Aim: December 2019 witnessed the emergence of a worldwide outbreak of a novel strain of coronavirus (CoV) termed SARS-CoV-2. Several preventive strategies are being developed, such as vaccines, to stop the spread of infection. Materials & methods: A comprehensive immunoinformatics approach was used to map conserved peptide sequences on the receptor binding domain of SARS-CoV-2 for their B-cell, T-helper & T-cytotoxic cell epitope profiles. Results & conclusion: The antigenic B-cell epitopes were LFRKSN and SYGFQPT. Among T-cell epitopes, CVADYSVLY and FTNVYADSF exhibited affinity for MHC class I, while YRLFRKSNL and VYAWNRKRI exhibited affinity for of MHC class II alleles. The overlapping epitope between B- and T-cells was YRLFRKSNL. The deployment of these epitopes in potential vaccine development against COVID-19 may help in slowing down the SARS-CoV-2 spread. The COVID-19 pandemic ,3. BeforReceptor-binding protein has been previously targeted for vaccinable solutions ,19. Howeo) as per various estimates is around 1.1–5.5 [Numerous studies have reported bats as the primary reservoirs of SARS and MERS viruses ,26–29; h 1.1–5.5 ,42. SincVaccinable peptide sequence for epitope based vaccine in case of alphaviruses, hepatitis B and C, HIV, HPV, and influenza viruses for recognition of potent immunogens has given propitious results . SeveralThis study reports key findings vis-a-vis SARS-CoV-2 RBD for its variable and conservative residues in comparison with BatCoV RaTG13 strain, which can be considered immunogenic epitopes for potential multi-epitope vaccine candidate for SARS-CoV-2 in the backdrop of its binding orientation.in silico analysis was performed  were retrieved from National Center for Biotechnology Information. The sequences reported from China, Australia, USA, Taiwan, India, Pakistan, Nepal, Italy, Sweden, Brazil, Vietnam, Spain, Colombia, Peru and Japan were selected for analysis. The sequence of S glycoprotein from BatCoV RaTG13 (GISAID accession no. EPI_ISL_402131) and SARS CoV ZJ02 (Accession No. ABB29898) were used as reference for comparison. Sequence analysis was performed to ascertain the changes in the RBD of S glycoprotein. Multiple sequence alignments were performed using Clustal X. The consensus sequence of SARS-CoV-2 was used as input for epitope prediction.For identification of B-cell epitopes, Immune Epitope Database (IEDB) and BepiPred-2.0 were used ,59. The For vaccine development, cytotoxic T-lymphocyte (CTL) epitopes play an important role. Hence the T-cell epitopes were identified that have the ability to bind with MHC class I (MHC-I) and class II (MHC-II). CTL epitopes were identified through NetCTL 1.2 server. It is based on artificial neural network and trained on different human MHC alleles for prediction of epitopes . The IEDB- and T-cell epitopes were further scrutinized for their enzymatic digestion, toxicity, hydrophobicity and physiochemical properties. The digestion of peptides with enzymes is an important parameter in vaccine development as the peptides that are digested by many enzymes are usually rendered unstable. Hence, the digestion of peptides by different enzymes was predicted through protein-digest server. AntiangioPred was used to predict the mutational variability and other physicochemical properties of peptides. It is based on machine learning model that is generated on the basis of already reported anti-angiogenic peptide . ClanTOXTo avert autoimmunity, vaccine contenders were screened for human and nonhuman homologues. The nonhuman homologues were identified by scrutinizing selected epitopes sharing <30% identity with human proteome, via BLASTp analysis.HLA-A*0101 was downloaded from Protein Databank . Same criterion was followed for MHC-II alleles and for that the crystal structure of HLA-DRB5*01:01 (PDB ID: 1FV1: resolution = 1.9 Å) was also downloaded from PDB. Both the structures after ligand removal underwent protonation followed by energy minimization by AMBER99 force field. In order to analyze the inhibitory potential of peptides the docking of MHC-I specific peptides was executed with the HLA-A*0101 alleles, while the MHC-II specific peptides were docked with HLA-DRB5_0101. The docking studies were accomplished with induced fit docking protocol implemented in molecular operating environment (MOE) version 2016.08. By using, triangle match as placement method with London-dG scoring and GBVI/WSA dG rescoring function, 50 poses of each peptide were generated. Interaction analysis was done using MOE and PyMOL v2.3.The peptides that showed affinity for maximum number of MHC-I and MHC-II alleles were selected for interaction analysis. The structure of peptides was modeled through PEPFOLD server  followedet al [After identification of vaccine epitopes, we further explored whether these vaccine epitopes harbor any important residues that could be involved in binding of SARS-CoV-2 with human ACE2 (hACE2) and BatCoV RaTG13 with bat ACE2 (bACE2). The interactions of SARS-CoV-2 with hACE2 were recently reported by Lan et al . The intet al . PROSA ret al . Qualitaet al . MolProbet al . ERRAT get al . The beset al . Analysiet al  and PyMOSupplementary Figure 1). The important changes were observed at position 439 (Lys→Asn), 440 (His→Asn), 441 (Ile→Leu), 443 (Thr→Lys), 445 , 449 (Phe→Tyr), 459 (Ala→Ser), 478 (Lys→Thr), 483 , 484 (Thr→Glu), 486 (Leu→Phe), 490 (Tyr→Phe), 492 (Ile→Leu), 493 (Tyr→Gln), 494 (Arg→Ser), 498 (Tyr→Gln), 501 (Asp→Asn) and 505 (His→Tyr). The changes at positions 441, 486, 492, 493, 498 and 505 may not have any obvious effect on binding due to similar properties of amino acids.RBD of SARS-CoV-2 is 192 amino acids long (within position 330–522 amino acids) lying in S1 region of S glycoprotein. When comparing the receptor binding motif with the BatCoV RaTG13 there was variation between the two virus strains . The next peptide showing the binding with maximum number of alleles was FTNVYADSF.The default setting in the NetCTL server was used to predict T-cell epitopes. On the basis of highest combinatorial scores, five epitopes (with NetCTL score = 1.1–2.5 nM) were opted for subsequent analysis . On the DRB1_0103, DRB1_0701, DRB1_0801, DRB1_0802, DRB1_1602, DRB4_0103, DRB1_1001, DRB1_1101, DRB1_1501, DRB4_0103 and DRB5_0101.A total of nine peptides were predicted which exhibited strong affinity for MHC-II alleles . Among tTo identify the best epitope for vaccine construction, different features of T-cell epitopes were determined . The ideHLA-A*0101 was selected as representative allele for interaction analysis. The docking of HLA-A with the top ranking peptide resulted in generation of 50 complexes for each peptide. The peptide 3 (CVADYSVLY) binds with HLA with binding energy of -11.3 KJ/mol, while a detailed interaction analysis revealed that peptide had H-bonding interactions with Asp-115, Arg-156, Tyr-99 and Asn-77 (Two peptides (CVADYSVLY and FTNVYADSF) with the maximum number of bindings with HLA alleles were selected for interaction analysis. PEPFOLD created five models for each peptide and the model with the best score was selected for further analysis. d Asn-77 A. Peptidd Asn-77 B.DRB5_0101. Peptide 5 (YRLFRKSNL) bound with the energy of -10.2 KJ/mol while peptide 6 (VYAWNRKRI) bound with the energy of -11.3 KJ/mol. The residues Asp-70, Asn-62, Arg-71 and Glu-55 of HLA-DRB5 were having H-bonding interactions with peptide 5 (Two of the peptides (YRLFRKSNL and VYAWNRKRI) that showed affinity with maximum number of MHC-II alleles were selected for interaction analysis. Docking of peptides with HLA-DRB5 depicted both the peptides to be binding with strong affinity. The peptide 6 and peptide 5 with maximum number of alleles were used to study affinity of these peptides with eptide 5 C. Interaeptide 5 D.Supplementary Table 1 shows the Z scores of all the seven clusters, size of each cluster, root-mean-square deviation (RMSD) from the overall lowest energy structure, and energy values of electrostatic, Van der Waals, and desolvation. The cluster with the best HADDOCK score (-178.9 ± 3.6) was further used for analysis. Detailed interaction analysis showed that 26 residues of bACE2 and nine residues of BatCoV RaTG13 were present at the interface. These residues were involved in 12 H-bonded, two salt bridges and 157 nonbonded contacts. The detailed interactions are shown in The interactions of BatCoV RaTG13 with bACE2 were done using HADDOCK. There were 155 different complexes of BatCoV RaTG13:bACE2 that were generated, which clustered into 12 groups. The important residues of BatCoV RaTG13 that were involved in interactions with bACE2 are Lys-417, Leu-455, Phe-456, Asn-487, Tyr-489, Asp-501 and His-505. The important conserved residues of BatCoV RaTG13 that involved in interactions and demonstrated overlapping with epitopes were Leu-455 and Phe-456. These residues lay within the B-cell epitope (455-LFRKSN-461) and T-cell epitope (453-YRLFRKSNL-461). The important interacting residues of BatCoV RaTG13 with bACE2 and a comparison with SARS CoV2:hACE2 are shown in Research on various CoV species earlier has been continually reported since the last one and a half decade after the emergence of SARS, for annotating signatures and virulence factors . Viral eIn case of SARS-CoV and MERS-CoV, S protein and specially S1 regions have been the prime focus in developing immune strategies against these strains . SimilarDRB5*0101 was used to study possible interactions of peptide with HLA-DR. In case of HLA-DRB5*0101 (PDB ID: 1FV1) the crystallized structure displayed interactions with Asp-9, Phe-12, Glu-55, Met-23, Ser-53, Asn-62, Asp-66, Asn-69, Asp-11, Tyr-13, Asp-70, Arg-71, Tyr-78 and Asn-82 [Current results from analysis of RBD of SARS-CoV-2 revealed that 423-YKLPDD-428, 455-LFRKSN-461 and 494-SYGFQPT-500 are the B-cell epitopes that are highly antigenic. The two epitopes lie in receptor binding motif and also muddled in interactions with hACE2 . The Leud Asn-82 . When wed Asn-82  as well in vitro and in vivo testing is required for the proposed vaccinable targets of SARS-CoV-2. Based on the study results, the predicted epitopes harbor attractive capability to be considered for ascertaining therapeutic potency. The identified epitopes from this study can further be investigated along with conserved region epitopes of S protein for molecular simulation studies, in construction of experimental vaccine constructs and for considering its potential as a peptide based vaccine.On the basis of different properties of these selected B- and T-cell epitopes, it can be observed that these epitopes were 100% conserved from the reported data, as predicted by conservation analysis. These peptides also did not exhibited homology with any human protein hence may not incite any autoimmunity. These peptides did not display any toxicity. The digesting enzyme data showed these peptides to be indigestible by a range of enzymes and hence are safer to use for vaccine development . The B- Cumulatively, the detailed interaction analysis of BatCoV RaTG13 has shown that hydrophobic and charged residues have been involved in binding with bACE2. Analogous pattern has been observed in binding of SARS-CoV-2 with hACE2 receptor . The impo estimates. As bats are considered primary hosts for CoV species, it will be interesting to scrutinize how bats evade viral entry as previous studies have identified bats evolving a mechanism for defying interferon pathway activation by the STING interferon pathway [Designing an effective vaccine against viral infection such as COVID-19 is tricky. On one hand, it has to be ensured that the vaccinable epitopes hold enough antigenic potential to mount a befitting yet specific immune response so as to rapidly clear the infection if the need arises; on the other hand, the host immune response should not be strong enough to trigger chronic inflammation which in case of COVID-19 might significantly deteriorate lung infection. Lung as an organ is highly sensitive to inflammatory changes initiated by a surge of cytokine response . Mutatio pathway .The current study proposed the identification of potential multiple epitopes for vaccine development against SARS-CoV-2. The potential vaccine epitopes have been rigorously screened for multiple HLA, B-Cell, CTL and helper T lymphocyte epitopes thus augmenting its capability in inducing both humoral and cellular immune responses. Furthermore, it can be co-opted with adjuvant treatment in further enhancing viral disease control. The epitopes were further screened and validated for 100% conservancy with nonoverlapping human proteome thus additionally reducing the chances of autoimmunogenic side effects. The molecular docking of epitopes with HLA alleles were further validated for their mode of binding patterns and analysis of binding energy affinities.The December of 2019 witnessed emergence of worldwide outbreak by a novel strain of coronavirus (CoV) termed SARS-CoV-2.There is no therapeutic or preventive strategy like vaccine developed so far to overcome infection.The receptor binding domain of SARS-Cov-2 for any potential vaccine epitopes were explored using immunoinformatics approach.The B-cell epitopes LFRKSN and SYGFQPT, were found to be highly antigenic. Among T-cell epitopes, the epitope CVADYSVLY and FTNVYADSF were antigenic and exhibited affinity for maximum number of MHC class I alleles. The T-cell epitopes YRLFRKSNL and VYAWNRKRI displayed affinity for maximum number of MHC class II alleles. The overlapping epitope among B- and T-cells was YRLFRKSNL.The epitopes were further screened and validated for 100% conservancy with nonoverlapping human proteome thus additionally reducing the chances of autoimmune side effects.The important conserved residues of BatCoV RaTG13, the Leu-455 and Phe-456 that have been involved in interactions, also demonstrated overlap with epitopes. These residues lay within the B-cell epitope (455-LFRKSN-461) and T-cell epitope (453-YRLFRKSNL-461).The deployment of these epitopes in potential vaccine against COVID-19 may help in sweeping the COVID-19 infectious spread.