Among the groups, only the TSC-OP–silane treatment effectively enhanced the bond strength and maintained the adhesion after thermocycling. With these results, the OP aided the silicatization protocol effectively, generated silane crosslinking, and resulted in superior resin–zirconia bond strength and durability compared to the current treatments.The combinations of alumina particle air abrasion (AA) and a 10-methacryloyloxyidecyl-dihyidrogenphosphate (MDP) primer and a tribochemical silica coating (TSC) and a silane–base primer are contemporary pre-cementation treatments for zirconia restorations for bonding with resin cements. However, the stability of zirconia resists the mechanical or chemical preparations. The purpose of this study was to develop an atmospheric-pressure oxygen plasma (OP)-aided silicatization method to enhance the adhesion of resin cements to zirconia. Zirconia discs were prepared to receive surface treatments of different combinations: (1) AA or TSC (2) with or without OP treatment, and (3) a chemical primer . The surface morphology, hydrophilicity, and chemical compositions were characterized, and the resin–zirconia bond strengths were examined either after 24 h or a thermocycling test. The results indicated that the OP treatment after the TSC facilitated the homogeneous distribution of silane and crosslinking of silica particles, and effectively improved the hydrophilicity. The OP increased the O and Si and reduced the C elemental contents, while the combination of TSC, OP, and silane induced SiO In recent decades, dental metal–ceramic restorations have been replaced with ceramic systems due to the rising costs of precious metals and the unpleasing opacity of metal copings . Yttria-Silicatization is another approach to increase the surface reactivity of zirconia. Selective infiltration etching was developed based on heat-induced nano-structural changes to allow the diffusion of a specific molten glass into the zirconia. The fused silica phase is subjected to HF etching to be retentive, whereby the adhesive resin monomers can infiltrate and interlock . Other s2O and CO2 [Plasma technology has been widely applied in biomedical engineering and is recognized as an economical and effective method for surface modifications . Plasma  and CO2 ,27. Base and CO2 ,26,28,29 and CO2 ,31.The use of a TSC together with silanization has been a common silicatization approach for adhesion substrates, regardless of the non-uniformity and small fraction of surface coverage . The plaCercon zirconia blank cylinders  were cut into disks (19.5 mm in diameter and 2.5 mm in thickness) and divided into eight pieces. A total of 200 pieces were generated and fully sintered. These specimens were polished serially using 320-, 400-, and 600-grit silicon carbide papers. The polished specimens were placed in 95% alcohol, subjected to ultrasonic cleaning for 5 min, and then dried. Afterward, the specimens were divided into ten groups to receive different combinations of surface treatments: (1) grit blasting treatment: alumina particle air abrasion (AA) or TSC, (2) with or without APOP treatment; (3) primer treatment: no primer, with a silane–base or silane–MDP mixture primer. The treatments for the experimental groups are illustrated in 2O3 particles  at a distance of 10 mm for 15 s at 2.5 bar pressure. The specimens were ultrasonically cleaned in deionized water three times each for 10 min. Subsequently, they were dried using an oil-free air gun. For the TSC treatment, the zirconia specimens were grit-blasted at the same distance with 30 μm silica-coated Al2O3  at 2.8 bars for 15 s.For the AA treatment, the zirconia disks were grit-blasted utilizing 45 µm AlIn the 2nd step of the surface treatments, half of the specimens were subjected to APOP treatments using a pen-style atmospheric-pressure oxygen plasma jet . The plasma jet consisted of two concentric cylindrical stainless-steel electrodes. The outer electrode was 18 cm long, with a 25 mm inner diameter and a converging nozzle with a diameter of 2.5 mm. The plasma was generated using an oxygen gas flow at 9.5 L/min, under a pulse power excitation of up to 80 W. The assigned zirconia disks were placed 5 mm under the plasma jet to receive the gas stream for 30 s. During the plasma treatment, the surface temperature of the specimens reached 160 °C. The OP-treated specimens received their assigned primers after 2 s, when their temperature decreased to approximately 80 °C.The last step of the surface treatment involved the chemical modifications. To investigate the silicatization caused by different agents, the specimens were divided into three subgroups: no primer, a silane–base primer (S), or an experimental primer in the form of a mixture (M) of silane and MDP. For groups AA, AA-OP, TSC, and TSC-OP, no primer was applied. For groups AA-S, AA-OP-S, TSC-S, and TSC-OP-S, the zirconia disks were coated with 12 µL of the silane–base primer Clearfil Porcelain Bond Activator  for 5 s using a micropipette, and dried using the oil-free air gun. For groups AA-M and AA-OP-M, an experimental primer of the MDP–silane mixture was prepared following our previous work . Equal vThree treated specimens in each group were gold-sputtered and then observed under a low–variable vacuum scanning electron microscope . In the preliminary micromorphologic findings, only TSC-S and TSC-OP-S showed the presence of surface coatings. Therefore, the zirconia specimens in these two groups were further examined under a field-emission SEM  at high magnification.n = 5). The contact angles were then analyzed using ImageJ software.To examine the changes in the hydrophilicity of each specimen, 2 µL of deionized water was dispensed on the specimen surfaces as the probing liquid. A charge-coupled device camera was employed to capture the images of the contact angles on the specimen surfaces . The specimen was placed in a vacuum chamber under 5 × 102 with a quartz halogen light unit  for 40 s from different directions. The light intensity was verified using a radiometer . The specimens were left to set for 30 min and placed in a 37 °C water bath for 24 h. Subsequently, half of the specimens (n = 10) in each group received the early (T0) SBS test, and the other half (n = 10) were subjected to 6000 thermocycles (T6000). The thermocycles consisted of alternative soaking in 5 °C and 55 °C deionized water baths, with a 30 s dwelling time at each temperature and a 5 s transfer time between the two baths. For the SBS test, the resin–zirconia assemblies were fixed on the jig of a universal material test machine . Force was applied near the bond interface using a chisel at a crosshead speed of 1 mm/min until fracture. After the SBS test, the zirconia specimens were sectioned to inspect the bond interface between the zirconia and the adhesive layers.The resin–zirconia bonding was examined using early and post-thermocycling shear bond strength (SBS) tests. In each group, twenty zirconia disks were prepared and treated with the assigned treatments. Pre-cured resin composite tablets (3-mm-diameter and 2-mm-thick) were fabricated using a microhybrid resin composite named Filtek Z250 . An adhesive cellulose acetate tape with a 3 mm hole was attached to the zirconia surface. A dual-cure resin luting cement  was filled in the hole, and the composite tablet was cemented and loaded under 5 N of force. The excess resin cement was cleaned, and the tape was carefully removed. The cemented specimen was light-cured at 850 mW/cmt test was conducted to examine the effects of the thermocycling on the SBSs. The significance level was set at p < 0.05.The statistical analysis was performed with SPSS 20.0 . The contact angles in the wettability test and the SBSs of all experimental groups were analyzed using a one-way analysis of variance (ANOVA) followed by a post hoc Duncan test. An additional three-way ANOVA test was performed to assess the individual effects of the grit blasting, APOP processing, primer agents, and their interactions on the SBSs. An independent samples Under SEM, the treated zirconia specimens showed roughened surfaces . All AA 2 particles.The micromorphology of the TSC-treated specimens was further examined at higher magnification  . TSC-alop < 0.05) (The results from the contact angle test identified significant differences among the groups ( < 0.05) . AA-alonAll of the TSC-treated groups showed high hydrophilicity (contact angles less than 12°). The TSC-OP group even showed superhydrophilicity, with unmeasurable contact angles. Similar to AA-S and AA-OP-S, the silane conditioning following TSC also increased the contact angles, but only to a minor degree.2O3 particles. Weak P 2p signals were present in AA-M and AA-OP-M.The XPS spectra of the eight experimental groups are illustrated in 3)2)O) at 533.6 eV.Fine scanning of the individual O 1s peaks was performed to analyze the chemical state of the treated zirconia b. The O The elemental compositions are listed in 2O3 (102.5 eV), SiO2 (103.5 eV), and SiO4 (104.1 eV) , SiO (101.5 eV), Si04.1 eV)    [29,30,3p < 0.05) improved the bonding strengths. In the T0 SBS test, the AA-OP-M and TSC-OP-S groups exhibited higher SBSs, followed by AA-M and TSC-S . AA-alont test showed that all groups had dramatic reductions in SBSs after thermocycles, with the exception of the AA-OP-S and TSC-OP-S groups. Actually, TSC-OP-S exhibited a comparable SBS to its T0 counterpart.After 6000 thermocycles, all AA-alone specimens failed before the test. The AA-OP and AA-S groups also showed low bond strengths , while AA-OP-S exhibited a much higher SBS (16.7 MPa). The p < 0.001) influenced the SBS at both T0 and T6000 stages. For the T0 SBSs, there were interactions between the mechanical abrasion and OP treatment, as well as between the primer and the OP treatment. In terms of the T6000 SBSs, there was no interaction between any two factors.According to the three-way ANOVA test, either the priming conditions, OP treatment, or thermocycling test substantially  and light-cured immediately after sitting. A previous study revealed that the light activation of the dual-cure cement resulted in better bonding performance compared to the self-curing mode . Even wiAmong the groups, only TSC-OP-S effectively enhanced and sustained the SBSs after thermocycling. The OP-aided silicatization method has multiple benefits in enhancing the resin cement’s adhesion to zirconia. As the TSC generated a partially fused silica coating on the surface, the APOP gas flow removed the loose silica coatings but left the bonded ones. The heat melted the coatings and transformed them into dense clusters or tightly silicate layers . These pWith the above findings, the first hypothesis was rejected. The type of grit blasting, use of APOP, and use of a chemical primer all affect the bond performance. The second hypothesis was also rejected, since the APOP had a synergistic effect on improving the adhesion when combined with the TSC or a silane agent, but had an antagonist effect when combined with the MDP. Among the groups, the protocol using the TSC then APOP and silane lead to strong resin–zirconia bonding and favorable durability. The incorporation of the APOP between traditional TSC–silane treatments facilitates surface silicatization, which could be an effective pre-cementation treatment for dental zirconia restorations to establish reliable adhesion with resin cements. With the advances in plasma technology, various micro-plasma jet systems can be applied for this purpose. In this regard, this study still presents limitations, since various AP conditions were not examined. Future research about the source gas and operating parameters may provide in-depth information about the plasma species concentration, and the species’ effects on the surface modifications and mechanical properties of the zirconia. Additionally, this study did not assess the combination of a TSC, APOP, and silane–MDP mixture. The interpolation between the primers and APOP-assisted silicatization should be explored for further improvement of the adhesion of resin cement to zirconia restorations.This study developed a new silicatization method for dental zirconia restorations via serial treatments of a TSC, APOP, and a silane coupling agent. This method avoids the problems of adhesion hydrolysis associated with AA-M treatments and incomplete silica coatings caused by TSC. The reactive oxygen species generated by the APOP may clean the organic pollutant and loosen the silica coating, and the residual heat strengthens the crosslinking of the silica coating and silane. Accordingly, an intact bond interface is formed, which leads to strong and durable resin–zirconia bonding. With the initial findings, the incorporation of APOP with AA or a silane–MDP mixture primer may enhance the initial bond strength, but the APOP has no effect on the performance against thermocycling. In this regard, the APOP is not recommended to be combined with MDP-based or universal adhesives.


