Zika virus (ZIKV) is an arbovirus from the Flaviviridae family and Flavivirus genus. Neurological events have been associated with ZIKV-infected individuals, such as Guillain-Barré syndrome, an autoimmune acute neuropathy that causes nerve demyelination and can induce paralysis. With the increase of ZIKV infection incidence in 2015, malformation and microcephaly cases in newborns have grown considerably, which suggested congenital transmission. Therefore, the development of an effective vaccine against ZIKV became an urgent need. Live attenuated vaccines present some theoretical risks for administration in pregnant women. Thus, we developed an in silico multiepitope vaccine against ZIKV. All structural and non-structural proteins were investigated using immunoinformatics tools designed for the prediction of CD4 + and CD8 + T cell epitopes. We selected 13 CD8 + and 12 CD4 + T cell epitopes considering parameters such as binding affinity to HLA class I and II molecules, promiscuity based on the number of different HLA alleles that bind to the epitopes, and immunogenicity. ZIKV Envelope protein domain III (EDIII) was added to the vaccine construct, creating a hybrid protein domain-multiepitope vaccine. Three high scoring continuous and two discontinuous B cell epitopes were found in EDIII. Aiming to increase the candidate vaccine antigenicity even further, we tested secondary and tertiary structures and physicochemical parameters of the vaccine conjugated to four different protein adjuvants: flagellin, 50S ribosomal protein L7/L12, heparin-binding hemagglutinin, or RS09 synthetic peptide. The addition of the flagellin adjuvant increased the vaccine's predicted antigenicity. In silico predictions revealed that the protein is a probable antigen, non-allergenic and predicted to be stable. The vaccine’s average population coverage is estimated to be 87.86%, which indicates it can be administered worldwide. Peripheral Blood Mononuclear Cells (PBMC) of individuals with previous ZIKV infection were tested for cytokine production in response to the pool of CD4 and CD8 ZIKV peptide selected. CD4 + and CD8 + T cells showed significant production of IFN-γ upon stimulation and IL-2 production was also detected by CD8 + T cells, which indicated the potential of our peptides to be recognized by specific T cells and induce immune response. In conclusion, we developed an in silico universal vaccine predicted to induce broad and high-coverage cellular and humoral immune responses against ZIKV, which can be a good candidate for posterior in vivo validation. Brazil is the country with the highest number of ZIKV infection cases reported in the Americas, with the first registered autochthonous case in 20152. The number of suspected and confirmed cases of autochthonous infections in Brazil reached more than 346 thousand people in the period of 2015 and March of 20173. Even though the death rate is low (< 0.003%)3, there were 14,558 suspected cases of microcephaly or congenital neurological malformations, from which 2,952 were confirmed4. Although the epidemic appeared to be waning after 2017, 31,576 cases were reported in the Americas in 2018, of which 3,589 were confirmed. In 2019, this number decreased to 12,079 reported cases and 3,482 confirmed cases1.Zika virus (ZIKV) infection has been documented in 87 countries globally (by July of 2019)5 that has increased global attention in recent years due to the severe symptoms in certain populations. Other forms of transmission include blood transfusion, sexual, intrauterine, perinatal and laboratory manipulation. ZIKV-infected individuals are generally asymptomatic but can manifest rash, body itching, fever, headache, myalgia and arthralgia3. Abortion6, neurological malformation including microcephaly and other anatomical defects in fetus have also been associated with pregnant women being infected with ZIKV in 5–30% of cases8; in addition, in rare cases, Guillain-Barré syndrome (GBS), a neurodegenerative disease, has been reported after ZIKV infection9.The ZIKV genome is composed by a positive-stranded RNA which codes a polyprotein that is subsequently cleaved into 3 structural proteins  and 7 non-structural proteins . ZIKV is a mosquito-borne flavivirus12. As this humoral response is dependent on CD4 + T cells, such cell population is also required for complete protection against ZIKV infection, specially Th1 cells15. This finding was corroborated by Hassert and colleagues, who observed that in absence of CD4 + T cells, there is an increased disease severity in mice and higher viral titer in the nervous tissues16. Another important cell population in the control of ZIKV is CD8 + T cells, as demonstrated when IFN receptor-competent mice lacking CD8+ T cells had increased susceptibility to ZIKV pathologies17 and transfer of CD8+ T cells from infected Ifnar1-/- mice to naïve mice lead to protection of the central nervous system after ZIKV challenge18. A screening of T cell epitopes revealed that the T cell response is directed mostly against structural proteins, such as prM and E, but also include epitopes from NS1, NS2A-B, NS3, NS4A-B and a high number of epitopes from NS55. CD4 + T cells have been observed against all 10 ZIKV proteins, although most are directed towards the capsid and envelope proteins. CD8 + T-cell responses were mostly directed to NS3, NS4B and NS5 proteins19. These findings suggest that the correlates of protection in ZIKV infection are mediated by the complex interplay between specific B and T cells, providing insights into new strategies to guide protective immune responses against the infection.Protection against ZIKV is greatly associated with neutralizing antibodies against structural proteins, such as the Envelope E, particularly against the domain III7, elderly20 and immunocompromised individuals21 deserve special attention. Several vaccine types against ZIKV are currently in phase I clinical trials, including DNA vaccines, mRNA-based, inactivated virus, peptide, and recombinant viral vectored vaccines22. However, hitherto only one DNA and one mRNA vaccine coding prM-E have succeeded to enter a phase 2 clinical trial, according to WHO vaccine pipeline tracker in April 2020 (https://www.who.int/). The most predominant form of antigens among them are the prM, E and NS1 proteins, but none of the listed vaccines are multiepitope vaccines or directed to other non-structural proteins24.Due to the risks related to the infection, including congenital malformations, prophylaxis against ZIKV infection is still an urgency, and the best answer is vaccinating people in risk areas, mostly susceptible populations. Pregnant women25. Multiepitope vaccines containing both B and T cells epitopes against Hepatitis C virus (HCV) have been tested in animal models and demonstrated increase in antibodies and specific T cell response26, and an HIV vaccine containing multiple CD8 + T cell epitopes has entered clinical trial phase I (NCT00076037)27. Furthermore, there are several multiepitope vaccines in clinical trials phase I and II for neoplasia, including melanoma , breast cancer , glioblastoma28, prostate  and ovarian cancer (NCT02764333) . Altogether, the safety profile and the success of multiepitope vaccines in entering clinical trials are encouraging features for a vaccine against ZIKV.Multiepitope vaccines are composed of individual or overlapping epitopes flanked by linkers  and have attracted scientific interest because they are a safer alternative for pregnant women and other individuals at risk as they contain only portions of the pathogen proteins, and therefore cannot cause infection. Furthermore, they are able to induce long-lasting humoral and cellular immunity, have the potential to include a repertoire of epitopes with potential to bind a high variety of Human Leukocyte Antigens (HLA) types, and therefore a high population coverageAiming to develop such vaccine, we performed extensive literature and bioinformatics analysis to select promising ZIKV protective epitopes derived from all structural and non-structural proteins, seeking promiscuous sequences that cover the highest number of type I and type II HLAs. We assembled a novel  in silico chimeric ZIKV vaccine (ZIKVac), composed of three main portions. At the N’ terminal, there is the ZIKV Envelope protein domain III (EDIII), followed by CD4 + T cell epitopes and finally by overlapping CD8 + T cell epitopes. The vaccine was evaluated with different protein adjuvants at the C’ terminal. Analysis of the biochemical and stability parameters were performed, as well as safety and immunogenicity indicators.Aiming the selection of conserved epitopes, a consensus sequence was generated from 40 complete Brazilian ZIKV sequences available on NCBI (GenBank access number on Supplementary 1). A Phylogenetic tree of all 40 complete sequences was generated, showing high conservancy between them  and GAAFGVMEALGT NS4A1-12) alone, for example, cover 22.5% and 30% of the available HLA, respectively, but together they cover 52.5%, an average coverage for a CD8 epitope. CD4 epitopes, on the other hand, presented greater HLA coverage and binding affinity in general. Also, 9 out of 12 CD4 peptides were shown to induce IFN-γ production  and IFN induction . However, these epitopes are important for the population coverage of our vaccine, so we performed additional analysis to make sure these epitopes would not bring negative outcomes for the whole vaccine immunogenicity. Therefore, we evaluated the capacity of the mentioned epitopes to induce IL-10 and IL-4, to activate APCs, and to induce proinflammatory response. None of these 4 epitopes were considered inducers of the immunoregulatory cytokines IL-10 and IL-4 and all of them were considered capable of activating APCs. Moreover, all the four epitopes were considered proinflammatory. These results are summarized on Supplementary 3.29; therefore, we used it to separate EDIII from the rest of the protein. GPGPG and AAY motifs were used to flank CD4 and CD8 epitopes, respectively. We evaluated seven designs (Supplementary 4) regarding their quality of tertiary structure, physical–chemical parameters and antigenicity. We opted to continue the analysis with the structure 6 because it presented the best antigenicity while being predicted to be stable and to have a high expected half-life (Supplementary 5). The EDIII tertiary structure of the final vaccine construct had a high overlap (RMSD 0.845 angstrom) with the EDIII from the crystal structure of ZIKV Envelope protein (PBD: 5JHM), suggesting that the addition of the CD4 + and CD8 + T cell epitopes to the protein structure did not impact the EDIII folding (Supplementary 6). We