The peanut allergens Ara h 2, h 6, and h 7 are potent allergens and can trigger severe reactions. Ara h 7 consists of three isoforms differing in their ability to induce basophil degranulation, whereas the ability of Ara h 7.0201 is comparable to Ara h 2 and 6 as shown in previous literature.To identify linear epitopes of Ara h 7.0101, Ara h 7.0201 and Ara h 7.0301 recognized by IgE and IgG4 from patients sensitized to Ara h 7 and to investigate their potential to elucidate divergent abilities of the Ara h 7 isoforms in inducing basophil activation.Linear epitopes recognized by IgE and IgG4 were mapped by peptide microarray analysis containing 15‐mer peptides of Ara h 2.0201, 6, 7.0101, 7.0201 and 7.0301 and 39 peanut allergic patients sensitized to Ara h 7 (discovery). For validation, 20‐mer peptides containing the minimal epitope and surrounding amino acids were incubated with 25 sensitized patients and 10 controls .Three out of 14 linear epitopes were unique for each isoform  but scarcely recognized by IgE. The main linear IgE epitope (aa 51‐57) located in the long flexible loop of all Ara h 7 isoforms was bound by antibodies from 31% of the patients . Regarding IgG4, 55% of the patients recognized an epitope present on all isoforms (aa 55‐65), whereas epitope aa 129‐137, only present on Ara h 7.0101/0.0301, was recognized by 38% of the patients. Recognition was highly individual, although 20% of the patients recognized any linear epitope neither by IgE nor by IgG4 despite a low mean z‐score of ≥ 1.7. Remarkably, only 50% of the patients recognized one or more epitopes by IgE.Ara h 7 isoforms share many linear epitopes being easily accessible for antibody binding. Unique epitopes, essential to elucidate divergent potencies, were scarcely recognized, suggesting a crucial involvement of conformational epitopes. While sensitization to Ara h 8 often results in mild reactions such as oral allergy syndrome, Ara h 2 and 6 can induce severe reactions, including anaphylaxis.For diagnosing peanut allergy, IgE antibodies against Ara h 2 and Ara h 6 are known to have a very high positive predictive value.22.1For identifying linear epitopes, sera with specific IgE to peanut extract (ImmunoCAP) were screened by EUROLINE  for sIgE‐binding to heterologously expressed Ara h 7.0201. Overall, 39 sera with sIgE levels ≥16 intensity units (EL) for Ara h 7.0201 were applied for the peptide microarray analysis, and peanut allergy was confirmed by food challenge according to the international consensus protocol or experienced physician diagnosis (discovery cohort).2.2For the discovery phase, a peptide microarray with overlapping 15‐mer peptides was commercially obtained (PEPperPRINT), comprising the sequences of Ara h7.0101, Ara h 7.0201 Ara h 7.0301 (offset: 1), Ara h 2.0101, and Ara h 6.0101 (offset: 2). According to the experiences of PEPperPRINT, 15‐mer peptides have the optimal length to provide sufficient sensitivity without significant induction of secondary structures. All peptides were printed as triplicates with a 3 amino acids linker  to prevent binding of negatively charged fluorescent dyes to positively charged amino acids close to the array surface. For validating the discovered minimal epitopes, a new microarray layout was applied. For each epitope, a 20‐mer peptide was designed containing the minimal epitope and surrounding amino acids, replenished with glycine and serine amino acids.2.3All dilutions and washing steps were performed in working strength universal buffer . Patient samples were diluted 1:4 and incubated on the microarrays at 4°C overnight. For the detection of bound IgE and IgG4 antibodies, the arrays were incubated with biotinylated anti‐IgE antibody  and biotin anti‐human IgG4 coupled with Neutravidin DyLight 680  for one hour at room temperature. After washing, the arrays were incubated for one hour at room temperature with fluorescent Neutravidin DyLight 800 (Thermo Fisher), diluted 1:5000 for IgE detection. After washing with dipping buffer (1 mmol/L Tris‐HCl pH 7.4), the peptide microarray slides were dried and scanned with a Licor Odyssey Imager at a wavelength of 800 nm (intensity: 10). Image focus was set to 0.8 mm, and an image resolution of 21 µm was chosen.2.4Fluorescent signals were extracted using PepSlide Analyzer Software (SICASYS) and exported to CSV files. For data evaluation, logarithmic signal‐noise‐ratios (S) were calculated for each peptide according to:These S‐values were normalized against the S‐values of blank spots on the array, resulting in a z‐score defined as:P < .05;*); Z > 2.4 ; Z > 3.0 ; Z > 4.0 (P < .0001). Peptides were only considered if the coefficient of variation for the triplicate of each peptide was lower than 50%. Recognized peptides were defined as epitopes if 3‐5 contiguous peptides with a mean z‐score ≥ 1.7 were detected.Significance levels of positive peptide binding were defined based on z‐scores as followed: Z > 1.7  according to manufacturer's instructions. Briefly, sera were applied  overnight, and after washing three times with universal buffer, bound IgE was detected by an anti‐human IgE and anti‐human IgG2.63D structure of Ara h 7.0201 was assessed by Modeller software.2.7P‐value (P < .0001). Overall, the structure was of high quality, apart from the flexible loops which are also experimentally difficult to assess. The quality assessment is shown in the Supplementary  and SPSS Statistics 21 (IBM Corporation). P values ≤ .05 were considered as statistically significant.The baseline data were statistically analysed using one‐way ANOVA or Mann‐Whitney 33.1To define unique epitopes of Ara h 7 isoforms, we mapped linear epitopes of these proteins by peptide chip analysis using patients’ sera with IgE levels for Ara h 7 ≥ 16 intensity units (corresponding to CAP‐class > 2). Patient characteristics are shown in Table 3.2All patients showed individual linear epitope recognition patterns as shown in Figure 3.3In a previous study, the ability to induce basophil degranulation was studied for all Ara h 7 isoforms.3.4A complete overview of identified linear epitopes is shown in Figure 3.5The location of epitopes was shown by applying epitope recognition patterns of four individual patients on the 3D model of Ara h 7.0201 , located in the long flexible loop, was recognized by only 32.5% of the patients. Other epitopes were detected by even less patients. Limited IgE‐binding to linear epitopes was also observed for Ara h 6 and to a fewer extent for Ara h 2. For Ara h 6, seven linear IgE epitopes were identified whereof the main epitope  was recognized by only 30% of the patients, comparable to our data for Ara h 7 (31% for epitope E). Regarding Ara h 2, linear epitopes were recognized by a greater number of patients, varying from 75% to 100% of the included patients depending on the study considered .Recognition of linear Ara h 7 epitopes by individual patients was highly distinct, stretching from multiple to none linear epitopes recognized by IgE. Individual recognition patterns were also observed for Ara h 2 in previously conducted studies.Linear epitopes were mostly located in the flexible loops of the 3D protein model. Due to these loci, the epitopes are accessible for antibody binding, but also for enzymatic degradation. Mainly epitope E was located on the longest flexible loop of the Ara h 7 isoforms. This is in line with the main epitopes of Ara h 2 and 6 showing similar loci. However, unique epitopes of Ara h 7.0101 and Ara h 7.0301 were found in the α‐helices, making them only accessible after enzymatic digestion.To validate the applied technique, we additionally mapped linear epitopes of Ara h 2 and 6. Compared with previous literature, the same main epitopes were found, although two epitopes of Ara h 2 and two of Ara h 6 were not detected Table . HoweverThe identification of conformational epitopes might help elucidating the divergent potencies of the Ara h 7 isoforms. Since detecting and characterizing conformational epitopes is more sophisticated than the identification of linear epitopes, especially with polyclonal serum, human monoclonal antibodies might be a suitable tool for the identification