A aterosclerose é a principal causa da maioria das doenças cardiovasculares, e novos biomarcadores para essa condição são sempre necessários. O domínio da peptidase M20 contendo 1 (PM20D1) está associado ao metabolismo lipídico e à obesidade. No entanto, nenhum estudo se concentra no papel do PM20D1 na aterosclerose carotídea. O objetivo deste estudo foi investigar o papel do PM20D1 em pacientes com aterosclerose carotídea. Estudo observacional prospectivo conduzido com um total de 231 pacientes com aterosclerose carotídea que estiveram em nosso departamento entre julho de 2018 e dezembro de 2019. Amostras de sangue e dados médicas foram obtidos de outros 231 indivíduos saudáveis com o mesmo índice de massa corporal (IMC) dos pacientes com aterosclerose carotídea. O PM20D1 sérico foi determinado por ensaio imunossorvente ligado a enzima (ELISA). As características clínicas e demográficas de todos os pacientes foram listadas, incluindo idade, sexo biológico, IMC e histórico médico. Os níveis de proteína C reativa (PCR), fator de necrose tumoral, homocisteína, colesterol total, triglicerídeos, leptina-colesterol de alta densidade e leptina-colesterol de baixa densidade foram registrados. Realizou-se análise estatística no software SPSS, com p<0,05 considerado estatisticamente significante. Os níveis séricos de PM20D1 foram marcadamente mais baixos em pacientes com aterosclerose carotídea comparados aos controles saudáveis, sendo significativamente mais baixos em pacientes com aterosclerose carotídea grave e pacientes com aterosclerose carotídea/acidente vascular cerebral. Pacientes com placas instáveis apresentaram PM20D1 marcadamente menor quando comparados a pacientes com placas estáveis. Nenhuma diferença significativa foi encontrada entre pacientes com aterosclerose carotídea com diferentes IMC. Pacientes com níveis mais elevados de PM20D1 apresentaram expressão significativamente menor de PCR, fator de necrose tumoral, homocisteína, triglicerídeos, colesterol total e colesterol de baixa densidade. PM20D1 correlacionou-se negativamente com PCR, fator de necrose tumoral, homocisteína, colesterol total e leptina de baixa densidade em pacientes com aterosclerose carotídea, podendo ser usado como biomarcador para pacientes com aterosclerose carotídea grave ou com aterosclerose carotídea e acidente vascular cerebral. Sexo biológico, fator de necrose tumoral, homocisteína e PM20D1 foram considerados fatores de risco para aterosclerose carotídea. O PM20D1 estava diminuído em pacientes com aterosclerose carotídea e foi associado com gravidade, estabilidade da placa, níveis de PCR, fator de necrose tumoral, homocisteína, triglicerídeos, colesterol total e colesterol de baixa densidade em pacientes com aterosclerose carotídea. No entanto, ainda é necessário um entendimento mais profundo sobre a manifestação da AC e seus resultados clínicos.Como principal causa da maioria das doenças cardiovasculares, a aterosclerose pode ocorrer precocemente e permanecer assintomática por longos períodos antes de se manifestar.11 Um estudo recente mostrou que o PM20D1 está ligado ao metabolismo lipídico e pode estar associado à obesidade,12 ambos fatores de risco para AC. Verificou-se também que o PM20D1 poderia regular os desacopladores de aminoácidos lipidados das mitocôndrias e aumentar o gasto energético de PM20D1.13 No entanto, até o momento, nenhum estudo focou no papel do PM20D1 na AC.O domínio da peptidase M20 contendo 1 (PM20D1) é uma enzima secretada, recentemente identificada, que é rica em proteína de desacoplamento 1 (UCP1+), em relação a adipócitos UCP1.Este é um estudo observacional prospectivo cujo intuito foi investigar o significado clínico do PM20D1 em pacientes com AC. Esta pesquisa pode fornecer evidências clínicas sobre o papel do PM20D1 na AC.14 O EMIEste estudo observacional prospectivo recrutou 231 pacientes com AC assistidos em nosso departamento entre julho de 2018 e dezembro de 2019. Os critérios de inclusão foram: 1) todos os pacientes diagnosticados com AC de acordo com achados da ultrassonografia com Doppler colorido dos vasos sanguíneos do pescoço e angiotomografia computadorizada intracraniana (ATC) do arco aórtico; 2) Pacientes com AC e AVC foram admitidos em até 72 horas após a manifestação, e o diagnóstico de AVC foi confirmado por ressonância magnética (RM) e tomografia computadorizada (TC); 3) os pacientes concordaram em participar da pesquisa observacional. Os critérios de exclusão foram: 1) pacientes com aneurisma cerebral, malformação arteriovenosa, dissecção, arterite ou doença de Moyamoya; 2) pacientes com embolia cerebral cardiogênica; 3) pacientes com outras doenças graves do sistema, incluindo câncer e disfunção cardíaca, renal ou hepática. A gravidade da AC foi definida da seguinte forma: 1) grupo AC leve/moderado, que apresentou espessamento médio-intimal (EMI)>1,0 mm, ou placa com estenose arterial <70% e sem AVC; 2) grupo AC grave, que apresentou uma ou mais placas com estenose arterial ≥70% e sem AVC; 3) grupo AC combinado com AVC, apresentando AC combinado com AVC isquêmico aterosclerótico de grandes artérias e estenose vascular cerebral >50%. As placas foram divididas em estáveis e instáveis, conforme amplamente aceito na prática clínica: 1) EMI≥1,2 mm foi considerado uma placa; 2) as placas estáveis possuem gradiente-eco forte uniforme ou gradiente-eco médio; 3) as placas instáveis são moles ou ulcerativas com gradiente-eco misto ou baixo.foi medido por meio de um instrumento de diagnóstico por ultrassom LOGIQ C9 Color Doppler  com frequência de sonda de 7~14 MHz. Foram medidos EMI da artéria carótida comum bilateral, proximal, distal e a 1 cm da bifurcação da artéria carótida comum. Todas as medidas foram realizadas pelo menos três vezes e o valor médio foi considerado o valor final do EMI.2; grupo com sobrepeso, com 28 kg/m2>IMC≥24 kg/m2; e grupo obesidade, com IMC>28 kg/m2.15 Também foram obtidas amostras de sangue e características médicas de 231 indivíduos saudáveis que compareceram ao exame físico de rotina, tendo a mesma distribuição de IMC dos pacientes com AC. Todos os pacientes assinaram o termo de consentimento livre e informado. Este estudo foi aprovado pelo comitê de ética do Fourth People’s Hospital of Chengdu (CDH-2018-057).Além disso, os pacientes foram divididos em diferentes grupos de índice de massa corporal (IMC): grupo normal, com IMC<24 kg/mAs amostras de sangue de todos os casos foram coletadas em até 24 horas após a admissão ou chegada ao ambulatório. Resumidamente, 5 ml de sangue foram coletados em tubos sem qualquer anticoagulante. Após uma hora, a centrifugação foi realizada a 2.000 g por 15 minutos, em temperatura ambiente, e foram obtidas as amostras de soro. Os níveis séricos de PM20D1 foram medidos por ensaio imunoenzimático (ELISA), usando um kit PM20D1 , em total acordo com as instruções do fabricante.As características clínicas e demográficas de todos os pacientes foram coletadas, incluindo idade, sexo biológico, IMC e histórico médico. Foi realizado hemograma total de encaminhamento com um analisador bioquímico automático , bem como dosados os níveis de proteína C reativa (PCR), fator de necrose tumoral (TNF-α), homocisteína (Hcy), colesterol total (CT), triglicerídeos (TG), colesterol de leptina de alta densidade (HDL-ch) e colesterol de leptina de baixa densidade (LDL-ch).Os dados com distribuição normal foram expressos por média ± desvio padrão (DP), e os dados com distribuição não normal foram expressos como mediana . As variáveis categóricas foram apresentadas como número (taxas). A distribuição dos dados foi analisada pelo método de Kolmogorov-Smirnov. Para dados com distribuição normal, foi realizada a comparação entre dois grupos pelo teste t não pareado e por análise de variância unidirecional (ANOVA), seguidos pelo teste post hoc de Tukey, usado para comparação entre três ou mais grupos. Para dados com distribuição não normal, utilizou-se o teste de Mann-Whitney para comparação entre dois grupos e o teste de Kruskal-Wallis para comparação entre três ou mais grupos, seguidos do teste post hoc de Dunn. As taxas foram analisadas pelo teste qui-quadrado. A correlação entre PM20D1, Hcy, metabolismo lipídico e fatores inflamatórios foi analisada por meio da correlação de Pearson. Uma curva receiver operating characteristic (ROC) foi usada para análise de PM20D1 em pacientes com AC. A regressão logística foi utilizada para análise de risco de AC. O valor de p<0,05 foi considerado estatisticamente significativo. Todo o cálculo foi realizado no programa SPSS 25.0  ou no GraphPad 6.0 .Esta pesquisa incluiu 231 pacientes com AC no total, sendo 152 casos de AC leve/moderada, 50 casos de AC grave e 29 casos de AC com AVC. A razão da soma dos pacientes com sobrepeso e obesidade foi significativamente maior nos pacientes com AC grave quando comparada à dos pacientes leves/moderados . A propoEm seguida, foi determinada a expressão sérica de PM20D1 em pacientes com AC. Verificaram-se níveis séricos de PM20D1 marcadamente mais baixos em pacientes com AC quando comparados ao controle saudável . PacientPara investigar mais detalhadamente o papel do PM20D1 em pacientes com AC, também avaliamos os níveis de PM20D1 em pacientes com AC e diferentes distribuições de IMC. Não foi observada diferença significativa .Em seguida, os pacientes foram divididos em grupo de alta expressão de PM20D1 e grupo de baixa expressão, de acordo com o nível médio de PM20D1 . Pacientes com níveis mais elevados de PM20D1 apresentaram expressão significativamente menor de PCR, TNF-α, Hcy, TG, CT e LDL-ch . Foi feiPor fim, uma curva ROC foi desenhada para observar o valor diagnóstico de PM20D1 para AC, bem como AC grave ou AC/AVC. Verificou-se que o PM20D1 poderia ser um potencial biomarcador diagnóstico de AC com valor de corte de 5,94 ng/ml, área sob a curva ROC (AUC) 0,876, IC95% , sensibilidade de 80,1% e especificidade de 73,6% . O PM20DO diagnóstico precoce e predição da AC podem melhorar muito a eficácia e o prognóstico do tratamento. Assim, biomarcadores para diagnóstico e prognóstico da AC são de grande importância. Neste estudo, demonstramos que PM20D1 estava mais baixo em pacientes com AC e se associou com a gravidade e condição de placa dos pacientes com AC. Além disso, o PM20D1 correlacionou-se negativamente com PCR, TNF-α, Hcy, TC e LDL-ch nesses pacientes.16 Outra pesquisa constatou que o tratamento com atorvastatina ou ezetimiba diminuiu os níveis de TC, TG e LDL-ch em pacientes com AC.17 Em estudo recente, Pan et al. mostraram que, em residentes rurais de baixa renda na China, LDL-ch e TC foram fatores de risco para aterosclerose em estágio inicial e foram ligados ao risco aumentado de placa carotídea em 24% e 62% para cada aumento de 1 mmol/L em TC e LDL-ch.18 Em nossa pesquisa, também encontramos que TG, TC e LDL-ch foram expressos em níveis altos em pacientes