also added different adjuvants to the vaccine construct to perform our analysis. The final construct (ZIKVac) is shown in Fig. We rearranged CD4 + and CD8 + T cell epitopes, separated them by proper linkers in order to elect the best vaccine structure based on allergenicity, antigenicity and autoimmunity analyses, secondary and tertiary structure quality and discontinuous epitope scores. EDIII was added to the structure in order to induce B cell response. The DNTAN linker is a loop in protein (LiP) and is appropriate to separate BCR epitopes as there is a need of flexibility between epitopes so they can be recognized directly by B-cellsOnly Flagellin and HBH had sequences with more than six identical amino acids. Flagellin has eight identical amino acids to the human Myosin VI isoform 3, and HBH presents 8 identical amino acids to Histone H1.5. ZIKVac presented sequences with a maximum of only five identical amino acids compared to human proteins (Supplementary 8).ZIKVac alone and associated with each of the evaluated adjuvants  were considered non-allergenic by AllergenFP server Table . AntigenThe secondary structure as predicted by RaptorX reveals that ZIKVac is composed of 32% alpha helices, 26% beta-strands and 40% of coils .ZIKV infection was considered a public health concern in tropical countries, especially in South America due to a rapid growth in the number of microcephaly cases in Brazil caused by the flavivirus in 2015. Although the number of reported cases has waned in the last two years, it could increase anytime with the spread of the mosquito vector during rainy seasons34. The ZIKV EDIII has been used in experimental subunit vaccines, and it was shown to induce neutralizing antibodies against different strains of ZIKV, as well as significant reduction of viral replication when used in combination with alum and Monophosphoryl lipid A (MPL)35. Conversely, ZIKV EDIII failed to induce satisfactory inhibition of viral replication in mice when given in the form of DNA, protein or when expressed by a chimpanzee adenovirus36, indicating that the choice of adjuvant, for instance, can impact the efficacy of the vaccine. Interestingly, immunization with a specific fragment of EDIII (E298-409) was sufficient to protect BALB/c pups against lethal challenge with ZIKV strains R103451 and FLR37.For an effective humoral response, it is fundamental that the vaccine is capable of inducing neutralizing antibodies that lead to inhibition of viral replication, protect against the infection and disease. Flaviviruses structural proteins, especially the Envelope protein, are the main targets for antibody production 40. Similarly, ZIKV EDIII specific antibodies have also shown neutralizing activity, while domains I and II have presented either modest or none42. EDIII was therefore a promising choice for a vaccine construct. The inclusion of the ZIKV EDIII to the multiepitope vaccine structure is a novelty of our work.With that in mind, we chose ZIKV EDIII to cover our vaccine’s B cell response. EDIII from Dengue virus and other flaviviruses have been previously reported to induce neutralizing antibodiesMoreover, a chimeric protein-peptide structure may be more efficient in eliciting neutralizing antibodies than the peptides alone, due to the fact that it is more probable to find discontinuous epitopes in a protein structure, while peptides usually contain only linear epitopes. Our vaccine structure revealed ten discontinuous epitopes and fourteen continuous epitopes. Interestingly, the two discontinuous epitopes (1st and 3rd best discontinuous epitopes) with the best scores were located at EDIII.43. Salvador et al. (2019) found by IEDB NetMHCpan method epitopes for CD8 + (MMLELDPPF) and CD4 + T cells (QMAVDMQTLTPVGRL) that are present in the EDIII of our vaccine44. By utilizing different methods (NetCTL1.2 and Ellipro Protrusion index), Mirza and colleagues found 4 epitopes present in EDIII, of which 3 are CD8 epitopes , and 1 is a linear B cell epitope (LKGVSYSLCTAAFTFTKI)45. Furthermore, Cunha-Neto and collaborators explored a new mathematical model of structural entropy and found that KMMLELDPPF is a CD8 epitope, and interestingly this epitope is also part of EDIII46. Several epitopes derived from EDIII were found in previous reports to induce CD8 + and CD4 + T cell responses, demonstrating that the protective potentialities of ZIKVac can extrapolate our predictions47.Even though the main purpose of EDIII in our vaccine is to induce neutralizing antibodies, it can also be degraded by different types of proteasomes and/or by cellular enzymes and be presented to T cells after binding to proper MHC48, which suggests that a CD4 + T cell epitope-based vaccine may contribute to eliciting a cytokine inducing response. Apart from binding affinity and promiscuity, we also considered IFN-γ induction capability in order to select our vaccine’s CD4 + T cell peptides, since high levels of this cytokine during the acute phase of ZIKV infection appears to be related to viral clearance and mild disease49. Moreover, some of the selected CD4 + T cell epitopes were described previously as being also CD8 and B cell epitopes, and interestingly, AAGAWYVYV derived from NS2B, induced IFN-γ as detected by IFN-γ ELISPOT50.Regarding the cellular immune response of ZIKVac, we included 12 HLA class II peptides derived from all structural and non-structural proteins of ZIKV. CD4 + T cell differentiation into effector memory and terminally differentiated cells was observed in patients in the acute phase of ZIKV infection 50. CD4 + and CD8 + T cells proliferation assessment in mice infected with ZIKV induced a strong adaptive immune response, which helped prevent infection in the brain and testes. Moreover, in ZIKV-infected pregnant mice, CD8 + T cell responses were decreased in comparison with non-pregnant mice, which suggests that the cell-mediated immunity during pregnancy may contribute to preventing virus spread to the fetus51. In accordance with our study, Cunha-Neto et al. (2017) described the epitope QEGAVHTAL (from E protein) as potentially capable of inducing CD8 + T cell response by using structural entropy-based predictions46. Furthermore, some epitopes placed in the CD8 portion of ZIKVac  were found to induce IFN-γ response in vitro50. Due to the limited amount of CD8 epitopes and aiming to increase population coverage as much as possible, we opted to use overlapping CD8 peptides as it would make possible to include a higher number of epitopes in a shorter amino acid sequence; however, this resulted in some peptides longer than 11 amino acids. In these cases, we would expect that intracellular peptidases, such as Endoplasmic reticulum aminopeptidase 1 and 2 (ERAP1 and 2) would play a role in cleaving these epitopes in order to allow their coupling with the HLA type I binding groove43. Some of the epitopes we selected showed negative scores for some of the parameters we analyzed. However, we believe they will not bring negative outcomes to the vaccine immunogenicity, since none of them were considered inducers of the immunoregulatory cytokines IL-10 and IL-4. Prediction results showed these epitopes are also capable of activating APCs, which can be a booster for activation of innate immunity by our vaccine. Moreover, all the four epitopes were considered proinflammatory. This can indicate that even if they are not able to induce IFN, they can be inducers of other proinflammatory cytokines such as TNF-α.CD8 + T cell epitopes are critical components of our vaccine construct, since most studies support the idea that these cells help control infection through viral clearance. In mice with IFN receptor-competent T cells and dendritic cells, CD8 + T cells demonstrated a protective role during ZIKV infection56. Differently from our vaccine structure, some of them were not based on consensus sequences56. Alam et al., (2016) and Franco et al., (2017) developed vaccines based only on the Envelope protein and NS5 protein, respectively, and Pandey et al., (2018) focused on E, C prM and NS1 proteins54. Similar to our approach, in which we included at least one peptide from each ZIKV structural and non-structural protein, Prasasty et al., (2019) and Shahid et al., (2020) explored almost all the ZIKV proteins56. Prasasty et al., (2019) did not have the whole vaccine construct analyzed for parameters such as autoimmunity, allergenicity and stability, but only the peptides alone55. Our analysis showed that the whole protein assessment is important to determine the assembly viability, since addition, deletion or changes in the order of peptides disposition can affect vaccine’s efficacy and safety. Finally, our vaccine assembly is the only one that includes a protein domain, which we believe is an important differential feature and can be advantageous when it comes to the presence of discontinuous B cell epitopes and induction of a more robust and specific antibody response.Other in silico vaccine structures based on epitope prediction have been designed since the association of ZIKV with severe disease and fetus malformation55. One of our CD4 + T cell epitopes (HGPIRMVLAILAFLR) and two of our CD8 + T cell epitopes  are contained in CD4 + T cell epitopes selected by Pandey et al., (2018) . Similarly, three CD8 + T cell  and two CD4 + T cell epitopes  selected by Prasasty et al., (2019) are contained in five of our CD4 epitopes . The epitope YRIMLSVHG selected was also selected by Alam et al., (2016)52.Interestingly, some of our epitopes were also found by Pandey et al., (2018) and Prasasty et al., (2019), even though they used different methods than ours57. These proteins were chosen based on other studies that showed that they presented promising results as adjuvants. For instance, flagellin is a TLR5 agonist derived from Bacillus subtilis subsp. spizizenii strain W23 (GenBank access number: ADM39502.1) retrieved from NCBI. It has been previously shown that flagellin induced robust humoral response and pro-inflammatory cytokine production when administered with a whole inactivated influenza vaccine in mice58. Heparin-binding hemagglutinin (HBH) (UniProtKB—P9WIP9) is a TLR4 agonist derived from Mycobacterium tuberculosis H37Rv, while 50S ribosomal protein L7/L12 (UniProtKB—P9WHE3) is also a molecule derived from Mycobacterium tuberculosis H37Rv and is described as a TLR4 binder. When used as adjuvant for a therapeutic cancer vaccine, HBH favored Th1 polarization and dendritic cell (DC) function59. 