Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), which has been reported worldwide. The expression of viral structural proteins: surface glycoprotein (gp51) and three core proteins - p15 (matrix), p24 (capsid), and p12 (nucleocapsid) induce a strong humoral and cellular immune response at first step of infection. CD4+ T-cell activation is generally induced by bovine leukocyte antigen (BoLA) region– positive antigen-presenting cells (APC) after processing of an exogenous viral antigen. Limited data are available on the BLV epitopes from the core proteins recognized by CD4+ T-cells. Thus, immunoinformatic analysis of Gag sequences obtained from 125 BLV isolates from Poland, Canada, Pakistan, Kazakhstan, Moldova and United States was performed to identify the presence of BoLA-DRB3 restricted CD4+ T-cell epitopes. The 379 15-mer overlapping peptides spanning the entire Gag sequence were run in BoLA-DRB3 allele-binding regions using a BoLA-DRB- peptide binding affinity prediction algorithm. The analysis identified 22 CD4+ T-cell peptide epitopes of variable length ranging from 17 to 22 amino acids. The predicted epitopes interacted with 73 different BoLA-DRB3 alleles found in BLV-infected cattle. Importantly, two epitopes were found to be linked with high proviral load in PBMC. A majority of dominant and subdominant epitopes showed high conservation across different viral strains, and therefore could be attractive targets for vaccine development. BLV, together with human T-cell leukemia viruses type 1 and 2 , belong to the genus Deltaretrovirus of the family Retroviridae. BLV infection has a worldwide distribution and causes substantial economic losses in the livestock industry3, infection with this virus result in a negative effect on dairy production and cow longevity, which is very likely based on the resulting impaired immune function following infection5.Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leucosis (EBL), a chronic, lymphoproliferative disease associated with persistent lymphocytosis and B-cell lymphomasenv, gag, pol/pro) and nonstructural, regulatory genes . The env gene gives rise to two glycoproteins: extracellular surface subunit  implicated in receptor recognition and virion attachment, and transmembrane subunit  responsible for anchoring the SU-TM complex into lipid bilayers (reviewed in6).The complex BLV genome encodes structural genes , responsible for initiating the process of virion budding from the infected cell and RNA packaging in the viral particle formation process8. During viral maturation, the precursor Gag is processed into three separate proteins: matrix , capsid  and nucleocapsid , which undergo substantial conformational rearrangements—and confer infectivity to virions9. The tax gene encodes Tax protein, involved in activation of transcription of viral mRNA.The 14. With respect to cellular immunity, CD4+ T-cell response to BLV, mediated through recognition of short viral peptides presented by bovine leukocyte antigen (BoLA) class II molecules on the surface of antigen-presenting cells (APC), is required for multiple anti-viral processes. These anti-viral processes confer protection from progression to persistent lymphocytosis and tumor development during BLV infection16. The CD4+ T-cell response can be directed toward any virion protein; yet, many studies into T cell immunity to BLV have focused on the external glycoprotein gp51. Indeed, existing molecular studies on CD4+ T-cells and BLV are limited to the gp51 CD4+ T-cell epitopes: peptide 98–117, peptide 169–188, and peptide 177–19217; peptide 51–70 and peptide 61–8018; peptide 98–11719. Recently, CD4+ T-cell epitopes on gp30 protein of the env gene were mapped 19. Furthermore, peptide 131–150 and peptide 111–13020; and peptides tax16/17, peptides tax19/20 and peptides tax 22–2419, were recognized as T-cell epitopes for Tax. To date, there are no structurally defined CD4+ T-cell epitopes from the internal gag proteins–MA (p15) and NC (p12). Experimental data regarding Gag polypeptide are limited to CA (p24). An in vitro study based on lymphocyte proliferation assays two non-universal CD4+ T-cell epitopes: peptide 31–55 and peptide 141–165 were defined13.Both humoral and cell-mediated immune responses strongly limits BLV replicative cycle in cows naturally infected with the virus22. Cattle express only two BoLA class II proteins, DR and DQ23. The only source of diversity in DR molecules is from the polymorphic DRB chain24. So far, 384 BoLA-DRB3 alleles have been identified according to the BoLA Nomenclature Committee of IPD-MHC Database [Available from: https://www.ebi.ac.uk/ipd/mhc/group/BoLA/species/]. Polymorphisms in the BoLA-DRB3 gene can influence immune response by peptide binding, antigen presentation, and T-cell receptor (TCR) gene sequences in a T-cell population and group of cytokines that mediate and regulate immunity26. Furthermore, the affinity of BLV epitopes to bind to certain BoLA-DRB3 molecules was suggested to correlate with the capacity to induce T-cell proliferation28. However, the BLV epitope peptides responsible for this binding remains unknown.Resistance or susceptibility to progression of BLV infection in cattle was linked to the polymorphism in the BoLA class II geneThe aim of this study was to determine epitopes on Gag-derived proteins  binding to different BoLA-DRB3 alleles and link them with blood proviral load (PVL) levels of BLV-infected cattle.*12:01, *14:01:01, *27:03, *07:01, *18:01, *41:01, *105:02, *116:01, *160:01, *09:02, *24:33, *25:01:01 and *57:02) had frequencies ranging from 1.7% to 3.8%; 20 alleles  had frequencies ranging from 0.8% to 1.3%; and the remaining 36 alleles had frequencies below 0.5%  plot exhibited 49 peaks with values ranging from 0.05 to 1.13 . Analysis revealed 22 putative regions within Gag proteins with high binding affinity to BoLA-DRB3 alleles. The binding affinities of the epitopes and complete calculations are presented in Supplementary Table Detailed analysis indicated that the highest number of BoLA-DRB3 alleles, 44 out of 73 (60.3%), had significant binding affinity to peptide 320#QPAILVHTPGPKMPG and additionally to 5 overlapping peptides sharing the same ILVHTPGPK core sequence , was sequence 72#RVPLVLATLNEVLSN plus 5 adjacent peptides with common core sequence LVLATLNEV. Completed sequence 69-KFGRVPLVLATLNEVLSNDE-88 for the group of peptides was designated epitope 5. The ninth peptide 42#NYIHWFHKTQKKPWT, for which 24 of 73 (32.9%) different alleles showed binding affinity together with 3 overlapping peptides with common core sequence FHKTQKKPW, created sequence 42-NYIHWFHKTQKKPWTFTS-59, which was designated epitope 6. The tenth peptide 225#LWLQAWKNLPTRPSV, to which 23 of 73 31.5%) BoLA-DRB3 alleles had binding affinity together with the other 6 peptides with common core sequence WKNLPTRPS, represented the combined sequence 222-YQNLWLQAWKNLPTRPSVQPW-242 designated as epitope 7. The eleventh peptide 346#GPCYRCLKEGHWARD showing binding affinity for 16 of 73 alleles (21.9%), together with three adjacent peptides with YRCLKEGHW core sequence, presented summarized sequence 344-PPGPCYRCLKEGHWARDC-361 and was designated epitope 8  were located in the matrix; one epitope (11B) within the matrix-capsid; 14 epitopes  in the capsid; one epitope (1A) at the capsid-nucleocapsid; and one epitope in the nucleocapsid domain (8) .Next, the numbers of epitopes were analyzed with respect to proviral copy numbers in BLV-infected cattle. Figure 5), and cattle with susceptible alleles may be at a high risk of BLV transmission via direct contact with healthy cows. In contrast, BoLA-DRB3*09:02, *02:01 and *014:01:01 alleles comprise resistant markers associated with the development of low PVL in blood cells , and cattle with resistant alleles may be low-risk spreaders for BLV transmission33. To determine possible associations of BoLA-DRB3 alleles related to susceptibility or resistance to BLV proviral load with BoLA-DRB3-restricted CD4 + T-Cell epitopes, the five (5/73) BoLA-DRB3 alleles like *012:01, *012:03, *015:01, *015:05, *016:01 were placed in a single group (Group A) and marked in red. Group A contained those BoLA-DRB3 alleles associated with a high BLV copy number, as previously described. The five BoLA-DRB3 alleles like *09:02, *09:01, *09:04, *02:01 and *14:01:01 were compiled as a second group (Group B) marked in gray and consisted of the alleles previously correlated in BLV-infected cattle with low proviral load.We tested 73 alleles for their affinity to particular epitopes and BLV proviral load. Supplementary Fig. t-value = 2.06255, P value = 0.040269)  Fig. .Figure 4The 13 codons, which were identified as the major sites with a process of positive selection were evaluated for epitope affiliation. Out of these 13, 11 codons 84.6%) were located in the following epitopes: codon 48 in epitopes 4A and 6; codons 61 and 63 in epitope 11A; codons 69, 87 and 88 in epitope 5; codons 108, 109 and 112 in epitope 11B; and codons 318 and 323 in epitope 1A. Therefore, to assess the observed mutations’ impact on the binding affinity of BoLA-DRB3, the individual sequences of 125 isolates with corresponding BoLA-DRB3 genotypes were submitted for analysis using NetBoLAIIpan. As result, 58 out of the 125 BLV strains had different amino acid changes (n = 25) in the epitope sequences that was predicted to alter the binding affinity of BoLA-DRB3 to the epitopes. Out of 25, 13 changes were located in positive selection sites , and an additional 9 other changes  have predicted a significant effect on the binding affinity of BoLA-DRB3 in combination with different BoLA-DRB3 alleles  sequence of the predicted 15-mer epitope that is flanked by three aa residues on the N- and