com AC, principalmente em casos graves, corroborando as pesquisas supracitadas. Também observamos que fatores inflamatórios como PCR, TNF-α e expressão de Hcy estavam elevados em pacientes com AC, o que também foi demonstrado em diversas pesquisas.22O metabolismo lipídico e a obesidade estão associados ao desenvolvimento da AC. Verificou-se que os níveis de TG, TC e LDL-ch estavam significativamente maiores em pacientes com placa na artéria carótida.13 Benson et al. demonstraram que a diminuição do PM20D1 estava associada à relação genética entre obesidade e doenças neurodegenerativas em humanos.23 Li et al. descobriram que a inibição do microRNA miR-324-5p aumentou o consumo de oxigênio das células primárias dos tecidos adiposos branco e marrom, aumentou o consumo de gordura e, assim, reduziu o peso dos camundongos, aumentando os níveis de PM20D1.24 Long et al. observaram que, em camundongos com deficiência de PM20D1, a atividade da hidrolase/sintetase do N-acetil aspartato estava diminuída no soro e nos tecidos, bem como uma variedade de fenótipos metabólicos e de dor, incluindo resistência à insulina, mudanças de temperatura fria e comportamento antinociceptivo, que estão associados à obesidade, diabetes e outras doenças.12 Assim, podemos especular que níveis mais altos de PM20D1 podem ser benéficos para a obesidade e redução de lipídios. Embora não haja nenhum estudo que demonstre o papel de PM20D1 na AC, a regulação diminuída de PM20D1 em pacientes com AC em nosso estudo pode se dever, em parte, à disfunção do metabolismo lipídico e obesidade de pacientes com AC. Como os pacientes apresentaram níveis mais baixos de PM20D1 do que os controles saudáveis com mesma distribuição de IMC, e os pacientes com AC e IMC diferente não apresentaram diferença significativa de PM20D1; ou seja, o IMC pode não estar associado ao nível de PM20D1 em pacientes com AC. A correlação negativa entre PM20D1 e Hcy, TC e LDL-ch pode ser uma das razões para a expressão anormal de PM20D1 em pacientes com AC. No entanto, para fundamentar todas essas especulações, são necessários mais estudos para obter mais evidências.O PM20D1 é um fator associado tanto ao metabolismo lipídico quanto à obesidade. Verificou-se que camundongos com aumento de PM20D1 circulante apresentaram aumento da frequência respiratória e aumento sérico dos aminoácidos N-acil, o que melhorou a homeostase da glicose e aumentou o consumo de energia e, portanto, pode regular a obesidade.O presente estudo teve, no entanto, algumas limitações. Primeiro, trata-se de um estudo observacional de um único centro, com apenas 231 casos. Assim como o mecanismo molecular do PM20D1 exercendo influência no desenvolvimento da AC ainda não está claro.Em conclusão, este estudo observacional demonstrou que a diminuição de PM20D1 estava associada à gravidade, condição da placa e expressão de PCR, TNF-α, Hcy, TG, TC e LDL-ch em pacientes com AC. Este estudo pode fornecer um novo alvo de pesquisa para PM20D1 em relação à AC. 2 Among kinds of atherosclerosis, carotid atherosclerosis (CAS) is thought to be a predictor for ischemic stroke.3 It is reported that carotid plaque is a risk factor for ischemic stroke, which is also associated with the cadmium, and increased carotid intima-media thickness and presence of carotid plaque are associated with increased risk of ischemic stroke in individuals with atrial fibrillation.5 Many risk factors are reported to be associated with CAS, including dysfunction of lipid metabolism,6 hypertension,7 diabetes,8 age,9 smoking10 etc. However, deeper insights are still needed for CAS onset and clinical outcomes.As the main cause of most cardiovascular diseases, atherosclerosis (AS) can occur early in lifetime and remain asymptomatic for long periods before onset.11 A recent study found PM20D1 is associated with lipid metabolism and might be associated with obesity,12 which are all risk factors for CAS. It was also found that PM20D1 could regulate lipidated amino acid uncouplers of mitochondria and increased PM20D1 augmented energy expenditure.13 However, up to now, no study focuses on the role of PM20D1 in CAS.Peptidase M20 domain containing 1 (PM20D1) is a newly identified secreted enzyme enriched in uncoupling protein 1 (UCP1+), versus UCP1- adipocytes.The present research performed a prospective observational study in order to investigate the clinical significance of PM20D1 in CAS patients. This research might provide clinical evidence for the role of PM20D1 in CAS.14 The measurement of IMT was conducted using a LOGIQ C9 Color Doppler ultrasound diagnostic instrument  with probe frequency of 7~14 MHz. The IMT of bilateral common carotid artery, proximal, distal and 1 cm from the bifurcation of common carotid artery were measured. All measurement was conducted at least three times and the mean value was considered as the final IMT value.The present prospective observational study enrolled a total of 231 CAS patients, who went to our department between July 2018 and December 2019. The inclusion criteria were: 1) all patients were diagnosed as CAS according to color Doppler ultrasound of neck blood vessels and aortic arch intracranial computed tomography angiography (CTA); 2) CAS patients with stroke were admitted within 72 hours after onset, and the diagnosis of stroke was confirmed by magnetic resonance imaging (MRI) and computed tomography (CT) scan; 3) patients agreed to participate in the observational research. The following patients were excluded: 1) patients with cerebral aneurysm, arteriovenous malformation, dissection, arteritis or moyamoya disease; 2) patients with cardiogenic cerebral embolism; 3) patients with other severe system diseases, including cancer and heart, renal or liver dysfunction. The severity of CAS was defined as: 1) mild/moderate CAS group, who showed thickening of the intima-media, with intima-media thickness (IMT) >1.0 mm, or plaque information with arterial stenosis <70% and without stroke; 2) severe CAS group, who showed one or more plaques with arterial stenosis ≥70% and without stroke; 3) CAS combined with stroke group, who showed CAS combined with large artery atherosclerotic ischemic stroke and cerebral vascular stenosis >50%. The plaques were divided into stable and unstable plaques as widely accepted in clinic: 1) IMT≥1.2 mm was considered as plaque; 2) the stable plaques are plaques with uniform strong echo or medium echo; 3) the unstable plaques are soft plaque or ulcerative plaque with mixed echo or low echo.2; the overweight group, with 28 kg/m2>BMI≥24 kg/m2; and the obesity group, with BMI>28 kg/m2.15 Blood samples and medical characteristics were also obtained from 231 healthy individuals, who came to routine physical examination with the same BMI distribution of CAS patients. All patients signed the informed consent. The present study was approved by the ethical committee of the Fourth People’s Hospital of Chengdu (CDH-2018-057).Additionally, patients were further divided into different body mass index (BMI) groups: the normal group, with BMI<24 kg/mThe blood samples of all cases were collected within 24 hours of admission or coming to outpatient. Briefly, 5 ml of blood was collected into tubes without any anticoagulant. After 1 hour, centrifugation was conducted at 2,000 g for 15 minutes, in room temperature, and the serum samples were obtained. The serum PM20D1 levels were measured by enzyme-linked immunosorbent assay (ELISA), using a PM20D1 kit , strictly according to the manufacturer’s instruction.Clinical and demographic characteristics of all patients were collected, including age, sex, BMI and medical history. Routing whole blood test was performed using an automatic biochemical analyzer , and the levels of C-reactive protein (CRP), tumor necrosis factor (TNF-α), homocysteine (Hcy), as well as total cholesterol (TC), triglyceride (TG), high-density leptin cholesterol (HDL-ch) and low-density leptin cholesterol (LDL-ch), were recorded.Data distributed normally was expressed by mean ± standard deviation (SD), and non-normal distributed data was expressed as median (interquartile range). Categorical variables were shown as number (rates). The distribution of the data was analyzed by Kolmogorov-Smirnov method. For normally distributed data, comparison between two groups was conducted by unpaired t-test and one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, which was used for comparison among three or more groups. For non-normal distributed data, Mann-Whitney test was used for comparison between two groups, and Kruskal-Wallis test was used for comparison among three or more groups, following with Dunn’s post hoc test. Rates were analyzed using chi-squared test. Correlation among PM20D1, Hcy, lipid metabolism and inflammatory factors was analyzed using the Pearson’s correlation. A receiver operating characteristic (ROC) curve was used for analysis of PM20D1 in CAS patients. Logistic regression was used for analysis of risk of CAS. p<0.05 was considered as statistically different. All calculation was performed using SPSS 25.0  or GraphPad 6.0 .This research included a total of 231 CAS patients, with 152 cases of mild/moderate CAS, 50 cases of severe CAS and 29 cases of CAS with stroke. The ratio of the sum of overweight and obesity patients was significantly higher in severe CAS patients when compared to the mild/moderate patients . The ratsignificantly lower levels of CRP, TNF-α and Hcy, as well as TG, TC and LDL-ch than all CAS patients. No other significant difference was found.Then, the expression of serum PM20D1 in CAS patients was determined. It was found that serum PM20D1 levels were markedly lower in CAS patients when compared to the healthy control . Severe PM20D1 was not associated with carotid atherosclerosis patients with different body mass indexTo further investigate the role of PM20D1 in CAS patients, we also evaluated the levels of PM20D1 in CAS patients with different BMI distribution. No significant difference was observed among patients with different BMI .Then, patients were divided into PM20D1 high expression group and low expression group, according to the mean level of PM20D1 (4.53 ng/ml). It was observed that patients with higher PM20D1 levels showed significantly lower expression of CRP, TNF-α, Hcy, TG, TC and LDL-ch . PearsonAt last, a ROC curve was drawn in order to see the diagnostic value of PM20D1 for CAS, as well as severe CAS or CAS/stroke. It was found PM20D1 could be a potential diagnostic biomarker of CAS with cutoff value of 5.94 ng/ml, area under the ROC curve (AUC) 0.876, 95%CI (0.845~0.906), sensitivity 80.1% and specificity 73.6% . PM20D1 Early diagnosis and prediction of CAS can greatly improve the treatment efficacy and prognosis. Thus, biomarkers for diagnosis and prognosis of CAS are of great significance. In the present study, we demonstrated that PM20D1 was