculated for S103-113, S868-876, M36-46, S1094-1102, M102-111, S1051-1061, S360-368, S191-199 and S686-696 had the highest average of interaction similarity score, respectively and For HTL epitopes, M107-128, N48-63, S689-704, S1057-1074, S196-231, N328-349, S32-53, N126-143, M163-181 and S114-130 had the highest average of interaction similarity score, respectively. Overall, CTL epitopes showed better quality of docking in comparison with HTL epitopes. Finally, the vaccine construction was completed after joining the LBL, CTL and HTL epitopes with KK, AAY and GPGPG linkers, respectively.For CTL epitopes, NThe molecular weights of the constructed LBL, CTL and HTL epitopes were obtained as 36.5, 28.6 and 49.5 kDa, respectively which were low molecular weights for a multiepitope vaccine. All constructs were soluble and stable indicating that the designed constructs had high solubility and stability for the initiation of an immunogenic reaction.In the case of 3D modeling, we used I-TASSER server to predict the tertiary protein structure. The accuracy of the selected models was evaluated by C-score. The C-scores of the models for LBL, CTL and HTL polypeptide constructs were -2.39, -4.42 and -0.63, respectively. The higher value of the C-score is the better quality of prediction. Thus, HTL with the C-score of -0.63 showed higher accuracy of the predicted epitopes. Also, the quality of the predicted constructs was improved by refinement which leads to a higher quality of final models. Over the 96.8% of the residues were found in favored and allowed regions. At last, we used Ellipro server to predict potential discontinuous B-cell epitopes. Ellipro servers identified 3 discontinuous B-cell epitopes for CTL with 143 residues, 4 for HTL with 225 residues and 3 for LBL with 72 residues indicating the ability of the designed constructs for robust induction of humoral response. Also, peptide-protein docking between three vaccine constructs and TLRs 2, 3 and 4 were performed by ClusPro server, and all data showed strong interactions between the designed constructs and TLRs 2, 3 and 4 supporting the hypothesis of SARS-CoV-2 susceptibility to TLRs 2, 3 and 4 like other Coronaviridae family. All three constructs showed better interactions with TLR 3.et al. paper using the same server of Bepipred [686-696 (VASQSIIAYTM), S1051-1061 (FPQSAPHGVVF), N103-113 (LSPRWYFYYLG), N304-313 (AQFAPSASAF) and all of M epitope of CTL including M36-46 (FAYANRNRFLY), M168-180 (TVATSRTLSYY), M195-204 (YSRYRIGNYK), M90-99 (MWLSYFIASF) and M102-111 (FARTRSMWSF) have not been reported in any literature. Also, among HTL epitopes reported in 303-323 (LKSFTVEKGIYQTSNFRVQPT), S1009-1030 (TQQLIRAAEIRASANLAATKMS), S801-817 (NFSQILPDPSKPSKRSF), N328-349(GTWLTYTGAIKLDDKDPNFKDQ), N126-143 (NKDGIIWVATEGALNTPK), N342-361 (KDPNFKDQVILLNKHIDAYK), N48-63 (NTASWFTALTQHGKED), N167-181 (LPKGFYAEGSRGGSQ), N405-419 (KQLQQSMSSADSTQA), M107-128 (RSMWSFNPETNILLNVPLHGTI) and M163-181 (DLPKEITVATSRTLSYYKL) have not been reported in any literature. The rest of the epitopes mentioned in Tables et al. [et al. [360-368 (CVADYSVLY) related to receptor-binding domain (RBD) region for S protein which was referred to the fragment of 347 to 520 amino acids [Overall, we tried to consider three major structural proteins including S, N and M proteins of the virus and design three different constructs including LBL, CTL and HTL constructs to elicit more robust humoral and cellular immunity. Comparing our study with other studies in the field of multi-epitope vaccine design for SARS-CoV-2, all LBL epitopes obtained in Bepipred . However [et al.  for MHC no acids –35. All in vivo and clinical experiments. Generally, with the help of in silico studies, experimental researches can march rapidly with higher probabilities of finding the desired solutions and controlling the current outbreak.In conclusion, we determined three vaccine constructs against three major structural proteins of SARS-CoV-2 designed based on robust vaccine design criteria including non-allergenicity, conservancy, affinity measurement to multiple alleles of MHC, worldwide population coverage, 3D prediction, refinement and validation, discontinuous B-cell epitope prediction, docking and effectiveness of molecular interaction with their respective HLA alleles and TLRs. These constructs require validation by S1 Table(DOCX)Click here for additional data file.