C-terminal ends. Therefore, the conservation of the 22 identified putative CD4+ T-cell epitopes was evaluated based on the number of changed amino acid residues in their binding-core sequences Table . Epitope38. The in silico prediction methods NetMHCIIpan and NetBoLAIIpan developed to predict HLA and BoLA class II restricted peptide binding, respectively, have proven to be among the best methods currently available40. Here, we used NetBoLAIIpan prediction method to determine BoLA-DRB3- restricted BLV peptides on p15, p24 and p12 of the Gag polyprotein, with broad BoLA allelic coverage. Of all tested epitope candidates, 11 top-scoring epitopes  were selected to identify broadly reactive BLV-specific CD4+ T-cell responses, by up to 60% of the analysed BoLA-DRB3 alleles. Another 11 subdominant epitopes  were predicted to be more restricted to one or more of the donor’s BoLA class II alleles, up to 17.8% of all examined BoLA-DRB3 alleles.The utilisation of bioinformatics to identify T-cell responses to retroviral infections has increased over the last few years43. Moreover, the binding core sequences of the other 12 epitopes  had a high conservancy—from 99.2 to 100%. These results show that the majority of the detected epitopes provide a solid guide for vaccine development.These identified epitopes often overlapped, thus creating long regions within the Gag protein that had BoLA-DRB3 binding affinity. In MA, dominant epitopes occupied 40%, in CA 57% and in NC 47% of the protein sequences. Importantly, the Gag peptide-binding motifs were detected for all bovine BoLA-DRB3 proteins (n = 73) recognized for the 125 cows used in the current studies. Each of the alleles had at least three of the identified target epitopes. The presented data demonstrates a high promiscuity of Gag protein to BoLA-DRB3. BoLA-DRB3-restricted CD4+ T-Cell epitopes 1A and 1B were considered the most promiscuous binders as they contained binding cores of epitopes predicted for 44 different BoLA-DRB3 types. They were located within a conserved part of the p24 region, C-terminal domain, which is required for capsid dimerization, Gag oligomerization and viral formation13 were compared with the newly identified epitopes in this study. The first epitope (residues 31–55) region corresponded to T-cell epitopes 4C (144-VWIQTLRLAILQADPTPADLE-164), 13A (142-GSQVWIQTLRLAILQAD-158) and 10 (126-LRELQDIKKEIENKAPGS-143) (specific residues are underlined). The second epitope (residues 141–165) region corresponded to T-cell epitopes 1B (255-EFVNRLQISLADNLPDGVPKE-275), 3 (247-QGPAESYVEFVNRLQISLADN-267), and part of 13B (238-PSVQPWSTIVQGPAESYVE-256). We noted that the previously defined epitopes were nested within newly identified epitopes. Therefore, we strongly suggest that these regions may be alternatively extended by the amino acids contained in the epitopes defined by BoLA-DRB3 II peptide-binding prediction method. Interestingly, in contrast to the second epitope (residues 141–165) region, epitopes 1B, 3 and 13B cover the full length of the major homology region (MHR) 244-IVQGPAESYVEFVNRLQISL-263, which was found to be essential for the stability and folding of the monomer, and hence for viral assembly, maturation and infectivity. This region is conserved throughout the whole retrovirus group and thus offers a novel and stable target for viral vaccines.The two previously defined regions of p24 protein, being recognized by specific T-lymphocytes, represented by amino acids 31-PGSQVWIQTLRLAILQADPTPADLE-55 and 141-AESYVEFVNRLQISLADNLPDGVPK-16513. This is likely related to the fact that the previously defined epitope regions are based on PBMCs from only four animals, which represented a particular allele of the BoLA-DRB3 genes interacting with the two specific p24 regions. The type of BoLA-DRB3 alleles in the tested PBMCs in prior published experiments are not defined. Nevertheless, based on our data, it appears that there indeed exist BoLA-DRB3 alleles which have affinity only for one or the other identified epitope regions; there are inter alia: BoLA-DRB3 *007:01, *009:04, *020:01:01 for residues 31–55 and BoLA-DRB3 *002:01, *004:01, *008:01, *010:03, *015:01, *018:01, *025:01:01, *043:03, *081:01, *116:01 and *160:01 for residues 141–165.In the current study, we detected a greater number of BoLA-DRB3-restricted epitopes than Mager and coworkersWe observed that a higher number of Gag protein epitopes recognized by certain BoLA-DRB3 alleles accompanied the alleles associated with BLV resistance in cattle. Interestingly, relatively few of them were observed in the population of BLV-infected animals. Likewise, fewer epitopes recognized by particular BoLA-DRB3 alleles were associated with the BLV susceptible alleles. Noteworthy, the percentage of these alleles in the examined population of virus-infected cows was relatively high. Indeed, the affinity for the interaction of certain BoLA–DRB3 alleles with a longer region of the Gag protein (where epitopes overlap) or more Gag regions may elicit a stronger cellular response. Thus, our results confirm the hypothesis that disease-susceptible cattle may have fewer epitopes than resistant cattle, resulting in weaker immune responses. Moreover, these results indicate a significant role of bovine MHC II polymorphisms in the mapping of BLV epitopes recognized by CD4+ T-cells on viral proteins.19. Takeshima and colleagues suggested that the BoLA-DRB3 gene may regulate both antigen epitope recognition and the magnitude of the antigen-specific T-cell response that is processed after exposure to infection23. Accordingly, our studies confirm that BLV antigens are restricted according to BoLA-DRB3, and that genotyping of cattle is important for determining antigenic epitopes recognized by the bovine immune system.Bai and coworkers studied gp51, gp30 and Tax protein epitopes related to the BoLA–DRB3 genotype and found that fewer CD4+ T-cell epitopes were observed in susceptible cattle than in resistant cattle19. This discrepancy may be due to the fact that the current study used a 25-fold larger group of cattle for the analysis. It is well known that the BLV proviral load varies greatly as it is the result of many different factors such us the time of exposure to the virus, biochemical and hematological factors of the cow, or the age of the cow, to name a few, which can generate erroneous results when experiments are conducted on a small number of animals46. In comparison, another retrovirus, HIV, specifically targets the HIV Gag peptides by CD4+ T-cells has been associated with lower viremia in both adults and children49. Ranasinghe and coworkers demonstrated an inverse correlation between viral load and the number of Gag peptides targeted by CD4+ T-cells50. Buggert and coworkers confirmed this finding, suggesting that broadly reactive Gag-specific CD4+ T-cell responses could have an impact on HIV disease progression51. However, whether the frequent targeting of Gag peptides is the cause or the consequence of the reduced viremia remains to be clarified.In this work, we also analyzed whether the number of BoLA-DRB3-restricted epitopes in Gag protein is related to the number of BLV proviral copies in PBMCs in the analyzed DNA samples; however, we did not find a significant correlation. Additionally, Bai and colleagues observed that the number of CD4+ T-cell epitopes was positively related to proviral load, which depended on the BoLA class II genotype53. Published data indicates some BoLA-DRB3 alleles such as *15:01, *12:01 and *16:01 are associated with high PVL in BLV-infected cattle but BoLA-DRB3 alleles like *09:02, *02:01 and *14:01:01 are associated with low PVL32. Of the 22 epitopes, we found two epitopes—1A (317-KIKQPAILVHTPGPKMPGPR-336) and 2 (293-ILQGRGLVAAPVGQKLQACA-312) that were significantly related to cattle resistant to developing high BLV proviral load. These epitopes were located in CTD-CA and between CA/NC proteins, respectively, highly conserved regions for retroviruses , sequence variation was still present, in which most sequence variation can be attributed to a single mutation. Based on the resulting proviral mutation profile, we revealed that the mutations are driven by immune selection pressure, suggesting mechanisms of positive selection and mutation hotspots.59. However, it has been shown that peptide flanking regions (PFRs) on either side of the binding core affect peptide–BoLA-DR binding and thereby ultimately also influence the peptide immunogenicity40. Indeed, 4 hotspots were located in PFRs and have input on binding affinity. These mutations may upend the presentation of virus-derived peptides via BoLA-DR. Based on the obtained results, certain mutations reduced while other mutations increased the affinity to bovine MHCII. Additionally, some mutations exhibited a neutral affinity. On the basis of our analysis of mutations, we selected 12 mutant peptides with predicted decreased BoLA-DR-binding strength 19 and mutant peptides with predicted increased BoLA-DR-binding strength for further biophysical and functional analyses. Our study provides evidence that single nonsynonymous mutations in BLV can subvert the immune response to CD4+ T-cell epitopes.Diversified positions were preferentially located within bovine CD4+ T-cell epitopes. Of 13 hotspots, 7 were located in the 9-mer core epitopes and had predicted a significant effect on the binding affinity of BoLA-DRB3 molecules. This is consistent with what is known about the peptide-binding core of epitopes that primarily interact with the BoLA-DR antigen binding groove. The peptide–BoLA-DR binding affinity is primarily determined by the amino acid sequence of the peptide binding coreOur hypothesis was that the substitution of single amino acids