of conformational epitopes. Moreover, human monoclonal antibodies directed to one specific epitope can also be characterized for their exact affinity.In conclusion, recognition of the 14 new mapped linear epitopes belonging to all three Ara h 7 isoforms was highly individual, and epitopes predominately bound by IgG4 varied from epitopes bound by IgE. These recognition patterns scarcely elucidated the divergent potency of Ara h 7 isoforms, indicating the importance of the addition conformational epitopes.W. Suer and Y. Weimann are employees of EUROIMMUN AG, Lübeck, Germany and M. Klinge and F. Besa are former employees of EUROIMMUN AG, Lübeck, Germany. The research position of A. Ehlers is partially funded by EUROIMMUN AG, Lübeck, Germany. Other authors have no conflicts of interest to declare.AE, MK, WS, YW, FB and HO involved in experimental design; AE, AK and TML involved in patient selection; AE, MK and FB involved in experimental performance; AE and MK collected and analysed the data; AE and MK drafted the manuscript; AE, MK, WS, AC and HO: contributed to data interpretation; WS, YW, FB, AC, TML and HO critically revised the manuscript.This study was carried out in accordance with the University Medical Center Utrecht, Biobank Regulations, which are in compliance with the applicable national and international laws and regulations. These regulations permit the use of “residual material from diagnostic testing” for research, unless the patient objects . None of the included patients objected the use of their serum. The protocol was approved by the Biobank Research Ethics Committee of the University Medical Center Utrecht under the protocol number 18‐428. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Global aging has proceeded at an unprecedented and accelerating rate. The aging of the population creates both opportunities and challenges for elders, their families, and society in general. Importantly, there is substantial variation in the effects of and response to global aging both within and across nations depending, in part, on prevailing cultural expectations and values, political and economic imperatives, and social and demographic characteristics. Thus, while some regions and countries have responded with innovative policies and programs to better enable the growing cohort of older adults to remain active and engaged in the community, other regions and countries have struggled with their response or barely begun to plan for the rising population of elders. This symposium assembles editors at five leading gerontological journals to demonstrate the role that peer-reviewed scholarship can play in disseminating knowledge that informs gerontological research, policy, and practice internationally. Editors include: Jeffrey Burr, PhD, Research on Aging; Deborah Carr, PhD, Journal of Gerontology: Social Sciences; Edward Alan Miller, PhD, Journal of Aging & Social Policy; Julie Hicks Patrick, PhD, International Journal of Aging & Human Development; and Julie Robison, PhD, The Journal of Applied Gerontology. Each presenter will review the scope, content, and focus of their journals and the role and opportunities for international scholarship. Michael Gusmano, PhD, a leading expert on the economic, political, and social consequences of global aging and International Editor of the Journal of Aging & Social Policy, will serve as discussant.


Experimentally identified epitopes were compared to a Gag reference sequence. Amino-acid-substitutions (AAS) were assessed for their impact on Gag’s budding-function using a trained classifier that considers structural models and sequence conservation. Experimental assessment of Gag-variants harboring selected AAS demonstrated an apparent classifier-precision of 100%. Compatible epitopes were assigned an immunological score that incorporates features such as conservation or HLA-association in a user-defined weighted manner. Using a genetic algorithm, the epitopes were incorporated in an iterative manner into novel T-cell-epitope-enriched Gag sequences (TeeGag). Computational evaluation showed that these antigen candidates harbor a higher fraction of epitopes with higher score as compared to natural Gag isolates and other artificial antigen designs. Thus, these designer sequences qualify as next-generation antigen candidates for induction of broader CTL responses.The partially protective phenotype observed in HIV-infected long-term-non-progressors is often associated with certain HLA alleles, thus indicating that cytotoxic T lymphocyte (CTL) responses play a crucial role in combating virus replication. However, both the vast variability of HIV and the HLA diversity impose a challenge on elicitation of broad and effective CTL responses. Therefore, we conceived an algorithm for the enrichment of CD8 Depending on geographical location, various clades (or subtypes) of the HIV-1 M group are circulating with varying frequencies, with clade C accounting for roughly half of all HIV infections2. Within-clade amino acid (aa) diversity is about 12%, and between clades 18%3. This enormous diversity is a major hurdle in developing a vaccine against HIV-1.In 2015, 36.7 million people worldwide were infected with HIV4. Approaches that aim at the elicitation of neutralizing antibodies are directed towards the envelope protein (Env) on the surface of viral particles5. In contrast, approaches aimed at eliciting T cell responses are often focused on the group-specific antigen (Gag) that drives the formation of virus particles also referred to as “budding“6. The preference for Gag as antigen for T cell vaccines7 is due to its abundance in HIV infected cells8, its comparably higher conservation (within-clade diversity below 10% on average)9, and the association of Gag-specific CD8+ cytotoxic T lymphocytes (CTLs) with reduced viral load, or even control, in some HIV-infected patients even without therapy12.In general, such a vaccine should elicit both potent humoral and cellular immune responsesThe specificity of a vaccine is fundamentally determined by the pathogen-derived antigens that are employed. The elicited immune response should be of sufficient breadth, i.e. directed towards multiple determinants, to increase the chance that some of the CTLs induced by vaccination later match the infecting virus. The determinants (epitopes) comprise peptides with a length of about 8 to 12 aa that are presented on MHC class I molecules on the cell surface. Thus, an “optimal” T cell antigen should contain a high number of epitopes that ideally match a large fraction of circulating viruses.14. This problem can be mitigated by using consensus, ancestral, or center-of-tree sequences that aim at minimizing the genetic distance, thus presumably being capable of eliciting broader immune responses17. Furthermore, the promising concept of so-called mosaic antigens specifically aims at broader CTL responses by enriching potential 9-mer T cell epitopes (PTEs)21. A limitation is, however, that the PTEs are predicted from HIV sequence analyses as 9-mer aa stretches with certain frequencies in natural isolates. Therefore, they may not represent genuine T cell epitopes, neither are they selected based on favorable immunological features. Other approaches aim at increasing the breadth by using artificially generated antigens that focus CTL responses on conserved parts of HIV proteins, like the HIVconsv22 or the p24CE design24. An alternative concept is to string together selected antigenic determinants flanked by spacers facilitating epitope processing27. However, such designs may give rise to T cell responses toward neo-epitopes generated by artificial junctions.However, considering the high diversity, antigens derived from an arbitrarily selected viral isolate may be quite unrelated to others+ T cell epitopes, thus potentially eliciting broader CTL responses in a vaccination setting. As a prerequisite, these novel Gag variants shall retain their function to drive formation of virus-like particles (VLPs), as this may have a major impact on the immune response. For instance, VLPs might be taken up by professional antigen presenting cells (APCs) for cross-priming. It