down-regulated in CAS patients and was associated with the severity and plaque condition of CAS patients. Besides, PM20D1 was negatively correlated with CRP, TNF-α, Hcy, TC and LDL-ch in CAS patients.16 Another research found that treatment of atorvastatin or ezetimibe obviously decreased the levels of TC, TG and LDL-ch in CAS patients.17 In a recent study, Pan et al. showed that, in low-income rural residents in China, LDL-ch and TC were risk factors for early-stage atherosclerosis and carotid plaque risk increased by 24% and 62% for each 1-mmol/L increase in TC and LDL-ch.18 In our research, we also found that TG, TC and LDL-ch were highly expressed in CAS patients, especially in severe CAS cases, which was consistent with the aforementioned researches. Besides, we also observed that inflammatory factors such as CRP, TNF-α and expression of Hcy were elevated in CAS patients, which was also demonstrated in several researches.22Lipid metabolism and obesity are both associated with development of CAS. It was found that levels of TG, TC and LDL-ch were significantly higher in patients with carotid artery plaque.13 Benson et al. demonstrated that decreased PM20D1 was associated with the genetic relationship of obesity and neurodegenerative diseases in humans.23 Li et al. found that inhibition of miR-324-5p increased the oxygen consumption of primary white and brown adipose tissue cells, increased fat consumption, and thus reduced the weight of mice by enhancing the levels of PM20D1.24 Long et al. observed that, in PM20D1 deficiency mice, knockdown of PM20D1 decreased the N-acetil aspartato hydrolase/synthetase activity in serum and tissues, as well as a variety of metabolic and pain phenotypes, including insulin resistance, cold temperature changes and antinociceptive behavior, which are associated with obesity, diabetes and other diseases.12 Thus, we can speculate that higher PM20D1 levels might be beneficial in the improvement of obesity and the reduction of lipid. Although there is no study demonstrating the role of PM20D1 in CAS, the down-regulation of PM20D1 in CAS patients in our study might be partly due to the dysfunction of lipid metabolism and obesity of CAS patients. Since CAS patients showed lower PM20D1 levels than healthy control who had the same BMI distribution, and CAS patients with different BMI showed no significant difference of PM20D1, this result indicated that BMI might not be associated with the level of PM20D1 in CAS patients. The negative correlation between PM20D1 and Hcy, TC and LDL-ch might be one of the reasons for the abnormal expression of PM20D1 in CAS patients. However, to substantiate all these speculations, more studies are needed in order to obtain further evidence.PM20D1 is a factor associated with both lipid metabolism and obesity. It was found that mice with increased circulating PM20D1 showed increased breathing and increased serum N-acyl amino acids, which improved glucose homeostasis and increased energy consumption, and thus might regulate obesity.The present study has some limitations. First, this is an observational study from a single center, with only 231 cases. Secondly, molecular mechanism for how PM20D1 influences CAS development is still unclear.In conclusion, this observational study demonstrated that decreased PM20D1 was associated with the severity, plaque condition, and expression of CRP, TNF-α, Hcy, TG, TC and LDL-ch in CAS patients. This study might provide novel research target for PM20D1 in CAS.


In vitro digestibility of starch is a common analysis in human nutrition research, and generally consists of performing the hydrolysis of starch by α-amylase in specific conditions. Similar in vitro assays are also used in other research fields, where different methods can be used. Overall, the in vitro hydrolysis of native starch is a bridge between all of these methods. In this literature review, we examine the use of amylolysis assays in recent publications investigating the complex starch structure-amylolysis relation. This review is divided in two parts: (1) a brief review of the factors influencing the hydrolysis of starch and (2) a systematic review of the experimental designs and methods used in publications for the period 2016–2020. The latter reports on starch materials, factors investigated, characterization of the starch hydrolysis kinetics and data analysis techniques. This review shows that the dominant research strategy favors the comparison between a few starch samples most frequently described through crystallinity, granule type, amylose and chain length distribution with marked characteristics. This strategy aims at circumventing the multifactorial aspect of the starch digestion mechanism by focusing on specific features. An alternative strategy relies on computational approaches such as multivariate statistical analysis and machine learning techniques to decipher the role of each factor on amylolysis. While promising to address complexity, the limited use of a computational approach can be explained by the small size of the experimental datasets in most publications. This review shows that key steps towards the production of larger datasets are already available, in particular the generalization of rapid hydrolysis assays and the development of quantification approaches for most analytical results. The study of starch hydrolysis by amylase, or amylolysis, in human health is central for assessing the role of starch in foods . AmylolyStarch comprises two polymers of glucose residues: amylose and amylopectin. Amylopectin represents by far the major fraction of native starch—75–90% of the relative dry weight of wild-type starches—while amylose is the minor fraction . Both amNative starch exhibits a large range of structural  and physicochemical  properties that can be used to meet various end-uses, in particular in food applications. Recent advances in crop selection and genetic approaches extend the potential properties of starch in crops even further . More thHence, in vitro starch amylolysis is commonly used in different research contexts. However, whatever the research context, the in vitro assay is generally used to investigate the mechanism by which the starch structure controls its enzymatic degradation. Many publications and review papers address this topic e.g., ,9,10,11),10,119,1Different methods exist for conducting starch digestibility analysis depending on the research branch and on the pioneering works the methods are based on, such as the Englyst et al.  classifiThe objective of this paper is to present a systematic review of recent publications investigating the relationship between amylolysis and starch structure, including starch materials, factors investigated, in vitro assay techniques and data analysis techniques. The review provides an overview of dominant research strategies, bottlenecks and emerging strategies in view to outline promising methods for a benchmark starch digestibility analysis. This review paper has two main sections; (i) the first section presents an overview of the intrinsic starch structural features and the experimental conditions that influence the native starch amylolysis results, and (ii) the second section presents a systematic review of the publications on the topic from 2016 to 2020. The processing of native starch generally affects its susceptibility to enzymatic degradation significantly, to the point that it can get more important than the native structure; here we used “starch” to refer to native starch unless specified otherwise. The starch enzymatic hydrolysis is a heterogeneous reaction as the catalytic act takes place at the interface between the solid phase (starch) and the liquid phase that conveys the enzyme. It involves different steps, including the initial diffusion of enzyme through the different levels of starch organisation followed by the adsorption on the surface (formation of enzyme-starch complex) and finally the hydrolytic . DiffereThe factors determining the enzymatic susceptibility of native or processed starch  have been investigated extensively. While starch is generally processed in human food, native starch still raises much scientific interest as a natural substrate for most amylolytic enzymes. In addition, the combination of multiple processed methods with water, high temperature, milling, etc., associated with the starch structural parameters generate a very complex analysis with endless sources of variability .Native starch is present in the form of granules made of concentric alternating amorphous and semi-crystalline growth rings of carbohydrate polymers. While, this general organization is common across the plant kingdom, the overall shape size and the polymers’ organizations vary greatly with the botanical origin . The starch granule can be described through four organizational levels: (i) the chemical structure of starch polysaccharide constituents (~1 nm), (ii) the crystalline and lamellar structure ~9 nm), (iii) the growth rings (120~500 nm) and (iv) the granular level 1~100 μm). The relations between starch structural features and the enzymatic hydrolysis are briefly described in this section, starting from the granular level to the chemical structure of the polymers. The aim was to provide a broad overview; the additional and thorough information can be found in several dedicated review papers ,9,10. nm,  digestion pattern, where α-amylase creates and enlarges pits from the core to the surface ,33. GranNative starch granules are layered structures made of crystalline regions with orderly packing of amylopectin chains double-helices and amorphous regions made of amylose and amylopectin without detectable molecular ordering . An x-raThere are two basic polymorphic types of crystalline structures in native starch, the A-type encountered mainly in cereal starches and the B-type found in potato or high amylose maize variants. A-type crystallites are made of double-helices of amylopectin chains ordered in a monoclinic unit cell, while B-type crystallites are made of double-helices ordered in a hexagonal unit . An addiIn native starch, the distribution of amylopectin chains, represented by unit Chain Length Distribution (CLD) or the short:long amylopectin side-chains ratio, is generally well correlated with the starch hydrolysis ,40,41. TThe ratio amylose/amylopectin is usually negatively correlated with the susceptibility to amylolysis, which is counter-intuitive as the α-1,6 linkages of the amylopectin should hamper the binding of α-amylase . Lopez-RBesides amylose and amylopectin, purified starch contains additional minor components, mainly proteins and lipids associated with the starch granule located on the surface or imbedded within the native starch granules ,45,46,47Starch lipids present a high diversity. They can be detected as Free Fatty acid (FFA), or