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Indica rice lines comprising of ~2.2 million single nucleotide polymorphisms. Low‐to‐intermediate RS phenotypic variations were identified in the rice diversity panel, resulting in novel associations of RS to several genes associated with amylopectin biosynthesis and degradation. Selected rice lines encoding superior alleles of SSIIa with medium RS and inferior alleles with low RS groups were subjected to detailed transcriptomic, metabolomic, non‐starch dietary fibre (DF), starch structural and textural attributes. The gene regulatory networks highlighted the importance of a protein phosphatase alongside multiple genes of starch metabolism. Metabolomics analyses resulted in the identification of several metabolite hubs  in the medium RS group. Among DF, mannose and galactose from the water‐insoluble fraction were found to be highly associated with low and medium RS lines, respectively. Starch structural analyses revealed that a moderate increase in RS is also linked to an elevation of amylose 1 and amylose 2 fractions. Although rice lines with medium RS content negatively affected textural and viscosity properties in comparison to low RS, the textural property of medium RS lines was in the same acceptable range as IR64, a rice mega variety popular in Asia.Resistant starch (RS) is the portion of starch that escapes gastrointestinal digestion and acts as a substrate for fermentation of probiotic bacteria in the gut. Aside from enhancing gut health, RS contributes to a lower glycemic index. A genome‐wide association study coupled with targeted gene association studies was conducted utilizing a diverse panel of 281 resequenced  The results of the present study are discussed in terms of the insights gained into the molecular physiological mechanisms of native RS formation in rice and its impact on texture, cooking and taste quality traits.indica (Oryza sativa L. indica) landraces and pre‐breeding lines in order to assess phenotypic plasticity of RS and to delineate its relationship with the in vitro glycemic index (GI), amylose content (AC) and gelatinization temperature (GT). We observed low to moderate phenotypic diversity for RS content ranging from 0.17% to ~3.37% while for GT the range was between 66.75 and 80.98 °C  > 7.67 (PGWAS) as the genome‐wide significance threshold after Bonferroni correction to identify a fine‐mapped genetic region of chromosome 6 at the interval 6.53–7.05 Mb containing at least 50 genes and delineating significant association with the RS and GT phenotypes. GWAS associations were assessed using the mean value of three replicates  and GT traits , while LD block 5 exhibited significant association with only GT phenotype only  was performed, by associating RS and GT phenotypes of 281 resequenced rice lines to 2,210 939 high‐quality single nucleotide polymorphisms (SNPs) using an efficient mixed model association expedited (EMMAX) model  present in block 1, and Phosphofructokinase (pfkB) present in block 2 showed significant associations with RS , three highly significant SNPs were identified with a superior haplotype (GGC) linked with moderate RS (mean of 1.31%) and higher GT content (mean of 77.1 °C), while inferior haplotype (ATT) explains lower RS (mean of 0.85%) and lower GT  and higher GT . Additional SNPs include snp_06_6752887 (T → G) located in exon 8  and snp_06_6752888 a non‐synonymous SNP variant in exon 8  linked with increased RS  and GT content  represent a mean of RS 1.28% and GT 77.0 °C, its alternative haplotype TCAATAGGG associated with mean of RS 0.84% and GT value of 71.72 °C. Analysis of the 3000 Rice Genome Project suggest that TCAATAGGG inferior haplotype is over represented in temperate japonica and tropical japonica populations  were conducted from the target genes identified from the fine‐mapped region of chromosome 6, which enabled us to further narrow down the candidates to the two most important genes present in the two consecutive LD blocks. e Figure b locateds Figure b. Withins Figure c,d. The s Figure d.SSIIa gene in influencing RS content in the diversity panel but it only explained a phenotypic variation of 15.88%. This indicates that the genomic basis of RS is more complex and hence cannot be attributed to a single gene alone. To further delineate the contribution of other minor quantitative trait nucleotide (QTNs) influencing RS content, we identified 100 starch biosynthesis genes based on publicly available functional annotation. Targeted associations were conducted for all the starch metabolism genes using the 310 diversity rice lines by linking the RS phenotype with genotype using identified SNPs. A total of 41 SNPs were found to be significantly associated (at P < 0.01) with RS (Table SSIIa (GGC haplotype) from LOC_Os06g12450 and SNPs found in loci LOC_Os06g12600 encoding pfkB (GTGTGTCAA haplotype) from the fine‐mapped regions of chromosome 6  found in LOC_Os06g04200 encoding the GBSSI or Wx1 genes on chromosome 6 explained moderate positive association . Of which, seven SNVs were found in the upstream promoter region, one SNV in the 5′ UTR variant, 15 SNVs in intronic region and one in a downstream gene variant (Table pfkB) from chromosome 1 and LOC_Os02g58480 (Sucrose Synthase) from chromosome 2 (Table SSIIa (GGC haplotype) and two alpha amylases (TG haplotype), the PVE explained for RS is 19.98%. These results suggest the importance of minor QTL effect contributing SNVs from starch biosynthesis and degradation controlling RS formation and accumulation.Association studies pointed to the importance of  6 Table . The addSSIIa (GGC) and pfkB (GTGTGTCAA) conferring medium RS content (3.30%–1.81% RS) with intermediate GI and high GT  or the heterozygous allele (GQ02212) exhibit lower RS content (0.60%–0.30%), lower GT and high GI. These contrasting moderate versus low RS groups were used to understand (i) the gene regulatory networks and metabolic pathways, (ii) the molecular physiological mechanisms related to the contribution of metabolites, non‐starch DF and starch structure; and, (iii) the impact of RS on various viscosity and textural properties. The stability of the RS phenotype was tested by growing the lines in independent years across dry and wet seasons. While the moderate RS lines tend to possess consistent increase in RS content across both wet  and dry seasons , the inferior lines showed lower RS content  and pyrophosphate–fructose‐6‐P phosphotransferase (PFP) , branching  and starch degradation  were found to be down‐regulated (log2 fold change) in MRS lines with a threshold P value < 0.01  remains higher in MRS lines compared to LRS lines with P value of <0.05 , EIN3‐like (EIL) and C2H2 transcription factors gene expression were down‐regulated in MRS lines  lines and six low resistant starch (LRS) lines from developing seeds at 16 days after fertilization (DAF). A total of 1982 differentially expressed genes (DEGs) were identified using limma package between the two groups Table . Of thes) Figure . Genes t) Figure . The gen1 Figure . The exp05 Table . The exp05 Table . Among ts Figure . Transcrs Figure .P value of ≤0.05, which resulted in the identification of 128 loci influencing RS content (Table PFK loci (LOC_Os05g10650) from glycolysis, transketolase (LOC_Os06g04270) from the oxidative pentose phosphate pathway, carbonic anhydrase (LOC_Os08g36630) associated with the TCA cycle  genes identified from starch metabolism, encodes 3 non‐synonymous SNVs with amino acid alteration linked with RS content  with nt Table . Severalle Table . In addile Table . Besidesnt Table . Among rAS Table . InteresAS Table .bHLH transcription factors  were found to have maximum interaction with various starch biosynthesis genes encoding for starch synthaseIIa (SSIIa), granule‐bound starch synthase (GBSS), sucrose synthase (RSUS2), ADP glucose pyrophosphorylase (OsAGPS2) and alpha amylases , here shown as red coloured nodes . These loci were LOC_Os01g60690 (4SNPs), LOC_Os02g21100 (3SNPs), LOC_Os06g12660 (4SNPs), LOC_Os06g43530 (1SNP) and LOC_Os07g47840 (1SNP) with unknown function. Among them, LOC_Os06g12660 was found to be present within the LD region of chromosome 6 of GWAS  led to the identification of seven distinct modules , P value |<0.01|  and water unextractable (WU) dietary fibre (DF) in the polished grains of the rice lines that belonged to the MRS and LRS groups Figure . The DF Molecular weight distribution (MWD) of total starch (TS) from whole grain and the RS fraction were analysed by size exclusion chromatography (SEC), which revealed the importance of both long and short glucan chains in conferring RS formation Figure a,b. MWD Pasting profiles of MRS and LRS analysed using a Rapid Viscosity Analyzer (RVA) provided an indirect measure of cooking quality and provided insights into the viscosity properties of the two groups. In this study, the peak viscosity (PV), trough viscosity (TV), final viscosity (FV) and breakdown showed no significant difference between the MRS and LRS lines Figure . This im. Comparing MRS textural attributes with IR64 a bench mark mega variety with superior grain quality that is widely preferred in Asia, did not reveal any observable differences for HRD, COH and SPR suggesting that a modified MRS content will be less likely affect consumer preference in South and Southeast Asia  is mechanically designed to simulate the first and second bites on a food sample as a two‐cycle, force‐versus‐distance compression programme. The texture profile of rice lines from MRS and LRS cooked grain analysed by TPA exhibited significant differences for the textural features such as hardness (HRD), adhesiveness (ADH) and gumminess (GUM) Figure . The MRSa Figure .2) except for one with very soft (1.0 kg/cm2) and two with medium (2.0–2.3 kg/cm2). Consumer sensory panel’s per cent acceptability ranged from 50.0% to 93.3%, mean overall rating of 2.2–3.6 and mean rating texture ranging from 2.0 to 4.1. The Pearson correlation analysis among resistant starch, cooked rice hardness and sensory properties suggested significant correlations for tenderness  and smoothness . All of them had soft Instron hardness , this would translate to 18 g of DF and in cooked grain DF reaches up to 27 g.Rice is considered to be a high GI food when eaten as white (polished) rice, and thus, profiling the composition of starch to maintain optimal glucose homeostasis is an important step to mitigate the current health challenges of non‐communicable diseases  of GBSSI in the japonica background  that were previously associated with amylopectin structure and GT in rice were validated  and more resistance to enzyme digestion, leading to enhanced RS content in Indica germplasm. In this study, targeted association studies revealed that RS formation is regulated by multiple QTNs in SSIIa with a PVE of 15.88% as well as several small effect novel alleles of GBSSI, two different PFK genes, sucrose synthase and