has been shown that VLPs can be taken up by monocyte-derived dendritic cells in an ex vivo assay28 and also induce CTL responses in mice29. Principally, a VLP resembles the pathogen more closely, and uptake of a VLP would at once deliver several hundred copies of Gag30 thus overcoming processing limitations31. Indeed we have shown that budding-competent Gag within a larger antigen is more immunogenic in mice than a budding-defective variant32. Moreover, DNA and viral vectored vaccine candidates harboring functional Gag antigens were highly immunogenic in non-human primates34 and clinical trials35.Given these limitations, here we describe an algorithm for the generation of full-length Gag variants that are enriched with experimentally identified, genuine CD8+ T cell epitopes. These should be selected based on a combination of immunological quality parameters in a way that also allows user-defined weighting of these criteria to tailor the sequences toward desired properties. At the same time, epitopes predicted to interfere with Gag’s budding-capacity should be excluded.The aim of this study was to develop an algorithm for the generation of sequences encoding artificial variants of the HIV-1 Gag protein that are enriched with experimentally identified, and thus validated, genuine CD8The projected algorithm for the generation of T-cell-epitope-enriched Gag (TeeGag) variants follows four basic steps: (i) Mapping: allocation of any given epitopes’ position within a Gag reference sequence and identification of amino-acid substitutions (AAS). (ii) Functional assessment of AAS: employment of a classifier to decide whether an AAS would cause a budding defect. (iii) Epitope scoring: calculation of an immunological quality score from several weighted epitope-specific properties. (iv) Antigen generation: assembly of sets of TeeGags, optimized regarding overall number and score of epitopes.+ T cell epitopes were retrieved from the Los Alamos National Laboratory (LANL) HIV Molecular Immunology Database. In total, 2688 epitopes were downloaded as raw data. The list of epitopes was then subjected to an initial quality control and was manually curated to remove or correct erroneous and undesired entries, leading to exclusion of 99 epitopes A multi-dimensional classifier was conceived that takes structural and sequence-based properties into account to predict whether an individual AAS is likely to be compatible with, or detrimental to the budding-capacity of Gag. Formation of Gag-VLPs is desired because of potential immunological benefits of particles such as the capacity to be taken up by APCs for cross-presentation.37 for each AAS modelled into a structure of the respective Gag-domain using homology-modelling with the MODELLER-tool38. For this, HXB2 reference structures for each of the five domains were built initially with MODELLER (see Methods). For p1, no template structure was available, thus one AAS could not be classified. In detail, the four features are (i) the DOPE value at the AAS position , (ii) the DOPE-value-difference between the wildtype and the mutant structure at this position , (iii) the DOPE-value-difference summed up for all aa positions , and (iv) the DOPE-score-differences summed up for a window of twelve amino acids around the AAS position (window ΔDOPE).Four different structure-based discriminatory features were envisaged that are based on the calculation of discrete optimized protein energy values (DOPE)39. For an AAS, the score is calculated as logarithm of the normalized frequency of this amino acid in the PSSM.For the sequence-based discriminatory feature, a position-specific substitution matrix (PSSM) based on a pre-computed, curated Gag-alignment from the LANL database  was built. For each position within Gag, this matrix contains the respective counts of all 20 amino acids occurring in the alignment. As even these 5001 sequences only represent a small subset of all sequences occurring in nature, non-observed substitutions were accounted for by adding artificial pseudo-counts to the PSSM40). Five mutations were left out of the training set as they lie within six aa of the p17 ends, so that structural feature 4 could not be calculated. The scores for each feature were calculated for the 32 AAS and the group separation of the 17 budding-compatible and the 15 budding-incompatible AAS was evaluated. As shown in Fig. Next, it was assessed how well these five features can discriminate a set of 37 AAS with known effect on budding of Gag . Here, 4839 out of 9196 (52.6%) were predicted to be budding-incompatible. This shows that – as expected – experimentally identified epitopes are significantly depleted of budding-incompatible AAS .The classifier then identified 18 of 158 (11%) AAS to be budding-incompatible, leading to the exclusion of 21 epitopes (3%) harboring at least one of these detrimental AAS. As shown in Fig. The set of 670 epitopes that were predicted to be free of budding-incompatible AAS was used further. As the epitopes differ in their immunological and virological characteristics, epitopes should also be selected based on such properties, thus conditioning the probability of an epitope’s incorporation into the finally assembled antigen sequence. For this, a scoring scheme based on the annotation data that is available in the LANL database was conceived. The following parameters were considered for each epitope: (i) HLA-association, (ii) subtype of the parental virus, (iii) response in long-term non-progressors (LTNPs), (iv) experimentally determined recognition rate, (v) conservation.43. It is important to emphasize that the scores are most likely biased because not all epitopes have been characterized to the same degree and with comparable methods.As HLA alleles are not uniformly distributed, it might be beneficial to preferentially incorporate epitopes that are restricted by more common HLAs. The HLA restriction reported for the epitopes in the LANL database is, however, quite heterogeneous. For instance, the HLA association is only given for 59.5% of the epitopes. By assigning the HLA-association of sub-epitopes also to their corresponding super-epitopes and using only allele groups, this is harmonized to a limited degree. The HLA-association-score is then calculated by summing up the global HLA population frequencies44.It might be relevant for some applications to adapt a potential antigen to one or several clades that for example are matched to viruses circulating in the target population. Thus, a parameter relating each epitope to the virus subtype from which it is derived was included. This information is again part of the LANL-metadata for each epitope. By assigning individual weights for each clade, the relative contribution can be defined by the user. The subtype-score is then calculated for each epitope by summing up the weights assigned to the clades with which the epitope is associated. Finally, the overall score gets normalized. For the proof-of-concept, we assigned weights that represent the relative world-wide frequency of each cladeThe boolean LTNP parameter receives a value of 1 if a T cell response against this epitope has been found in LTNPs or elite-controllers according to the LANL metadata.For some epitopes it has been investigated how many people in a study population do have a T cell response against the respective peptide. If this information was provided among the metadata of the epitope’s database entry, this fraction was used as the “experimental recognition” score.Finally, to assign a measure of conservation to each epitope, it was assessed how frequently the exact epitope sequence occurs in the filtered web-alignment of full-length HIV-1 Gag sequences. As the fraction of sequences from a specific subtype in the alignment does not correlate with the natural subtype frequency , the epitope frequency was assessed for each subtype individually, weighted according to the natural subtype distribution, summed up for all subtypes and finally normalized.Eventually, the overall epitope score was then calculated by summing up the five weighted individual scores. The weight (w) can be user-defined. For the