lipids can form amylose-lipid complexes in native starch amorphous or crystalline arrangements, i.e., V-type crystallinity . An amylWhile starch structure and composition are universally recognized for influencing amylolysis, the actual conditions defining the reactions can have a significant additional impact on the hydrolysis results. Substrate concentration. According to Tawil et al. [l et al. , both thl et al. ,43. Source and concentration of enzyme. Enzyme type, biological origin and concentration influence the kinetic and hydrolysis final extent as well as the degradation products. The α-amylases particularly have diverse modes of actions and product specificities depending on their origin [Bacillus licheniformis α-amylase, extensively used in industrial processes, produces mainly maltopentose [r origin ,56. For opentose . The difopentose . For in opentose  as a subopentose . Other experimental conditions. As for any enzymatic reaction, the amylolysis must be conducted within a range of incubation temperatures and pHs that would ensure optimal enzymatic activity. The incubation temperature and duration need to accommodate with the nature of the substrate, as modification starch granule structure, especially starch annealing and starch gelatinization, may occur in the conditions of a hydrolysis assay [is assay . Other ois assay  showed tObservational studies of starch degradation during hydrolysis have greatly contributed to our current understanding of the mechanisms. The development of new analytical techniques allows scientists to monitor continuously the dynamic changes of the starch granule during the course of amylolysis ,62. ThisDeciphering the impact of each and every factor affecting the amylolysis profile is critical but challenging when those factors synergistically interact. Potato starch, a tuber starch, has rather long amylopectin side-chains, B-type crystallinity and large-size granules without visible surface pores while rice starch, a cereal starch, presents an almost opposite profile. All the potato starch features can account for the low susceptibility to amylolysis (not mentioning phosphate groups bound to amylopectin) and, vice-versa, for the high susceptibility of rice starch. Hence, determining to which extent each of these factors individually or synergistically accounts for the overall susceptibility to the enzymatic degradation is challenging. Besides this, factors measured in the context of a research study captures only a fraction of the real starch structure. A significant fraction remains unknown . Hence, the starch structure-amylolysis conundrum is a multi-dimensional problem affected by uncertainty due to unknown parameters. Purely observational investigations have limitations in addressing this type of problem, and other research orientations are proposed, such as the identification of dominant rate-determining factors of the amylolysis ,11,63. The following section reports on the experimental designs and analysis employed by recent studies to address the starch structure-amylolysis conundrum. Starch enzymatic degradation has been investigated for decades with continuing interest since 2016. Using a literature research engine  and the procedure described in In the first stage of the selection procedure, a list of 2053 journal articles covering the last 30 years was obtained from the WoS using a general research equation about starch hydrolysis by α-amylase, explicitly excluding acid hydrolysis . Stage 2The information about the 57 articles of the panel used for the following analysis is supplied as an excel table; in the wx (waxy), ae (amylose-extender) and su2 (sugary), with various granular and macromolecular structures [Cereal starch from maize, rice and wheat, together with potato starch, were extensively studied in our publication panel , most liructures . Other sThe experimental designs found within the panel include on average eight distinct samples , which aThe number of studied factors described in the publication panel varies from one to nine, with an average of four . No trenThe most frequent factors describe distinct organizational levels of native starch (from granular to molecular level) as well as few functional properties . At the The functional properties reported in the panel publications are mainly related to starch gelatinization (28%) measured by Differential Scanning Calorimetry (DSC). DSC reflects on the swelling, the leaching of soluble polysaccharides, the disruption of the molecular order and the melting of crystallites , thus prEach of the uncommon factors  assay  Table 4Table 4. e.g., in ). Neverte.g., in , which lFor 91% of the panel, an enzymatic assay is used to obtain kinetic data of the starch degradation by an α-amylase (30%), sometimes in association with an amyloglucosidase (61%). Several methods were used to interpret the kinetic data. Twelve percent of the panel used a starch classification method, more precisely derived from Englyst’s method, to identify starch fractions with decreasing digestibility based on their digestion time . Figure t, k is the reaction rate constant. Although the first-order kinetics describes well the hydrolysis profile, it needs an accurate estimation of k. Seventy percent of the panel applied the empirical first-order kinetics  using Eqis assay . The apph et al.  allows tKinetics data do not always follow a first-order kinetics, which led to adaptations of Equations (1) and (2). Two main adaptations of the first-order kinetics model have been proposed to obtain a better fitting of kinetic data, a sequential model that describes a fraction of the starch digested before others and a parallel model that describes two or more fractions of starch digested simultaneously at different rates . EdwardstY is the normalized starch concentration and RS, RDS and SDS; RDS and SDS. Compared to the sequential model, the parallel model avoids breakpoints at the times of intersection between two hydrolysis phases [s phases .k is affected by a parameter h that reflects the time-dependency of the reaction rate coefficient: A Weibull model proposed by Kansou, Buléon, Gérard & Rolland-Sabaté  is anothThe majority of the panel publications employed standard statistical analysis methods . Up to 7The conditions of application of the usual linear models, including the linear regression models or the ANOVA, are often problematic in the context of starch structure-degradation studies because of the collinearity of the data . DespiteDespite many publications, the study of “structure-degradability” relationships remains challenging, as the starch hierarchical structure is complex and multidimensional and our understanding is incomplete. The interdependencies between the structural features of starch hinder the identification of their respective contribution to the enzymatic degradation. How is this challenge currently addressed by the scientific research? Predominant sources of starch are a few major starch-rich crops of the human diet: cereals  and tuber (potato). Focusing on selected crops limits the splitting of the research effort and favors the understanding by accumulating knowledge.A typical starch-degradability study investigates the digestibility of eight different samples via four structural/functional factors characterizing the starch. The most frequent factors in the panel involve the crystallinity, the granule morphology via SEM, the amylose content, the chain length distribution and the gelatinization properties. The selected starch materials present marked distinctive characteristics, such as high amylose vs. wild-type vs. no-amylose starches, the presence or absence of granule pores and A-type vs. B-type crystalline polymorph starches. On the one hand, this generates contrasted digestibility profiles, while on the other hand, this tends to overlook the role of the accompanying structural alterations, which are possibly critical to discriminate amongst conventional starches.The size of the average experimental dataset  might be insufficient to exploit recent data analysis techniques . This is a limitation to addressing complexity, as computational approaches are necessary to address multifactorial systems.Quantitative approaches exist for analyzing most structural factors of starch. Even though quantification can be indirect and uncertain—the determination of crystalline proportion from X-ray diffractometers, the quantification of granule characteristics from SEM and the continuing development of quantitative approaches is an important lever to generalize the use of computational techniques.Several assays and methods can be employed for the measurement and the analysis of starch hydrolysis, in relation to the research topics. While diversity in this matter is necessary—studying starch digestibility in human diet is different from investigating the action of a specific amylase—using the same method for a given topic can favour the comparison of inter-publications. This review presents the most frequent assays and methods used in recent works; this can guide the selection of methods in future studies.To answer this question, our systematic review of a panel of 57 recent publications on this topic outlines the following aspects of the experimental research: Overall, this review shows that the dominant strategy for conducting starch digestibility analysis is to circumvent the complexity of the underlying mechanism by careful selection of contrasted substrates. The most promising works in that regard use mutants and transgenic lines from a generic line to obtain starch samples displaying variations for specific targeted parameters, thus limiting the variability for unknown parameters. A second strategy that is in the minority so far addresses the interrelated factors issue using multivariate analysis techniques. Both approaches have drawbacks: the former requires more efforts from the plant genetic domain to produce starch with interesting characteristics and depends on a forward genetic approach, while the latter demands larger datasets in terms of number of samples and number of measured factors. The second strategy appears closer at hand for the research community, as the average size of the datasets will naturally increase with the development of high-throughput techniques and the growing quantification capability of the analytical results. This review has shown that key steps towards scaling up experimental designs are already available, in particular the generalization of rapid hydrolysis assays with the dosage of reducing sugars and the application of kinetic models to interpret the data. These combined would provide a reliable quantification and analysis of the amylolysis profile. A remaining bottleneck is certainly the development of high-throughput relevant analytical techniques to describe the starch properties.