alpha amylases, contribute to the cumulative effect explaining the PVE of 42.9%  to mimic intermediate chain amylose AM2, rendering the starch granule in these rice grains more resistant to enzymatic digestion. In support of this hypothesis, SSIIa has been characterized as being involved in elongating short‐chain to medium‐chain amylopectin  identified based on linkage disequilibrium mapping of the association peak on chromosome 6  and pfkB (LOC_Os01g66940) genes identified through targeted association analysis , thus reducing the concentration of fructose, a feedback inhibitor of sucrose synthase (SuSy), an enzyme involved in the synthesis of plant cellulose . While up to 90% of milled rice is starch, only trace amounts are present as NSPs, in the form of DF such as cellulose, hemicellulose and beta‐glucans  or diverted to nitrogen metabolism in glycolysis to produce amino acids  or polyamines , identified in the MRS metabolic network  had increased the hardness of cooked rice  from Megazyme . For further details, refer Appendix RS profile of the et al., et al.  was made using Pearson all pairwise correlation. Statistical analysis was carried out to check the variation among MRS and LRS lines in SEC, TPA, RVA, GI and GT comparison using Wilcoxon rank sum test at G.M. and R.A. conducted the GWAS and haplotype analyses. S.P. performed TGAS, gene and metabolic network analysis and measured amylose, amylopectin and texture parameters. V.B. standardized the RS measurement technique and J.J.A. conducted the RS measurements across the diversity panels. C.L., Y.B. and A.R.F. conducted the metabolite analysis. O.K. and A.L. measured DF content. M.V.R. and E.H.B. measured sensory parameters. N.S. conceptualized the work and supervised the Phd work of SP together with M.S.M. N.S. wrote the manuscript with contributions from S.P. All authors read and contributed to the revision of manuscript.The authors declare no conflicts of interest.Figure S1 Manhattan plots and Q–Q plot of the genome wide association studies on RS from individual replicates.Figure S2 Targeted association of genes underlying at the significant region of Chromosome6 showing RS phenotype distribution (bloxplot), gene structure and haplotype distribution in the 3K panel.Figure S3 Selected rice lines with associated contrasting haplotypes at the significant region of Chromosome 6. The box plots of resistant starch (%RS), glycemic index (GI) and gelatinization temperature(GT).Figure S4 Resistant starch content of 15 medium and low resistant starch lines from different seasons.Figure S5 Map Man pathway‐I depiction of differentially expressed genes (DEGs) in medium and low resistant lines.Figure S6 MapMan pathway‐II depiction of differentially expressed genes (DEGs) in medium and low resistant starch lines.Figure S7 Pearson all pairwise correlation of healthier and grain quality traits among 15 contrasting resistant starch lines.Figure S8 Cooking quality and textural attributes comparison of medium and low resistant starch lines with IR64.Figure S9 Pearson pair wise correlation of resistant starch and sensory properties among medium resistant starch (MRS) and low resistant starch lines (LRS) and its box plots.Figure S10 Schematic overview summarizing the overall methodology used in the study.Click here for additional data file.Table S1 Significant SNPs present at Chromosome 6 region based on single locus GWAS and targeted association. Table S2 9 medium resistant starch and 6 low resistant starch lines, microarray data from 16 days after fertilization (DAF) of rice grain.Table S3 Chain length distribution of rice starch in fraction and in total grain across medium resistant starch and low resistant starch lines.Table S4 Pasting profile of medium resistant starch and low resistant starch lines by Rapid Viscosity Analysis (RVA).Table S5 Pasting profile of medium resistant starch and low resistant starch lines by Texture Profile Analysis (TPA).Table S6 (a) The node and edge values of medium resistant starch lines (MRS) of Dietary fibre (DF) correlation network.Table S7 (a) The node and edge value of medium resistant starch lines (MRS) metabolite correlation network.Table S8 Differentially expressed genes between MRS and LRS at log fold ±1 and P value = <0.05 along with their annotation and normalized value.Table S9 Differentially expressed genes significantly associated with Resistant starch (RS) at P = <0.05.Table S10 The node and edge value of turquoise co‐expressed module network.Click here for additional data file.Appendix S1 Detailed supplementary method details.Click here for additional data file. Click here for additional data file.


Light microscopy (LM) is commonly used in the study of biological materials to determine the morphology of cells and tissues. The potential of this technique for studying the structure of food products is also recognized but less known. Especially rare are information regarding LM studies of the supramolecular structure of starch. The aim of the work was to fill this gap by providing data on the possibilities for application of LM in starch studies. It was shown that in spite of an enormous progress in the development of microscopic techniques, including both increase of resolution and improvement of image analysis methods, light microscopy still has a huge potential for starch studies. The advantage of LM over other microscopic techniques is the possibility of differentiating between amylose and amylopectin by iodine staining. That makes LM especially useful in the analysis of the process of gelatinization of starch, the extent of molecular dispersion of its macromolecules, and the changes in its structure caused by modification. Moreover, it can be particularly useful for studying the changes in the supramolecular structure of starch in a food product matrix, providing more information than scanning electron microscopy (SEM)–the most common technique used for these purposes. The history of the use of microscopy in starch research dates back to the early days of microscopy in XVII century and is closely related to the findings of Antonie van Leeuwenhoek who confirmed the presence of starch in various parts of plants . MoreoveThe topography of granules i.e., presence of pores, channels or non-starch compounds, may distinctly modify the physicochemical properties of starch with emphasis on its susceptibility to chemical or physical treatment. Atomic force microscopy (AFM) has been successfully used to probe surface structures, determine the surface properties of native and/or modified starches  and the interactions of polymers at the nano-structural level ,11,12. AThe combination of various microscopic and/or spectroscopic techniques allows scientists to analyse not only the structural alterations but also the changes in the chemical profile of the processed granules ,18. On tThe aim of this work was to fill this gap by providing data that regards the possibilities for application of LM in starch studies. The gelatinization process of the most popular commercial starches that differ in amylose content and in pasting viscosity profile was investigated. This made it possible to show that thickening capability is related to the extent of molecular dispersion of starch. Moreover, microstructure of the most important modified potato starches was studied . It was shown that the incorporation of hydrophilic and hydrophobic moieties into starch macromolecules affected not only the viscosity but also significantly changed the ability to complex iodine within amylose and amylopectin helixes. The application of light microscopy to the studies of cross-linked starch allowed to observe the effect of acids or the sterilisation process on the molecular dispersion of starch and the resulting applicability of different modified starches as texture forming agents for different food products.Commercial native potato starch  was the initially studied material. Commercial pregelatinised drum dried potato starch , oxidised potato starches of different degree of substitution (DS)  and native starches of other botanical origin i.e., tapioca super high grade (Thailand), wheat (Roquette), corn (Cargill) and waxy corn (Cargill) were also used in this study.All modification processes were performed using potato starch as a raw material. Starch extrusion was performed in an industrial scale twin screw extruder (custom made) operating at temperature of 135 °C and 80 rpm. Initial moisture content of starch subjected to modification was 13%. The extrudate was grinded and sieved through a 60 mesh.Acetylated starches were prepared in water suspension with acetic anhydride according ≥98%  to the procedure described in . CrossliPasting characteristics were analysed with a Brabender viscograph under the following conditions: measuring cartridge 0.07 Nm; heating/cooling rate 1.5 °C/min; thermostating 20 min. pH value of starch suspensions subjected to viscographic analyses were adjusted with citric acid. To record light microscopy images, native and modified starches (0.8 g) were suspended in water (10 mL), and depending on the preparation, the starch slurry was incubated or heated at an appropriate temperature for 15 min with vigorous stirring. The temperature conditions, at which the slurry was treated, were determined based on the pasting characteristics of each individual preparation analysed with Brabender viscograph. The temperatures corresponding to the changes in the Brabender profile of the analysed sample, were used for the specimen preparation, and they were given in the captions of the figures presented in the work. A drop of the heated sample was transferred onto glass slides, smeared, and, following cooling, stained with Lugol’s solution (standard procedure). The preparations were examined with an OLYMPUS BX60  microscope.Degree of substitution of food grade modified starches were determined according to JECFA recommendations . For catStarch in its native granular form has very limited applicability, as it is insoluble in cold water. Therefore, the process of its dissolution (gelatinisation or pasting) is a subject of keen interest of technologists. Standard methods for the study of the pasting process involve using viscographs, such as Brabender viscograph or Rapid Visco Analyser. They provide useful information regarding changes in viscosity of starch dispersion in water during heating and cooling. Quantitatively, the process of gelatinisation is studied with differential scanning calorimetry. This technique provides information on the temperature-dependent enthalpy changes that accompany the decay of the crystalline phase in starch granules . HoweverAt room temperature potato starch granules remained unchanged and were visualised in the micrographs as black spots A. This cLight microscopy makes it also possible to visualise the differences between starches of various botanical origin. As presented in Many methods of starch modification have been developed in order to enhance the positive attributes and eliminate the shortcomings of the native starches . The mosSignificant differences were visible between drum dried and extruded starches. Both of these starches reveal lower viscosity when compared to their not processed counterpart. However, this difference is more pronounced in extruded starch . LM pictThe reactivity of hydroxyl groups present in the structure of starch macromolecules creates the possibility for various chemical reactions which results in a vast range of modified starches. Despite the enormous variety of modified starches offered on the market, the applied chemical reactions can be classified to three types: oxidation, esterification, and etherification. The use of multi-functional reagents  enables cross-linking of starch macromolecules in the course of esterification or etherification. This creates almost limitless possibilities of precise tuning of the properties of modified starches to specific applications .Oxidation of starch can be performed with different agents, such as hydrogen peroxide, ozone or sodium periodate. Nonetheless, the process that employs sodium hypochlorite is of the greatest practical importance. Hypochlorite modified starches were initially used primarily as sizing agents . NeverthChanges in the starch pasting process and in the starch-water interactions that were related to the oxidation of starch were also reflected in the LM images . At the However, the most important observation coming from the LM studies of oxidised starch is probably