proof-of-concept, the following values were used: w(HLA-association) = 6; w(subtype) = 3; w(LTNP) = 6; w = 1; w(conserved) = 3.Finally, a genetic algorithm was employed to assemble artificial T-cell-epitope-enriched Gag variants with the maximal overall score by including the highest-possible number of highest-scoring epitopes. Overlapping epitopes can be combined if the overlapping part has identical aa sequence. Here, the genetic algorithm is important for approximating the best solution for combinations of such compatible epitopes. Finally, unassigned aa positions were filled with the respective aa from HXB2 to come up with a full-length Gag protein.The genetic algorithm was executed in iterations. Epitopes integrated into the first Gag variant were removed from the input data set for the subsequent assembly. By this, all 670 epitopes could be integrated into a set of 11 TeeGags. The overall number of epitopes, as well as the cumulative score of combined complementary TeeGags, rapidly increased and soon reached a plateau Fig. . Whereasgag gene with a single one of the 84 AAS were constructed. As reference, another 18 plasmids with AAS predicted to cause a budding-defect were generated.Next, the performance of the classifier regarding the correct prediction whether inclusion of an AAS into the Gag reference protein causes a budding-defect was experimentally assessed. For this, the influence of each AAS contained in either TeeGag1, TeeGag2, or TeeGag3 on VLP-formation was analyzed. Plasmids carrying the codon-optimized HXB2 All plasmids were transfected in parallel into HEK293T cells and supernatants were analyzed 48 h later. As shown in Fig. In addition, plasmids encoding the three TeeGags were generated, and, as negative control, a myristoylation-deficient HXB2-Gag variant. To rule out that an AAS affects recognition by Gag-specific antibodies, a His-tag was added for detection of the proteins. All three TeeGag variants did form particles despite the inclusion of up to 40 point mutations Fig. . HoweverComparing the experimental results with the theoretical predictions by the classifier shows that apparently a precision  of 100% was obtained. As described above, the highest value of this analytical parameter guided selection of the feature-combination for the classifier, and achieved 98% based on the training-set-data. The negative predictive value was only 50%, reflecting that false exclusion of some AAS is accepted in favor of not including detrimental AAS. The sensitivity and specificity were 90% and 100%, respectively.A computational analysis was performed to validate the newly designed TeeGag variants regarding the absolute numbers of epitopes contained and their overall score. We compared these parameters for the TeeGags, natural Gag-isolates, as well as the artificial consensus (con), ancestral (anc), center-of-tree (cot), and mosaic-Gag (mos) designs that all represent full-length Gag-proteins.As shown in Fig. The quality of natural isolates is surpassed by TeeGag1 with 402 epitopes (56.5% score). The M group consensus (conM) has a coverage of 283 epitopes (40.6% score), the ancestor (ancM) of 293 (40.9% score), the center-of-tree (cotM) of 311 (43.1% score), and the mosaic (mosM) 277 (44.7% score). Values for coverage and scores relating to the whole epitope set for artificial Gag sequences designed toward individual clades are given in Supplementary Table As a large fraction of the higher-scoring epitopes are contained in TeeGag1 and thus are excluded from the subsequent complementary TeeGags, the epitope coverage is lower in TeeGag2 and TeeGag3, with 266 epitopes (37.6% score) and 222 epitopes (31.8% score), respectively. The advantage of using complementary antigen sets becomes evident when the quality of trivalent antigen-sets is compared. Here, the combination of TeeGag1, TeeGag2, and TeeGag3 covers 588 epitopes with 91.0% of the score. The combination of mosaics mosM.1, mosM.2, and mosM.3 which similarly consist of complementary sequences, achieves a coverage of 524 epitopes (77.4% score). By virtue of their design, complementary con and anc sequences do not exist. However, combining the sequences of the three most dominant clades  leads to a coverage of 551 epitopes (83.1% score), and 493 epitopes (72.0% score) for consensus and ancestral sequences, respectively. Taken together, although all these alternative sequence designs reach substantial theoretical coverage in this computational analysis, the TeeGags are superior in relation to the analyzed epitope set.In addition to TeeGags 1 to 3, we also generated sets of clade-specific TeeGags for the most frequent clades, i.e. TeeGagC, TeeGagA, and TeeGagB  were randomly selected respecting world-wide clade-frequencies. Then, the number of epitopes present in the respective Gag-antigen and the pathogen at the same time and their combined pathogen-score were determined Fig. . TeeGag116. Gag is the prime antigen for approaches aimed at eliciting CTL responses7. Such responses have the capacity to reduce viremia after infection, especially if the response broadly targets several Gag peptides45. Moreover, there is an inverse relationship between the breadth of Gag-specific responses and the set-point viral load46.An efficacious HIV-1 vaccine will have to elicit broadly reactive T cell and antibody responses to address the variability of circulating viruses20, there are two major differences: First, the TeeGag-algorithm incorporates epitopes that have been experimentally identified in humans. Second, the TeeGag-algorithm evaluates all epitopes for compatibility with Gag’s budding-function.Here, we describe a novel algorithm for the design of T-cell-epitope-enriched Gag antigens (TeeGags) that harbor more and, according to several criteria, better epitopes than arbitrarily selected natural Gag sequences. Compared to the design approach for mosaic antigens47. This leads to a very good coverage of highly conserved sequences including T cell epitopes, but less frequent ones or those with different length may be missed. In contrast, the TeeGag-algorithm considers all epitopes in the pre-specified set, in this case all the entries from the LANL database. Yet, a number of relevant epitopes is of course expected to be missed, simply because they have so far not been experimentally identified. A bias of the database entries is for instance evident by the strong overrepresentation of epitopes derived from B clade viruses  antigens have been shown to elicit broader responses to the CE-antigens in animal models as compared to full-length antigens24, but here as well, the antigens are not specifically tailored toward peptides that are presented on HLA molecules but any reactivity in a CE region generally indicates immunogenicity of the respective CE element. Thus, to assess whether the novel TeeGag antigens can elicit broader CTL responses, it will be necessary to either test their stimulatory capacity with a large panel of T cells from HIV-infected people, or even to assess the breadth of CTL responses that get primed in humans in a clinical trial.Preclinical evaluation in animal models will be necessary to further develop TeeGags as antigens. Analyses in mice or macaques will be informative regarding the safety of the antigens and in determining optimal vaccination regimens. However, such models would only yield very limited information on whether the epitope-enrichment of TeeGag’s does lead to broader CTL responses mainly due to the non-human specifics of peptide processing and presentation. For mosaics, it has been shown in mice and macaques that broader T cell responses get primed as compared to antigens from natural isolates53. However, as there are at the moment only few SIV-Gag-derived CTL epitopes identified in macaques in the LANL database, only a limited enrichment of epitopes in an SIV-TeeGag would currently be possible. However, once more epitope sequences become available, this might be a viable model to test whether epitope enrichment translates into broader T cell responses.An interesting alternative would be to transfer the concept to SIV antigens that could be evaluated in macaques, as for