50S ribosomal protein L7/L12 also enhanced Th1 polarization in a DC-based tumor immunotherapy60. Finally, RS09 (APPHALS) is a short synthetic LPS peptide mimics designed using Phage Display Libraries and described as capable of binding to TLR461. RS09 was previously used as adjuvant in an HIV-1 vaccine that showed promising results in mice62. Our autoimmunity analysis showed that the only portion of the vaccine that had more linear amino acids similarity with human proteins was present on the Flagellin adjuvant portion (MAKEMSEF). However, this peptide is not long enough to induce a CD4 type response and is not predicted to bind to any of the HLA class I molecules considered in this study (the sequence is too short to be assessed in respect to its CD4-inducing properties). Without the help of T-cells, it is not likely that this peptide will induce a B cell response63. Furthermore, flagellin has been used as adjuvant in vaccines against bacterial pathogens65, parasitic organisms67, cancer68 and virus infection, such as influenza69, WNV70, DENV71 and HIV72. This adjuvant was also the one that increased vaccine antigenicity score the most in relation to the other 3, indicating that it is a potent adjuvant, however safety experimental evaluation is necessary.Adjuvant use in vaccine constructs is important for immune response enhancement. We tested our vaccine with 4 different adjuvants in order to find one that could increase its antigenicity. We selected 4 TLR agonists as adjuvants because activation of these receptors leads to innate cell stimulation, cytokine production, receptor expression, dendritic cell maturation and subsequently T cell priming73. ZIKVac could also be used with aluminum, which is the most frequent adjuvant in licensed vaccines, showing a great safety record75. The combination of both adjuvants can lead to a more balanced Th1/Th2 response and resulted as the best adjuvant scheme when compared to either adjuvant alone or MF5935. Thus, we initially plan to express the proposed vaccine in eukaryotic cells and test it as a protein adjuvanted with alum and MPL in mice. However, it would also be particularly interesting to test it as a nanoparticle encapsulated mRNA or carried by a Hepatitis B core Antigen (HBcAg)-VLP since both strategies have been used for ZIKV vaccines and showed promising data76.Even though we presented our ZIKVac in combination with 4 protein TLR agonists, its potential application is not limited to them. ZIKVac could also be expressed or synthesized without the adjuvant domain and be administered with other types of adjuvants, such as MPL. MPL is a TLR4 agonist dependent on TRIF signaling that has been used for influenza vaccination in Brazil due to its not so aggressive immunogenicity in comparison to LPS and for reducing the dose of the antigen in vaccination regimens77, our vaccine’s peptides covered 94.9% of the population. The highest coverage was in South Asia (98.71%), and the lowest coverage was found in South Africa (18.36%). However, the coverage in North and Central Africa were 77.17% and 95,44%, respectively. In general, world coverage was 96.81%, indicating that these epitopes are promiscuous and might be recognized by various populations worldwide. This is important because the virus may spread to unexpected regions, and a universal vaccine would be crucial to stop any novel epidemics. Similarly, when we evaluated HLA coverage of our peptides using Propred1 and Propred server, we found that ZIKVac’s CD4 peptides covered 100% of the HLA molecules available in the server and CD8 epitopes covered 46 of the 47 available molecules. HLA-A*1101 (predominantly found among the Asian population) was the only molecule to which none of our peptides were predicted to bind. Experimental validation of predicted CD8 + T cell DENV and hepatitis B virus (HBV) epitopes for this molecule showed that the predicted and laboratory results did not match78. This could be explained by the fact that HLA molecules frequently observed among the Caucasian population (e.g. HLA-A*0201) have a larger amount of data available than Asian predominant HLA molecules, and the efficacy of epitope prediction algorithms depend directly on the training data available for each HLA molecule78.An ideal vaccine should have global coverage. In South America, where ZIKV infection has been more prevalent79. Similarly, Farrell et al., (2016) found that predicted promiscuous Mycobacterium bovis epitopes significantly induced IFN-γ secretion from infected cattle T cells when compared to epitopes with low promiscuity80. Moreover, Ribeiro et al., (2010) developed a DNA vaccine which encodes 18 HIV subtype B CD4 + T-cell epitopes81. The DNA vaccine induced robust CD4 + and CD8 + T cell responses in mice expressing different class II HLA alleles, and 16 of the 18 coded epitopes were recognized by PBMC from HIV infected patients. These studies suggest that in silico prediction approaches can guide the selection of conserved and antigenic epitopes that can be recognized by the target population, thereby reducing the time to develop effective vaccines. In order to test whether our peptides are capable of inducing immune response, we performed a preliminary in vitro assay and evaluated the production of cytokines after stimulation of PBMCs of individuals with previous ZIKV infection with a pool of the 25 ZIKV peptides. Both CD4 + and CD8 + T cells showed significant production of IFN-γ upon stimulation, confirming what was predicted using the IFN epitope prediction server. IL-2 production was also detected by CD8 + T cells. The results indicate that the ZIKV peptides of our in silico designed vaccine were presented to T cells in the course of natural ZIKV infection and generated memory responses that could be boosted by peptide stimulation in vitro leading to cytokine production by CD4 + and CD8 + T cells. These data support our epitope prediction strategy and reinforce the vaccinal potential of these peptides.Despite the limitations of prediction servers, various studies have been successful with experimental validation of predicted epitopes. Iwai et al., (2003) tested synthetic peptides of Paracoccidioidomycosis (PCM) gp43 protein selected using the TEPITOPE algorithm and found that peptide recognition by PCM patients correlated with the promiscuity of HLA-DR binding predictionZIKV infection is especially concerning in pregnant women, and more elaborated approaches are needed in order to attend this population. We designed a chimeric multidomain Zika vaccine intending to cover all the protective immune responses against ZIKV, targeting specific CD4 + , CD8 + T cells and B cells. This in silico approach gave us many promising hits. In vitro tests showed that our ZIKV peptides can be recognized by T cells and induce immune response. We intend on later synthesizing the whole vaccine structure to validate its potential in vivo as well.https://www.ncbi.nlm.nih.gov/) and downloaded in FASTA protein format. The sequences access numbers are listed on Supplementary 1. The alignment was done by the software ClustalX2.1, and the consensus sequence was obtained with Jalview (version 2.11). This sequence was later separated into single proteins, which were used as input in FASTA format for the selection of potential HLA type I and II immunogenic epitopes. After aligning the sequences with ClustalX, we built a phylogenetic tree using FigTree v1.4.4.All complete Brazilian ZIKV strains (by March of 2018) were obtained from NCBI (http://crdd.osdd.net/raghava/propred1/), IEDB Class I Immunogenicity (http://tools.iedb.org/immunogenicity/) and IEDB MHC-I Binding Predictions (http://tools.iedb.org/mhci/), following this order. Epitopes that were found promising in all 3 platforms were considered for further investigation. Propred1 highlights antigenic epitopes in a given protein sequence in 47 MHC alleles including HLA and H2 alleles82. All available alleles were selected for maximum population coverage, and the threshold of 3% was selected for the immunoproteasome and the proteasome filters. IEDB MHC-I Binding Predictions combines information from several binding prediction algorithms. The IEDB recommended method was used as it includes the Artificial Neural Network (ANN), Stabilized Matrix Method (SMM) and Combinatorial Library methods. The IEDB server has a list of the 27 most frequent class I alleles available (97% population coverage)83, which were used for the prediction considering 9mer peptides. ANN and SMM indicate the predicted binding affinity between HLA class I and a given epitope in nM, so low values (< 500 nM) of ANN and SMM indicate that the peptide and the HLA molecule may have a stable interaction. Finally, to measure peptide immunogenicity, IEDB Class I Immunogenicity resource was used. Peptides with immunogenicity score (> 0.15) were considered as immunogenic. In this tool, the amino acids properties as well as their position inside a given peptide are used to calculate the immunogenicity score of a given epitope84.In order to find the best potential ZIKV type I epitopes for immunization purposes, three binding peptides prediction platforms were compared: Proped1 (http://crdd.osdd.net/raghava/propred/). 15-mer length epitopes were predicted for all 51 HLA class II alleles available in the server with 1% threshold. Propred server is based on matrices for the alleles obtained from a pocket profile database previously described85. Epitopes were selected considering the maximum number of alleles that bind to the epitope. The IEDB server (http://tools.iedb.org/mhcii/) was then used to determine epitope affinity to HLA class II alleles based on percentile rank and IC50 values. Percentile rank ≤ 20 was considered for selecting promiscuous peptides and IC50 ≤ 500 nM was considered for peptides with intermediate to high binding affinity. Epitope immunogenicity was evaluated using the IEDB server (http://tools.iedb.org/CD4episcore/), considering percentile rank lower than 66 for immunogenic peptides86. Finally, epitope potential to induce IFN-γ was evaluated using IFNepitope server (http://crdd.osdd.net/raghava/ifnepitope/), and peptides with positive values were considered to be capable of inducing IFN-γ production.In order to select the best potential ZIKV CD4 + T epitopes, the binding affinity to HLA class II molecules, promiscuity, immunogenicity and predicted IFN-γ induction were evaluated. Epitopes found promising in all four parameters were selected for further analysis. Initially, CD4 + T cell epitopes for ZIKV structural and non-structural proteins were predicted using Propred MHC-II Binding Peptide Prediction Server (87 and with the IL-4Pred online server (https://webs.iiitd.edu.in/raghava/il4pred/index.php)88, respectively. The capacity to activate APCs and induce proinflammatory response was performed with the VaxinPAD online server (https://webs.iiitd.edu.in/raghava/vaxinpad/index.php)89 and ProInflam online server (http://metabiosys.iiserb.ac.in/proinflam/index.html)90, respectively. A 0.5 threshold was used for all these parameters as previously described91.The capacity to induce IL-10 and IL-4, activate APCs, and induce proinflammatory response was evaluated for epitopes that presented negative immunogenicity scores in order to check if they would bring a negative outcome to the overall vaccine immunogenicity. Prediction of induction of IL-10 and IL-4 were performed with the downloaded IL10pred platform92. In order to find a sequence of epitopes that could achieve the highest antigenicity while maintaining protein stability, we evaluated the best order in which the epitopes could be organized before choosing the final construct, such as EDIII-CD4-CD8, EDIII-CD8-CD4 and CD8-CD4-EDIII.The selected CD4 + and CD8 + T cell epitopes were chosen from a set of peptides predicted by the aforementioned servers considering population coverage, immunogenicity score and MHC binding affinity. ZIKV Envelope E Domain III (EDIII) (consensus) was added to the structure as well. The epitopes and EDIII were flanked by linkers, short amino acid sequences, which have been described to be cleaved by proteasomes and lysosomes so the epitopes can bind to HLA class I and HLA class II, respectively61. Antigenicity, stability, safety and some physicochemical parameters of all of them were predicted using different platforms described in the following methods.Aiming to increase vaccine antigenicity, its structure was evaluated with different combination of protein adjuvants, such as flagellin, Heparin-binding hemagglutinin (HBH), 50S ribosomal protein L7/L12 (50S rib) and RS09, a synthetic LPS peptide mimics designed with Phage Display Libraries93 while GPGPG linker was used to separate CD4 epitopes94, and AAY was used to individualize CD8 epitopes95. We chose GPGPG linkers considering it induced broad MHC class II-restricted T cell responses in mice vaccinated with an epitope-based HIV vaccine previously designed by our group81. The DNTAN linker was chosen to give flexibility to the different parts of the vaccine construct because it is considered a loop in protein (LiP)96, which is important to mediate biological processes such as signaling and protein–ligand binding97.To separate the epitopes, protein domain and adjuvant, proper linkers were used. DNTAN linkers were used to separate the adjuvant from the vaccine epitopes, and/or the EDIII from the other parts of the vaccinehttp://tools.iedb.org/population/) developed by Bui et al., (2006)98, utilizing the Allele Frequencies Net Database that contains a study of 1081 populations and 1066 alleles of the HLA gene101. The HLA class I and class II restricted epitopes category was selected, and the 12 CD4 + and 13 CD8 + T cell epitopes were included. All world regions were selected to assess all possible areas that could be covered by the vaccine.Assessment of vaccine epitopes coverage is important to predict which population or populations are going to benefit from the selected epitopes. To this end, we used the IEDB online server Population Coverage (http://ddg-pharmfac.net/AllergenFP/index.html). Allergen FP v.1.0 method is based on four steps algorithm considering the physicochemical properties of individual amino acids in a given protein. It applies a universal fingerprint approach that is alignment-free and has shown accuracy of 88%102.If not evaluated properly, vaccines can become allergens and/or induce autoimmunity. In order to assess if the proposed vaccine could be a potential allergen, the protein FASTA sequences were used as input in the allergenicity prediction server Allergen FP v.10 (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins), using the parameters described elsewhere103. Identical continuous human sequences smaller than 6 amino acids were discarded as the likelihood of such pairing is high, but not significant103.Additionally, if self-antigens are found in the vaccine sequence, the humoral and cellular responses induced against the vaccine may target self-proteins, leading to autoimmunity. In order to increase the chances that ZIKVac would not present such complication, blastp was performed with the NCBI platform (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html), with a threshold of 0.4 and “virus” as target organism or with ANTIGENpro (http://scratch.proteomics.ics.uci.edu).Antigenicity of the final vaccine construct was predicted using VaxiJen v2.0 .Evaluation of physicochemical characteristics of proteins are crucial for proper protein expression, separation, purification, storage and additional analyses. Thus, the vaccine's physicochemical properties were analyzed with ProtParam ExPASy Serverhttp://raptorx.uchicago.edu/StructurePrediction/predict/). This server was chosen because it is capable of predicting tertiary structures of proteins that have less than 30% similarity with proteins deposited in PDB (Protein Data Bank), i.e. without any similar homologs; additionally, it also provides information related to the protein secondary structure, solvent accessibility and a quality score, which can be used to select the best model. RaptorX performance in CASP9  was superior than the other participating servers when complex targets were evaluated105.Tertiary and secondary structures of all constructs were predicted using RaptorX Structure Prediction (http://sysbio.rnet.missouri.edu/3Drefine/). The PDB file obtained with RaptorX was used as input. After complete running, the server provides 5 refined models with values of 3D refine score, GDT-TS GDT-HA, RMSD (root-mean-square-deviation), MolProbity, RWPlus. The 3D refine and RWplus scores are the model potential energy after refinement; GDT-TS and GDT-HA indicate how conservative was the refinement; RMSD indicates in angstroms the deviation score of the model; MolProbity indicates how physically realistic the refined model is.Tertiary structure refinement was conducted with 3D Refine web server , which provides the Ramachandran plot. The Ramachandran plot will present if the Ѱ and Φ angle clusters are well positioned and points out if there is any abnormality resulted from the protein refinement. The torsion angles Ѱ and Φ characterizes the spatial conformations of each amino acid residue, and the Ramachandran plot groups information about the number of residues in favored region, allowed region or outlier region107.After refinement, protein structure was validated by RAMPAGE , and minimum score was set as 0.5 (default) and maximum distance was 6 Angstrom (default). ElliPro shows as output potential sequences of discontinuous epitopes as well as their location in the tertiary protein structure. This prediction is based on Thornton’s method108 and also includes residue-clustering algorithm. It is taken into consideration that, in case the protein is considered an ellipsoid, sequences that protrude from the ellipsoid are more freely recognized by antibodies109. ElliPro has the best AUC, 0.732, when compared with six other structure-based methods after testing of discontinuous protein epitopes from a benchmark dataset110.Elucidation of conformational vaccine epitopes are important as BCR recognizes epitopes without presentation or previous processing. In addition, protein folding may hide linear B-cell epitopes. Tertiary structure of the protein was used as input in the ElliPro server , considering the peptides with purity > 95%. Peptides were dissolved in dimethylsulfoxide (DMSO). All 25 peptides were pooled together and after testing different concentrations, 25 µg/mL was determined as the best concentration for usage.In order to validate the immunogenicity of the selected peptides, blood samples from 10 convalescent donors previously infected with ZIKV were used to carry out in vitro assays (Supplementary 2). ZIKV infection was confirmed by plasma and/or urine detection of ZIKV RNA by RT-qPCR during the acute phase of infection. Blood collection was conducted between two and three years after the acute phase of infection diagnosis at Federal University of Goiás . IgM and IgG serology for ZIKV was performed after blood collection using the double-path TR DPP® Zika IgM/IgG immunochromatographic test at the Bio-Manguinhos Recombinant Technology Laboratory in Osvaldo Cruz Foundation . Peripheral Blood Mononuclear cells (PBMCs) were isolated by standard density gradient centrifugation on Ficoll-Paque™ Plus  and cryopreserved in a 90% fetal bovine serum (FBS)  and 10% DMSO  solution. Cells were stored in liquid nitrogen until usage.2 for 2 h. Cells were then washed and distributed at a concentration of 2 × 106 cells/mL in polypropylene tubes with RPMI 1640 medium  supplemented with 0.1% penicillin, 0.1% streptomycin  and 5% FBS. Cells were stimulated for 5 h with 25 µg/mL of the ZIKV peptide pool containing all 25 CD4 and CD8 peptides at 37 °C with 5% CO2. After 1 h of stimulation, 1 µg/mL of Brefeldin A was added (BD Biosciences). DMSO was added to non-stimulated cells as negative control and Staphylococcal enterotoxin B (SEB) was used as positive control. Cells were washed with PBS and then with FACS buffer and stained with surface antibodies ; CD4-PerCP-Cy5.5 ; CD8-PE . Cells were incubated at 4 °C for 30 min and then washed twice in FACS buffer. 100 µl of Cytofix/Cytoperm  was added and cells were incubated at 4 °C for 20 min. Cells were then washed with Perm/Wash 1X  and stained with intracellular cytokine antibodies ; IL-2-APC ; TNF-α-PE-Cy7  at 4ºC for 30 min. Cells were washed twice with Perm/Wash 1X and fixed in 2% paraformaldehyde solution . Acquisition was performed on a BD FACS Canto II flow cytometer (BD Biosciences) and a minimum of 400,000 events were recorded from each tube. Data were analyzed using FlowJo software .PBMCs from patients previously infected with ZIKV were thawed and incubated at 37 °C with 5% COKolmogorov–Smirnov and Shapiro–Wilk were used to test the normality of the data. The non-parametric Mann–Whitney test was used to analyze differences between non-stimulated and peptide stimulated conditions. P values < 0.05 were considered statistically significant. Statistical analysis was performed using Prism version 10 .Ethical approval for this study was obtained from the Institutional Review Board of Pontifical Catholic University of Goiás (CEP—Research Ethics Committee) under the protocol number 46073815.9.0000.00370. All patients signed a written informed consent form before the interview and sample collection. The authors also declare that all methods were carried out in accordance with relevant guidelines and regulations.Supplementary Information.