Lemna turionifera 5511) as the research object and used a stable genetic transformation system to heterologously over-expressing Arabidopsis AtPSAT1 . Our results showed that, under nitrogen starvation, the transgenic plants grew faster, with higher values of Fv/Fm, rETR, and Y(II), as well as fresh and dry weight, than the wild-type. More promisingly, the accumulation of starch was also found to be significantly improved when over-expressing AtPSAT1 in the transgenic plants. qRT-PCR analysis results showed that the expression of genes related to nitrogen assimilation, carbon metabolism, and starch biosynthesis was up-regulated, while the expression of starch degradation-related genes was down-regulated by AtPSAT1 over-expression. We propose that the increased starch accumulation caused by AtPSAT1 over-expression may result from both elevated photosynthetic capacity and nitrogen utilization efficiency. This research sheds new light on the mechanism underlying the ability of PPSB to coordinate nitrogen and carbon metabolism, and provides a feasible way to improve starch production, that is, through engineering PPSB in crops.It has been demonstrated that the phosphorylation pathway of L-serine (Ser) biosynthesis (PPSB) is very important in plant growth and development, but whether and how PPSB affects nitrogen metabolism and starch accumulation has not been fully elucidated. In this study, we took the energy plant duckweed (strain  Duckweeds are among the smallest aquatic flowering plants, consisting of only a leaflike structure , either with one to several pseudo-roots or without roots. Considering their high multiplication rate, high starch content, and various other practical characteristics, they have been recognized as promising biofuel plants for the generation of hydrogen, ethanol, butanol, and biogas ,2,3. To AtPSAT1 and AtPSAT2) have been identified which encode PSAT [Phosphoserine aminotransferase  is the second enzyme in the so-called phosphorylated pathway of serine biosynthesis (PPSB), which synthesizes serine from 3-phosphoglyceric acid (3-PGA) in plastids. 3-PGA is converted to Ser in three steps, sequentially catalyzed by phosphoglycerate dehydrogenase (PGDH), PSAT, and phosphoserine phosphatase (PSP). PSAT plays a key role in the biosynthesis of serine in plants and other organisms ,19, whicode PSAT ,26,27. Iode PSAT .AtPSAT1 in Lemna turionifera 5511, providing solid evidence that PSAT/PPSB regulates the vegetative growth and accumulation of starch in duckweed fronds, at least under nitrogen starvation conditions.In this paper, we report the stable transformation and over-expression of AtPSAT1 and AtPSAT2—in Arabidopsis [AtPSAT1 over-expression vector based on pCAMBIA 1301, replacing the GUS coding sequence with the full-length CDS sequence (1293 bp) of AtPSAT1 from Arabidopsis  and different levels of nitrogen starvation. There were almost no differences in phenotype between transgenic lines and WT in D (Datko) after 9 days of incubation  of WT and transgenic lines were measured in the samples treated under nitrogen starvation and full-nutrient conditions  for 9 days. There was no difference between WT and transgenic duckweed under Datko conditions . Meanwhirameters . Interesrameters e. rETR  and different nitrogen content culture conditions a,b. The 2-KI-stained sections of WT and PSAT-7 further illustrated starch accumulation at the cellular level , NIR (nitrite reductase), GS (glutamine synthetase), and GOGAT (glutamate synthase) after nitrogen starvation for 6 days , LtASP1 was up-regulated 5-fold on average, LtGAD1 was up-regulated close to 10-fold on average, and LtGLN1 and LtGS1 encoding GS were significantly up-regulated 2.6-fold and 2.5-fold on average, respectively. The expression pattern of LtGLT1 encoding GOGAT was up-regulated but not significantly different, and LtGLU1 was up-regulated approximately 2-fold, with PSAT-1 being the most significant.The above results suggested that the growth in over-expression elopment . How arer 6 days . The NR r 6 days a,b. The r 6 days c. The acr 6 days d. Then, r 6 days . The resLtAPS1 was significantly higher in the transgenic lines than WT, with a 5.5-fold increase in nitrogen starvation, while the expression of LtAPL1 did not significantly differ between the transgenic lines and WT. The key enzymes GBSS and SSS  in starch synthesis provide the glucose substrate for straight-chain starch elongation and transfer glucose units to straight-chain starch molecules, respectively [LtSSS1, also showed a different expression pattern, with significantly higher expression in the transgenic lines than WT, with the highest expression of PSAT-1 among the three transgenic materials, increasing 6.5-fold compared to WT , soluble starch synthase (SSS), and granule-bound starch synthase (GBSS). Starch degradation is mainly driven by isoamylase (ISA), α-amylase (α-Amy), and β-amylase (β-Amy) . To inveectively ,34,35. Ted to WT . The expan in WT . α-amylaic lines . The expic lines .LtPFK1 (phosphofructokinase) and LtFBA1  did not differ significantly between the transgenic lines and WT after nitrogen starvation for 6 days, while the expression of LtPK1 (pyruvate kinase) was significantly higher in transgenic lines than in WT by about 3-fold, and LtPDC1 (pyruvate dehydrogenase complex) expression was also significantly higher than WT, by about 1.7-fold , LtIDH1 (isocitrate dehydrogenase), LtMDH1 , and LtCS1 (citrate synthase) were significantly down-regulated, while Lt2OG-DH1 (2-OG dehydrogenase), LtSDH1 (succinate dehydrogenase), and LtFUM1 (fumarase) were significantly up-regulated (The TCA (the tricarboxylic acid) cycle is a common oxidation pathway for the sugars, lipids, and proteins, which also has some association with the glycolytic pathway . In addiegulated  in transAtPSAT1 over-expression lines in duckweed under normal conditions; however, under nitrogen starvation conditions, the relative growth rate, fresh weight, and dry weight in AtPSAT1 over-expression of L. turionifera were significantly higher than those of the WT , nitrite reductase , glutamine synthetase , and glutamate synthase . The transcription profiling of these key genes, as well as the activities of their corresponding enzymes, were all promoted in AtPSAT1 over-expressing transgenic lines, compared with WT, under nitrogen starvation conditions  [AtPSAT1 over-expression would be helpful in increasing the NUE in transgenics lines. Meanwhile, the lower content of protein in AtPSAT1 transgenic lines suggested that, under nitrogen deficiency, the transgenic duckweed probably met the nitrogen source requirement for its growth by increasing endogenous protein degradation  . The higradation l, confirAtPSAT1 transgenic lines than in WT. Meanwhile, compared with the wild-type, the PSII-mediated relative electron transport rate (rETR) and the actual photochemical efficiency of PSII (Y(II)) were both substantially higher in the transgenic lines. This further indicated that over-expression of AtPSAT1 is helpful for maintaining photosystem function to prompt the growth of duckweed under nitrogen starvation.Fv/Fm denotes the maximum quantum efficiency of photosynthetic tissue and is an important indicator of plant photosynthetic activity. Generally, Fv/Fm values are about 0.8 in healthy plants. Both for the wild-type and transgenic duckweeds, their value of Fv/Fm was above 0.8 when they were cultured in the Datko medium, suggesting that they were healthy under full-nutrition conditions . When thAtPSAT1 can effectively promote starch accumulation in duckweed under nitrogen starvation conditions.Starch is one of the main storage compounds in duckweed, and is usually accumulated under stress conditions . This is an adaptive strategy acquired during long-term evolution that allows plants to survive in unfavorable environments . In our LtAPS1 is the gene encoding the small sub-unit of AGPase, which plays an important role in regulating starch levels and determining the starch deposition pattern of plants [LtAPS1, LtSSS1, and LtGBSS1 were all significantly up-regulated in the transgenic lines under nitrogen starvation  and LtFBA1  did not significantly differ between transgenic lines and WT, while LtPK1 (encoding pyruvate kinase) and LtPDC1 (encoding pyruvate dehydrogenase complex) were significantly higher in transgenic lines than WT. Pyruvate kinase (PK) is a key regulatory enzyme that catalyzes the conversion of phosphoenolpyruvate (PEP) to ATP and pyruvate in the glycolytic pathway [LtPK1 and LtPDC1 in transgenic lines likely prompts the production and metabolism of pyruvate, as a carbon backbone, to increase amino acids and intermediates in the TCA cycle. Furthermore, the 2-oxoglutarate (2-OG) released by PSAT activity can be directly recycled to glutamate by the GOGAT enzyme in the plastid [AtPSAT1 is helpful in maintaining the metabolic balance of TCA cycle intermediates. In conclusion, the expression patterns of genes involved in TCA seem to regulate the content of 2-ketoglutarate in a coordinated manner, prompting it to enter the pathway of carbon and nitrogen metabolism, which may result in decreasing sugar content and starch accumulation after over-expression of AtPSAT1 under nitrogen starvation.The phosphorylated serine pathway (PPSB) is known for its role in regulating the glycolytic flux and tricarboxylic acid cycle (TCA) . Glycolyn stress . The rel pathway . The up- plastid . 2-OG prLemna turionifera 5511 were cultivated in aseptic Datko medium, according to Wang and Kandeler [−2·s−1. At first, fresh fronds were grown on Datko medium for 7 days. Then, 20 fronds with constant growth status were transferred to Datko medium that lacked nitrogen or fresh Datko medium in a 100 mL conical flask. The plants were cultivated in the same long-day light period conditions and temperature, with a light intensity of 100 μmol photons m−2·s−1 at the plant level.Kandeler . The ducAtPSAT1 (first encoded gene of 3-phosphoserine aminotransferase) was obtained from the Arabidopsis thaliana cDNA by PCR. It was inserted into the plasmid of pCAMBIA1301 with hygromycin resistance gene, and modified with CaMV-35S promoter at 5′ and the terminator of the nopaline synthase gene at 3′ end, in order to construct the plant binary expression vector pCAMBIA 1301-35S-AtPSAT1. According to the freeze–thaw method, the vector was transformed into the Agrobacterium tumefaciens strain EHA105. The transgenic duckweed of AtPSAT1 was acquired using the callus transformation system described by Yang at al. [TMRT reagent Kit with gDNA Eraser . The DNA and cDNA for Lt18s and AtPSAT1 were amplified using PCR and RT-PCR with specific primers  as the initial fresh weight (taking 10 lines of duckweed with the same growth status and taking fresh weight) and at the end of the growth day . The relative growth rate (RGR) was calculated according to Su and Ziegler [The growth rate of duckweed was calculated according to the change in fresh weight, which was weighed at the first growth day .Next, 0.1 g of duckweed material treated for 9 days was immersed in 1 mL of 95% ethanol and placed in an incubator at 28 °C until the duckweed turned completely white. Then, 200 μL of the extract was taken and its OD values at the wavelengths of 663 nm, 645 nm, and 440 nm were measured under a plate reader, and the photosynthetic pigment content was calculated according to the Arnon method . Chlorop2-KI-stained sections, fronds were prepared in paraffin sections. According to the method described by Zhu [2-KI staining solution for 5–10 min and the slices were sealed directly with staining solution for observation and comparison of tissue structure by light microscopy. For TEM samples, plant tissues were collected and fixed overnight with 2.5% glutaraldehyde solution at 4 °C. The fixed solution was poured out, rinsing the sample with 0.1 M pH 7.0 phosphoric acid buffer three times, each time for 15 min. The samples were fixed with 1% osmium solution for 1–2 h. Osmium acid waste liquid was carefully taken out, again rinsing the sample with 0.1 M pH 7.0 phosphoric acid buffer three times, each time for 15 min. The samples were dehydrated with a gradient of ethanol solutions . Each concentration was treated for 15 min, after which it was treated with 100% ethanol twice for 20 min and finally treated with pure acetone for 20 min. The mixture of embedding and acetone (v/v = 1/1) was used to treat the sample for 1 h, then another mixture of embedment and acetone (v/v = 3/1) was used to treat the sample for 3 h. The sample treated with pure embedding agent was covered overnight. The embedded sample was then embedded and heated at 70 °C overnight, then sliced using a Leica EM uc 7 ultra-thin microtome, with which 70–90 nm sections were obtained. The sections were stained with a lead citrate solution and 50% alcohol saturated solution of uranyl acetate