in CD4+ T-cell epitope may influence the BoLA-DRB3 binding affinity and that nsSNP might be associated with variations in individual immune responses to antigens and susceptibility or resistance to disease. There are therefore many factors that make it difficult to predict peptide binding affinities to BoLA-DRB3 molecules, including the polymorphic sites of Gag epitopes. However, without functional analysis, the impact of single anchor residue substitutions on the response of CD4+ T-cells is still unclear. This study does not allow direct conclusions to be drawn concerning potential selection pressures, which shape the mutational landscape of CD4+ T-cell epitopes. This would invariably involve accounting for the BoLA-DRB3 genotype of all individuals from whom BLV genomes were sequenced. Moreover, how T-cell escape mutations within BLV are maintained during virus transmission between individuals with differing BoLA types and how viruses carrying epitope mutations affect disease severity requires further investigation.60. This may be of particular importance for BLV subunit vaccines to induce responses against an unlimited number of CD4 epitopes. These results highlight the capacity of BLV to evade cellular immune responses through sporadically emerging mutations in BoLA-BRB3 epitopes.Many CD4+ T-cell epitopes for BLV have been described in this study. The CD4+ T-cell response against BLV was associated with broad epitope recognition of, on average, 6 CD4+ T-cell epitopes per antigen per BoLA-DRB3 allele, which raises the question whether and how mutations in single epitopes affect virus control61. Moreover, preventive vaccinations based on selected peptide immunogens could become an integral part of BLV eradication programs63. Finally, the importance of immunopeptidomics should be emphasized in subsequent studies taking into account other exotic and local cattle breeds as well as the circulation of endemic BLV variants.Taking into account the very conservative and wide range of identified epitopes and, on the other hand, the lack of progress in obtaining an effective vaccine, the new discovery has a high chance of success. The new vaccine could be an important element in protecting herds against BLV infections, especially in dairy cattle, where this category of cattle is especially susceptible to BLV infectionIn the present study, BLV Gag protein was characterized by immunoinformatic techniques to identify potential T-cell epitopes. Twenty-two BoLA-DRB3 class II epitopes were available across the entire BLV Gag polyprotein, however the p24 protein was identified as the main target for recognition by antigen-specific CD4+ T-lymphocytes. The thirteen broadly conserved BoLA-DRB3-restricted CD4+ T-cell epitopes shared between BLV isolates from different countries and 9 epitopes with changes in the binding core were identified. Among them two promiscuous conserved pBoLA-(gag)peptides, 1A and 2, related to hosts that mounted a successful host–pathogen immune response  were discovered. We believe the newly-identified pBoLA-(gag) peptides, together with additional peptides that have been shown within gp51, gp30 and Tax proteins, will be important for inclusion in a multivalent antigen peptide vaccine for BLV that can provide protection against BLV infection caused by geographically distant viral strains in cattle that express different BoLA class II DRB3 haplotypes.The study was approved by the Veterinary Sciences Animal Care Committee No. AC21-0210, Canada; the Institutional Animal Care and Use Committee No. PROTO202000096 from 4/13/2020 to 4/14/2023, Michigan State University, United States; the Ethics Review Board, COMSATS Institute of Information Technology, Islamabad, Pakistan, no. CIIT/Bio/ERB/17/26 and the Bioethics Commission No. 06-18 on 30 January 2018, Almaty, Kazakhstan. Blood samples from Polish and Moldovan cattle, naturally infected with BLV, were selected from collections at local diagnostic laboratories as part of the Enzootic bovine leukosis (EBL) monitoring program between 2012 and 2018 and sent to the National Veterinary Research Institute (NVRI) in Pulawy for confirmation study. The approval for collection of these samples from ethics committee was not required according to Polish regulation .65, Moldova (n = 14)66, Pakistan (n = 20)67 and Kazakhstan (n = 21)68. Between 2020 and 2021 48 peripheral blood and serum samples from naturally BLV-infected cattle were obtained from three dairy farms of Alberta, Canada and two dairy farms of Michigan, US 69. Reactions were carried out in 30 µl final volume containing 1 × Pol Buffer B, 2.5 mM MgCl2, 115 µM of each dNTP, 0.3 μM of each primer, 0.4 U of OptiTaq DNA Polymerase and 12 ng genomic DNA. The thermal cycling protocol was initial denaturation at 94°C for 3 min; 35 cycles at 94°C for 20 s, 60°C for 20 s, and at 72°C for 1 min, followed by final extension at 72°C for 5 min. Reactions were carried out in a TAdvanced Twin PCR Thermal Cycler (Biometra). PCR products were clean-ed up using ExoSAP-IT (Applied Biosystems) by incubation at 37°C for 15 min, followed by ExoSAP-IT inactivation by heating to 80°C for 15 min. The DNA was directly sequenced (Genomed SA Company) with BoLA-DRB3 exon 2 forward and reverse primers HL030 (5′-AGATCTATCCTCTCTCTGCAGCACATTTCC-3′) and HL031 (5′-CGCGCTCACCTCGCCGCT-3′) respectively69.A 247 bp fragment of BoLA-DRB3 exon 2 containing the hypervariable domain was amplified on all DNA samples (n = 125) by a PCR as described in a previous studyhttps://www.ebi.ac.uk/ipd/) using Haplofinder script (http://bioinformatics.roslin.ed.ac.uk/haplofinder/haplofinder.py) and Python 2.7.18 software (https://www.python.org)70. To confirm that the assigned alleles did indeed match the pairwise sequence combination, a custom BLAST database was created in Geneious Prime and the data from IPD-MHC were implemented and the query centric alignment was generated using Megablast configuration for highly similar sequences and Low Complexity Filter with Scoring: 1–2 and max E-value of < 0.05.The raw sequences of the 247 bp fragment of BoLA-DRB3 gene were visualized and aligned in Geneious Prime software. Consensus sequences were generated, in which the heterozygous positions were assigned ambiguity codes according to the IUPAC coding system (Supplementary file gag gene was amplified from DNA samples (n = 125) by nested PCR using oligonucleotide primers as previously reported71  according to the manufacturer’s instructions. The following pairs of primers were used to direct sequencing as shown in Supplementary Fig. 72 for each position in the amino-acid alignment, and the entropy values were summed for each Gag domain. The DnaSP v. 6.12.03 program was used to quantify strength of selection by comparing synonymous substitution rates (dS) with nonsynonymous substitution rates (dN)73. Ratio of dN/dS < 1 was interpreted as negative or purifying selection and > 1 was interpreted as positive selection pressure.The raw sequence reads in both directions were proofread and analyzed in Geneious Prime 2021.0.1 (Biomatters Ltd). The consensus sequences were determined and deposited in the GenBank database under accession numbers OP146492-OP146601 Table . The amiFASTA format. For this analysis, a comprehensive list of BoLA molecules available in the server for prediction was updated with new additional sequences deposited in the IPD-MHC database. The prediction values represented by likelihood for BoLA antigen presentation and %Rank score were calculated. In detail, the percentile rank for each Gag peptide was generated by comparing its score against the scores of 100,000 random natural peptides74. For example, if a peptide was assigned a rank of 1%, it meant that its predicted affinity was among the top 1% scores for the specified molecule. The %Rank score of < 1.0, ≥ 1 to < 5.0 and ≥ 5.0 were interpreted as strong binders, weak binders, and non-binders, respectively. All %Rank score predictions ≤ 5.0 were considered as epitope peptides.A BoLA-DRB- peptide binding affinity prediction method, NetBoLAIIpan—1.0 was used to predict the Gag protein peptides presented by the BoLA-DRB3 molecule. The consensus Gag protein sequence calculated for the 125 sequences alignment was submitted to the server in pol gene and H3F3A gene were performed according to previously published methods76. Briefly, genomic DNA was amplified using primers and probes for pol gene and H3F3A gene, and QuantiTect Multiplex PCR NoROX master mix (Qiagen AG GmbH) according to the protocol: 95 °C for 15 min, followed by 45 cycles each of 94 °C for 60 s and 60 °C for 60 s. Ten-fold dilutions of the pBLV1 and pH3F3A plasmids from 1 × 106 copies/μl to 100 copies/μl were used as the standard to estimate BLV copy numbers. The BLV proviral load (copies/1000 cells) was calculated as [copies of BLV pol gene/(copies of H3F3A gene/2)] × 1000 cells75.The qPCR assays for the BLV The correlation between number of CD4 + T-cell epitopes and proviral load in BLV-infected cattle was calculated using the Spearman non-parametric test . The difference in proviral DNA copy number between two groups of samples with different BoLA-DRB3 genotypes was calculated using the Student’s t-test, where a P value of < 0.05 was considered to be significant. The statistical analysis was performed using STATISTICA ver. 10 (StatSoft).77 was used to identify the location of the epitopes. The structure was opened in UCSF Chimera78 and the surface representation was used to display epitopes by color.A PDB file of a putative BLV Gag structureSupplementary Figure S3.Supplementary Figure S4.Supplementary Information 3.Supplementary Information 4.Supplementary Table S1.Supplementary Table S3.Supplementary Table S4.Supplementary Table S6.