The day we have dreaded for several years has finally come. Our Editor-in-Chief (EIC), Professor Arne Andersson, has decided to retire after serving that role since 2006. During those years, Upsala Journal of Medical Sciences (UJMS) experienced a remarkable transformation from a local journal with an impact factor of 0.476 to a global open access player with a 2-year impact factor of 2.384 (5-year impact factor is 3.080). Ever since it was founded in 1865, UJMS has been our precious ‘little gem’ (Arne Andersson is a distinguished diabetes researcher who made important contributions with particular focus on pancreatic islet transplantation. He shared the role as EIC with Gunnar Ronquist from 2006 to 2012 after which he became a sole EIC. He grasped at an early stage the ongoing evolution of medical science with its diversification and specialization, and consequently, he implemented changes in journal policies to address this. This resulted in the establishment of an editorial team of associate editors and an editorial board including international members. He accepted 493 manuscripts as EIC after scrutinous review, always with the highest’s scientific standards. His personal commitment to this process includes supervision of linguistic text revision in order for manuscripts to reach impeccable standards. The extreme diversity of high-quality publications recently accepted can be illustrated by publications in the fields of antibiotic resistance –7, microThe journal has experienced additional changes during Arne Andersson’s leadership. It has become an open access online journal, initially with both electronic and printed paper versions of all articles but since 2021 only with electronic publications. That transition was a necessity due to conditions imposed by the COVID-19 pandemic, and Arne Andersson had mixed feelings about this change, always preferring the printed version for reading.We are immensely grateful to Arne Andersson for his sublime input to our Journal, and our intentions are to maintain the course set out by him.Institute of Medical Sciences, Uppsala University Hospital, Uppsala, SwedenGerhard WikströmDepartment of Medical Cell Biology, Uppsala University, Uppsala, SwedenMichael Welsh


The aim of this research is to contribute to a better understanding the destructuration of three native starches and a wheat flour in mixtures of water and choline chloride. Model systems have thus been defined to allow a better approach to hydrothermic transformations related to the interactions between choline chloride and starch. We have observed that choline chloride has an impact on the gelatinization of starch which corresponds to the stabilizing salts phenomenon. The depolymerization and dissolution of the starch have also been demonstrated and can there dominate the gelatinization. However, the results obtained in X-ray diffraction by heating cell have shown that the exotherm which appeared was not only related to the depolymerization of the starch, but that a stage of crystalline rearrangement of the starch coexisted with this phenomenon. Choline chloride, because of its unique nutrient functionality and its ability to enhance flavor, provides a new option as a partial substitute for sodium chloride in reformulations of processed foods to reduce their sodium content. To date, very few studies have explored the potential of choline chloride to act as a substitute for salt. Locke and Fielding were the first to identify the properties of choline chloride to improve the salty taste of food products ,2,3.Starch is a natural polymer with particular properties unlike those of traditional polymers. As a heterogeneous material, it has macromolecular structures bound in a granular superstructure.When native starch granules are heated in water , their semicrystalline nature and three-dimensional architecture are gradually disrupted, resulting in a phase transition from the ordered granular structure to a disordered state in water, which is known as “gelatinization” ,5. GelatCholine chloride solubilized in water gives an ionic liquid. Several studies have shown the effects of ionic liquids (solvents and plasticizers) based on the effects of choline chloride on starch ,12,13,14This work presents the mechanism of choline chloride penetration into the starch grain and its impact on gelatinization and the structural evolution of starch during heating kinetics.5H14ClNO) were supplied by Sigma-Aldrich (France).Wheat flour (type 65) was supplied by Evelia, La Varenne, France. Wheat starch was purchased from Loryma GmbH , and waxy corn starch was purchased from Roquette Italia SpA . Potato starch and choline chloride (Cw) and wheat, potato, and waxy corn starch  solubilized in an aqueous solution containing different concentrations of choline chloride.The measurements were carried out on wheat flour , choline chloride, and water content, respectively.The model systems studied being composed of three ingredients  and were presented under the form of Equation (1) with FA mixing design has been used (1). The factors are the concentrations of each component of the mixture. Responses are expressed as a function of these concentrations with the sum of the mass fraction of each component being equal to 100 (x + y + z = 100). For each analysis, 5 g of mixture was prepared.x[CcyWz].The mode of preparation (quoted ILS = “Ionic Liquid + Starch” for the rest of the paper) consisted in adding the choline chloride under the form of ionic liquid  (previoup < 0.005) to detect significant differences.All analyses were repeated in triplicate. A statistical analysis was performed with variance analysis  for one hour and then placed in the oven of the appliance. The pans were heated at a rate of 1 °C/min from 10 to 120 °C by using the SETARAM microcalorimeter (µDSC) III (France). Scans were run on the following starch suspensions: SGe (peak temperature gelatinization), and ΔHGe denotes the transition enthalpies for different endotherms. The enthalpies were determined by endotherm integration, and all traces were normalized to 1 mg of dried sample. The partial enthalpies were calculated from the onset of the endotherm to the end (every 1 °C).Thermal transitions were defined as T56W24] were prepared as follows: SWC20[Cc56W24], SP20[Cc56W24], SW20[Cc56W24], and FW20[Cc56W24]. The appropriate amounts of choline chloride, water (when required), and starch were weighed. The same formulations without choline chloride were studied: SWC45W55, SP45W55, SW45W55, and FW45W55, corresponding to the same F/W ratio as the previous mixtures.The samples containing flour and starch suspended in  systems, i.e., when choline chloride is added to the aqueous phase. All the samples exhibited an endotherm on their thermograms, which corresponded to the gelatinization of the starch at the following temperatures: FW 61.7 ± 0.2 °C, SW 58.1 ± 0.1 °C, SWC 70.8 ± 0.1 °C, and SP 65.7 ± 0.1 °C (Ge (SW) < TGe (FW) < TGe (SP) < TGe (SWC), whereas the gelatinization enthalpies increased in the following order: ΔHGe (FW) < ΔHGe (SW) < ΔHGe (SWC) < ΔHGe (SP).The thermograms of wheat flour . This phenomenon was not visible for SW and FW. A DSC study was then carried out on the SWC20[CcxWy] systems (with x included between 56 and 70 and y between 24 and 10) to understand this phenomenon  increased as well. There was a critical concentration, which depends on the type of starch used, for which both exothermic and endothermic transitions took place. For this concentration, both phenomena seem to happen at very close temperatures, at which they overlapped, resulting in a neutralization with no visible thermal effect. The same behavior was found by Sciarini et al. and Mateyawa et al. working with choline acetate and EMIMAc and by Koganti et al. using N-methyl morpholine N-oxide (NMMO) ,16,26. Tenomenon .The thermogram of pure Cc (data not shown) shows an endotherm near 73.6 ± 0.1 °C. With the addition of water, this endotherm moves to lower temperatures and undergoes a strong shift from the baseline until disappearing. In the presence of waxy maize starch (20%), the concentration of Cc was increased in steps of 1% (from 56 to 70%), and water was decreased likewise (from 24 to 10%), which produced an endotherm at low temperatures for the sample with 17% water.During these investigations, the Cc was solubilized in water to form the plasticizer, and then the starch was added to the plasticizer. When the amount of water was sufficient, complete solubilization of the Cc was achieved. When the water concentration was less than 17%, the solubilization of Cc was partial, and we observed a solubilization endotherm that broadened with a decrease in the water content. For the 14% water sample, there was enough unsolubilized choline chloride for the allotropic change in choline chloride at 68 °C to be observed.It was assumed that the solution (water + Cc) became more viscous with increasing Cc concentrations, resulting in more difficulty for the Cc solution to penetrate inside the starch granule; in turn, a shift to higher gelatinization temperatures was observed.The effect of chlorine choline (Cc) on the gelatinization of starch is very different from that of NaCl observed in the literature. Indeed, if the gelatinization temperature follows the same trend when adding Cc or NaCl, the enthalpy of gelatinization shows the opposite behavior. The decrease in the total enthalpy of starch gelatinization at high NaCl concentrations suggests a destabilization of the ordered regions of the starch in the presence of NaCl. As explained by Chiotelli et al., sodium chloride may be hindered by polymer–polymer interactions in favor of water–polymer interactions, resulting in a lower enthalpy for fusion of organized regions. The addition of Cc causes a reorganization of the internal structure of the starch grain .To understand the penetration kinetics of the ionic liquid (Cc + water) in the starch granule, the cumulative enthalpy curves were calculated and reported as a function of temperature.The curves of the partial enthalpy of gelatinization for all the plasticizer contents studied are shown in P and SW: the melting of the potato starch ordered zones was more abrupt than it was for the wheat starch and the wheat flour. These observations are in good agreement with the results of Waigh et al. on the two-stage destructuration of SW  Wz. These mixtures were studied by μDSC.As previously described, choline chloride in the absence of water is not considered as an ionic liquid. Therefore, it seemed interesting to observe the action of Cc on starch when Cc is no longer acting as a plasticizer, but when it enters into the formulation as dry matter, Cc is placed directly in competition with water in front of starch. The relevant suspensions were formulated as follows: the starch (or flour) was mixed with the choline chloride (dry powder), and the water was added to the mixture. The obtained mixes were quoted using the following script:  system, an increase in the peak intensity occurred at 5.6° in 2Θ at a temperature of 60 °C, followed by the onset of collapse at 100 °C and the total disappearance of the structure at 110 °C. This phenomenon corresponds to the appearance of the exotherm on the thermogram from the DSC study.For the SWC20[Cc56W24] and SW20[Cc56W24], the transition to an increase in crystallinity before melting is much less visible, and it is even absent for the FW20[Cc56W24] system. This explains the absence of the exotherm for the FW20[Cc56W24] system. In the literature, the exotherm is related to the solubilization of the starch in the plasticizer. However, during the X-ray diffraction study, we showed that the exotherm also corresponded to the increase in the intensity of the diffraction peaks and therefore to the increase in the crystallinity of the starch before its solubilization and then its melting.For systems SP20[Cc56W24] system at low rates (0.1 °C/min). Two peaks were clearly observed (To complete this work, a μDSC study was conducted on the Sobserved , indicatThe starch/water system shows a gelatinization accompanied or unaccompanied by a fusion, according to the water content of the matrix. The choline chloride/water system possesses the characteristics of an ionic liquid and exhibits an allotropic change at low water concentrations and solubilization for water contents greater than 30%.Choline chloride is an ionic compound, which, like NaCl, has a “structure making” effect: that is, it increases the viscosity of the aqueous solution while decreasing the water fraction with high mobility. As a result, the gelatinization temperatures are displaced to higher temperatures. In contrast to the addition of NaCl, the addition of choline chloride entails a significant increase in the gelatinization enthalpy, which suggests stabilization or better organization of the ordered regions of the starch in the presence of choline chloride.An X-ray diffraction study in a heating cell demonstrated the crystalline rearrangement of the structure of the starch grain, which takes place simultaneously with the solubilization phenomenon of amylose.