the one concerning colour upon iodine staining. The colour was significantly altered in comparison to micrographs of native starch. This observation is important because of the popularity of iodine colorimetry for determination of amylose content . SubstitAcetylation of starch, like oxidation, has been known for over 150 years and still is of industrial importance. This reaction is applied either to obtain exclusively acetylated starches or dually modified acetylated crosslinked derivatives. Acetylation performed in anhydrous media makes it possible to achieve a very high degree of substitution, up to 2.9. For this reason, starches modified by this method have significantly changed morphology, decreased crystallinity, and are soluble in non-polar solvents . For appEven by the presence of less than one acetyl group per hundred anhydroglucose units, no more blue colour that corresponds to amylose-iodine complex could be observed A. This oCrosslinked starches are readily used in food production. A very slight change in the structure of starch macromolecules causes huge changes in the properties of this biopolymer. The degrees of substitution of food grade crosslinked starches are about an order of magnitude lower than those of monostarch derivatives . CrossliLow cross-linked distarch phosphate, the most popular variant of E1412 in food technology, revealed higher viscosity than native potato starch in pH = 5.5. Microscopically, it was manifested by the formation of a paste at the temperature of 90 °C A that waAcetylated distarch phosphate samples revealed gelatinisation patterns similar to their non-acetylated counterparts . When coStarch modified by the etherification reaction are primarily hydroxypropylstarch used both in food production and other industries as well as cationic starches designed for paper sizing. Hydroxypropylation results in a significant decrease in pasting temperature, time to attain peak viscosity accompanied by increased solubility, and paste clarity. Moreover, hydroxypropylated starches reveal excellent freeze-thaw stability as well as reduced retrogradation tendency which is important in food applications. SEM studies proved that hydroxypropylation results in deep changes in the morphology of starch granules, including the main alteration – formation of deep grooves in the central core region. The extent of these changes depends on the amount of the hydroxypropyl groups incorporated into starch macromolecules ,37,38. TTo obtain products with higher degrees of substitution, dry or semi dry reaction conditions are required. Starches subjected to such conditions not only change their morphology but also lose their crystal structure ,39. The Despite the great progress in microscopy, visual light microscopy remains a potentially useful technique for studying the structure and functionality of starch. While advanced microscopic techniques, such as AFM, CLSM or SEM, provide more detailed information regarding the morphology of the starch granule, LM can be successfully used to demonstrate the structural changes of starch in dispersed systems. With the use of LM, amylose can be easily distinguished from amylopectin by iodine staining. Moreover, the extent of molecular dispersion of starch in pastes as well as the changes in the polymer-solvent interaction caused by the substitution of hydroxyl groups with polar or non-polar moieties can also be successfully studied under LM. These features make the technique particularly useful for investigating the changes in the supramolecular structure of starch in the matrix of food products as they allow to obtain essential and detailed information.


Respiratory syncytial virus (RSV) is primarily associated with respiratory disorders globally. Despite the availability of information, there is still no competitive vaccine available for RSV. Therefore, the present study has been designed to develop a multiepitope-based subunit vaccine (MEV) using a reverse vaccinology approach to curb RSV infections. Briefly, two highly antigenic and conserved proteins of RSV (glycoprotein and fusion protein) were selected and potential epitopes of different categories (B-cell and T-cell) were identified from them. Eminently antigenic and overlapping epitopes, which demonstrated strong associations with their respective human leukocyte antigen (HLA) alleles and depicted collective ~70% coverage of the world’s populace, were shortlisted. Finally, 282 amino acids long MEV construct was established by connecting 13 major histocompatibility complex (MHC) class-I with two MHC class-II epitopes with appropriate adjuvant and linkers. Adjuvant and linkers were added to increase the immunogenic stimulation of the MEV. Developed MEV was stable, soluble, non-allergenic, non-toxic, flexible and highly antigenic. Furthermore, molecular docking and molecular dynamics (MD) simulations analyses were carried out. Results have shown a firm and robust binding affinity of MEV with human pathogenic toll-like receptor three (TLR3). The computationally mediated immune response of MEV demonstrated increased interferon-γ production, a significant abundance of immunoglobulin and activation of macrophages which are essential for immune-response against RSV. Moreover, MEV codons were optimized and in silico cloning was performed, to ensure its increased expression. These outcomes proposed that the MEV developed in this study will be a significant candidate against RSV to control and prevent RSV-related disorders if further investigated experimentally. Paramyxoviridae and has been categorized into the genus Orthopnemovirus  and F [GENBANK: BAA00105.1] proteins were retrieved from GENBANK in FASTA format sequence. VaxiJen v.2.0 was then used for checking antigenicity. According to the results, both proteins were significantly antigenic with values 0.5791 and 0.5173 respectively. Besides, secondary structure evaluation and other physicochemical properties such as stability profiling, half-life, theoretical pI, molecular weight, and aliphatic index were also analyzed . The 3D IEDB-AR v.2.22 consensus network servers were used to predict CTL epitopes, and the NetMHCIIpan 4.0 server was used to predict the HTL epitopes. ABCPred server was used for the prediction of linear epitopes of B-cells, and Ellipro server was used to determine conformational B-cell epitopes. The SVM hybrid algorithms excluding Motif were used to predict IFN-γ inducing potential of the predicted epitopes by the IFN epitope server. The criteria designed for selecting the best possible epitopes was that their conservation among the proteins should be 100%, should have the best binding affinity, they should not overlap with the human proteins, significantly antigenic/immunogenic and should not lie inside the post-translational-modification sites and glycosylation-sites of the particular proteins. According to all these specifications, some promising epitopes were recognized in the current study. Total 43 CTL epitopes (G 19 and F 24), 13 HTL epitopes (G 7 and F 6), 51 linear B-cell epitopes (G-20 and F 31) and 12 conformational B-cell epitopes (G 3 and F 9) were identified . To construct a subunit vaccine, the chosen epitopes should be 100% conserved, overlapping and antigenic. Therefore, a total of 15 (13 MHC I and 2 MHC II) overlapping, conserved and highly antigenic epitopes were selected and their binding affinity with their respective HLA alleles was determined utilizing the molecular docking approach . It was found that all selected epitopes  bind deeThe population coverage was collectively calculated for selected 13 CTL and 2 HTL epitopes with their relative HLA alleles. HLA alleles diversify according to world ethnic groups and areas. Therefore, it affects the development of an epitope-based vaccine. Analysis revealed combined coverage of ~70% of the world’s population for selected epitopes . The higAll 15 selected epitopes (G 8 and F 7) were further used to develop an MEV construct. An adjuvant (45 amino acid long β-defensin) was bound with the aid of the EAAAK linker at the start . EAAAK linker reduces connection with other protein areas with efficient detachment and increases stability ,89. The Homo sapiens proteome, and results showed MEV has no similarity  with any human protein. Next, allergenicity, antigenicity and toxicity of the vaccine construct were evaluated. Results described that MEV is highly antigenic (0.6027 at 0.5% threshold), non-allergenic and non-toxic. Furthermore, ProtParam was used to evaluate the physicochemical properties of the MEV construct. The theoretical PI and vaccine molecular weight were 9.48 kDa and 30537.22 kDa, respectively. The mean half-life of the construct was calculated as 30 h in vitro, >20 h in vivo and >10 h in yeast. The estimate of the grand-average-hydropathicity (GRAVY) was −0.122; the negative sign in the score shows the hydrophilic nature of the MEV. The above results indicated MEV as a potential vaccine candidate.First, Blastp analysis  was execTo analyze the secondary structure of MEV, PSIPRED had been used. According to the findings, 74 amino acids participated in the development of α-helix consisting of 26.24% of the overall sequence, for β-strands formation 25 amino acids which are 8.87% and for coils formation 102 amino acid which constitute 36.17% of the entire vaccine construction participated .https://servicesn.mbi.ucla.edu/PROCHECK/). The refined model score was 82.6087 in quality check analysis through ERRAT. These results show that the refined model is of good quality.To predict the tertiary structure of MEV, the CABS-fold server was used. The structure was refined by the GalaxyRefine2 server . The imphttp://biocomp.chem.uw.edu.pl/A3D2/) [In addition, the flexibility of the MEV structure was evaluated using CABS-flex 2.0 server with 50 cycles’ simulation at 1.4 °C temperature. In between the 10 final retrieved 3D structures, regions near to N-terminal depicted lesser fluctuation compared with the regions near the C-terminal A. Resultl/A3D2/)  was usedB-lymphocytes besides secreting cytokines, also produce antibodies, which in return provide humoral immunity . TherefoAn appropriate association between immune receptor molecules and the antigen molecule is essential to activate immune responsiveness. The HADDOCK v.2.2 server has thus been used to perform the docking of the MEV with human immune receptors TLR3. TLR3 can efficiently induce the immune response after virus recognition. The docking analysis showed the strong interactions between the MEV and TLR3. The binding score of TLR3-MEV was −151.20 kcal/mol. The statistics of docking are shown in TLR3 is shown in the dark orange-brown color, while the MEV is shown in the cyan color, in MD simulation is a popular method used to analyze the micro-interactions between the ligand/vaccine and protein/receptor structures ,94. To fΔGbinding of the TLR3-MEV complex is shown in ΔGbinding reveal that the formation of the TLR3-MEV complex is driven mainly by the electrostatic interaction energy (ΔEelec) and the van der Waals (ΔEvdW) interaction. ΔEelec with −3451.087 ± 187.883 kJ/mol energy has high contributions than the other energies, which is also consistent with the hydrogen bond interactions between the TLR3 and vaccine.To clarify the energetics of the binding of MEV and TLR3 quantitatively, we carried out the MM/PBSA calculations. The calculated The in silico mediated immune responses were adequate with real-life phenomena as shown in E. coli hosts. First, codons of MEV construct were adapted as per codon utilization of E. coli expression system. JCAT server was used to optimize the MEV codons according to E. coli (strain K12). The optimized MEV construct contained 858 nucleotides  had the highest antigenicity. Surface glycoproteins of RSV command the early phases of infection. G focuses on the ciliated cells of airways, and F makes the virion layer to join with a target cell membrane . There iA perfect multiepitope vaccine would include epitopes of both B- and T-cells for encouraging a complete system of immune reactions. Thus, both kinds of epitopes were forecasted from RSV proteins and overlapping epitopes were further chosen depending on their immunogenic characteristics . Many HLA binding-alleles were also considered for comprehensive performance. B-cells are key components of the adaptive immune system which produce antibodies against invading pathogens. Antibodies are becoming increasingly popular and useful therapeutic agent to treat infectious diseases ,101. HowConsequently, a vaccine construct was created utilizing the strongest epitopes with suitable linkers and adjuvant. The adjuvant β-defensin is a thoroughly basic 45 amino acids long peptide that behaves both as an antimicrobial-agent and as an immunomodulator . The adjThe designed vaccine construct was profoundly immunogenic, antigenic, non-toxic, and non-allergic which shows its efficacy in inducing vigorous immune reactions without causing unwanted responses. The designed vaccine contains 282 amino acid residues (30.57 kDa), where the adjuvant alone has a length of 45 residues. Interestingly, the MEV has three conformational and 21 linear B-cell epitopes overall in its domains. The final vaccine is moderately basic and can give a stable connection in the physiological pH range. The mean half-life of the construct was calculated as 30 h in vitro, >20 h in vivo and >0 h in yeast, which is consistent with already reported subunit vaccine studies ,107. FurTo effectively transport the candidate antigenic protein within the body, it should have a stable connection with immune receptors such as TLR3 . In the Hypothetically, a multiepitope vaccine having B- and T-cell epitopes should manage to activate both humoral and cellular immune reactions . Our vacE. coli K12 system. In silico restriction cloning was also performed using the pET30a (+) vector to synthesize possible candidate vaccines. For the ease of purification, that vector has both S- and His-tags as the fusion partners which are significant for easy purification [The foreign gene expression may vary inside the host cell genome because of the mRNA codon inconsistency, thus, codon optimization is essential for higher expression . Fortunafication . Furtherfication .This type of multiepitope-based subunit vaccines possesses remarkable qualities that give them an advantage over traditional vaccines such as: (a) it consists of B-cell, CTL and HTL epitopes, therefore it could produce both humoral and cellular immune reactions in the host; (b) it consists of epitopes focusing various HLAs, it can be identified by various T-cell receptors, thus can be effectual in a large population; (c) the risk of autoimmunity or other adverse effects is decreased as the protein sequences/epitopes which cover by human proteins and other undesirable proteins are eliminated; (d) different proteins may be targeted by a single vaccine because it consists of immunogenic areas of different proteins fused as a single peptide fragment, thus enhancing their efficacy; (e) these vaccines may provide durable immunity in hosts as they are additionally linked to an adjuvant; and (f) if these vaccines administrated through oral, intranasal or sublingual route, they can stimulate the mucosal immune response that result in stopping the pathogens before they get enter into the host body by producing defensive both B- and T-cells in mucosal and systematic environments ,114,115.RSV infection has been a mysterious issue for a decade and is now considered a worldwide health problem. As such, there is no effective vaccine available for the treatment of RSV infections or any eternal cure yet. Many antiviral medications have been studied but none have clearly demonstrated effective results against the infection. Reverse vaccinology and computational techniques have been used to build a multiepitope-based subunit vaccine that could activate humoral and cellular immune responses. The proposed MEV model, coupled with computational analysis and immuno-information data, could lead to the development of a potential vaccine against RSV infection. We assume our predicted vaccine model will exert a positive effect on curing RSV infection research. However, the current study is the result of an integrated vaccinomics approach. Therefore, further lab experiments are necessary to demonstrate the efficacy and safety of the designed MEV.


It is generally accepted that migrants are favorably self-selected for labor market skills such as higher schooling and greater cognitive capacity, which are highly correlated with early-life nutrition. However, the influence of early-life nutrition on later-life migration is understudied.z scores (HAZ) at 24 mo and subsequent international migration in a cohort of 2392 participants born between 1962 and 1977 in 4 rural villages in eastern Guatemala.The objective of this study was to examine prospectively the association between height-for-age Information on nutritional status and covariates was collected between 1969 and 1977 and migration status was determined as of 2017 (at ages 40–57 y). We used proportional hazards and logistic regression models to assess whether HAZ was associated with international migration, adjusting for early-life and adult characteristics.Between 1978 and 2017 there were 297 international migrants  during 99,212 person-y of follow-up. In pooled models that were adjusted for early-life characteristics, a 1-SD increase in HAZ was associated with a 19% increase in the risk of international migration . Further adjustment for village characteristics did not alter the estimate substantively , while additional adjustment for schooling attainment attenuated the estimate somewhat . In all models, effect sizes were stronger for men than for women.Our results indicate that early-life nutrition is positively associated with subsequent international migration. As of 2020, there were an estimated 272 million international migrants worldwide, a 24% increase since 2010 , a census was conducted in each village. As part of this work, the survey teams ascertained the current location  of each living participant in the original cohort. The approximate date of international migration and country of destination were obtained from the migrant's families and trusted intermediaries residing in the original study villages. For each participant, we created a variable, “international migrant,” that was given a value of 1 when the respondent was identified as having ever lived outside Guatemala, or 0 if not.n = 48); in participants identified as international migrants in 2002–2004 who did participate in the 1988–1989 follow-up, the age was imputed to the midpoint between 1988 and 2001 . In participants first identified as international migrants in 2017–2019, the age was imputed to the midpoint between 2002 and 2017 .The exact age at migration was known for 131 (44%) participants who migrated internationally. In the remaining 166 (56%) participants who were international migrants with unknown age at migration, we imputed age at migration as follows. In participants identified as international migrants in 2002–2004 who did not participate in the 1988–1989 follow-up, age at migration was imputed as the midpoint between 1978 and 2001  using the WHO growth standards (n = 960). The robustness of this approach has been assessed elsewhere  we used data collected in the 2017–2019 follow-up. Informal education data consists of literacy programs available in Guatemala for adults who wished to complete primary or secondary school.n = 181), resulting in 2211 participants who comprise our main analytical sample. In some analyses, we further excluded individuals who had died by 2017–2019 (n = 199) or were lost to follow-up (n = 102) resulting in 1910 participants  were excluded from the analysis . Last, because schooling is an important predictor of human capital that may have increased local labor market opportunities and economic resources, facilitating the occurrence of international migration, we additionally adjusted for the participant's completed years of schooling (model 4). We present pooled and sex-stratified results. Pooled models additionally controlled for sex.For covariates with missing values, we used multiple imputation techniques, which produce unbiased estimates under the assumption of missing at random. The proportion of missing information was 15.9% for HAZ at 24 mo, 25.5% for mother's height, 8.9% for mother's schooling, 14.3% for father's schooling, 2.1% for mother's age at childbirth, and 26.2% for attained schooling.In sensitivity analyses, we additionally modeled the association of linear growth and international migration using logistic regression models adjusting for the same set of covariates previously described and using multiple imputation techniques for missing covariates. The proportion of missing information was 13.9% for HAZ at 24 mo, 22.6% for mother's height, 5.4% for mother's schooling, 10.2% for father's schooling, 1.1% for maternal age at childbirth, and 18.6% for attained schooling.P < 0.05. All analyses were conducted using STATA 16.All statistical tests were 2-sided and considered statistically significant at Supplemental Table 1). Mean HAZ scores at 24 mo, socioeconomic status in childhood, and years of schooling were all higher among international migrants . Age at migration ranged from 14 to 46 y, and most international migration occurred to the United States  . The further adjustment for village characteristics resulted in an estimated 19% increase in the likelihood of international migration  and the estimate was attenuated with additional adjustment for schooling attainment   with international migration, adjusting for potential confounders.Our study findings show that better early-life nutrition is associated with international migration, and the association is attenuated when schooling attainment is included in the model. We did not examine explicitly the pathways through which early-life growth influences international migration, mostly because we lacked the data in migrants to undertake such an approach. However, our findings suggest that one of the pathways through which early-life growth associates with subsequent international migration may operate via schooling. Growth failure in early childhood is a marker of malnutrition and other early-life deprivations occurring during critical periods in brain development . ConsequOur study also shows that the association between HAZ and international migration was robust to confounding factors , which have already been widely identified as important predictors of international migration. We found that international migrants came from families with higher socioeconomic status in childhood than nonmigrants, which is well aligned with previous studies conducted in similar populations . MoreoveMoreover, our findings support the healthy migrant hypothesis, which is based on the notion that those who migrate are healthier than the population they leave behind . This hyThere are important social and political factors that help contextualize our study results. The INCAP  longitudinal study took place during a civil war that lasted almost 40 y (1960–1996). The civil war was mostly targeted toward Mayan populations who were subject to political persecution . The regThere are some limitations to this study. Age at migration was imputed in more than half of the international migrants (∼56%), potentially affecting the precision of estimates. However, estimates using logistic regression models (which do not require age at migration) were very similar to those obtained using Cox proportional hazard models. Also, there is a possibility of nondifferential misclassification of international migration, especially because study follow-ups were widely spread, and some instances of short-term migration could have been underidentified . However, given the close relationship that the research team has built with the study participants and the study villages in almost 50 y of collaboration, (which includes frequent updates of information of members of the cohort), we believe this factor had minimal impact on the estimates since it is unlikely that a case went unidentified. An additional limitation is that we did not control for unobserved characteristics in childhood and adolescence that could potentially be associated with childhood linear growth and are likely to have an influence on international migration.This study also has strengths. To our knowledge, our analysis is unique for 2 reasons. First, we used individual level rather than aggregate data. Second, we assessed information collected during infancy and early childhood, which distinguishes this study from many others that lack such information. In addition, the use of survival analysis coupled with multiple imputation techniques ensured efficient use of available data improving precision around the estimates without introducing bias.nxaa379_Supplemental_FileClick here for additional data file.