Pleurotus placentodes (PPL) and Pleurotus cystidiosus (PCY) are economically valuable species. PPL grows on conifers, while PCY grows on broad-leaved trees. To reveal the genetic mechanism behind PPL’s adaptability to conifers, we performed de novo genome sequencing and comparative analysis of PPL and PCY. We determined the size of the genomes for PPL and PCY to be 36.12 and 42.74 Mb, respectively, and found that they contain 10,851 and 15,673 protein-coding genes, accounting for 59.34% and 53.70% of their respective genome sizes. Evolution analysis showed PPL was closely related to P. ostreatus with the divergence time of 62.7 MYA, while PCY was distantly related to other Pleurotus species with the divergence time of 111.7 MYA. Comparative analysis of carbohydrate-active enzymes (CAZYmes) in PPL and PCY showed that the increase number of CAZYmes related to pectin and cellulose degradation  in PPL may be important for the degradation and colonization of conifers. In addition, geraniol degradation and peroxisome pathways identified by comparative genomes should be another factors for PPL’s tolerance to conifer substrate. Our research provides valuable genomes for Pleurotus species and sheds light on the genetic mechanism of PPL’s conifer adaptability, which could aid in breeding new Pleurotus varieties for coniferous utilization. Coniferous trees are a primary source of forest resources, generating ten million tons of conifer waste each year due to tree aging, cutting, and industrial waste. Unfortunately, most of this waste is only used as fuel, resulting in low utilization and environmental pollution . DespiteEdible mushroom production is an important component of fully utilizing agricultural and forestry waste . China iBetula . Additionally, Pleurotus species, thereby expanding our understanding of the genus. Among these 13 genomes, seven were sequenced for the first time, and they belong to the following species: P. abieticola, P. djamor, P. eryngii var. ferulae, P. giganteus, P. nebrodensis, P. populinus, and P. sapidus. While many Pleurotus species are known to grow on broad-leaved trees or Apiaceae plants, some can also be found on conifers. Despite several studies available on the phylogeny  medium overlaid with cellophane sheets. The cultures were grown in a light-free environment at 25°C for 10 days. Afterward, 300 mg of hyphae was collected and transferred to Eppendorf tubes, where they were subjected to enzymatic hydrolysis using 2% lysozyme  at 30°C for 4 h. Mononuclear hyphae were obtained by regenerating the protoplasts and staining the nucleic acid with 4′,6-diamidino-2-phenylindole  before identification under an optical microscope. The mononuclear hyphae were then cultured on a PDA medium overlaid with cellophane. The mononuclear hyphae were cultured in PDA medium overlaid with cellophane for 25°C for 15 days, and 100 mg hyphae was collected separately for each strain. Finally, the highly efficient NuClean Plant Genomic DNA extraction kit  was used to extract the genomic DNA for each strains. Genomic integrity, purity, and concentration were evaluated using 0.8% agarose gel electrophoresis, Nanodrop 2000 , and Qubit , respectively.De novo genome sequencing of PPL and PCY was performed with a 20 k library size using a PacBio sequel platform in the Engineering Research Center of the Ministry of Education of Jilin Agricultural University, China  (https://figshare.com/projects/Genome_sequencing_of_Pleurotus_placentodes_and_Pleurotus_cystidiosus/165418).y, China . The subn 1.0.0) . The Corn 1.0.0)  and Bencn 1.0.0)  were useDe novo and homologous prediction strategies were used to annotate the two genomes of Pleurotus, including the wild strain PPL from the Tibetan Plateau and the PCY strains. Four reference species, including Agaricus bisporus, Coprinopsis cinerea, Pleurotus ostreatus, and Schizophyllum commune were used for homologous prediction. Augustus (version 3.3.2), Genescan (version 3.7), GlimmerHMM (version 3.0.4), and SNAP software (semi-hmm-based nucleic acid parser) were used for de novo prediction. Then, GLEAN (http://sourceforge.net/projects/glean-gene) was used to integrate the results obtained by the two methods. The integrated results were used for subsequent analysis: 1) For functional annotation, diamond software with e value < 1e-5 was used to search genes against Nr (Non-Redundant Protein Sequence Database), KOG (Clusters of Orthologous Groups), Interpro, and SwissProt databases. Interproscan software with parameters--applications Pfam - Gene Ontology (GO) terms was used for GO annotation, and Kobas (KEGG orthology based annotation system) software with parameters default was used for KEGG (Kyoto Encyclopedia of Genes and Genomes) annotation. 2) For repeat components annotation, the assembled genome was compared with the Repbase database using Repetmasker  to determine the transposon sequence; the tandem repeats were predicted using TRF  software, including microsatellites, etc.,; 3) For non-coding RNA annotation, tRNAscan-SE software was used for annotation of transfer RNA (tRNA); rRNAmmer software (version 1.2) were used homology prediction and de novo prediction of Ribosomal (rRNA); non-coding RNAs such as small nuclear RNA (snRNA) and micro RNA (miRNA) were annotated by the Rfam database .http://www.cazy.org/). The input data consisted of proteomes of PPL and PCY. A threshold was set for the search results, such that if the alignment length was greater than or equal to 120 amino acids, the E-value had to be less than 1e-5.Carbohydrate-active enzymes (CAZYmes) in the PPL and PCY genomes were annotated using profile hidden markov models  by searching against the CAZY database (P. tuoliensis (PT), P. eryngii var. ferulae (PC), P. eryngii var. eryngii (PE), P. ostreatus (PO), P. florida (PF), Coprinopsis cinerea (CC), Laccaria bicolor (LB), Schizophyllum commune (SC), Serpula lacrymans (SL), Coniophora puteana (CP). Orthomcl software (version 1.4) was used to cluster gene families and obtain single-copy orthologues  . Firstlyhttp://abacus.gene.ucl.ac.uk/software/paml.html) was used for positive analysis. KEGG annotation of gene families were conducted by aligning the genes to KEGG database. Blast2GO was used to identify the associated GO terms.Gene families were identified using an all-against-all blastp method and clustered with the OrthoMCL software (version 1.4) . ExpansiTo obtain high-quality genome assemblies for PPL and PCY, we used monokaryon strains obtained through protoplast mononuclearization. We generated 6.7 Gb (186 x) and 4.8 Gb (113 x) of data using the PacBio Sequel platform for PPL and PCY, respectively. The assembly genome size of PPL and PCY was 36.12 and 42.74 Mb, and the contig was all 21. Our assembly also represents good contig N50 length, 3.7 M, and 3.3 M, respectively . CEGMA adenovo annotation. The result showed that PPL and PCY genomes include 10,851 and 15,673 protein-coding genes, accounting for 59.34% and 53.70% of the genome size, respectively  and 190 (43.3%) of these two species, followed with the Auxiliary Activities (AAs) and Carbohydrate-Binding Modules (CBMs), annotated to 97 (22.4%) and 88 (20.3%) in the PPL, and 106 (24.1%) and 81 (18.5%) in PCY. The number of genes annotated in GlycosylTransferases (GTs), Carbohydrate Esterases (CEs) and Polysaccharide Lyases (PLs) was relatively small, annotated to 38 (8.8%), 10 (2.3%) and 17 (3.9%) in PPL and 36 (8.2%), 13 (3.0%) and 13 (3.0%) in PCY. Further comparison with the PCY found that, AA9, CBM13, CE16, GH30, GH31, GH47, GH7, GH71, and PL1 were significantly increased, and CBM21, CBM48, GT30, GT31, and GT50 were unique CAZYmes genes in PPL. PCY contains more AA1, GH10, GH11, GH15 CAZYmes and CBM14, CE8, GH105, GH135, and GT2 were unique CAZYmes gene family. We identified differences in the number of specific pectin lyase gene modules and genes in the genomes of PPL and PCY. Both fungi have four gene modules for pectin lyase, including PL1, PL3, PL4, and PL14. While PL4 and PL14 contain the same number of genes in both fungi, PPL has a greater number of genes for PL1 and PL3 compared to PCY. Specifically, PPL has a total of 8 copies of the PL1 gene and 3 copies of the PL3 gene, while PCY has 5 copies of the PL1 gene and 2 copies of the PL3 gene.To study the difference in composition of enzymes mainly related to lignocellulose degradation, we conduct comparative CAZYmes analysis of PPL and PCY. A total of 434 and 439 CAZYmes genes were annotated in PPL and PCY genome using hmmer software, respectively . GlycosiAlthough the main components of hemicellulose are xylan and mannan, the composition of hemicellulose in coniferous wood is quite different from that in broad-leaved wood. Firstly, coniferous tree xylan do not contain acetyl groups, but have arabinose branched chains that require corresponding debranching enzymes for degradation. A total of 91 GHs (PPL 45 and PCY 46) with related functions were annotated in PPL and PCY, namely, GH1, GH5, GH30, GH43, and GH51. Among them, no difference occured in the number of GH1 enzymes. GH3, GH30, and GH51 in PPL contain α-L-arabinofuranosidase (EC 3.2.1.55) and glucuronoarabinoxylan endo-β-1,4-xylanase activities, while GH5 and GH43 in PCY contain L-arabiofuranosidase activities, which indicating PCY also has the ability to degrade hemicellulose of coniferous wood, and hemicellulose degrading enzyme is not the main reason for determining whether fungi can grow on coniferous substrate. Secondly, in general, in addition to glucose and mannose, mannan in coniferous trees also contains acetyl- and galactose branched chains, which are absense in broad-leaved trees. Compared to PCY, GH31  in PPL is 2.5 times than PCY. In addition, CBM1, CBM5, CBM13, CBM35, and CBM43 all have the ability to bind and assist in the degradation of cellulose or hemicellulose. PPL contains 75 genes, and PCY contains 69 genes, indicating PPL has more advantages in the co-enzyme of cellulose degradation. The number of GTs is relatively large in PPL, especially three unique enzymes related to mannose transfer, GT30, GT31, and GT50, which assist in the degradation of mannan. No quality difference in AA7 and AA8 related to cellulose degradation, while AA1, AA2, AA3, and AA9 with differences are Laccase, Manganese peroxide, cellobiose dehydrogenase, and lytic polysaccharide monooxygenases, all of which are related to lignin degradation. In conclusion, although the number of cellulose and hemicellulose degradation related enzymes in PCY and PPL is similar, the number of co-enzymes in PPL is significantly more than that in PCY. Therefore, PPL has the ability to degrade cellulose and hemicellulose faster.Plant pectin is mainly composed of Homogalactiuronan and Rhamnogalactiuronan. A total of 50 enzymes (PPL 27 and PCY 23) related to the degradation of these two substances were annotated, namely, GH28, GH78, CBM67, CE8, CE12, PL1, PL3, and PL4, which indicating PPL has a higher pectin degradation ability than PPL. Among the 50 enzymes, the number of PL4 genes is consistent in PPL and PCY, CE8 is a unique pectin methylesterase in PCY, and the remaining enzymes are more abundant in PPL than in PCY. Homogalacturonan is a polymer which was composed of galacturonic acid residues connected by α- 1,4-linked. GH28 is endopolygalacturonases, which can cleave the α-1,4-linkage. PL1 and PL3 are two pectate lyase, which can degrade the α-1,4-linkage between galacturonic acid residues at the non-reducing end of homogalacturonan. In terms of rhamnogalacturonan degradation, the main enzymes involved include GH78, CBM67, CE8, and CE12. Among them, GH78 isα-L-rhamnosidase enzymes, which can act on the terminal non reducing end of the rhamnogalacturonan backbone. CBM 67 can bind L-rhamnose and enhance the degradation rate of rhamnose. CE12 and CE8 are pectin acetyl esterases and pectin methylesterase, which target the backbone of rand homogalacturonan, respectively.Lignin is an important component of plant cell walls, which content is higher in coniferous trees than broad-leaved trees. Therefore, laccase, manganese peroxidase and lignin peroxidase, which are mainly related to lignin degradation, have also been annotated. A total of 70 enzymes related to lignin degradation were annotated, including 40 in PPL and 30 in PCY. The quantity in PCY is much higher than that in PPL, indicating PCY has stronger lignin degradation ability, and also reflected enzymes related to lignin degradation are not the main reason why PPL adapts to coniferous tree substrates.P. tuoliensis (PT), Pleurotus eryngii var. ferulae (PC), P. eryngii var. eryngii (PE), P. ostreatus (PO), P. florida (PF), Coprinopsis cinerea (CC), Laccaria