Information of the edges, trabecular thickness, and spacing are summarized in functions on the unitary sphere used as inputs for the SphCNNs. The concomitant dimensionality reduction makes it possible to train neural networks on relatively small datasets. The predicted tensors were compared to the stiffness tensors computed by using the micro-finite element method (μFE), which was considered as the gold standard, and models based on fourth-order fabric tensors. Combining edges and trabecular thickness yields significant improvements in the accuracy compared to the methods based on fourth-order fabric tensors. From the results, SphCNNs are promising for replacing the more expensive μFE stiffness estimations.The apparent stiffness tensor is relevant for characterizing trabecular bone quality. Previous studies have used morphology-stiffness relationships for estimating the apparent stiffness tensor. In this paper, we propose to train   •Characteristic stiffness tensors are derived from trabecular bone micro-CT samples.•Previous approximation methods fall short on heterogeneous data-sets.•gradient, trabecular thickness and spacing are mapped to a spherical domain.The •Spherical convolutional neural networks are used for the prediction.•The prediction error is significantly reduced compared to the state-of-the-art. The staD nature . Many stD nature . One of D nature . Stiffnefinite element (FE) modeling with appropriate boundary conditions , have gained popularity to solve problems in the field of biomechanics. For example, In recent years, machine learning techniques, and in particular convolutional neural networks (CNNs) with 3D volumes of trabecular bone as input and the entries of the apparent stiffness tensor as output might require hundreds of thousands of training samples due to the high number of parameters to be trained in the neural network. Rather than using transfer-learning for the domain adaptation of such a high-parametric model, we suggest an alternative strategy to tackle this problem.The main drawback of using DL in any application is that it typically requires a lot of training data, which is a big challenge for stiffness estimation. Here, the implementation of an end-to-end solution with extended Gaussian images (EGI) as the input of the neural network. The EGI is a function on the unitary sphere S2 that captures the orientation distribution of the gradient of the image. Since the EGI is a function on S2, it is suitable to use spherical convolutional neural networks (SphCNNs)   instead SphCNNs) .trabecular thickness (Tb.Th) and trabecular spacing (Tb.Sp). Thus, we also propose to combine EGI with Tb.Th and Tb.Sp in order to improve the accuracy of the neural network for stiffness prediction.While the EGI conveys the edge information of trabecular bone, it completely disregards the distribution of bone material in the volume. To some extent, that information is captured by the 1.1•To the best of our knowledge, this is the first attempt to use DL, specifically SphCNNs, to estimate the apparent stiffness tensor. The results show that DL is more accurate than fabric tensor-based approaches.•A novel way of combining edge information encoded in the EGI with Tb.Th and Tb.Sp is proposed, that is meaningful for DL models.The main contributions of this paper are:2σ = 1.2, support = 2). After dividing each specimen into the cubic subregions with a side length of 5.3 mm, the volumes were segmented via the application of a single-level threshold of IPL . Furthermore, for each subsection, structures that were not connected to the main region after thresholding were removed from the volumes. Subsequently, each subregion was transferred into a piece-wise linear domain by considering each voxel as a hexahedral eight-node element. The Young modulus and the Poisson's ratio assigned to each element were 12 GPa and 0.3, respectively. Finally, FE-simulations were executed for six different loading scenarios under kinematic boundary conditions as described in http://www.dr-pahr.at).The present study utilized the data from 33.1apparent stiffness tensor C of a material describes its mechanical properties by relating its strain (ϵ) and stress tensors (σ) through the Hooke's law:In general, the The apparent stiffness tensor is represented as a fourth-order tensor with 3 × 3 × 3 × 3 = 81 coefficients. However, symmetry constraints reduce the number of independent entries of the apparent stiffness tensor to 21 . Alterna3.2p be a point in the image. Local thickness (Tb.Th) at that point is derived according to  is a ball centered at t with radius r, that lies completely inside the trabecular bone. Tb.Th is zero for points outside the trabecular bone. Local spacing Tb.Sp is defined analogously for points outside the trabecular bone:T, and Tb. Sp(p) = 0 for pϵT.As mentioned, we wanted to explicitly expose the DL model to information about the local thickness of trabeculae. Furthermore, we hypothesized that the spacing between the trabecular structures contains valuable information for predicting the mechanical properties. Let rding to :(2)Tb.ThNotice that we use local maps of Tb.Th and Tb.Sp instead of the customary use of the mean in the analysis of bone specimens, e.g., 3.3extended Gaussian image (EGI). The surface normals are mapped onto the sphere by putting their tails at the center of the Gaussian sphere and their heads at the appropriate place on the surface of the Gaussian sphere  being the unit impulse function, < · , · > the dot product, and ϵ a small constant to avoid the division by zero. Thus, the EGI can be seen as the orientation distribution function of the gradient of the image. Since the gradient of x can only take values of zero or infinity, it is customary to perform a Gaussian smoothing with a very small standard deviation before the gradients are calculated.For a segmented image α, β) with αϵ being the azimuth βϵ being the elevation. One example of a resulting EGI on a sphere and unwrapped on the α-β-plane is visualized in In this paper, the EGI is parameterized by the spherical coordinates (mean intercept length (MIL) tensor, the generalized MIL (GMIL) tensor, and the global structure tensor (GST)  . Thus, t3.4As already mentioned, the EGI contained the edge information of trabecular bone. In order to consider the distribution of bone and marrow material in the image, Tb.Th and Tb.Sp maps are combined with the EGI.g being either Tb.Th or Tb.Sp maps. Since the gradient is non-zero only in the interface between bone and marrow, the effect of this weighting is to create an orientation distribution function of the trabecular thickness (or spacing). Thus, the regions with the thickest trabeculae (or marrow) will contribute more to the EGI.The first idea is to use the Tb.Th and Tb.Sp maps as weighting functions in the estimation of the EGI:Alternatively, one can create EGIs by using the Tb.Th and Tb.Sp maps instead of the segmented image. In this case, the EGI is computed directly from the gradient of Tb.Th and Tb.Sp using Eq. 3.52-sphere (S2). Thus instead of traditional CNNs, we opted to use SphCNNs f^ml=f with a zonal kernel h can be computed as y^ml=S2 is taken into account. Further details of SphCNN can be found in After the last convolution layer, a weighted global average pooling is applied in which the difference in the area of the different areas of the sampling of the 3.644.14.1.1The input of the neural network consists of one or multiple EGIs. In the first experiments, only the EGI of the segmentation is used. In further experiments, we add EGIs that contain thickness and spacing information as additional channels of the same size. Every EGI is represented as a 2D pixel array of size 64× 64.4.1.2The activation signals of the SphCNN pass a stack of three spherical convolutional layers with 64, 128, and 256 convolutional filters, respectively. Each filter is parameterized by 32 coefficients. Weighted average pooling layers are added after each spherical convolutional layer to reduce the size of the feature maps by a factor of two. Here, the implementation of the spherical convolutional layers by Esteves et al. (2020)4.1.3parametric rectified linear units (PRELU) . This enables us to output the continuous stiffness values. All other layers employ  (PRELU)  as nonli4.1.4All experiments described in this report share the same training configuration see . These hCo and Cp are the observed and predicted apparent stiffness tensors and ‖·‖F is the Frobenius norm.The Frobenius error serves as both, the performance metric and the loss function. It is defined by:4.2In R2 is lower when using only the EGI for prediction than the experiments where additional thickness and spacing local maps were incorporated (5–6% improvement in R2), with the scaling option being slightly better.To further support the assumption that the additional feature channels are beneficial, we analyze the correlation of the predicted and observed tensors' entries. In 4.3We compared the Frobenius Error of the predictions from the three SphCNN models with the fourth-order tensor models proposed by Notice that the results reported in 4.4In this section, we investigate the effect of samples taken from different bone regions on the resulting accuracy of the stiffness predictions. We trained multiple versions of the model that only inputs the EGI on different subsets of the training data. Each of those subsets contains only samples from a specific region, i.e., from the femur, radius, vertebra, or a combination of all of them. As before, we evaluated the resulting predictors on a hold-out set from the respective region with 5-fold cross-validation. In this experiment, we only used the EGI of the segmented image as the input of the SphCNN.As shown in 4.5SO(3) . In this section, we investigate how this equivariance property translates to the complete prediction model. We performed a qualitative evaluation by analyzing one good  since different sample orientations resulted in various errors. Second, rotations around the y-axis seem to affect the prediction less than rotations around the other directions. A possible explanation for this effect might be related to how we represent the spherical EGI on a 2D grid. At some specific rotations, the presented method might lead to an oversampling of the data on the sphere's poles versus points closer to the equator. Therefore, a rotation aligned with the sphere's central axis (pole to pole) only translates the resulting EGI, while other rotations might perturb the data. It might be possible to tackle this problem by analyzing the poles of the EGI. In case much information of the EGI is concentrated on the poles, the procedure would be to estimate the EGI from the rotated image, predict the apparent stiffness tensor from the rotated image, and rotate back the prediction. It is, however, not obvious after which criteria the poles need to be analyzed . Therefore, we leave this question open here for future research.4.65In this work, the application of a DL-based pipeline to approximate complex volumetric FEM-based apparent stiffness predictions of trabecular bone samples was introduced. A significant constraint was the absence of a massive dataset, which is a requirement to train a deep 3D CNN from scratch. Instead, we projected our data into a reduced spherical domain and trained specific SphCNNs thereafter.The experiments showed that the proposed spherical mapping is a reasonable candidate due to its low number of internal parameters. An additional advantage of the spherical mapping is that the method is able to estimate the apparent stiffness tensor independent of the imaging resolution or shape of the trabecular bone sample . Moreover, extensions of the method to in vivo acquisitions are feasible by using estimations of Tb.Th and Tb.Sp in grayscale . HoweverOur results also suggest that the trabecular bone at different sites might be heterogeneous. Thus, for our neural network to have a good performance, it is necessary to use training samples from all skeletal sites. The results also suggest that fourth-order tensor models might require a specific parameter tuning for every skeletal site and dataset. This is also true for other models . In turnSO(3) transformations . The accuracy of the prediction can be affected when the EGI contains much information close to the poles of the unitary sphere. The equivariance property can be used for estimating the stiffness tensor from a rotated (and more stable) version of the sample.One specific theoretical aspect of stiffness tensor prediction is that it must be equivariant against Finally, we found out that the prediction error is reduced when trabecular thickness and spacing information is added to the network, especially for specimens with low bone density. Still, the error is higher for specimens with a small Frobenius norm of the apparent stiffness tensor. This suggests that it is necessary to enhance the neural network's input with additional features that better capture the variability of the stiffness tensor in this case.The best performing neural network was the one that only included the EGIs of the segmented image and the thickness map, with errors of around 20% regardless of the skeletal site. One possible reason to explain the better performance of this strategy compared to scaling the gradients is that, since the gradient of the segmented image is only non-zero in the surface of trabecular bone, thickness (or spacing) information is disregarded elsewhere in the scaling approach cf. .From the results, including thickness information both from the surface and inside the trabecular bone seems relevant for the estimation of stiffness. While this is true for thickness, only trabecular spacing information along the trabecular bone surface seems relevant for stiffness prediction since including spacing information from inside the marrow led to worse performance.Unlike previous studies, the EGI was used instead of fabric tensors to model bone anisotropy . Notice Previous studies have also shown that bone density is relevant for stiffness predictions . Our resOne of the main challenges of using DL in biomechanical applications is that this technology requires plenty of training data, which is usually unavailable . To solv5.1In addition to adding more local features to the neural networks, we plan to use other neural network architectures. Instead of SphCNNs, we plan to test graph CNNs , which m6In this paper, we propose the use of SphCNNs to approximate the apparent stiffness tensor of trabecular bone samples. The neural network makes use of local edge, thickness and spacing information to perform the prediction, which results in a reduction of more than 30% in the Frobenius error compared to state-of-the-art methods. Thus, SphCNNs are promising for replacing the more expensive μFE estimations.10.13039/100013297Eurostars, grant E11626. We thank NVIDIA Corp. for the donation of the Titan Xp GPU we used in this study. The funding sources had no involvement in the research and preparation of this article.This work was partially supported by All authors have given their consent for this publication.FS: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Software; Validation; Visualization; Writing original draft. JvK: Methodology; Software; Writing - review & editing. DHP: Conceptualization; Data curation; Investigation; Methodology; Software; Validation; Writing - review & editing. RM: Conceptualization; Funding acquisition; Investigation; Methodology; Project administration; Resources; Software; Supervision; Validation; Writing - review & editing.The authors declare that they have no conflict of interest.