Terrestrial gamma ray flashes (TGFs) are a class of enigmatic electrical discharges in the Earth’s atmosphere. In this study, we analyze an unprecedentedly large dataset comprised of 2188 TGFs whose signatures were simultaneously measured using space- and ground-based detectors over a five-year period. The Gamma-ray Burst Monitor (GBM) on board the Fermi spacecraft provided the energetic radiation measurements. Radio frequency (RF) measurements were obtained from the Global Lightning Dataset (GLD360). Here we show the existence of two categories of TGFs − those that were accompanied by quasi-simultaneous electromagnetic pulses (EMPs) detected by the GLD360 and those without such simultaneous EMPs. We examined, for the first time, the dependence of the TGF-associated EMP-peak-amplitude on the horizontal offset distance between the Fermi spacecraft and the TGF source. TGFs detected by the GBM with sources at farther horizontal distances are expected to be intrinsically brighter and were found to be associated with EMPs having larger median peak-amplitudes. This provides independent evidence that the EMPs and TGFs are produced by the same phenomenon, rather than the EMPs being from “regular” lightning in TGF-producing thunderstorms. TGFs are one of the highest-energy (10–20 MeV or more) natural photon emissions on Earth. The mechanism for creating the causative high-energy electrons in the source region still remains unclear. Some models assume that charged particles are accelerated in the relatively-high electric field region concentrated at the tips of lightning leaders3, while other models consider the particle-acceleration to occur in large-scale electric fields in thunderclouds5. A leading hypothesis for TGF-production is the Relativistic Runaway Electron Avalanche (RREA)7 process followed by bremsstrahlung photon emission occurring in the presence of thundercloud electric fields. However, some important parameters of the processes leading to the production of TGFs, such as the spatial-scale of the electron-acceleration region and the source of the seed particles necessary for the RREA processes, as well as the current and charge-transfer characteristics of TGFs remain unknown. TGFs are typically detected worldwide by satellite-based instruments8, but several ground-based14 and two aircraft-based16 observations have been made. TGFs reported from ground-based observations are associated with  cloud-to-ground discharges effectively transporting negative charge to ground. The fact that TGFs are often accompanied by radio-frequency electromagnetic pulses (EMPs) was not known until very recently17. These accompanying relatively-high-amplitude EMPs have been measured by sensors operating in the low and very low frequency (VLF) ranges19. These EMPs are also geolocated by ground-based lightning locating systems (LLSs)26. Connaughton et al.21 found that the rate of association between Fermi GBM-TGFs and LLS-reported VLF EMPs depended strongly upon TGF durations and they interpreted EMPs that occurred almost simultaneously with TGF photon-count peak-times to be the VLF signatures of relativistic electrons and their resulting ionization. However, further evidence is needed to determine whether these EMPs are signatures of lightning that “triggers” the TGFs or whether they are RF signatures of the TGFs themselves. Also, not all TGFs are accompanied by EMPs that are detected by LLS. Note that LLSs are tuned to report EMPs from lightning having durations of about a microsecond to several hundred microseconds and estimated source-peak-currents of few kiloamperes or more27. The question remains as to why some TGFs are not accompanied by LLS-detectable EMPs. This could be related to the source-intensity, discharge-duration, frequencies of emission, radiation pattern (orientation), or location of the source region within thunderclouds of such TGFs being significantly different than TGFs whose accompanying-EMPs are reported by LLSs.Terrestrial gamma ray flashes or TGFs, a phenomenon discovered relatively recently by Fishman In this paper, we examine the characteristics of the two categories of TGFs: those for which associated EMPs were reported to occur simultaneously (within ±200 μs of a TGF’s start-time) and those for which associated EMPs were reported to occur non-simultaneously (in the 200 μs–3.5 ms time-window before or after TGFs). See also the Methods section for further discussion. EMPs and TGFs were reported, respectively, by a ground-based LLS  and a satellite-based detector (Fermi Gamma Ray Burst Monitor or Fermi-GBM). See the Methods section for details on how EMPs and TGFs were correlated. We examined an unprecedentedly large dataset of 3860 TGFs reported by the Fermi-GBM during five years 2013–2017) over its entire coverage region . The peak currents for 721 TGF-associated non-simultaneous EMPs ranged from 4 to 435 kA, with the median being 26 (±1) kA. As discussed in the Methods section, the GLD360-estimated peak current should be viewed as a quantity proportional to the distance-normalized peak-amplitude of the magnetic radiation field of EMPs, rather than the peak current of their sources. Therefore, the median peak-amplitude of TGF-associated EMPs is 3.2 times higher for simultaneous versus non-simultaneous EMPs.Figure We examined the dependence of the GLD360-estimated TGF-associated EMP-peak-current on the horizontal offset distance between the Fermi spacecraft-nadir and the TGF source-location determined by GLD360. From Fig. et al.30 show using observations and modeling that for relatively compact  cloud lightning flashes with channel lengths on the order of 100 m occurring 15 km above the Earth’s surface, the RF bipolar radiation EMP measured at ground is produced by a unipolar source current traveling over the relatively short channel length. Additionally, they show that since LLSs assume that the peak current is proportional to the peak field (which is accurate for cloud-to-ground lightning), they tend to significantly underestimate the peak current of compact intracloud discharges (CIDs), for which the peak of the RF radiation field is proportional to the peak of the time-derivative of the source current (dI/dt). If we assume that TGFs have a vertical extent of a few hundred meters and if runaway electrons and the resulting ionization are directly generating the TGF source current, then we can view the TGF EMP measured by LLS-sensors at distances of many tens to hundreds of kilometers as the radiation field due to this TGF source current. In such a scenario, the peak amplitude of this EMP would be proportional to dI/dt and the LLS-estimated peak current would be an underestimate of the real TGF source peak current. If the above assumptions about TGF phenomenology are correct, the median peak current of 82 kA for TGFs with simultaneous EMPs reported by GLD360 discussed in the previous section should be treated as an underestimate. Also, non-simultaneous EMPs could be viewed as lightning-produced pulses in TGF-producing thunderstorms which were reported by GLD360, rather than signatures of the TGFs themselves. The absence of a simultaneous EMP accompanying a TGF could be due to a significantly-non-vertical propagation path of the TGF source current. This would result in the production of a TGF-EMP radiation field signature that would not be suitable for detection by multiple GLD360 sensors (as is needed for EMP-geolocation) which measure changes in the vertical component of electric and horizontal  component of magnetic field.Nag For 1489 out of 1502 TGFs with simultaneous EMPs reported by GLD360, TGF-durations, hardness ratios, and photon counts could be estimated (see Methods section). Also, these parameters could be estimated for 672 out of 686 TGFs with non-simultaneous EMPs.From Fig. For TGFs with simultaneous EMPs, we see from Fig. Figure 4c shows that for TGFs with simultaneous EMPs, the total photon counts ranged from 8 to 67, with the median (±standard error) being 17 (±0.4). For TGFs with non-simultaneous EMPs the total photon counts ranged from 8 to 219, with the median being 22 (±0.8). 2 and 16% of the TGFs with accompanying simultaneous and non-simultaneous EMPs, respectively, had total photon counts greater than 40. So, TGFs with accompanying simultaneous EMPs have a significantly smaller median total-photon-count reported by the Fermi GBM than that for TGFs with non-simultaneous EMPs. For comparison, the median BGO-detector total-photon-count for 3821 TGFs reported in 2013–2017 by the Fermi-GBM (for which photon counts were available), regardless of whether they were accompanied by EMPs or not, was 19.In summary, from the above analysis we conclude that TGFs with accompanying simultaneous EMPs tend to have shorter durations, smaller number of photons with energies greater than 300 keV reported by the Fermi-GBM , and lower total photon counts. The impact on these parameters from instrumental and other effects  are discussed in the Methods section.32. Additionally, electromagnetic wave travel-time corrections and a propagation model are applied to fine-tune the geolocation, and the measured magnetic field peak-amplitudes of EMPs. The peak currents of EMP-sources are estimated from their magnetic field peaks, after distance-normalization (to 100 km) and compensation for propagation effects on the electromagnetic waves in the Earth-ionosphere waveguide, using an empirical field-to-current-conversion equation appropriate for negative cloud-to-ground lightning subsequent return strokes32. The median location error and peak current estimation error for GLD360 are 2 km and 27%, respectively33. For a TGF-associated EMP, the GLD360-estimated peak current can be viewed as a quantity proportional to the distance-normalized (to 100 km) peak-amplitude of the magnetic radiation field.The Global Lightning Dataset (GLD360) is obtained from a global LLS which has been in operation since September 2009, with data being made available to users since May 2011. The GLD360 employs magnetic field sensors sensitive to the VLF range, located around the world. EMPs measured by multiple sensors are geolocated using a combination of time-of-arrival and magnetic-direction-finding methods in conjunction with a lightning waveform feature recognition algorithm34. Particles with effective energy ranges of 8–1000 keV and 0.2–40 MeV are measured by the NaI and BGO detectors, respectively. The timing precision of the measurements is 2 μs and the absolute accuracy is several microseconds. The Fermi-GBM is capable of measuring TGFs within a horizontal distance of up to about 800 km from the spacecraft’s footprint35. After implementation of new ground-search algorithms in 2012, the TGF detection rate improved to about 800 events per year29. The Fermi-GBM TGF catalog provides the photon counts per 2 μs for each of the two BGO detectors, cumulative photon counts for the 12 NaI detectors per 2 μs (10 μs if the NaI detectors are saturated), spacecraft position, TGF start-time measured at the spacecraft altitude, and the duration of the discovery-bin. The discovery-bin is the time-window in the ground-search algorithm corresponding to the most significant joint Poisson probability of occurrence of an identified TGF35. The discovery-bin start-time is a reasonable approximation for the TGF photon flux start-time24.The GBM is an auxiliary instrument onboard the Fermi Gamma-ray Space Telescope comprising of 12 sodium iodide (NaI) and two bismuth germanate (BGO) detectors36. The BB algorithm identifies statistically significant events by dividing the data into piecewise blocks of time and finding the optimal boundaries for each time-block using non-parametric analysis methods. The total photon counts shown in Fig. 29. This effect is seen in Fig. The TGF-duration shown in Fig. 38, which occurs when two or more lower energy photons arrive at the detectors within 2 μs (or 10 μs) of each other and are reported as a single higher energy photon. For short-duration TGFs with high photon-counts this will lead to higher values of hardness ratios, since the GBM could overestimate the counts of higher energy photons due to pulse-pile up. Pulse-pile up would also cause the BGO-detector-reported photon counts to be underestimated for shorter duration TGFs. Since the median duration of TGFs with simultaneous EMPs is significantly shorter than that for TGFs without simultaneous EMPs, pulse pile-up can therefore result in overestimation of the hardness ratios and underestimation of the total photon counts more often for the former class of TGFs than for the latter.The