bicolor (LB), Schizophyllum commune (SC), Serpula lacrymans (SL), Coniophora puteana (CP) , followed by PPL adapted to conifers substrate with the divergence time 62.7 MYA  . We used62.7 MYA . Other sPleurotus from the genomic level, we conducted a collinearity analysis of the genomes of the PCY adapted to the broad-leaved tree and the PPL adapted to the coniferous tree  to identify 264 and 496 positively selected genes in PPL and PCY, respectively. GO and KEGG analysis showed that the positively selected genes were significantly enriched in peroxisome, signaling pathway, AMPK signaling pathway, MAPK signaling pathway, protein processing in endoplasmic reticulum and other metabolic pathways in PPL  for degradation. Coniferous substrates have more lignin than broad-leaved tree substrates, which makes the role of glycoside hydrolase in lignocellulose degradation limited . HoweverPectin is one of the main components to make up plant cells, which was composed of homogalactiuronan and rhamnogalactiuronan. In cell wall degradation, pectin degradation is earlier than cellulose and hemicellulose, and cellulose and hemicellulose directional hydrolase genes with the highest transcriptional abundance level come from GH1, GH3, GH5, GH7, GH12 and GH30 CAZyme families . In thisConifers are rich in galactoglucomannan, a complex polysaccharide composed of mannosyl  and glucDrechslera oryzae, at concentrations as low as 0.2 μL/mL  serving as a common precursor that can form various terpenoid precursors via isopentenyl transferase. Terpene synthase then catalyzes the formation of different terpenoid skeletons, such as monoterpenes, sesquiterpenes, diterpenes, dipsesquiterpenes, and triterpenes . The annAgaricus bisporus, and tyrosine forms DOPA under the action of tyrosinase. It then reacts with polyphenoloxidase and other enzymes to form melanin  and broad-leaved substrate (PCY), and carried out phylogenetic and coniferous adaptability studies respectively. Species evolution studies reflect PPL and PCY have undergone significant differentiation in the genome. PPL is closer to the traditional Pleurotus species and differentiates from 62.7 MYA, which is 49 MYA later than PCY (111.7 MYA). Comparative CAZYmes between PPL and PCY indicate PPL has an advantage in the quantity of enzymes related to cellulose and pectin degradation, despite coniferous substrate contains higher lignin content than broad-leaved substrate, which may providing favorable advantage for faster coniferous substrate colonization. In addition, comparative genomic analysis identified geraniol degradation and peroxisome pathway should play an important role for PPL’s tolerance to conifer substrates in stress resistance. These findings provide valuable genetic resources for understanding the evolution and coniferous adaptation of Pleurotus species from the genomic level, and lay the foundation for developing new varieties of coniferous substrate utilization.Exploring the adaptability of species to coniferous substrate is an important prerequisite for the development and utilization of coniferous waste resources. In this study, we provided two genomes of


Anyone fortunate enough to have regular access to the pages of a newspaper knows that reading can become a valuable and faithful companion throughout life. A newspaper is a source of knowledge that is easily accessible, that can be consulted at any time of the day, and that always adds something to one’s experience. This statement applies to any type of magazine, be it political, sports, general information, and of course scientific too.The European Journal of Nuclear Medicine has been one of the most constant references in my professional life, and it happened since 1973 when I started working with professor Buraggi as a young assistant at the Istituto Nazionale dei Tumori in Milan. Month by month this journal helped me to increase my knowledge of Nuclear Medicine and gave me the opportunity to update my culture about innovations and technological advances in our discipline. At the beginning only the copy subscribed to by our Director arrived in our department. Professor Buraggi was jealous and kept the whole collection in his office. Each new issue was of immediate interest to the whole department and there was competition in our group for reading it. I soon came to the conclusion that it was essential to open a personal subscription, in order to have this source of news in my hands and to be able to anticipate a topic of interest to my colleagues. I remember that the direct receipt of a personal hard copy of the journal with my name printed on the package was a great achievement, a kind of emancipation in my carrier as nuclear medicine physician. How many impulses came from discussing the articles, how many ideas about the possibility of translating some recent discoveries or some original approaches into our daily routine, and how many frustrations from the fact that the realisation of some innovations would never become impossible in our realty!After almost half a century of working in our field, I look back and realise that the world of nuclear medicine has undergone major changes and developments over the past 30 years. Numerous radiopharmaceuticals have been developed, covering a wide range of diseases. In terms of technology we have seen several advances in rapid evolution: the introduction of SPECT and PET, the hybrid imaging revolution, and the introduction of artificial intelligence. Techniques, instruments, procedures, and legislation have all changed radically, including the role nuclear medicine which has become central both as subject of interest for basic and clinical research and for its integration into healthcare system. Of course the journal has also changed in many aspects and contents over the last decades. I remember the first editorial look with the white and blue cover; the replacement with the new elegant colour cover; the change of the title integrated with Molecular Imaging; the modifications of the editorial lay out; the succession of the Editors in Chief Peter Ell, Ignasi Carrio, and Arturo Chiti who marked the history of the journal; and the continuous improvement of the Impact Factor to become the most qualified international journal of Nuclear Medicine.What makes our journal so special and gives it particular importance? The European Journal of Nuclear Medicine and Molecular Imaging is the official organ of the European Association of Nuclear Medicine (EANM). The great success of the journal parallels the success of the EANM, a major organisation representing the discipline at European level, recognized by public authorities and medical and scientific societies. All members of the EANM are reached by the journal, but it gives space to a much larger community, as it also gathers voices from other scientific associations and brings together virtually all professional groups active in nuclear medicine and related disciplines: radiologists, radiotherapists, physicists, radiochemists, radiopharmacists, and technologists. Both the readers and authors represent a big community engaged in a lively exchange of information, knowledge, and proposals, with an enormous impact on clinical procedures and in particularly in the fields of oncology, cardiology, and neurology.111In-pentetreotide, 67Ga, 131I/123I MIG, and 18F FDG PET scintigraphy). I invited as co-authors the most prestigious European colleague expert in oncology . We spend nice time in several intense meetings, and we presented these guidelines as a special educational session of the journal named “blue pages.” This was an example of a joint project between the EANM and the journal to make common clinical protocols available for the international nuclear medicine community.I have had the pleasure and the honour to contribute to the European Journal of Nuclear Medicine and Molecular Imaging in various ways during my professional career. I have been a member of the editorial board. I had the opportunity to publish several original papers together with my good collaborators. I have written several letters to the Editor, many reviews, and editorials. In particular I would like to remind that in 2003, when I was the coordinator of the EANM Oncology Committee, I proposed and obtained the publication of six EANM Procedure Guidelines for Tumour Imaging, dedicated to the standardisation of different clinical protocols currently used in the clinical routine (breast and bone scintigraphy, On the occasion of 25th anniversary of the EANM, together with my friend Savas Frangos, as editors of a commemorative book, we wrote that the spirit of our discipline is based on the use of radioactivity according to the rules and ethics of good medical practice Fig. . Democri


Type 2 endoleaks remain the Achilles heel of abdominal aortic endografting. They drive imaging costs and repeat intervention. We believe that after two endovascular interventions, patients should be considered for either graft explantation or graft salvage through an open abdominal exploration. Graft explantation has been associated with increased morbidity and mortality but remains necessary in the face of non-correctible type 1a endoleaks, graft failure, or graft infection. In the majority of cases AAA expansion due to persistent type 2 endoleak is the culprit. In this situation, open repair, with oversewing of the lumbar or inferior mesenteric arteries, can be accomplished providing the seal zones and component overall zones are adequate. This approach does not require aortic clamping. We provide detailed descriptions and videos to facilitate the surgeon in performing these complex procedures. For this reason, I include links to the extensive video library of DeBakey Cardiovascular Education. The videos herein can be found on the DeBakey Cardiovascular Education YouTube channel at https://www.youtube.com/c/HoustonMethodistDeBakeyCVEducation.Recent years have seen a dramatic increase in the number of aneurysms treated with abdominal aortic endografts. Indeed, up to 70% of primary repairs are now performed with an abdominal aortic endograft. At the present time, a significant number of open aortic procedures reflect remedial secondary interventions. This represents two broad groups of patients: first, those in whom aneurysm enlargement continues to occur most often due to type 2 endoleaks; and second, those who have nonremedial type 1 endoleaks or graft infections . These o5 Typically, these patients present many years after placement of an endograft and have had frequent and numerous interventions for aneurysm growth directed at occluding either the lumbar arteries or the inferior mesenteric artery. The indication for exposing and salvaging an endograft is an enlarging aneurysm from a type 2 endoleak. Of the two types of open aortic procedures requiring remedial secondary interventions, this type is a bit more frequent and less technically challenging.When evaluating the patient who has failed catheter therapy for type 2 endoleaks, we carefully evaluate the proximal and distal seal zones to ensure that there is a good seal. Typically, this is a minimum of 10 mm proximally and ideally 20 mm distally. A good seal requires a minimum length, over which there is complete wall opposition between the endograft and the wall of the aorta or iliac artery. It is especially important to ensure there is no proximal type 1 leak. Confidence in the integrity of the seal zones is fundamental to the approach described here. Increasingly, we have been using dynamic computed tomography (CT) scans to evaluate this. Additionally, the surgeon must evaluate overlap zones within modular devices. Spending time preoperatively with a good CT angiogram (CTA), allowing the surgeon to map out where the offending lumbars enter the back of the aneurysm, proves very beneficial once the aneurysm is opened. Lumbar bleeding, which occurs at the proximal end of the aneurysm immediately below the neck, can be particularly difficult to expose due to the presence of the endograft. Having a plan for where to look for these lumbar arteries saves time and minimizes blood loss. Once these factors have been evaluated, a decision for open repair with graft salvage is based on patient comorbidities and abdominal hostility.Our hospital’s most common approach is a long midline incision with standard exposure of the abdominal aorta. We expose the neck of the aneurysm in order to place a clamp in the infrarenal position. If this is necessary, we typically use padded Fogarty clamps. As we have gained more and more experience with graft salvage, we minimize the dissection of the iliac arteries if we are satisfied with the seal