The accumulation of high amounts of petro-based plastics is a growing environmental devastation issue, leading to the urgent need to innovate eco-safe packaging materials at an equivalent cost to save the environment. Among different substitutes, starch-based types and their blends with biopolymers are considered an innovative and smart material alternative for petrol-based polymers because of their abundance, low cost, biodegradability, high biocompatibility, and better-quality film-forming and improved mechanical characteristics. Furthermore, starch is a valuable, sustainable food packaging material. The rising and growing importance of designing starch-based films from various sources for sustainable food packaging purposes is ongoing research. Research on “starch food packaging” is still at the beginning, based on the few studies published in the last decade in Web of Science. Additionally, the functionality of starch-based biodegradable substances is technically a challenge. It can be improved by starch modification, blending starch with other biopolymers or additives, and using novel preparation techniques. Starch-based films have been applied to packaging various foods, such as fruits and vegetables, bakery goods, and meat, indicating good prospects for commercial utilization. The current review will give a critical snapshot of starch-based films’ properties and potential applicability in the sustainable smart (active and intelligent) new packaging concepts and discuss new challenges and opportunities for starch bio composites. Packaging of eatable materials is considered a broad-spectrum tool that indicates its nutrients detail, manufacturing, and expiry information, making it convenient for customers. Food products are available in many forms, such as semi-solid, solid, and liquid phases, making it necessary to pack them accordingly. Efficient packaging enables protection against dust, undesirable pollutants, and protects the food material from microbial contamination . To makeFurther, paper packing is recommended for a wide range of applications; however, they have a lower moisture barrier with non-significant mechanic strength. After that, plastic was used as an alternate material for packing purposes as it has both mechanical strength and moisture barrier. However, their long-term environmental persistence creates pollution problems and an unnecessary burden on food processing industries. Therefore, it raises the need for an alternate packing material that is safe for consumption and is biodegradable.Starch, a biologically important macromolecule, is considered the most promising polymer of natural origin as it has overwhelming biodegradability, significant abundance, and cost-effective nature ,3. Starc(i)Pregelatinized: physically modified by extrusion or drum drying;(ii)Derivatized: chemically modified with modifying agents such as acetyl, hydroxypropyl, phosphate;(iii)Crosslinked: chemically modified using epichlorohydrin, trimetaphosphate;(iv)Dextrinized: physically modified by irradiation, heat (pyrodextrinization); chemically modified: oxidizing agents, acid hydrolysis; enzymatically modified: amylolytic enzymes.Modified starches, based on physicochemical characteristics and modifying agents, and on their production, are classified into the following categories:By combination, chemically modified derivatization and crosslinking obtain double-derived starches. Starch in food products imparts physicochemical and functional characteristics  .Edible films and coating are attracting attention at an industrial scale as they have enough potential to combat rising pollution problems. Starch has remarkable properties, including biodegradability, sustainability, abundance, and can be modified or blended with other polymers. Various starches have been investigated for food packaging applications, including cereals, millets, and pulses starches. Starch could be used as base blending material to prepare eco-friendly packing material ,6. AdequA thin layer of the edible film may be applied to protect the food material from dust and preserve them in a fresh stage. To decrease the waste production rate, biodegradable polymers may be used .Besides their biodegradability, biopolymers have other important properties/features, such as low temperature, seal ability, air permeability, availability, and low cost. Biopolymers, especially different types of starch, chitosan, whey, gums ), plant-origin gums , and microbial gums , are recommended for coatings and packaging purposes alone or in blends to improve the mechanical properties ,10,11,12Biopolymers, as model systems, have an incredible ability to undergo gelation upon the addition of multivalent cations, leading to the crosslinking and aggregation of both when in contact with cations. Due to the exchange of divalent ions during the reticulation process, the creation of a strong network via gelation occurs, which, in turn, leads to a stronger matrix  ,14,15,16The ionic crosslinking of biopolymers can successfully produce aerogels, typically produced by the sol–gel technique. The aerogels are used for advanced intelligent food packaging materials applications. Starch suspensions are initially crosslinked to increase aerogel hardness , followed by gelling and then freeze-dried to corresponding aerogels ,18,19.The investigation of biopolymer interactions in gels is ongoing research. It has become increasingly critical for a better understanding of the behavior of biopolymer blends which is necessary for further applications. Due to these rapid form gels of the biopolymers, their interest and applications have increased in recent years in specific low-cost three-dimensional (3-D) shapes and patterns printing (3D printing) ,21,22.The film and coating layer features of starch enable its use for commercial purposes . Rising A recent study published this year has shown that the research on “starch food packaging” is still beginning, based on the low number of studies published in the last decade in Web of Science . TherefoThe capability of film formation is a fascinating feature of starch. Starch is not soluble in cold water and does not melt like conventional plastics since the starch degradation temperature is less than the melting point. Nevertheless, starch granules lose their semi-crystalline behavior irreversibly and are transformed into a continuous matrix when starch is subjected to force, heat, and plasticizers . This chgel [Solution casting is one of the commonly used techniques to prepare starch films. Solution casting involves (a) solubility of the biopolymer in a solvent/plasticizer, (b) casting in the mold and, (c) drying. The process requires starch gelatinization by mixing starch in water (3–12%) followed by heating above Tgel . The swegel . Drying gel . Quick-dgel .Water has been proven to be an idol plasticizer/solvent for starch; however, as it evaporates, films become brittle. Therefore, it is suggested to mix water with other plasticizers, including glycerol, sorbitol, simple sugars, and urea, to access conditions fit for solution casting. Plasticizers are essential for films since they enhance the flexibility of films by decreasing intermolecular H-bonding because it enhances the intermolecular space. In addition, they improve the mechanical characteristics of starch-based films for packaging applications. This process is considered an energy and time-consuming method since water is the only cost-effective solvent for starch. Since this method is not a commercial-scale technique for film formulations, it displays an advantage over all other film-forming methods; the films prepared by the casting method exhibit an excellent aesthetic quality, an essential feature for food packaging materials.Staphylococci in sliced cheese during 28 days of storage. However, without propolis, ethanolic extract and commercial PE film show no antimicrobial activity [S. aureus (22–100% inhibition), those prepared with cinnamon oil were effective against C. jejuni (19–22% inhibition), and growth of E. coli was inhibited (33–40% inhibition) to the maximum extent by potassium sorbate incorporated films [The nonionic, cationic, and anionic starch-based films obtained using the casting method activated with the cationic surfactant lauroyl alginate possess antimicrobial activity. They are ideal candidates to extend the shelf-life of the packaged food products . Cassavaactivity . Furthered films .Several intelligent starch-based films blends with PVA, and nanoparticles have been developed for simultaneous colorimetric indication or pH indication and at the same time with antimicrobial activity for food packaging applications. Changes in the chemical environment of food are very important for product safety, and incorporating such indicators is of great interest ,38.Significant mechanical and barrier enhancement have been reported by different studies using the casting method to obtain poly(lactic acid)/nanoclay composite film or starch/nanoclay composites prepared by employing organically modified montmorillonite (MMT) minerals (poor dispersion and presence of large agglomerates) ,40.The extrusion process is another widely used and generally preferred processing method to produce polymer films and is a commercially used polymer processing method. This technique alters structural properties and enhances the physiochemical characteristics of extruded substances . The extMechanical and thermal energy are the two key factors involved in the extrusion technique to formulate extruder-based film . Screw sFurther, the variation in screw speed allows the removal or addition of suitable additives (stabilizers). The decrease in torque value of edible films concerning screw speed depends on the thermoplastic behavior of polymers during heating and plasticization.g) and reduced water level conditions [This process depends on polymers’ thermoplastic behavior whenever plasticization and heating occur above the glass transition temperature  was significantly higher as compared to WVP values of plastics (high density polyethylene) (0.01 mg·mm/Pa·hr·m2) followed by low density polyethylene (0.03 mg·mm/Pa·hr·m2) and  (0.05 mg·mm/Pa·hr·m2), respectively.Water vapor permeability: Water and oxygen are the two important barrier characteristics of biodegradable packaging polymer. These barrier properties directly prevent moisture and oxygen exchange between the product and the surroundings. Delassus  investig2·h·Pa, however, when EF was added in corn starch, the WVP values rose significantly. Further, films prepared using pure EF showed the WVP value as 0.0027 g·mm/m2·h·Pa. Higher WVP values might be due to the porous structure of films and irregular surfaces, indicating lipids and protein, and starch in the matrix. Higher WVP values in any films limit their uses at a commercial scale as it may lead to deterioration of the quality of food products packed in them [2. The authors reported that crosslinking of faba bean starch at different levels  using sodium trimetaphosphate brings desirable changes in films as confirmed by lower WVP values (modified starch). WVP was observed as native (faba bean) (1.10 g·m/Pa·s·m2) and modified starch films (1.34 g·m/Pa·s·m2). Further, the level of modification is directly proportional to the changes in WVP values up to certain specific limits depending on the type of botanical starch used. Starch modifications result in improved mechanical strength of films and WVP value [Majzoobi et al.  observed in them . Ultimat in them . Studies in them  investigVP value .A study on films prepared with native rice starches did not evaluate for WVP because they showed cracks along with the film. The intensity of acetylation significantly influenced the water vapor permeability of high amylose starch films, once the acetylated starch films with 0.42 and 0.72 of substitution degree had a lower WVP than the acetylated starch films with DS = 0.24 . FurtherStarch nanocomposites incorporating montmorillonite