instance has been successfully demonstrated for conserved elements with a p27Gag-CE design46, i.e. the number of different epitopes that are targeted by T cells. However, most likely not all reactivities contribute equally to the overall response. Especially CTLs that are directed toward variable epitopes prone to rapid escape may have only little and transient impact on controlling HIV replication. T cell responses to such epitopes may be of high strength and impede priming of CTL responses toward other epitopes in a phenomenon called immunodominance. To avoid this, strategies have been proposed where the variable regions eliciting such responses are deleted, thus coming up with artificial polypeptides such as p24CE24 or HIVconsv22. Though these antigens lead to a focusing of the immune response on conserved regions that mostly elicit subdominant responses, the benefits of using the full-length, functional protein are of course lost. Moreover, the unnatural linking of sequences in the artificial antigens may cause further problems54, like the generation of neo-epitopes55, which is not the case for the full-length Gag designs (i.e. mosaics and TeeGags). Whereas a full-length Gag protein elicited responses of similar breadth but of higher magnitude than the HIVconsv antigen56, for p24CE, in contrast, it could be shown that it elicits broader responses recognizing more CE elements than when a full-length Gag was used for vaccination in mice24. Similarly, use of an analogous design for SIV, i.e. p27CE, in macaques led to robust CTL responses targeting subdominant epitopes of the conserved elements, thus altering the immunodominance hierarchy53. Similar results were obtained for HIV-Env-derived CE-antigens in macaques57. Even in the setting of a therapeutic vaccination in SIV-infected macaques, the p27CE was able to elicit focused responses toward the conserved element sequences58.As mentioned above, the overall efficacy of a CTL response correlates with the breadth53. Thus, even in the context of strategies aimed at focusing of immune responses to conserved regions, TeeGags might be useful as boosting agents as well. Alternatively, the TeeGag-algorithm could be modified to penalize epitopes known to elicit immunodominant responses to prevent their incorporation into the protein.Interestingly, co-delivery of a full-length SIV-Gag together with the p27CE antigen to boost responses initially primed by p27CE proved to be superior to a p27CE-only boost in increasing the magnitude of the response while the breadth was not negatively affected17. Put simply, a virus-like structure is what our immune system has learnt to deal with and therefore it is reasonable to assume that a VLP constitutes the best form for delivery of Gag as an antigen.The other important design feature of the TeeGag-algorithm is the prediction whether an AAS is compatible with VLP formation, as this may be of high importance for priming a Gag-specific CTL response59. An accurate assessment of the AASs is of prime importance when considering the realization probability of functional Gag-variants with many AASs combined. For example, if the prediction had a precision of only 98%, then a variant like TeeGag1 with 28 mutations would have a realization probability of just 57%, and TeeGag3 with 40 mutations only 45%. Yet, the experimental assessment of the three complementary TeeGags showed that they do form VLPs, although TeeGag2 may be slightly impaired . For such an extension, however, it would be necessary to modify the classifier by performing a training on a set of variants with known budding phenotype that harbor AAS combinations. Yet, the current approach already yielded very good results, with all of the three TeeGags that were experimentally assessed being budding-competent. In addition, an up-front curation step that manually excludes AAS known to interfere with budding based on literature-data could further improve the realization probability of budding-competent TeeGag-antigens.Therefore, the algorithm encompasses a classifier that judges whether an AAS is likely to be compatible with budding or not. This classifier excellently ruled out budding-incompatible AAS. Interestingly, the fraction of AAS that would cause a budding defect was extraordinarily higher when all theoretically possible AAS were considered. This is in line with an analysis showing that especially p24 is genetically extremely fragile in that it tolerates only very few substitutionsred Fig. . Of cour+ T cell epitopes can be achieved  is difficult. Therefore, analyses in human ex vivo assays or possibly also models of humanized mice will be required to determine whether the TeeGags are superior antigens in regard to eliciting broadly reactive T cell responses. In conclusion, only via an extensive immunogenicity analysis with special emphasis on human MHC-directed epitope-presentation, it will eventually be possible to verify whether the described design strategy translates into improved immune responses.By combining iteratively generated TeeGags into multivalent Gag-antigens quite a significant coverage of the set of known CD8ved Fig. . However+ T cells requires additional help by CD4+ T cells that recognize related antigens on the same APC, improved presentation of MHC-II-restricted T cell epitopes might be beneficial. In this regard, the algorithm also could easily be applied to enrich MHC-II-restricted epitopes by including the set of CD4+ T cell epitopes from the LANL database (currently about 400 entries) and amending the scoring function for a user-adjustable parameter to control the balance between MHC-I- and MHC-II-restricted epitopes. Incorporation of linear B-cell epitopes would also be possible, though this is likely not relevant for Gag, but could rather be used for the generation of a likewise TeeEnv antigen. However, it must be considered that B cell epitopes, especially those recognized by broadly-neutralizing antibodies, are often structural determinants that cannot be treated like linear T cell epitopes. However, if the residues contributing to the antibody-epitope are known, these can be kept constant, and T cell determinants could be enriched in the remaining sequence parts. Finally, otherwise designed Env-antigens retaining the transmembrane-domain could be incorporated into Gag-VLPs built from TeeGags. Such complexes might prove to be very useful immunogens, as they would deliver (i) TeeGag for elicitation of CD4+ and CD8+ T cell responses, (ii) Env for elicitation of antibody responses, and (iii) the physical combination in a VLP might direct CD4 T cells toward the Env-specific B cells by intrastructural help63.Since efficient priming of CD8http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html) and curated by excluding non-M-group and unclassified (“U”) sequences (84 of 5085). Consensus and ancestral Gag sequences (2004) were obtained from LANL, mosaic sequences from18. Center-of-tree sequences were kindly provided by James Mullins.The premade filtered web-alignment of Gag (2013) was downloaded from the LANL database , epitope’s viral subtype origin (subtype-field), association with LTNP and experimentally determined fraction of responders .CD838 using PDB structures as templates . To analyze the impact of an AAS, a 3D structure of Gag containing the respective AAS was modelled accordingly. Next, a per residue energy landscape profile was calculated by using the DOPE model37. Four different scores were then derived: (i) the DOPE free energy value of the AAS , (ii) the DOPE-value-difference between the reference and mutant structure at the AAS’s position , (iii) this difference summed up for all aa , and (iv) the difference summed up from −6 to +5 aa around the AAS (window ΔDOPE).Initially, HXB2 reference 3D-structures for the individual Gag domains p17, p24, p2, p7, and p6 were built by homology modelling using the automodel function of MODELLER version 9.7fka of the occurrence of all amino acids a at each position k were calculated from the curated Gag alignment. To account for unobserved substitutions, artificial pseudo-counts were added with frequencies of bka. These pseudo-frequencies