zone. Once adequate proximal control has been obtained, we make a vertical incision through the wall of the aneurysm, which is then extended circumferentially immediately below the distal extent of the proximal seal zone. This entire process is performed using electrocautery due to the aforementioned thickening and inflammation in the aneurysmal wall. As an adjunct, we frequently use a running locking stitch to provide hemostasis in the aneurysmal wall. After the aneurysm is opened, the operator is presented with organized thrombus. Typically, there is no active bleeding at this point; however, if the inferior mesenteric artery is one of the sources, then oversewing the orifice from inside the aneurysm may be required early.We use an empty sponge stick and finger to gently remove the organized thrombus, taking care not to grasp the endograft graft in this process. One needs to work around in the graft, which can lay anywhere within the sac. Most commonly, it lies in the anterior sac, but the operator must determine this by review of the preoperative CT scan. This is important because one must get behind the graft to address the lumbar arteries. As thrombus is removed, bright red blood is encountered when entering the endoleak cavity. Cell saver suction is recommended since bleeding can be fairly dramatic. Entering the cavity, which also can be located on the CT scan, allows one to follow the bleeding down to a source; when there are many, this can be confusing.We attempt to remove all the thrombus down to the wall of the aneurysm. The ligation of all the lumbar artery origins is performed with 2-0 silk pop-off sutures. Once again, knowing how many lumbar one is looking for, and their location, is very helpful. The most difficult lumbars to address are those that occur from the aneurysm wall immediately after the neck flares into the aneurysm because the operator is constrained by the presence of the endograft. It certainly crosses the operator’s mind that there is a type 1 endoleak, which is why a diligent review of the preoperative CT scan is reassuring. Once the immediate bleeding is controlled, it becomes necessary to spend time removing all the thrombus from the aortic wall to prevent delayed hemorrhage.Since the intervention is being performed for an enlarging aneurysm, there is usually more than adequate sack to cover the graft. If bleeding from the aortic wall is ongoing, this is the point when we run and lock a suture along the length of the wall. Prior to closure, the operator also may decide to trim some of the aortic wall to provide a tighter closure over the underlying device.We used to fill the aneurysm sack with omentum, but I do not believe this is always necessary. Another adjunct we routinely use, which can be performed early after exposure or after evacuation of the hematoma, is to place three or four horizontal mattress pledgeted 3-0 prolene sutures through the wall of the aorta, encompassing the underlying device. These are placed at least in the 12 o’clock, 3 o’clock, and 9 o’clock positions. Our thought process is to ensure secure anchoring of the proximal aortic device. Depending on the adequacy of the iliac arteries, we may place similar pledgeted sutures distally. Closure of the abdomen then continues in standard fashion.6 Other indications include persistent type 1 endoleak and failure of the integrity of the device. However, increasingly in the case of proximal leak or device failure, endovascular salvage techniques are available. This discussion primarily addresses aortic graft infection.8The absolute indication for explanting an abdominal aortic endograft is graft infection. This is associated with an aortoduodenal fistula in up to 25% of cases.Once the diagnosis of graft infection has been established, attention focuses on the technique for graft removal . This deCareful evaluation of the proximal seal zone, perirenal segment, and visceral segment of the aorta is fundamental to performing this procedure adequately and safely. These decisions must be made against the background of patient comorbidities and risk. In some cases, a full thoracoabdominal approach may be ideal to permit complete device explantation. However, many patients cannot tolerate such a procedure. In general, we find that leaving behind the metallic suprarenal struts is a reasonable compromise to permit this procedure to be performed through a transabdominal approach alone. A midline laparotomy should be adequate if the plan is to remove all material below the renal arteries, although a retroperitoneal approach provides a little more latitude in moving clamps up into the visceral and supraceliac segment. The following describes steps for graft explantation:Strongly consider placing ureteric stents.Obtain supraceliac control. Expose the aneurysm and the iliac arteries essentially in the same way as the open aortic aneurysm repair, although this is likely to be more challenging due to periaortic inflammation.In the overwhelming majority of cases, divide the left renal vein.Get circumferential control of each renal artery and prepare for suprarenal clamping.Perform clamping proximally and distally using padded Fogarty clamps, which mold better around the aorta and the underlying endograft.Open the aneurysm in a relatively standard fashion with longitudinal electrocautery, and extend circumferentially immediately distal to the seal zone.As the aneurysm is entered, be prepared to encounter purulence, infected thrombus, and occasionally bleeding from the lumbar artery, which need to be suture ligated.Take cultures from any purulence, thrombus, and the graft before sending for culture.Usually, divide the endograft using heavy scissors. This is done by choosing an area between the stent rings, if possible.Rotate the proximal end of the device toward the abdomen and gradually work along the stent graft to free it from the proximal attachment zone.9Free up the graft in the perirenal space. A number of techniques can be used to do this. We have no personal experience in dealing with infected devices that have deployed endoanchors, which would markedly increase the complexity of completely removing all fabric.Focus on ensuring that adequate aortic wall is left after the explantation to permit safe suturing of a proximal aortic anastomosis.https://www.youtube.com/watch?v=SZmjBSDZhTo.)Reconstructive options  include Create the proximal anastomosis by ensuring that adequate bites of healthy aortic tissue are available, which is key for this step.The distal anastomosis may be equally challenging although, in general, the explantation of the entire endograft is feasible. The surgeon must decide whether the tissue is healthy enough for an iliac anastomosis or whether the bypass should be extended to the common femoral arteries. Again, careful examination of the preop CT scan will guide the distal end of the stent graft relative to the bifurcation.Since this dissection around the iliacs also may be challenging due to the perivascular inflammation, the presence of ureteric stents can be very helpful. They can be palpated, but not easily.Almost universally, whenever feasible, mobilize the omentum to cover the graft and close the aneurysm sac. Because these complex procedures rarely follow a straightforward postoperative course, I recommend treating these patients with lifelong antibiotics tailored to the intraoperative cultures.10The postoperative management of these patients may be very complicated, and one needs to carefully follow the renal function. Main concerns long-term are for recurrent infection of any retained endograft and reinfection of the graft used for reconstruction. Retaining portions of the graft is associated with high risk of infection.11 For example, if sutures will not hold in the perirenal space, it may be necessary to move the anastomosis up closer to the superior mesenteric artery. In this situation, a hepatorenal and splenorenal bypass may be of value but will markedly increase the magnitude of the operation, so it must be determined in advance.12 If an anastomosis cannot be performed, then one alternative, although somewhat discredited, is to oversew the aortic stump (13Numerous alternate techniques may be of value in certain circumstances .11 For eic stump . This, o15The procedure, when performed for type 2 endoleak with graft salvage, can be performed safely. However, procedures performed for infected endografts, in which explantation is required, are associated with up to a 20% mortality rate.17 although it is beyond the scope of this article to address all of these variances. As endografts begin to be used for the thoracoabdominal segment and the arch, salvage operations will continue to evolve and become more complex. Techniques for graft explantation have been developed for almost every part of the aorta,


This systematic review assessed the available evidence on the survival and success rate of zirconia and titanium implants. As secondary outcomes, aesthetic, radiographic and clinical parameters, as well as biological and mechanical complications, were considered.A systematic search was performed up to March 2022 to identify CCTs/RCTs comparing zirconia and titanium implants with a minimum of 12 months of follow-up. Meta-analysis was performed when ≥ 2 articles with similar characteristics were retrieved.P = 0.0938). The success rates were 57.5–93.3% and 57.1–100% for zirconia and titanium implants, respectively. The pink aesthetic score (PES) was higher for zirconia (10.33 ± 2.06 to 11.38 ± 0.92) compared to titanium implants (8.14 ± 3.58 to 11.56 ± 1.0).Four published articles with two RCTs (2 different patient populations) with 100 zirconia and 99 titanium implants that were followed up over 12–80 months were selected out of the 6040 articles. A non-statistically significant difference between zirconia and titanium implant survival at 12 months was suggested . Future RCTs are warranted to evaluate the long-term outcomes of zirconia implants.Zirconia implants may be the procedure of choice, particularly in the aesthetic zone, since they show a similar survival and success rate as titanium implants on a short-term follow-up.—CRD42021288704 (PROSPERO).Systematic review registration numberThe online version contains supplementary material available at 10.1007/s00784-023-05242-5. The replacement of missing teeth with dental implants is a widely accepted treatment procedure, with well-documented long-term stable results –3. IntroAs a consequence of the increasing aesthetic standards in the field of implant dentistry and the concern of sensitivity to titanium, there has been a growing interest towards metal-free implant rehabilitations. Moreover, about 9.7% of implants display an aesthetic complication over a 5-year period . These aZirconia (zirconium dioxide) was introduced in the 1990s into the field of dentistry, and it showed superior biomechanical properties compared to other ceramics . Due to Commercial zirconia implants were initially available only as one-piece implants. However, they displayed various limitations such as the lack of angled abutments to correct misalignment and the fact that cementation was the only option for connecting prosthodontic elements to one-piece implants, with the risk of leaving excess luting cement in the submucosal area, particularly when inserting the implants deeper in the aesthetic zone . This lePrevious systematic reviews have concluded that one- and two-piece zirconia and titanium implants demonstrate no significant difference in terms of bone-to-implant contact, removal torque or implant survival , 25. ConThis review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement , and theIs there a difference in the survival and success rate of zirconia implants as compared with the titanium implants post 12 months of loading, as reported by randomised controlled studies and controlled clinical trials?The focus question for this review based on the PICOS  was, Is Type of participants (study population): systemically healthy, partially or completely edentulous subjects receiving one or more dental implants;Intervention (test group): zirconia implants;Comparison (control group): titanium implants;Outcomes: primary outcomes: implant survival and success (as defined by the authors of each study); secondary outcomes: marginal bone loss, implant failures, aesthetic outcomes , visual analogue scale), patient-reported outcome measures (PROMs) and incidence of biological complications and adverse events, including peri-mucositis, peri-implantitis and implant mobilityTypes of studies: randomised controlled trials (RCTs) and case-controlled trials (CCTs) with a minimum of 10 