and crosslinked  were found to reduce the moisture sorption and swell at high relative humidity, improving the barrier properties. Several studies have proved the crosslinking of starch with citric acid to produce a water-insensitive barrier applied for coatings. It is commonly used to produce hydrophilic films of carboxymethyl starch (CMS) and carboxymethyl cellulose (CMC) with improved physicochemical properties ,70,71,72It seems that the addition of CMC to different starch-based films , which were glycerol-plasticized enhanced water resistance as increased maximum tensile strength ,74,75,76Nanocomposite films obtained by extrusion from different combinations of cassava starch, xanthan gum, and nanoclays (sodium montmorillonite—MMT-Na) were more transparent and resistant, had lower water sorption capacities and lower water vapor permeability, good combination of mechanical properties. The addition of xanthan gum has improved the elongation of starch films .2·day·kPa. Inclusion of glycerol (20–30%) while making starch-based films results in a slight decrease in OVP value. Further, compared to other non-plasticized materials, when sorbitol (30–40%) was used, it resulted in a comparatively low OVP value [Oxygen vapor permeability: Oxygen vapor permeability (OVP) of starch-based films is well documented. Rompothi et al.  preparedVP value . ThirathVP value  promulgaVP value ,84. The VP value . Low OVPVP value . The parBasiak et al.  preparedIncorporating other kinds of oils  seems to have the same positive effect in reducing oxygen and reducing the hygroscopic of the starch-based films ,91.The addition of oil reduces the oxygen permeability because the hydrophobic characteristics of the components reduce the water content of the starch-based films and thus their oxygen solubility .The mechanical characteristics, including tensile strength (TS), elongation at breakage (EAB), Young’s and storage modulus, and loss factor (tan δ), are crucial in packaging materials. These mechanical attributes help predict their mechanical potential and evaluate the feasibility of their application as food packaging materials .TS and EAB are primary parameters to characterize film for packaging. TS and EAB demonstrate the potential of food packaging resistance to breakage and maintaining integrity under stress during processing, handling, and storage . The mecFurther, DMA analysis is used to demonstrate dynamic mechanical properties of starch-based films through the changes of storage modulus  and loss factor . Mechanical parameters that determine the commercial status of starch-based films are their tensile strength followed by thickness of the film, moisture level, WVP, elongation at break, and solubility, respectively. The variation in the above-said properties determines the use of films for different purposes. These factors help the researchers to provide relevant information regarding the use of starch films in the market as per the demand of consumers. Mechanical properties of starch-based films are presented in Tg, and higher thermal stability, compared to high amylose corn starch, 70:30; wheat starch, 25:75; or potato starch, 20:80. It seems that the ratio amylose:amylopectin regulates the orientation of molecular structure in the starch-based films.Cassava starch, mung bean starch, and a blend of cassava and mung bean starch with two types of plasticizers (glycerol or sorbitol) were used by Vu et al.  for filmSong et al.  evaluateStaphylococcus aureus (S. aureus), Escherichia coli (E. coli), Salmonella typhimurium, Listeria monocytogenes), to control and to release active ingredients, as colorimetric indicator template for intelligent packaging, and biodegradability stimulator [Incorporating nanoparticles as reinforcers and fillers into food packaging materials is reported to exhibit improved mechanical properties compared to the pure starch films. Nanoclay, nanocellulose, and nano silicon dioxide are examples of additive that is widely available, cost-effective, and biodegradable and has been shown to improve the properties of various polymer materials. Sadegh-Hassani and Nafchi  formulatimulator ,107,108.A biodegradable colorimetric indicator starch–clay nanocomposite film was developed to monitor milk spoilage and was attached to the milk bottle. The water solubility of the starch-based film was reduced by adding nanoclay and restrained a dye release phenomenon into the milk .The incorporation of nanoparticles occupies the sites on starch that normally would be occupied by water . MechaniHowever, there is still a restriction in using nanocomposite films in food packaging due to legislation surrounding nanomaterials lack of respect for consumer and environmental safety.Convenient, transparent, and easy-to-use starch films without any foreign particles (insoluble) could be prepared using yam starch (plasticized) . While pAlthough glycerol is required to achieve transparency in films (starch), it should be added in an optimized amount as the increase in glycerol concentration leads to less transparency. Geleta et al.  preparedFurther, drying temperature also affects the transparency of film up to certain specified limits. Prabhu et al.  supporteThe addition of oil extracted from seeds (sunflower) in starch films decreases transparency . This mi2 has been used extensively in food and cosmetic applications to block light and give a white appearance in compliance with the recommended safe dosage. Oleyaei et al. [2. The authors reported that the presence of TiO2 in the film matrix caused a remarkable absorbance of UV light even at the low contents of nanoparticles.Further, metal oxides are often used to extend or improve the functional properties of biodegradable films. TiOi et al.  modified2/potato starch films obtained by Zhang et al., showed resistance against UV light and restrained the deterioration of food caused by UV radiation due to nano-SiO2 causing low transmittance [The 100 nm Nano-SiOmittance . Potato mittance .Biodegradability defines the ability of a material to decompose into simpler ones after interacting with biological elements . For plaDominici et al.  investigStarch-based biodegradable materials need to meet specific mechanical, barrier, antibacterial, and antioxidant characteristics for food packaging materials. Improved food quality, shelf life, and safety are the essential attributes of food packaging materials. Many attempts have been made to improve the barrier (water and oxygen permeability) by blending with other polymers or adding antioxidants/antimicrobial agents in film-forming materials to extend the shelf life of foods. The films can suppress respiration and delay oxidation ,148,149.Starch-based films have been applied to packaging various foods, such as fruits and vegetables, bakery goods, and meat, indicating good prospects for commercial utilization .Coating of strawberries with 3% cassava starch (amylose content 17–19%) and potassium sorbate (0.05%) reduced the respiration rate, improved the water vapor permeability, and provided good sensorial attributes to strawberries . During 2 particles to the potato starch to formulate composite film and observed better barrier, mechanical, and antibacterial properties, which could be used as packaging material for white mushrooms preservation.Apart from fruits and vegetables, starch-based edible coatings are also applied to nuts and bakery products. Due to the better flexibility of rice starch-based films than wheat and cornstarch-based films, rice starch was suggested as a suitable coating for walnuts ,154. RicBrochothrix thermosphacta [Salmonella and Staphylococcus aureus [Listeria innocua and Escherichia coli depending on the internal diffusion of carvacrol through the bilayer. Garrido-Miranda et al. [Incorporating antimicrobial agents into starch-based materials can release a controlled amount of active ingredients and reduce their interaction with other food compounds . Starch-osphacta  and pomes aureus . In addis aureus  added Vis aureus . Requenas aureus  prepareda et al.  also inca et al.  blended a et al.  for yeasTo evaluate starch-based films’ antioxidant potential, Piñeros-Hernandez et al.  incorporA bio-hybrid material containing porous starch, halloysite nanotubes, and the antioxidant fucoxanthin was developed by Oliyaei et al. . FucoxanBio-based smart packaging is a potential option, where sustainability and real-time monitoring of food quality are combined, assuring health safety and providing economic and environmental benefits. Different intelligent starch bases for packaging applications have been developed in the recent decade. Already some examples have been highlighted in this review, and a couple of other examples will be presented. An intelligent function of packaging is related to communicating, detecting, recording, sensing, tracing, aiming to provide information, facilitating the decision to extend shelf life, enhance safety, improve quality, and warn about possible problems .Different components of plant extracts or natural dyes, such as chlorophyll and carotenoids, possess the property of giving yellow-green pigmentation and undergo color changes when exposed to fluctuating pH conditions. The changes in pH of a food product indicate changes in state and quality; therefore, pH is a valuable and essential indicator. In general, the natural bioactive compounds used to obtain intelligent films for packaging applications possess antimicrobial and antioxidant activities; therefore, intelligent packaging is, in most cases, active packaging at the same time, but few studies are evaluating both functions. If both active and intelligent functions are considered, then the packaging is presented as smart biodegradable packaging ,168.Starch-based intelligent food packaging can be used as a visual indicator of freshness to monitor the user to monitor shrimp freshness. Choi et al.  preparedThe purple sweet potato anthocyanins have been incorporated into carboxymethyl-cellulose (CMC)/starch as an indicator to monitor the real-time freshness of raw fish (grass carp) stored at 20 °C, shifting color from red to blue and green when exposed to different pH or ammonia .In another study, purple sweet potato was added to starch/PVA films for simultaneous colorimetric indication and antimicrobial activity for smart food packaging applications .Medina-Jaramillo et al.  preparedFurther, Zhang et al.  extracteBrassica oleraceae) anthocyanins added to chitosan/corn starch-based biopolymer mixture have been implemented as a visual indicator of fish fillet deterioration [Red cabbage  anthocyanins into starch/PVA film composite with zinc-oxide nanoparticles [Starch/PVA composite packaging film with Roselle anthocyanins has been developed to monitor raw fish freshness (silver carp). An intelligent pH sensing wraps for food packaging applications has been obtained by incorporating Jamun (articles . Starch Starch has received considerable attention for biodegradable film formulation due to its biodegradability, edible, low cost, ease of use, and thermoprocessable nature. Starch-based films and coatings have been considered an alternative to conventional packaging to improve food quality and safety. Additionally, starch-based films are used as carriers of functional ingredients to prepare active, antioxidant, and intelligent packaging by incorporating antimicrobial, antibrowning, and nutraceutical agents to improve shelf-life and quality.However, developing new technologies to improve the delivery properties of films and coatings still requires future research. Most studies on food applications have been conducted at a laboratory scale; thus, research on cost reduction and larger-scale production and stability and safety is necessary to promote the feasibility of commercialized biodegradable packaged or coated products. Further studies should optimize film formulation and processing conditions to enhance humidity susceptibility and film properties for specific applications.