were calculated by conditioning the general mutation probabilities qia for substitution of amino acid a by i (taken from64) with the observed frequency asA position-specific-substitution matrix (PSSM) over the length of Gag for all amino acids was built (500 × 20 matrix). First, observed frequencies Nk), and the number of added pseudo-counts (Bk), the probability pka of a occurring at k is estimated asBk was calculated as m × Rk, where Rk is the number of different aa at k, weighted by an empirical number m (chosen as m = 5 as recommended in65). Finally, the PSSM score wka was calculated as log-odds-ratio of pka and the background frequency pa of a (obtained from http://www.pseudogene.org/composition/index.cgi):Accounting for the number of sequences in the alignment . For the classification of AAS with unknown effect on budding, a trained classifier was implemented using Fisher’s linear discriminant analysis+ T cell epitopes were calculated by evaluating the epitope’s metadata. If applicable, annotations of superepitopes were amended by annotations of the corresponding subepitopes.Scores representing immunological and virological properties of CD8sh(e) of epitope e was calculated by summing up the population frequencies (from43) of all associated allele groups.As HLA-association is heterogeneously given either for the allele, or only the allele group, all annotations were harmonized to allele groups (e.g. HLA*A2:01:01 to HLA*A2). The HLA-association-score ss(e) is calculated by summing up user-supplied weighting parameters ws for the 12 individual clades  if the epitope is derived from a virus of the respective clade. Here, the world-wide frequencies of clades circulating between 2004 and 200744 were chosen as weights (ws).The viral-subtype-score sl(e) of 1, otherwise none.If an epitope’s description stated that it was identified in an LTNP, the epitope received an LTNP-score f(e) was taken as the experimental-recognition score sr(e). If the cohort was pre-selected regarding carrier-status of a certain HLA-allele, the allele frequency h was factored in:If the metadata contained information on the fraction of people from patient cohorts with responses toward a given epitope, this fraction fs(e), representing the fraction of sequences from the Gag alignment of each subtype s harboring the exact epitope sequence. The overall conservation score sc(e) was then calculated by summing up the weighted frequencies:Conservation of an epitope was first calculated per subtype as frequency s(e) of epitope e was eventually calculated as the sum of the normalized individual scores sk(e) outlined above , taking user-defined weighting parameters wk into account:The overall score wi of vertex i given by the epitope’s score s(e), and with edges connecting vertices of incompatible epitopes. A genetic algorithm (GA) was used to select an epitope set that approximates the theoretically best solution, i.e. as many and as high-scoring epitopes as possible. The GA was initialized with a population of 70 chromosomes x that each represent a bit vector of length v for all vertices. Each bit xi indicates the presence (1) or absence (0) of vertex i. Initially, the chromosomes were populated with random, unique selections of compatible epitopes, i.e. non-connected vertices. The fitness of the chromosomes was calculated by the fitness functionnsup and nsub are the numbers of superepitopes and subepitopes, respectively. The chromosome with the highest fitness value represents the current “solution representative”.Many epitopes span overlapping parts of the sequence. If two epitopes harbor different AAS in the overlap, such epitopes are mutually exclusive for incorporation into the same antigen sequence (“incompatible epitopes”). To select a compatible set, an incompatibility graph was initially created, in which vertices represent epitopes, with the weight In each evolution round, 24 chromosomes were generated and populated with newly initialized random chromosomes to maintain genetic diversity (probability 0.05), or by generating offspring as follows: (1) Selecting two chromosomes with a probability proportional to their fitness. (2) Replacing one with a newly initialized chromosome with probability 0.4. (3) Generating offspring chromosome by three-point crossover at random sites, restricted in a way that the offspring receives numbers of bits relative to the parents’ fitness. (4) Spontaneously mutating each chromosome by flipping random bits with probability 0.01. If the chromosome harbored incompatible epitopes (connected vertices), the offspring was repaired by repeatedly setting the bit with highest number of incompatibilities to 0 until all incompatibilities were resolved. Chromosomes in the subpopulation already contained in the overall population were rejected. Finally, offspring chromosomes were each compared to four randomly selected chromosomes from the parent population, and replaced the one with the lowest fitness, if the offspring scored higher. In total, 2000 rounds of this process were executed and the final solution representative was chosen as antigen candidate. Positions not covered by an epitope were filled in with the respective aa from HXB2. Then, all epitopes contained in this candidate were removed from the incompatibility graph and the GA was repeated with the reduced epitope set to come up with a second solution representative as complementary antigen candidate. This overall process was repeated until all epitopes were covered in the set of complementary antigens.s(e) of these epitopes were calculated and summed up to assign an overall score-parameter to every Gag-sequence. To avoid overestimations of epitope scores, subepitopes were not considered if a corresponding superepitope was present in the sequence.To generally compare Gag sequences by computational methods, the numbers of epitopes from the input set contained within the respective sequences were counted. In addition, the scores 43. Next, only if an epitope was known to be restricted by at least one allele, it was retained. The population coverage and score resemble the average number and overall score of retained epitopes.The population score was calculated by first simulating HLA haplotypes of 1,000 members of the world-wide general population by randomly picking two HLA-A, -B, and -C allele groups with a probability matching the natural allele frequencies44 and one sequence of this subset was selected. The number and overall score of epitopes in this sequence matching those present in the Gag sequence of interest were finally calculated.The pathogen coverage and score were calculated as average from 1,000 iterations of the following procedure: The sequences of the Gag alignment were split into clade-specific subsets. Then, a clade was randomly chosen with a probability resembling its global frequencyHuman codon-optimized DNA sequences encoding the Gag-variants were obtained from GeneArt  and cloned into pcDNA3.1(+) via KpnI and XhoI sites. Gag variants with single AASs were generated by PCR-mediated directed mutagenesis according to standard procedures.2. The day before the transfection, 1.4 × 105 cells in 1 ml were seeded per well of a twelve-well plate. Cells were co-transfected with 1 µg of the respective Gag-plasmid and 0.05 µg of the CMV-SEAP-plasmid  using PEI in a 1:5 (w/w) ratio66. Medium was replaced 6 h later by DMEM0. Conditioned media with Gag virus-like particles were harvested after 48 h, cleared by centrifugation and treated with 0.5% TX-100. The Gag content was quantified via a p24-ELISA as described in67. SEAP-activity was assayed colorimetrically as described in68.Human embryonal kidney 293 T cells (ATCC-# CRL-11268) were cultivated in DMEM medium with 10% FCS and 1% Pen/Strep at 37 °C and 5% COAll data generated or analysed during this study are included in this published article and its Supplementary Information files.Supplementary InformationSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3