hardness ratio shown in Fig. 41.Compton scattering can result in increased counts of lower energy photons as well as increased durations for TGFs occurring at farther distances from the detectors. Thus, for such TGFs, the hardness ratios would be underestimated. Compton scattering is expected to more significantly impact GBM NaI-detector measurements (with an energy-range lower-limit of about 8 keV) than those from the BGO detectors (with an energy-range lower limit of about 200 keV). The effect of Compton scattering on TGF spectral and temporal characteristics have been explored in detail in previous studiesIn our dataset, we found that, generally speaking, the median TGF duration, hardness ratio, and total photon count were all lower for TGFs with simultaneous EMPs than for those without such EMPs at the same horizontal distance ranges see Fig. . The onlIt remains to be seen, what the relative contributions to the observed differences in the Fermi GBM-derived characteristics of the two classes of TGFs see Fig.  are from24. The ±6.5-ms time-window was selected to ensure that after light travel-time correction was performed, any EMPs occurring within  ±3.5 ms of TGFs were not missed. EMPs were defined to occur simultaneously with a TGF if they occurred within ±200 μs of the TGF’s start-time. This definition has been used in previous GBM-TGF studies21. EMPs occurring outside this time-window were defined to be non-simultaneous with the respective TGF.To identify TGF-associated EMPs, we searched for GLD360-geolocated EMPs occurring within a ± 6.5-ms time-window around a GBM-reported TGF’s start-time. We assumed that the two-dimensional TGF source location was the same as that of the EMP that was closest in time to the TGF. Then, light travel-time correction was performed24. For 1545 simultaneous EMPs accompanying 1502 TGFs, the median absolute EMP-TGF time-interval was 55 μs. This is similar to the 50 μs EMP-TGF time interval reported by Mailyan et al.24 for TGF-simultaneous EMPs geolocated by the U.S. National Lightning Detection Network. 1244 (81%) of the 1545 simultaneous EMPs started after the TGF start-time. The median peak currents (orange dots in Fig. We examined the time intervals between the GLD360-reported pulse start-times and the GBM-reported TGF start-times for 2266 EMPs associated with 2188 TGFs. As can be seen from Fig.


CNS Neuroscience & Therapeutics provides a medium for rapid publication of original clinical, experimental, and translational research papers, timely reviews, and reports of novel findings of therapeutic relevance to the central nervous system. Its focus includes clinical pharmacology, drug development, and novel methodologies for drug evaluation in neurological and psychiatric diseases. We are pleased to announce that CNS Neuroscience & Therapeutics has become an Open‐Access Journal as of January 2019. This would allow wider dissemination of scientific knowledge and facilitate collaborative efforts toward advancing novel and solid research on the maintenance of brain homeostasis and repairing the aging and dysfunctional brain.The Over the past year, the Journal covered the latest advancements and future directions in clinical neuroscience with an emphasis on mitochondrial biology, cerebrovascular disease, brain stimulation, and microglia through four special issues. Specifically, the special issue on Mitochondria in Neurodegenerative Diseases highlights how modifiable factors like caloric restriction and exercise can influence mitochondria biogenesis and effective removal following damage, and how disruption of mitochondria quality control mechanisms can lead to neurodegenerative diseases like Parkinson's, Huntington's, and Alzheimer's disease.CNSNT will have a variety of special issues including Traumatic Brain Injury.In the coming year, We are delighted to invite proposals for Special Issues on focus topics with high current interest. Recognized experts are encouraged to organize and guest edit special issues with the journal.


Dear colleagues,th, 2020. He was 67 years old.We share with you the painful news of the death of Michel Brémont. Michel has succumbed to cancer on the night of November 10Michel was a researcher recognized for the quality and extent of his innovative work on infectious diseases of fish. He is known for his commitment to establish reverse genetics systems on fish RNA viruses and to develop original virus vectors and vaccine platforms. With his characteristic determination, he also invested in the creation of understanding and knowledge in the development of preventative solutions for fish farm species but also for mammals. His notoriety was international and we know that the news of his passing widely affects the community of his collaborators and colleagues in many countries.Among the Molecular Immunology and Virology (VIM) unit at INRAE, Michel was an inspiring leader and a flawless supporter for his close associates. He had a sense of the collective and a concern for others.He passed on his expertise, his values, and his tenacity to young scientists as the former leader of the Fish Molecular Virology team. Beyond the VIM unit, for his colleagues in the INRAE Animal Health Division, he remains a “character”, the symbolic figure of researcher honesty, passion, stood up for his convictions with vigor (and sometimes of bad mood) who has guided many of us in their careers.With his extensive and eclectic scientific knowledge, his determination to lead his projects, his motivation to give and to shape and his candid way of speaking, Michel reached success with each mission that makes up the range of jobs of Researcher.Among his exemplary involvement for the scientific community, the responsibility of Editor-In-Chief of the journal “Veterinary Research”, assumed a unique dimension. With clear-sighted strategy, Michel learned to guide the journal to be a beacon of the international infectious disease community in his move to go to “open access”, the change of his economic model expanded his renown and his readership. This important community of readers is totally in his debt.On this sad day of the news of the passing of Michel Brémont, our compassionate and kind thoughts go to his loved ones, particularly to his children: Adrien, Antoine, Camille, and Sarah.We keep the faithful and inspiring memory of Michel as a man of passion and huge generosity, a lively rare power of conviction always tinted with humor. The beautiful picture that we attached to this message, which was transmitted to us by his children, perfectly illustrates his generous and warm personality.


T1s, C2-7 lordosis, and C2-7 sagittal vertical axis (SVA) are the three most important sagittal parameters in the cervical spine. This study was conducted to investigate the relationships between classical sagittal alignment parameters and a new parameter, K-line tilt, and to study the impact of K-line tilt.A total of 72 patients who underwent adjacent two-level anterior cervical discectomy and fusion (ACDF) were retrospectively analyzed. Radiographic measurements included T1 slope (T1s), C2-7 lordosis, segment angle (SA), C2-7 SVA, and K-line tilt. The Neck Disability Index (NDI) scores were used to evaluate the clinical prognosis. Pearson correlation coefficients were calculated between radiographic measures. Linear regression analysis was used to analyze the relationship between follow-up K-line tilt and NDI.r = 0.620, p < 0.05), ΔC2-7 SVA , and ΔK-line tilt ; ΔK-line tilt was positively correlated with ΔT1s , ΔSA , and ΔC2-7 SVA ; ΔT1s was positively correlated with ΔC2-7 lordosis , ΔSA , and ΔC2-7 SVA ; ΔC2-7 lordosis was positively correlated with ΔSA  and negatively correlated with ΔC2-7 SVA . The preoperative and postoperative K-line tilt values were statistically significant (p < 0.01), increasing from (7.50 ± 6.48)° to (9.95 ± 5.09)°. Preoperative NDI was positively correlated with preoperative C2-7 SVA  and K-line tilt ; follow-up NDI was positively correlated with follow-up T1s , C2-7 SVA , and K-line tilt . The linear regression model showed that when the follow-up K-lint tilt was > 23.75°, NDI scores were > 25 .ΔNDI was positively correlated with ΔT1s (This study showed that the K-line tilt was strongly correlated with the C2-C7 SVA, indicating that the K-line tilt can be used as another cervical parameter to evaluate cervical alignment in adjacent two-level ACDF. K-line tilt is an important parameter similar to the classical parameter C2–C7 SVA. In particular, a K-line tilt greater than 23.75 corresponded to a worse clinical prognosis, which was defined as an NDI score greater than 25. Many studies have indicated that the sagittal balance of the cervical spine may play an important role in clinical prognosis –4. AdditKwon et al.  noted thThe K-line was first described by Fujiyoshi et al.  as a linp = 0.000) : tt test was used to evaluate the influence of the lateral position of the lower cervical spine on the sagittal position of the cervical spine. A value of p < 0.05 was defined as statistically significant.SPSS 20.0 software  was used for all statistical analyses. Measurement data are shown as ‾x ± s. The Pearson correlation coefficient was calculated for between-time-point changes in X-ray measurements. Linear regression analysis was used to analyze the relationship between sagittal parameters in the cervical spine and NDI scores. A paired Overall, we reviewed clinical data from 75 patients, 1 of whom was excluded because of traumatic injury and 2 of whom were excluded because T1 could not be measured. A total of 72 patients (M/F = 46/26) were included, and their mean age was 52.6 ± 5.2  years. The following segments were involved: C3–5 (12 cases), C4–6 (39 cases), and C5–7 (21 cases). The average follow-up period for which postoperative radiographic measurements and HRQOL scores were obtained was 25.8 ± 7.6 months.Table r = 0.620, p < 0.05), ΔC2-7 SVA  and ΔK-line tilt ; ΔK-line tilt was positively correlated with ΔT1s , ΔSA , and ΔC2-7 SVA ; ΔT1s was positively correlated with ΔC2-7 lordosis , ΔSA , and ΔC2-7 SVA ; and ΔC2-7 lordosis was positively correlated with ΔSA  and negatively correlated with ΔC2-7 SVA   and K-line tilt , and follow-up NDI scores were positively correlated with follow-up T1s , C2-7 SVA , and K-line tilt . The linear regression model showed that when the follow-up K-line tilt was > 23.75°, NDI scores were > 25   and K-line tilt (p = 0.000), but by analyzing the correlations among Δ values  can better evaluate sagittal balance. Furthermore, the T1s is not easy to measure because the thoracic spine is difficult to correctly identify on a simple lateral X-ray film due to anatomical interference from the shoulder contour density, especially in obese people with thick thoraces . Althougr = 0.832 vs 0.756), and K-line tilt was positively correlated with C2–7 SVA  and T1s . During the operation, the angle is more intuitive than the length because there is no need to consider the effect of scale. Therefore, we believe that K-line tilt may provide a more advantageous assessment method. Linear regression analysis was used to analyze the relationship between K-line tilt and NDI scores, and the results indicated that when the follow-up K-line tilt was > 23.75°, the NDI scores were > 25 . In this regard, we believe that if the patient’s K-line tilt is larger before surgery, it may be considered to increase the size of the cage  during surgery and to correct the regional lordosis, which will yield a better prognosis for the patient.Of the indicators evaluated in our study, we found that both K-line tilt and C2–7 SVA were substantially correlated with NDI scores  resulted in little change in sagittal parameters before and after surgery. There were more patients with two-level ACDF in our hospital. In principle, if there are sufficient segments to perform multi-segment ACDF, this approach is best. In this study, we only had one experienced doctor to measure the data, which inevitably leads to errors. We hope that a follow-up study will enhance and verify the value of this article by increasing the sample size and improving scoring standards.This study showed that the K-line tilt was strongly correlated with the C2–C7 SVA, indicating that the K-line tilt could be used as another cervical parameter to evaluate cervical alignment in adjacent two-level ACDF. The K-line tilt was also an important parameter, similar to the classical parameter C2–C7 SVA. In particular, a K-line tilt greater than 23.75 corresponded to a worse clinical prognosis, which was defined as an NDI score greater than 25.