for 5–10 min, then observed using a HITACHI H-7650 .For observation of the morphology and ultrastructure of fronds, the duckweeds were sampled after 9 days of cultivation without nitrogen. For Id by Zhu , the secA starch extraction and measurement kit was used to extract and measure the starch content, according to the manufacturer’s protocol . The total sugar was extracted and measured using a total sugar extraction and measurement kit, according to the manufacturer’s protocol . Soluble protein content extraction was carried out using 30 mM Tris-HCl (pH 7.0), and was measured using the manufacturer’s protocol .A nitrate reductase (NR), nitrite reductase (NIR), glutamine synthetase (GS), and glutamate synthase (GOGAT) extraction and measurement kit was used to extract and measure the enzyme activities, according to the manufacturer’s protocol .Lt18s, AtPSAT1, starch metabolism genes, nitrogen assimilation genes, and carbon metabolism genes was amplified using qRT-PCR with specific primers, designed from the duckweed genome sequence obtained by RNA-Seq , and plotted using GraphPad 8.0  software.All data were measured using at least three biological replicates, and the experiments were repeated at least three times independently. Data were collated using Excel, statistically analyzed using SPSS 22.0 , analyzed for significance using one-way ANOVA (*, AtPSAT1 in duckweed can facilitate its growth and prompt starch synthesis under nitrogen starvation conditions. The better growth and elevated photosynthesis capabilities were due to up-regulation of the expression of key genes involved in nitrogen assimilation in the AtPSAT1 over-expressing lines under nitrogen starvation. The high starch accumulation was due to the regulation of expression of genes involved in the glycolytic and TCA cycle pathways, as well as genes involved in the starch synthesis and metabolism pathway. This study provides a prospective pathway for the accumulation of starch through genetically engineering the PPSB pathway into the plant, and sheds light on the mechanism by which PPSB promotes starch accumulation under nitrogen starvation.Duckweed is well known for its potential as feedstock for bioenergy. This is not only because it is an aquatic plant, meaning that it avoids competition for limited arable land; more importantly, the duckweed plant can accumulate starch under nitrogen starvation conditions, which provides a unique advantage over other crops. This work reported, for the first time, that over-expression of


Epstein-Barr Virus (EBV) is a human pathogen that has a morbidity rate of 90% in adults worldwide. Infectious mononucleosis is caused by EBV replication in B cells and epithelial cells of the host. EBV has also been related to autoimmune illnesses, including multiple sclerosis and cancers like nasopharyngeal carcinomas and Burkitt’s lymphoma. Currently, no effective medications or vaccinations are available to treat or prevent EBV infection. Thus, the current study focuses on a bioinformatics approach to design an mRNA-based multi-epitope (MEV) vaccine to prevent EBV infections. For this purpose, we selected six antigenic proteins from the EBV proteome based on their role in pathogenicity to predict, extract, and analyze T and B cell epitopes using immunoinformatics tools. The epitopes were directed through filtering parameters including allergenicity, toxicity, antigenicity, solubility, and immunogenicity assessment, and finally, the most potent epitopes able to induce T and B cell immune response were selected. In silico molecular docking of prioritized T cell peptides with respective Human Leukocytes Antigens molecules, were carried out to evaluate the individual peptide’s binding affinity. Six CTL, four HTL, and ten linear B cell epitopes fulfilled the set parameters and were selected for MEV-based mRNA vaccine. The prioritized epitopes were joined using suitable linkers to improve epitope presentation. The immune simulation results affirmed the designed vaccine’s capacity to elicit a proper immune response. The MEV-based mRNA vaccine constructed in this study offers a promising choice for a potent vaccine against EBV. Epstein-Barr Virus is a member of the herpes viruses γ family. It has a linear dsDNA genome (approximately 171 Kb) that codes for around 90 genes . EBV is EBV is found in nearly 90% of the world’s adult population . EBV cauThe immune system can control the primary infection and then the reactivation phases of EBV, forcing it to remain latent in the memory of the B cells. There is currently no pharmacological cure for EBV, and no preventative or therapeutic vaccinations are available. Many efforts to develop a vaccine aimed at preventing EBV infection, have focused on the antigenic proteins, notably gp-350 because it is the primary target of neutralizing antibodies and is the amplest gp found on the viral particles ,13. SeveShigella spp., and Staphylococcus aureus [Conventional vaccines, such as inactivated and attenuated pathogens, provide long-lasting immune protection against the subject pathogen, but have limited large-scale and quick production protocols . Becauses aureus ,23.In our study, B and T cell epitopes that were predicted and filtered, were extracted from EBV proteins that were engaged in the self-assembly of the capsid, replication of DNA, and attachment of viral particles to the oral epithelial cells, were joined to construct a candidate mRNA vaccine against EBV. Furthermore, T cell epitopes were modeled for docking with respective HLA molecules to analyze their binding. HLA genes are highly polymorphic in humans, which presents a difficult challenge for designing a vaccine given that HLA molecules can bind a particular set of peptides ,25. The Six proteins  were selected for this study based on their role in EBV infection and their attachment to host cells. The selected EBV proteins were downloaded from the Uniprot database . The canThe selected candidate proteins were used for epitopes prediction using online servers. NetCTL1.2 server was used at a 0.75 threshold to predict MHC I binding epitopes . The serThe HTL epitopes prediction was made by the Immune Epitope Database (IEDB) , where aAntigenicity. The Vaxijen server  was emplImmunogenicity. It is the potential of MHC I-binding epitopes to elicit a cell-mediated and humoral immune response. The MHC-I Immunogenicity calculator of the IEDB database  predicteSolubility. The antigenic and immunogenic epitopes that were shortlisted from previous steps were investigated for solubility by the CamSol Intrinsic server .Toxicity. To make sure that we selected the non-toxic T cell epitopes only, the ToxinPred server was used to assess the toxicity of T cell epitopes. The server evaluates different physiochemical properties  of an epitope to assess its toxicity . The nonhttp://tools.iedb.org/mhci/) accessed on: 15 December 2021 [MHC-I binding. MHC-I alleles for the prioritized CTL epitopes were predicted by the IEDB server  to make sure that the selected epitopes used to construct the vaccine would cover most of the world’s population . RegardiLinear B cell epitopes were predicted for the candidate proteins and subsequently evaluated for antigenicity and allergenicity. B cell epitopes bind to B cell receptors present on the surface of B cells. B cells are critical for antibody production by the immune system as the linear B cell epitopes induce the B cell response. BCPreds  predictsThe selected T cell epitopes fulfilling the set parameters were modeled using the PEP-FOLD3 server . The 3D The presence of five critical components in the Open Reading Frame is required for a highly immunogenic mRNA vaccine formulation: 1. Kozak sequence; 2. Linkers; 3. B and T cell Epitopes; and 4. Stop codon. The AUG codon (the start codon) has to be part of the Kozak sequence , whereasAnother sequence that has been previously reported is a signal peptide which helps to increase antigen presentation (to the discharge of translated epitopes that need to leave the cell). As a result, the tissue Plasminogen Activator (tPA) secretory signal sequence was included in the ORF’s 5′ regions . The proIn silico simulation of the vaccine induced immune system response was carried out using the C-ImmSim server . The C-iWe assessed the stability of the HLA-epitopes by performing an all-atoms simulation using a molecular simulation tool known as AMBER 20. In this regard atom parameterization was achieved through the recruitment of the FF19SB force field. The systems were prepared by adding water molecules and no effect of charge was left. The next phase was to relax each system through energy minimization. In multiple steps the minimization was completed and followed by the heating of each system. The equilibration and production were carried out for each system and a cumulative 100 ns simulation was performed. A post-dynamics assessment of the complex was performed to compute the stability and flexibility using PTRAJ and CPPTRAJ ,61,62. Amino acid sequences of the selected proteins  were retrieved from the Universal proteins knowledgebase (UniprotKB). The Vaxijen server predictions at 0.4 thresholds affirmed that the selected proteins were able to induce a host immune response. The complete information of the selected proteins and corresponding scores are in NetCTL1.2 was utilized for the prediction of MHC I epitopes for chosen EBV proteins. The high combined score of an epitope corresponds to a high binding affinity for MHC-I receptors in terms of vaccine development. The epitopes predicted previously were filtered as per the combined score, immunogenicity > 0, antigenicity ≥ 0.4, solubility > 1, and toxicity < 0. Overall, six MHC I epitopes that were antigenic, immunogenic, soluble in water, non-toxic, and conserved were selected to construct the vaccine sequence. The selected epitopes with corresponding scores are provided in 876–890 and LERGLINTVLSVKLR172–186 (Major Caps Protein), GEVALIKARKKVSGL98–112 (Envelope Glycoprotein M), and LMLLRSQLLSYSDFI404–418 (Thymine Kinase). The selected HTL epitopes with corresponding scores are provided in MHC-II binding epitopes activate the helper T cells of the immune system. The IEDB MHC-II module predicted binding of the HTL epitopes corresponding to the reference set of seven human leukocyte antigens: HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB5*01:01, HLA-DRB3*02:02, and HLA-DRB4*01:01. MHC II epitopes that have the lowest adjusted percentile rank correspond to a high binding affinity for MHC II molecules. The predicted HTL epitopes were filtered according to the lowest percentile score, antigenicity ≥ 0.4, immunogenicity > 0, solubility > −1, allergenicity < 0.3, interferon-gamma inducing ability, and toxicity < 0. Overall, four HTL epitopes that were antigenic, immunogenic, soluble in water, non-toxic, conserved, non-allergenic, and interferon-inducing were selected for vaccine construction: PTVGMIRDLSASFMTHLA allele diversity and expression differs between ethnicities and geographical regions. As a result, it impacts on the development of an MEV. The IEDB population coverage tool was used to assess the population coverage and suitability of the selected T cell epitopes. The selected epitopes revealed a world population coverage of 99%. The majority of the countries for which data is available showed 100% population coverage. Region-wise, the population coverage analysis revealed 100% population coverage except for Central America, which has the lowest (12.38%) population coverage. The overall population coverage is further depicted in The BCPreds server predicted linear B cell epitopes for selected EBV proteins. Linear B cell epitopes with higher scores corresponded to the strong binding potential for B cell receptors. The predicted linear B cell epitopes were prioritized according to the high binding score, antigenicity ≥ 0.4, and allergenicity (<0.3). Overall, 10 linear B cell epitopes that qualify for the filtering parameters were selected for constructing the final vaccine. The selected linear B cell epitopes with corresponding scores are provided in Six CTL and four HTL epitopes were modeled for in silico molecular docking using PEP-FOLD3. T cell epitopes along with corresponding HLAs are listed in The HAWKDOCK server predicted the stable conformation of the modeled peptide into the binding groove of the respective HLA receptor . The CTLThe final vaccine construct comprised six CTL epitopes , three HTL epitopes  and 10 linear B cell epitopes . The final CTL, HTL, and LBL epitopes were joined by AAY, GPGPG, and KK linkers, respectively. The final mRNA vaccine started from tPA and ended at the stop codon added right before a 3′ UTR region, namely S27a+R3U and a Poly (A) tail of 120 nucleotides at the end. The CTL, HTL, and LBL epitopes included in the final construct are listed in The secondary