High density and high quality interconnects are necessary for the preparation of miniaturized and lightweight electronic products. Therefore, small-diameter and high-density through-holes in FPCs (Flexible Printed Circuits) are required. However, the current processing methods cannot further decrease the diameters and improve the quality of through-holes. Comparatively, ultrashort pulse laser is a good choice. In this paper, the processing technology for the microdrilling of through-holes in FPCs using a 10 ps pulse laser was systematically studied. The effects of laser parameters, including the wavelength, energy, pulses and polarization, on the drilling of through-holes were investigated. The various processing parameters were optimized and the plausible reasons were discussed. Finally, the desired small-diameter and high-density through-holes in FPCs were obtained. The experimental results showed that, through-holes with diameters of less than 10 µm and inlet interconnection pitches of 0~2 µm could be successfully drilled in FPCs using ultrashort pulse laser. Flexible printed circuits (FPCs) are composite laminated materials that are mainly composed of copper (Cu) and polyimide (PI). Cu foil is used as the conductive metal material, while the typical organic polymer PI serves as the dielectric or insulating layer due to its advantages of good dielectric property, thermal stability, excellent mechanical properties, plasticity and less fragility . Based o2 lasers with wavelengths in the range of 9.2–10.6 μm and solid-state UV nanosecond lasers with wavelengths in the range of 266–355 nm are the most commonly used at present [2 lasers involves heating ablation which leads to some obvious defects, such as excess carbide formation and collapse between layers [2 laser in FPCs. Therefore, a coating metal is required on the surface of Cu resulting in complex processing [2 lasers [2 lasers are approximately 75~150 μm which could not be further decreased [For drilling through-holes in FPCs, CO present . However2 lasers . Moreoveecreased . Regardiecreased . In otheUltrashort pulse lasers are lasers having pulse durations in the picosecond to femtosecond range . The advThe laser utilized for irradiation in this paper was a neodymium-vanadate laser  with the pulse duration of 10 ps, repetition rate of 1 kHz and wavelengths of 355, 532 and 1064 nm. Multiple optical lenses with the focal lengths of 25~150 mm were used to focus the laser beam on the surface of samples. The experiments were performed in air atmosphere. After the experiments, the size and morphology of through-holes were measured mainly using scanning electron microscopy . Furthermore, in order to obtain the cross-section morphology of through-holes, the samples with large area through-holes were inlaid using transparent gel and then were split using sandpaper and polishing agents. The absorptions of Cu and PI under various wavelengths were measured using ultraviolet and visible light spectrophotometer . The FPC samples composed of Cu and PI mainly include single-, double- and multi-sided boards, as shown in The evaluations of the through-holes drilled in FPCs are shown in When a metallic material is irradiated using ultrashort pulse laser, based on the inverse bremsstrahlung, laser energy is absorbed by the free electrons in the skin depth . At thisω0 (at 1/e amplitude) and the laser wavelength λ, as shown in Equation (1): ωs is the initial beam radius and is about 2.4, 1.3 or 0.78 mm for wavelengths of 1064, 532 or 355 nm in λ is the laser wavelength. f is the focal length of the optical lens and equals to 150 mm for the holes ablated in M2 is the beam quality factor and is 1.33 [ω0 can be calculated and is approximately 28.17, 26 and 28.91 µm for wavelengths of 1064, 532 and 355 nm in  is 1.33 . Then, ωThe laser wavelength also affected the saturation state of the inlets and outlets. As shown in For the inlet and outlet diameters, based on a large number of experiments, it was found that there are much more times for the inlets diameters saturation with the outlets diameters still increasing gradually for the wavelength of 532 nm and it is reverse for the wavelength of 355 nm. This phenomenon could be ascribed to the different absorptivities of the FPC materials for different laser wavelengths, as shown in In order to ensure the effective ablation, the laser energy must be larger than the material ablation threshold. At the same time, for the pulsed laser machining, the laser energy is transmitted to the material through the pulses. In short, the laser energy and pulses have important effects on microhole drilling. Based on the un-excessive ablation of outlet with the energy of 0.032 mJ, through-holes were drilled with the energy of 0.032 mJ and multiple pulses in single-sided board. The SEM images of through-holes are shown in The above experiments illustrated that the rips around the outlets resulted in poor roundness of the holes. Although the rips could be avoided effectively by reducing the laser energy or pulses, the other characteristics of the outlets, such as the sizes and tapers, could not meet the requirement. In order to further optimize the roundness of outlet, the laser beam was adjusted to the circular polarization and the contrast experiments including the circular- and P-polarization were carried out with the higher laser energy and more pulses. The results are shown in ∅ is the incident angle between the direction of the incident beam and the surface normal, as shown in n is the material refractive index.The effect of laser polarization on the outlet has been reported. It was presented that there were two bulges located around the outlet and they were placed opposite to each other and oriented perpendicular to the direction of the polarization of the laser beam, which was confirmed by turning the direction of the polarization by 90° ,31. The Based on the above experiments on the processing technology of microdrilling of through-holes in FPCs using picosecond ultrashort pulse laser, high quality through-holes were processed. It is essential to prepare small-diameter and high-density through-holes in FPCs for the sake of practical commercial applications.In order to explore the connectivity between the through-holes ablated using picosecond ultrashort pulse laser, large area through-hole groups with diversified pitches were processed in a single-sided board with 46 μm Cu and 30 μm PI. The main process parameters were: laser wavelength of 532 nm, energy of 0.063 mJ, repetition rate of 1 kHz, 1000 pulses, circular polarization beam and optical lens with the focus length of 25 mm. With further decrease in the inlet pitch to zero as shown in For drilling high-density micro through-holes in FPCs, the independence between the through-holes needs to be ensured and also, the independent conductivity and insulation of the Cu layer and PI layer also need to be guaranteed. Therefore, the side-walls should remain clean for the through-holes processing. Next, the side-wall morphology of large-area through-holes drilled using picosecond ultrashort pulse laser was studied, as shown in As electronic products are becoming increasingly miniaturized and light-weight, high density and high quality interconnects in FPCs are inevitable. In this paper, the microdrilling of small-diameter and high-density through-holes in FPCs using picosecond ultrashort pulse laser has been studied. First, the effects of laser parameters, including the wavelength, energy, pulses and polarization, on the drilling of through-holes drilling were investigated. It was found that visible light, especially ultraviolet light, was superior to near-infrared light for the drilling of small-diameter and high-quality through-holes. The use of excessively high energy and too many pulses resulted in ablation defects, such as spatters around the periphery of the inlets, collapses and fractures, poor roundness with some rips around the outlets and so forth. By optimizing the process conditions, the desired small-diameter and high-density through-holes in FPCs were obtained. Experimental results showed that through-holes with diameters less than 10 µm and inlets interconnection pitches of 0~2 µm could be achieved. Therefore, high density and high quality interconnects can be realized in FPCs using ultrashort pulse laser.