patients per arm and followed up for ≥ 12 months post loading.Inclusion criteria based on the PICOS were as follows:A systematic search was performed in MEDLINE via OVID, Embase, Scopus and Cochrane Database ) in October 2021 and updated again on 31st March 2022 using the same search strategy developed by one reviewer and that combined MeSH term and free text . Grey literature was searched in http://www.greynet.org/opensiglerepository.html, and soon-to-be-published manuscripts were searched by contacting research groups with an interest in adjunctive therapies. Clinicaltrials.gov was investigated to identify potential ongoing or already completed RCTs/CCTs meeting the inclusion and exclusion criteria. Conference abstracts were excluded, and no language restrictions were applied.In the attempt to include both published and unpublished data, a specific thesis database, A two-stage screening was carried out in duplicate and independently by two reviewers . Studies were assessed based on their titles and abstracts first, and those studies that met the inclusion criteria were then screened for full-text analysis by the same independent reviewers. Any disagreement was resolved by discussion, and if necessary, a third reviewer (EC) was consulted. Reasons for study exclusion at the full-text stage were reported, and agreement at each of the two-stage screening processes was calculated using Kappa statistics.Two reviewers  independently extracted and recorded study data on ad hoc forms. In case of missing or unclear information, the authors were contacted by email to provide clarification or missing information. In case of missing or incomplete data and the absence of further clarification by study authors, data were excluded from the analyses.In particular, the data extrapolated from each publication included authors’ names, year of publication, study design, trial registration, country of recruitment and treatment, funding status, population characteristics , implant characteristics  and prosthetic parameters . Moreover, data on the primary outcomes  and secondary outcomes  were extrapolated. Articles dealing with the same study population but simply reporting on different follow-ups were grouped together during data extraction as indicated in the Cochrane Handbook .Cochrane Handbook for Systematic Reviews. As such, the risk of bias in non-randomised studies of interventions (ROBINS-I) tool was employed for CCTs, and the revised Cochrane risk-of-bias tool for randomised trials (RoB 2) (updated October 2018) was employed for RCTs [Quality assessment and risk of bias for all included papers were conducted by two reviewers  following the recommendations of the for RCTs . Recommep*q*n/n − 1, where p is the probability of success (surviving implant), q the probability of failure (q = 1 − p), and n represents the number of implants placed. We compared titanium and zirconia implant survival by calculating the differences between the means of the two implant groups (titanium vs. zirconia) and their 95% confidence interval (CI). Combined analyses were assessed through a random effect approach, and the forest plot was drawn with StatsDirect 3.2.7 (StatsDirect Ltd). Heterogeneity was assessed using the I-squared tests. Given the small number of studies included, the publication bias was not assessed [I-squared for the same reason. A p value < 0.05 was considered statistically significant.For the primary outcomes , mean values along with 95% confidence intervals were used to summarise data for each treatment group. The unit of analysis was the implant. Quantitative data analysis was performed considering implant survival after 12 months of follow-up. Implant survival was obtained as the number of available implants at the follow-up divided by the number of implants placed at baseline. We calculated the survival standard deviation by extrapolating the square root of the variance, according to the formula assessed , and StaA total of 6040 potentially relevant articles was initially identified. After the first-stage screening based on titles and abstracts, 5982 articles were excluded, and 58 articles were selected for full-text analysis. After full-text assessment, 54 articles were excluded  Table . A higheSince the criteria applied to calculate implant success were heterogenous amongst the studies, no meta-analysis was performed. Nevertheless, the success rates tended to be lower for zirconia compared to titanium implants, ranging from 57.5 to 93.3% and from 57.1 to 100%, respectively  Table .Secondary outcomes are detailed in Table From the available data, only 1 titanium implant showed signs of peri-implantitis at the 80-month follow-up period, while 3 one-piece zirconia implants had mechanical complications (implant fracture at 1-year follow-up). The PES ranged from 10.33 ± 2.06 to 11.38 ± 0.92 and 8.14 ± 3.58 to 11.56 ± 1.01 for two-piece zirconia and titanium implants, respectively.Although our protocol included other secondary outcomes, such as interdental papilla fill, visual analogue scale and PROMs, no data were available in this respect from the included clinical trials.Risk of bias assessment was done using the ROB-2 assessment tool Fig. . OverallThis systematic review aimed to compare the survival and success rates of zirconia and titanium implants with a minimum of 12 months of follow-up after functional prosthetic loading. In contrast to previous systematic reviews that focused on animal studies or non-comparative studies , 36, 37,Previous literature  suggesteOne-piece implants have a rough body for intraosseous placement, while a machined smooth collar emerges through the soft tissues. On the other hand, two-piece implants are designed for submerged healing, which claims to reduce the initial bone resorption . Also, iIn a randomised trial by Cannizzaro et al. , a surviWhile implant survival was well defined, implant success criteria were heterogeneously described in the included studies. The studies by Payer et al.  and KollWhile previous short-term studies indicated success rates for zirconia implants ranging from 93 to 100% , 48, ourOver the past 4 decades, titanium has often been considered the material of choice for dental implants due to its high survival rate (97.2% at 5 years and 95.2% at 10 years), coupled with biocompatibility, low corrosion and high strength , 50. HowZirconia implants have shown to present a lower inflammation rate compared to titanium implants due to lower bacterial attachment . In our This systematic review presents some limitations. Since research for zirconia implants is in its incipient stage and still considered an emerging treatment modality, most available literature on this topic is based on animal model studies, case series or in vitro analyses. Considering that RCTs and CCTs offer the best evidence for therapy efficacy and effectiveness , we onlyFuture investigations are warranted to compare the long-term survival and success outcomes, along with any potential late mechanical and biological complications. It would be relevant also to assess the effects of guided bone regeneration  in association with zirconia implants. It is also suggested that three-dimensional radiographic assessments should be considered in future studies to allow a 3D evaluation of peri-implant bone stability, including buccal and lingual/palatal bone levels around zirconia implants. Moreover, it is also recommended that patient-reported outcome measures are investigated , since pThere is a limited number of CCTs/RCTs in the literature comparing titanium versus zirconia implants,; hence, no robust conclusions can be made. Despite a trend for increased failure rate in zirconia implants, results from the current review do not support an increased survival rate of titanium compared to zirconia implants in the short term (12 months). While zirconia implants may potentially be advantageous in the aesthetic area, no clear advantage could be inferred based on the few RCTs retrieved in the literature. Likewise, the limited data available do not allow us to draw robust conclusions in relation to the prevalence of biological complications and inflammatory scores. A higher incidence of fracture for zirconia implants was reported in one study. Future RCTs controlling for confounding factors and considering clinical, radiographic and patient-reported outcomes are needed to evaluate the long-term success and performance of zirconia implants.Supplementary file1 (DOCX 16 KB)Supplementary file2 (DOCX 17 KB)Below is the link to the electronic supplementary material.


Starch-based biodegradable films have been studied for a long time. To improve starch properties and to increase film characteristics, starch is commonly modified. Amongst different types of starch modifications, oxidation and heat moisture treatment are interesting to explore. Unfortunately, review on these modifications for film application is rarely found, although these starch modifications provide interesting results regarding the starch and film properties. This paper aims to discuss the progress of research on oxidized and heat moisture-treated-starch for edible film application. In general, both HMT and oxidation modification on starch lead to an increase in film’s tensile strength and Young’s modulus, suggesting an improvement in film mechanical properties. The elongation, however, tends to decrease in oxidized starch-based film, hence more brittle film. Meanwhile, HMT tends to result in a more ductile film. The drawback of HMT film is its lower transparency, while the opposite is observed in oxidized films. The observation on WVP (water vapor permeability) of HMT starch-based film shows that the trend of WVP is not consistent. Similarly, an inconsistent trend of WVP is also found in oxidized starch films. This suggests that the WVP parameter is very sensitive to intrinsic and extrinsic factors. Starch source and its concentration in film, film thickness, RH (relative humidity) of film storage, oxidation method and its severity, plasticizer type and its concentration in film, and crystallinity value may partly play roles in determining film properties.  Plastic packaging has become a growing environmental concern due to its extremely slow degradability characteristics, leading to soil, water, and crop contamination. Growing effort has been applied to solve the environmental problem through an application of biopolymer-based packaging, which is known to be degradable. Biopolymers commonly used as a degradable packaging matrix include carbohydrates, proteins, and lipids . AmongstAs a carbohydrate polymer, starch can be found in almost all regions throughout the world. Apart from the main sources of starch, such as wheat, potatoes, and rice, other sources are considered as potential alternatives, including corn , arrowroEdible film has recently gained increasing interest from researchers and food industry ,15. ThisAlthough starch is a promising biomaterial for edible packaging, native starch requires modification to possess these expected properties. This is because the film made from native starch often displays high sensitivity to water, brittleness, and low mechanical properties . To overEdible film can be defined as a thin layer of edible materials that is safe for consumption and used as food packaging to protect food against external factors that stimulate food deterioration, such as moisture, oxygen, light, contaminants, and many other aspects. Materials used to manufacture edible films are regarded as eco-friendly materials because they are made of edible, versatile, renewable biopolymers that can be degraded in the environment. Edible film materials are synthesized from plants, animals, and marine biota or derived from natural food-grade polymers, such as polysaccharides, proteins, or lipids ,32,33,34Apart from their ecofriendly characteristics, edible films also exhibit promising abilities to protect food products, improve organoleptic properties of packaged products, and provide nutritional supplements, flavors, dyes, antimicrobial substances, and antioxidants. Furthermore, the product’s original appearance can be maintained for a longer period .2, CO2, water vapor permeability, and to reduce the rate of destructive chemical reactions, of physical changes, and of microbial growth so that the quality of the packaged products can be maintained. Edible films are generally used as primary packaging. Therefore, the edible films are not directly exposed to factors, such as handling, storage, and transportation [Edible film serves to control Oortation . In the manufacture of edible films, plasticizers, such as glycerol and sorbitol are added to