response reported in the simulations was noted to be higher than the primary, and similarly for the tertiary immune response. The immune simulations results were as expected and are presented in The assessment of molecular stability of each complex during the simulation demonstrates essential knowledge regarding the stable interaction of biological molecules. It yields significant information about the inhibition and activation pathways. Hence, the significance of this approach was considered, and the stability was calculated as root mean square deviation (RMSD). Efforts to reduce EBV’s pathogenic activity have been persistent, necessitating a variety of therapeutic strategies ranging from small molecule inhibitor development to immunization. Vaccines containing lytic and latent EBV proteins are being developed to evoke an immune response . Many atTCRs identify antigens displayed on the surface of APCs that have MHC receptors (classified as class I and II) on their surface. These antigens are recognized by the cytotoxic and helper T-cells, respectively. BCRs bind antigens from foreign particles and induce B cell maturity and differentiation that leads to the release of antibodies .For instance, computational biology helps to unveil the molecular mechanism and discovery of new drugs, vaccines, or peptides for different pathogens ,78,79,80Each of the epitopes in our devised method, derived from various antigens, represents a distinct immunological component. The position and order of the T and B cell epitopes within the construct will need to be carefully considered because the final vaccine must incorporate both. The epitopes were arranged in “string-of-beads” design as presented in Likewise, even though the final construct’s VaxiJen v2 score was over the cut-off, their overall immunogenicity will need to be carefully investigated experimentally since the immunogenicity of isolated epitopes may vary when they have been joined in a string-of-beads array. Optimal spacers with a balance of flexibility and rigidity prevent inter-domain interactions and promote the mRNA vaccine’s proper functioning. Several studies have reported that poly(A) tails function synergistically with 5’ m7G cap sequences, with the optimal length of 120–150 base pairs. The mRNA ORF must be flanked by the 5’ and 3’ UTRs to enhance translation and stability . NCA-7d In silico immune responses, following repeated antigen exposure showed an overall rise in immunogenicity. The presence of humoral immunity, which is essential for a full immune response, was shown by increased B and T cell activity and also B cell memory that persisted for several months. High levels of IFN- and IL-2 release upon constant exposure suggested a cell-mediated immune response. An immune response can also be boosted by cytokine IFN-, which promotes B cell proliferation and isotype switching ,98. The The recent shift of interest from conventional to mRNA vaccines has proven its usefulness. The mRNA vaccine is able to induce a proper immune response towards the subject pathogen and provide long-lasting immunity. Despite no major advances to date, vaccinations remain a potential therapeutic strategy for suppressing the virulence of the Epstein-Barr virus, which is linked to infectious mononucleosis and various cancers in humans. In our study we have used an immunoinformatic and structural bioinformatics approach to rationally develop an mRNA vaccine for immune protection against EBV. The selection of candidate proteins and prioritization of epitopes were based on tested protocols. Moreover, T cell epitopes, both CTL and HTL, were predicted using the NetCTL1.2 and IEDB MHC-II webservers, respectively. Peptide modeling and in silico molecular docking were utilized to analyze the binding potential of selected T cell epitopes for corresponding HLA molecules. An advantage of the immunoinformatic over the conventional laboratory approaches is the low cost, quick identification, and facility for screening epitopes within the vaccine design process. Furthermore, to validate the outcomes of this in silico study, experimental studies are required to validate the effectiveness of vaccines constructed against EBV.


The Nasometer is a popular instrument for the acoustic assessment of nasality. In light of the currently ongoing COVID-19 global pandemic, clinicians may have wondered about the infection control procedures for the Nasometer. The current research investigated whether nasalance scores are affected if the Nasometer 6450 microphone casings are covered with a material such as rolled polyvinyl chloride household wrap. For the experiment, pre-recorded sound files from two speakers were played back through a set of small loudspeakers. Nasalance scores from two baselines and three wrap cover conditions were compared. While there was no statistically significant condition effect in a repeated-measures analysis of variance, the within-condition cumulative differences in nasalance scores were 2 for the initial baseline, 42 for wrap cover 1, 24 for wrap cover 2, 78 for wrap cover 3, and 8 for the final baseline. Mean differences between the wrap cover and the baseline conditions were 8.2 to 15.3 times larger, and cumulative differences were 8.3 to 16.6 times larger than between the two baselines. Based on the higher cumulative and mean differences observed, clinicians should not cover Nasometer microphones with household wrap as this increases variability of nasalance scores. Since there is evidence that the COVID-19 virus can survive for some time on metal surfaces, clinicians should be mindful of the fact that the Nasometer microphone housings can only be cleaned superficially and should be handled with gloves to minimize any possible risk of touch transfer of pathogens to the next speaker or the clinician. The Nasometer  is a popular instrument for the quantitative acoustic assessment of nasality disorders in different clinical populations, including individuals with cleft palate. To assess whether too much or too little sound is emanating from a speaker's nose, a sound separation plate is positioned on the speaker's prolabium, and microphones on the top and bottom of the baffle plate are used to measure sound pressure levels SPLs; . The nasAccording to the widely used Spaulding classification system , the NasIn light of the currently ongoing coronavirus disease 2019 (COVID-19) global pandemic, clinicians have wondered about the infection control procedures for the Nasometer. A hygiene practice that can be used in addition to cleaning and disinfection is physical shielding with a barrier material such as plastic wrap . CommonlAny barrier material between a sound source and a microphone membrane will affect the microphone's response, for example when a foam cover is used to reduce wind noise or air pressure spikes from plosive consonants. Recent research on the effect of face masks on speech has demonstrated acoustic dampening effects especially on higher frequencies. The research was explorative and not guided by a specific hypothesis. Differences between nasalance scores between covered and uncovered conditions as well as variability of nasalance scores served as outcome measures.In the present study, the Nasometer was tested with single layers of Glad Press ‘n Seal rolled polyvinyl chloride household wrap  over the microphones. Press ‘n Seal is textured on one side and has a light adhesive applied to the other side. The combination of the adhesive and the textured surface makes the material stick to itself as well as to the items to be wrapped. The material is air- and watertight. Among the range of readily available household wraps, it appeared to be the most obvious choice and the most convenient material to wrap the Nasometer headphones for infection control purposes.To create test recordings, a Nasometer 6450 headset was connected to a Tascam DR-05 digital recorder . Two typical adult speakers  were recorded producing two repetitions each of the non-nasal sentences “Look at this book with us. It's a story about a zoo,” the nasal sentence “Mama made some lemon jam” , and theA Nasometer 6450 was used for the experiment. The Nasometer software was run on a Dell Vostro 1000 laptop . A holder for the Nasometer sound separation plate was made from Sculpey molding clay . The Nasometer separation plate was positioned vertically in the holder, and the two loudspeakers were positioned so that the centres of the loudspeaker membranes were 3 cm away from the nasal and oral microphones, respectively . When thThe Nasometer was calibrated, and five sets of recordings were made. In the first baseline, each of the 12 test stimuli were recorded 3 times, so that 36 nasalance scores were obtained. The Nasometer plate was then removed from the holder and the two microphones were each covered with a single piece (8 cm by 8 cm) of household wrap as shown in Outcome measures were mean nasalance scores and absolute differences between repetitions and different conditions. The 72 nasalance scores from the two baselines and the 108 nasalance scores from the three recordings with the wrap-covered microphones were analyzed with a repeated-measures analysis of variance. Differences between repetitions and conditions were analyzed descriptively.F = 3033.72; P < 0.01). There was no significant effect for condition. There were significant differences for the 3 different speech stimuli . Bonferroni post-hoc tests showed that all 3 stimuli differed significantly from each other, with the highest nasalance scores for the nasal sentence and the lowest for the non-nasal sentences .In each of the experimental conditions, each of the 12 sound files (2 repetitions of 3 sentences by 2 speakers) was played back through the loudspeakers and re-recorded through the Nasometer headset 3 times. To assess variability between these nasalance scores of the repeated stimuli, the absolute differences in nasalance between the repetitions of the same stimuli were added up. For example, one such contrast were the 3 repetitions of the first recording of the Zoo sentences spoken by the female speaker. The sums of the within-condition cumulative differences in nasalance scores were 2 for the initial baseline, 42 for wrap cover 1, 24 for wrap cover 2, 78 for wrap cover 3, and 8 for the final baseline.In a final step, the mean and the cumulative absolute differences between the mean nasalance scores for the three sets of stimuli  were calculated between the two baselines and the 3 wrap cover conditions. The present study investigated the effect that covering the Nasometer 6450 microphones with thin layers of household wrap had on nasalance scores. Since human speakers show variability in speech and in their nasality levels over time , the tesVisual inspection of the results in While the analysis of variance did not show a significant effect of condition, it would have been imprudent to conclude that the wrap covers did not affect the nasalance scores. The detrimental effect of the wrap covers could be appreciated more clearly from the additional descriptive statistical measures that illustrated the variability of the nasalance scores. The mean and cumulative absolute differences in nasalance between the three repeated recordings of each sound file showed numerically larger variability in nasalance in the wrap cover condition, and smaller variability in nasalance between the two baseline measures. The fact that there were still small numerical differences between the repetitions of the baseline recordings despite the stable recording setup was likely explained by the fact that nasalance scores are reported rounded to the nearest whole number, so minute fluctuations in SPL can result in different rounding solutions. The test-retest differences in the baseline conditions were smaller than what is typically observed in human speakers . The aveTaken together, the findings showed that wrapping the Nasometer microphone even with something as thin as a layer of household wrap caused marked fluctuations in nasalance scores, within and between wrap cover conditions. In general, SPL measurements at a close range can be affected by small changes in distance, background noise, or calibration of microphones, so In conclusion, clinicians should not cover Nasometer microphones with Glad Press n’ Seal kitchen wrap as this affects nasalance scores. It is likely that other household wrap products would cause similar distortions. Other materials such as the acoustic foam used for microphone covers may be more suitable but may not provide the level of infection control clinicians might hope to attain. In the present study, face mask material was not considered because it would be much more difficult to fit over the microphones, and materials, design, thickness and absorption properties may differ between different kinds of masks . Since t