The aim of this study was to compare the clinical properties of tooth-colored computer-aided design/computer-aided manufacturing (CAD/CAM) materials for the fabrication of a 3-unit fixed dental prostheses (FDPs) in the same clinical scenario. A 53-year-old female patient was supplied with a 3-unit FDP to replace a second premolar in the upper jaw. Restorations were fabricated from 3 mol%, 4 mol%, and 5 mol% yttrium oxide zirconia, zirconia with translucency gradient, indirect composite resin, polyetheretherketone (PEEK), and polyetherketoneketone (PEKK). Milling time, weight, and radiopacity were investigated. Esthetics were examined following the US Public Health Service criteria (USPHS). The milling time for zirconia was twice as high as for the indirect composite resin, PEEK, or PEKK. The latter materials had a weight of 2 g each, while zirconia restorations yielded 5 g. Zirconia presented intense radiopacity. PEEK and PEKK required veneering and an opaquer was applied to the PEKK framework. All FDPs showed acceptable esthetics. PEEK and PEKK restorations were featured by a grayish shimmering. A variety of CAD/CAM materials are available to fabricate 3-unit FDPs with esthetically acceptable results. In the esthetic zone, PEEK and PEKK require veneering and an opaquer might be applied. Milling time, weight, and radiopacity were relatively high for zirconia FDPs. The conventional wisdom in restorative dentistry is that the esthetic appearance of dental restorations has gained increasing importance in the last decades, which is why the application of tooth-colored materials is highly favored by both patients and dentists. Recent surveys revealed that dentists often recommend ceramics, especially lithium disilicate and zirconia, for anterior and posterior single crowns ,2. HowevTooth-colored materials currently available on the market for the fabrication of 3-unit FPDs belong to different classes regarding their chemical structure and composition. It is therefore clear that their distinct properties may impact the individual esthetic appearance of the restoration. In addition, it has been highlighted that zirconia restorations may produce artifacts in X-ray examinations , which i3 have been reported for zirconia, while PAEK and composite resin materials feature far lower densities between 1.3 and 2.0 g/cm3 [The differences in chemical structure and composition of the materials available on the market account for differences in the individual material’s density. With regard to this aspect, densities of 6 g/cm.0 g/cm3 ,13,14. FAgainst this background, this clinical report illustrates the replacement of a second premolar by using a 3-unit fixed dental prosthesis fabricated from different restorative materials available on the market for this particular setting, comparing clinically relevant properties such as esthetics, radiopacity, and weight as well as parameters potentially relevant for the dental laboratory such as milling time. The working hypothesis was that there are no differences between the various materials and restorations regarding the properties investigated.A 53-year-old healthy female patient introduced herself at the Department of Prosthodontics and Materials Science at Leipzig University . The second premolar in the left upper jaw was missing. The patient history revealed that she was supplied with a dental implant that had to be explanted a year before the appointment, due to an inflammation. Therefore, she was looking for an alternative to replace the missing premolar.During clinical examination, healthy periodontal conditions were observed and a screening for temporomandibular disorders was negative. The patient denied being aware of bruxism, and during the clinical examination no signs of bruxism were identified. All teeth were naturally healthy or restored with sufficient restorations, except for a carious lesion in the left first molar of the upper jaw a,b.As the patient was not interested in any implant-supported restoration, the fabrication of a 3-unit FDP was proposed to replace the missing tooth. The patient agreed to the concept and gave her signed consent to be part of the clinical trial. For comparisons, 3 Y-TZP, 4 Y-TZP, and 5 Y-TZP supplied by the same manufacturer were chosen, and another zirconia with translucency gradient, one composite resin, one PEEK, and one PEKK material were selected .The abutment teeth were prepared according to preparation guidelines for ceramics a and an The tooth color was D3, which was determined by using the Vita classical shade guide . Working casts were prepared and digitalized with a laboratory scanner . Afterwards, the FDPs were designed  in accordance with the manufacturer’s instructions  and millThe FDPs fabricated from PEEK and PEKK materials were facially veneered with a composite resin using a conventional technique. For PEKK, an opaquer was priorly applied to mask the grayish framework.To keep tooth hard tissue removal to the necessary minimum and to better compare the original colors, the occlusal and oral sides of all FDPs were fabricated in a monolithic approach. Only PEEK and PEKK restorations were facially veneered. All materials were characterized with stains to imitate the adjacent teeth .The weight of the FDPs was measured . An X-ray was made  to evaluate radiopacity with gray values  of the dentine of a reference abutment tooth as well as three distinct locations of the individual restorations . An intraoral try-in of the FDPs was performed  and the patient was asked to choose her favorite FDP. Finally, all FDPs were evaluated for the parameters ‘anatomic form’, ‘marginal adaption’, and ‘color match’ according to USPHS guidelines  by an inThe approximated milling time for zirconia with translucency gradient was highest, while half the time was estimated to mill FDPs from PEEK, PEKK, and the indirect composite resin . The PEKZirconia restorations presented intense radiographic opacity with highest gray values of 255 in all points of interest , Table 2According to US Public Health Service (USPHS) criteria, the clinical evaluation of all FDPs revealed rankings of Alpha for the categories ‘anatomic form’ and ‘marginal adaption’ . From thRegarding the esthetic appearance of the 3-unit FDPs investigated in the current clinical report, the results suggest that a variety of CAD/CAM materials can be employed to supply patients with esthetically favorable 3-unit FDPs in the posterior area. However, PEEK and PEKK frameworks should be covered with an opaquer and require veneering with a composite resin or ceramic material to produce acceptable esthetic results. Moreover, the materials featured differences in their radiopacity, with the lowest radiopacity were identified for PAEK materials and highest radiopacity for zirconia. Regarding laboratory processing, approximated milling time was different between the various materials. Thus, the working hypothesis of this clinical report was rejected.This clinical report illustrates that acceptable esthetical results in the posterior area can be achieved with various formulation of zirconia. Composite resins yielded similar favorable esthetic outcomes, yet for PAEK materials, veneering seems necessary in the esthetic zone. Even if PAEK materials are available in colors that are described as whitish or universal by the manufacturer, these are not similar to those of natural teeth. However, for the veneering of PEEK with CAD/CAM composite resins or ceramics, similar colorimetric properties might be achieved than for zirconia or metal frameworks . BesidesRegarding cementation, it is possible that restorations fabricated from zirconia ceramics, PEEK, and PEKK are conventionally cemented. This might be beneficial in comparison to restorations fabricated from CAD/CAM composite resins that need to be adhesively cemented. Nonetheless, debonding of composite resins is still a frequently described complication; thus, the adhesive cementation technique should be processed with caution .The mechanical properties of CAD/CAM composite resins, PEEK, and PEKK are featured by a relatively low elastic modulus of 3 to 5 GPa. This value is close to that of dentine (15 GPa), which might yield a cushioning effect on tooth-supported restorations and might also be interesting in implant dentistry. Stress transferred to the abutment tooth or abutment/implant might be reduced in comparison to restorations fabricated from other materials such as zirconia with 200 GPa ,18. Due In X-ray examinations, margins of the FDPs fabricated from PAEK and composite resins were less radiopaque than dentine. Radiopacity of zirconia was most intense, which corroborates previous investigations . JudgingRegarding microbiological parameters, favorable results have been reported in previous investigations focusing on biofilm formation on any of the CAD/CAM materials investigated in this clinical report. For implant surfaces, zirconia showed a significant reduction in biofilm formation in comparison to titanium, while biofilm formation on PEEK was equal or lower than on zirconia or titanium ,21. LoweTo date, clinical investigations focusing on tooth-colored CAD/CAM materials for multi-unit FDPs are sparse. Only short-term investigations for monolithic zirconia are available, which revealed a survival rate of 96.7% after three years . The strZirconia, composite resins, PEEK, and PEKK were available to fabricate 3-unit FDPs that replace a second premolar. Milling time and weight of zirconia was twice as high as for the other investigated materials. Radiopacity of zirconia restorations was intense at restoration margins and the gray values reached 255. All fabricated restorations achieved acceptable esthetics; yet, PEEK and PEKK required veneering and an opaquer was additionally used to cover the PEKK framework. However, the observations should be supported by clinical trials that use validated instruments for the estimation of esthetical perception.