increase the flexibility of the film . SeveralEdible films have been widely used and applied to food, especially highly perishable products, such as horticulture products. In principle, edible film packaging protects food products during the supply chain, including distribution, storage, and marketing .Edible films can extend the shelf life of food products by inhibiting moisture migration and the loss of volatile compounds, reducing respiration rates, and delaying changes in texture properties. In addition, edible films are excellent coatings or membranes for fats and oils ,32,35,38Edible film preparation includes the dry method and the wet method. The dry method is carried out by processing biopolymers, such as proteins and polysaccharides, at low moisture content in such a way that the film is formed due to their thermoplastic properties. This method comprises extrusion, injection, and compression molding ,42. The The wet method is also known as the casting method. In this method, film is formed by solubilizing or dispersing biopolymers in suitable solvents, such as water or alcohol. The heating-while-mixing process is applied to solubilize and to homogenize the dispersion. The blended mixtures are then cast onto flat, leveled, non-stick plates, followed by a drying process at ambient temperature . In thisAnother emerging method, which is deemed as an effective approach to form films, is the electrospinning technique. This technique results in a fibrous film with interesting properties . ElectroStarch is one of the few renewable resources that can form films and meets all the key criteria, including ease of availability, high extraction yield, nutritional value, affordability, biodegradability, biocompatibility, and edibility, thus possessing functional qualities. It is, therefore, a potential substance for edible coatings and films ,52,53. IHowever, native starch has various undesired shortcomings due to its semi crystalline (20–45%) structure, including its hydrophilic nature, poor solubility, poor mechanical qualities, uncontrolled paste consistency, and low freeze–thaw durability during film development . Many mo2O2), sodium hypochlorite (NaClO), sodium periodate (NaIO4), nitrogen compounds , organic oxidants, as well as metal compounds (CrO3). However, among the wide choice of oxidizing agents, sodium hypochlorite (NaOCl) and ozone (O3) are some of the most used modifying agents [Oxidation is classified as one of the chemical modification methods widely used to modify starch. Oxidation modification involves the oxidation reaction of primary or secondary hydroxyl groups, which results in carbonyl or carboxyl groups . The schg agents ,58,59. Starch oxidized by ozone can be modified in dry form or in the form of aqueous suspension. In terms of processing practicality, dry starch ozonation possesses several advantages over its wet counterpart. Dry starch ozonation is not affected by pH of milieu during ozonation. Furthermore, it does not require a re-drying step following ozonation, thereby using less energy and having a shorter time of oxidation. Dry starch ozonation, also known as gaseous ozonation, has been proved to alter granular morphology of starch . CarbonyCommonly, starch modified with oxidation possesses low viscosity, high thermal stability, low retrogradation, high paste clarity, good film forming, and high binding properties. Besides being widely used as a food additive in the food industry because of its specific properties, oxidized starch also is commonly applied in other industries, such as in textiles or paper fabrication . When apHeat moisture treatment (HMT), a physical  method, is used to modify the starch structure and functional properties without damaging the granular structure of the starch. The process is strongly influenced by several factors, including the level of humidity, type of starch, heating time, and temperature . In prinHMT modification leads to the disruption of starch crystalline structure and dissociation of double helix structures. At the end of the process, rearrangement of the disrupted crystalline structure occurs. These changes strengthen the interaction between amylose and amylopectin branching ,63. As aThe increase in the physicochemical properties of HMT starch has broad potential for industrial and laboratory applications that require starch with high gelatinization temperature . When apFilm properties made from oxidized and HMT-starch, such as mechanical properties, permeability to water vapor and gas, film solubility, transparency, and mechanical properties, are presented in 2 gas are given by their lowest permeability [Adequate food packing depends on the packaging material’s capacity to prevent or reduce gas or moisture transfer between the food and the environment. The best barrier characteristics of edible films against WVP and Oeability . The valeability ,83.Films made from HMT rice starch exhibit higher WVP than native starch films . A similIt is not clear whether the starch source affects the WVP of HMT film. However, film thickness has been found to affect the WVP of film ,85. More2 are rarely found. A study on oxidized cassava starch-based film shows an increased permeability of film to O2 [Ozonized cassava starch-based film shows an increase in oxygen permeability and WVP . The inclm to O2 . Oxidation of starch allows the formation of carboxyl and carbonyl groups. Initially, hydroxyl groups of starch are oxidized to carbonyl groups. Further oxidation forms carboxyl groups, along with an increase in the hydrophilicity of oxidized starch. Oxidation level is estimated according to the number of carboxyl and carbonyl groups of the oxidized starch ,88.The discrepancy in the WVP trend of oxidized films may be explained by several factors. The first one is the presence of cracks in the film. Cross-section observation on ozonized cassava starch-based film shows the presence of cracks, despite its compact structure and smooth surfaces . These cControlling the wettability parameters, such as contact angle, surface energy, surface tension, and forces of adhesion and cohesion, is crucial for the success of edible films . ContactA study on ozonized cassava starch-based film shows that the contact angle of film decreases with the increase in ozonation time, suggesting that the film is more hydrophilic with ozonation. Interestingly, when analyzing its solubility, the film is less soluble with ozonation . Given tFilm with dually modified (OSA-HMT) sago starch matrix indicates its higher initial water contact angle compared to its native starch film, suggesting that dual modification increases the hydrophobicity of the film surface. A single OSA modification of starch leads to an increase in the hydrophobicity of the film, while the contact angle of single HMT starch film failed to be measured because the surface could not hold the water drop on the surface. Therefore, the increase in the hydrophobicity of film can be attributed to the role of OSA starch. The effect of HMT starch on the surface hydrophobicity is not clear . The transparency of starch paste is essential because it is related to the visual acceptability of edible film. Oxidation of starch results in more transparent film, as observed in the study on ozonized cassava starch-based film. Better clarity, however, was observed when the starch was ozonized at longer time, suggesting the requirement of more severe oxidation . A studyFilm matrix made from HMT rice, potato, and amaranth starch displays lower transparency ,70,73. SMechanical strength is an important parameter, referring to the ability of film to withstand an applied load without failure or deformation. The parameters used to describe the mechanical strength are Young’s modulus, elongation, and tensile strength. Tensile strength is the maximum force (stress) that a given substance can withstand without tearing apart. Young’s modulus indicates a stiffness of material expressed as the ratio between the stress and the strain of the material. Meanwhile, elongation, which is a measure of material ductility, is expressed as the ratio between changed length and initial length of the material.Film made of ozonized cassava possesses higher tensile strength and Young’s modulus, but less elongation . SimilarAlthough a study on oxidized potato starch-based film found a decrease in tensile strength , most stFilm prepared from HMT starch also generally shows an increase in tensile strength, elongation, and Young’s modulus, as observed from the studies using potato, amaranth, plantain banana, and sweet potato starch ,73,74,79Broadly speaking, as far as the mechanical properties are concerned, both oxidized and HMT starch tend to result in a film with higher tensile strength and Young’s modulus. The elongation tends to decrease in oxidized starch based-film, hence resulting in more brittle film. That is, HMT starch tends to result in a more ductile film. Crystallinity is a crucial factor in determining starch’s mechanical and thermal properties. Crystallinity is an essential property of polymers that shows the bonds between molecular chains to produce a more ordered molecular arrangement. High crystallinity properties cause increased stress and stiffness. Each polymer chain structure from different starch sources has a different crystallinity. The degree of crystallinity is directly related to the amount of amylopectin, while the amorphous phase is directly related to the amylose contained in this polymer . CrystalStarch modification could affect not only its crystalline type, but also crystallinity level. Observation on sago and sweet potato starch, modified with HMT, shows an increase in its relative crystallinity ,79. A crThe oxidation of sorghum starch does not change its crystalline type, nor its relative crystallinity . HoweverA study on the film made from oxidized cassava starch indicates that the film crystallinity increases compared to film from native cassava starch . This reHMT modification of sago starch increases starch thermal stability, as indicated by the increase in the onset (To), peak (Tp), and conclusion (Tc) of gelatinization temperature and the decrease in gelatinization temperature range (ΔT) and its enthalpy . This obThe parameters of gelatinization temperature measured by DSCs, such as To, Tp, and Tc, are the indicators of the degree of starch thermal stability. HMT and oxidized starch display a trend of higher thermal stability compared to their native forms. However, in the packaging matrix, not only starch affects the packaging thermal properties, but also other compounds added to the matrix, such as plasticizer. For instance, glycerol, which is commonly used as plasticizer, can decrease film thermal stability when added into film formulation .Modified starch (oxidation and HMT)-based edible film displays interesting features in terms of mechanical properties, permeability, transparency, and solubility. Starch modification by HMT and oxidation results in an increase in film’s tensile strength and Young’s modulus. Oxidized starch-based films tend to possess lower elongation, while the opposite holds true for HMT films. In terms of film transparency, HMT leads to less transparent films, which might be related to the high HMT temperature during starch modification. On the other hand, oxidized starch-based films exhibit higher transparency.In terms of WVP (water vapor permeability) of HMT and oxidized films, the variations of this property are not consistent from one study to another. This property is not necessarily related to the solubility parameter and the contact angle value, as the contact angle value represents the film surface property, while the WVP is governed by many other factors, such as film structure, thickness, area, etc.Broadly speaking, film properties are determined by factors, such as starch source, oxidation level, film thickness and crystallinity, relative humidity (RH) at mechanical properties measurement, plasticizer type used in the film, the presence of cracks or holes, etc. Oxidation levels could result in different degrees of crystallinity, leading to different film properties. Given the interesting result of, particularly, mechanical properties and the inconsistent results for other film properties, future research should be directed toward revealing and proving the factors affecting the film properties. Oxidation level must be taken into account. Furthermore, the film should be prepared in such a way that the thickness is similar for all tested films. As such, the film comparison properties would be more reliable. Application of both HMT and oxidized starch-based film is also interesting to further examine.