Chikungunya virus (CHIKV), causes massive outbreaks of chikungunya infection in several regions of Asia, Africa and Central/South America. Being positive sense RNA virus, CHIKV replication within the host resulting in its genome mutation and led to difficulties in creation of vaccine, drugs and treatment strategies. Vector control strategy has been a gold standard to combat spreading of CHIKV infection, but to eradicate a species from the face of earth is not an easy task. Therefore, alongside vector control, there is a dire need to prevent the infection through vaccine as well as through antiviral strategies.This study was designed to find out conserved B cell and T cell epitopes of CHIKV structural proteins through immuno-informatics and computational approaches, which may play an important role in evoking the immune responses against CHIKV.Several conserved cytotoxic T-lymphocyte epitopes, linear and conformational B cell epitopes were predicted for CHIKV structural polyprotein and their antigenicity was calculated. Among B-cell epitopes “PPFGAGRPGQFGDI” showed a high antigenicity score and it may be highly immunogenic. In case of T cell epitopes, MHC class I peptides ‘TAECKDKNL’ and MHC class II peptides ‘VRYKCNCGG’ were found extremely antigenic.The study led to the discovery of various epitopes, conserved among various strains belonging to different countries. The potential antigenic epitopes can be successfully utilized in designing novel vaccines for combating and eradication of CHIKV disease.The online version of this article (10.1186/s12967-018-1672-7) contains supplementary material, which is available to authorized users. From mid 1950s till now, chikungunya fever is posing a substantial global health threat. The infection caused by an arthropod borne virus namely chikungunya virus (CHIKV), was initially reported in Africa and later on disease outbreaks specifically restricted to the tropical regions of South-East Asia, however West Pacific Islands were also documented. In Pakistan CHIKV was first disseminated in 2016 and affected 30,000 people in coastal region of Karachi. Of 30,000 cases, only 803 cases came into the knowledge of World Health Organization (WHO) [Human epithelial, fibroblasts, macrophages and progenitor of muscle cells are primary targets of CHIKV. Chikungunya infection is characterized by an abrupt onset of headache, nausea, fatigue, muscles pain, crippling joint pains, high-grade fever and the prominent rashes. Wide variety of clinical appearances comprising myalgia, vomiting, muscular and joint pain has been observed in acute cases, while depression after several months of acute infection is considered the most lethal form of chikungunya infection. Severe acute arthritis is produced through the destruction of collagen which alters metabolism of connective tissues in cartilage –7. ChronTogaviridae family. It is predominantly spread by two mosquito borne vectors namely Aedes aegypti and Aedes albopictus, and that is why chikungunya fever and dengue infection shares analogous clinical manifestations [Genome of CHIKV is comprised of single stranded RNA which is approximately 11.8 kb in size and has 11,990 base pairs. It has been classified as an alphavirus of stations . StructuTill now, no licensed therapeutic or vaccine for the treatment of chikungunya infection is available in the market. Efforts for the discovery of antiviral drugs are at very early stages now. One of the successful approaches used for designing antiviral therapies is to target the non-structural proteins, so that multiplication of virus within the host can be controlled. But in CHIKV unlike other alphaviral proteins, non-structural proteins that make up active enzymatic complexes of its replication machinery are very difficult to target. Out of the four non-structural proteins, core polymerase subunits nsP4 is extremely challenging as it is inactive on its own . The nsPCurrently, recombinant CHIKV vaccine candidates have been proposed by using adenovirus, vesicular stomatitis virus and measles virus vectors which are focused on expression of CHIKV structural genes to induce robust immune responses within the host. These vaccines protected mice from arthritis and helped in clearing viral load. But none of them has been evaluated in clinical trials –17. Thushttp://www.rcsb.org/pdb) (PDBId: 3N44). Protein sequence was analyzed for its physical and chemicals properties including half-life, GRAVY (Grand average of hydrophathicity), stability index, molecular weight and amino acid atomic composition through an online server Protparam [Primary sequence of the CHIKV structural polyprotein was obtained from the Uniprot (ID: Q1H8W5). Experimentally known crystal structure of CHIKV structural polyprotein was retrieved from Protein Data Bank (rotparam . Secondarotparam .http://www.cbs.dtu.dk/services/DiscoTope/) was used to model B cell epitopes from 3D structure of CHIKV structural polyprotein. All predicted epitopes were given in plain format while the 3D structure of structural polyprotein was given in PDB format.Systematic strategy was used to design CHIKV structural polyprotein B and T cell epitopes. TMHMM was used for transmembrane topology prediction . Freely http://crdd.osdd.net/raghava/propred1/) and Propred tool (http://crdd.osdd.net/raghava/propred/), respectively. The predicted results from both these tools are quite significant because they use huge number of alleles of HLAs (human leukocyte antigens) during calculations. For class I allele’s prediction, the threshold value of Proteasome and immune-proteasome were set to 5%.By using two different online available tools, CTL epitopes of targeted protein were predicted. Major Histocompatibility Complex (MHC) class I and II were predicted by ProPred-1 (https://www.qiagenbioinformatics.com/products/clc-main-workbench/). The aligned files  were further used to construct phylogenetic tree through MEGA7 software (https://www.megasoftware.net/). Epitopes were selected based on their conserved regions. Moreover, Immune epitope database and analysis resource (IEDB) was used to cross-check the conservation of the selected epitopes.Sequences of CHIKV structural polyprotein, belonging to 23 different countries, were taken from Genbank and Uniprot databases. Multiple sequence alignment (MSA) was performed to find out the conservation of selected epitopes by using CLC work bench . This server compares query sequences of peptides against the database of already reported allergens to give significant results.Allergenicity analysis of predicted epitopes was done by Aller Hunter server (The primary structure of the target sequence (Uniprot ID:Q1H8W5) was analyzed via Protparam. Physiochemical parameters of the CHIKV structural polyprotein computed through Protparam shows that protein is comprised of 1248 amino acid (aa) and have molecular mass of 138.31 kD. Theoretical iso-electric point (PI) of our subject protein was 8.90 which show its positive nature. Briefly, out of 1248 residues, 138aa were found as positively charged and 106 were calculated as negatively charged residues while Methionine (Met) was selected as an N-terminal residue in our subject structure. Protparam computed instability index, aliphatic index and GRAVY of target protein were 40.75, 74.93 and − 0.305 respectively.Secondary as well 3D structure analysis of structural polyprotein through PSIPRED and UCSF Chimera respectively showed 50% beta sheet, 5% helixes and 45% loops , 0.988 (average) and 1.443 (maximum). Graphical representation of Chou and Fasman’s output is shown in Fig. T cell epitopes were predicted through the application of two different online servers. MHC class I alleles were predicted through ProPred while MHC class I alleles were predicted through Propred simply. Results of both MHC class I alleles and MHC class II alleles are given in Tables Sequence of CHIKV structural polyprotein isolates from 23 different countries was subjected to multiple-sequence alignment to analyze the conservation of selected epitopes through CLC workbench. It was observed that all the selected epitopes are highly conserved in all sequences used for analysis  was already available with co-crystallized peptide. So focused docking was performed by using same active pocket to dock our peptide library. Ten confirmations for each epitope were generated and top ranked conformations based on their dock scores were selected Table . Later oAllergenicity assessment of query protein was performed via Aller Hunter to predict whether it is allergenic or not based on the FAO/WHO standards. Results showed that our query sequence was non-allergenic with a score of 0.0 .Emergence of new viral diseases in resource poor countries of Africa and Asia are adding a huge global disease burden. Chikungunya infection is not often recognized as a risk in developing countries and much less attention is paid towards the development of therapeutic solutions. Chikungunya infection mimics the symptoms of dengue and often co-infection of both the infection leads to misdiagnosis. In addition to this various mutation in CHIKV strains makes it resistant to already available antiviral molecules. There is no FDA approved reliable and specific drug against CHIKV infection is available in the market. Various recombinant vaccines have been designed which induces robust immune mechanisms in mice, but these vaccines are still in their clinical evaluation process. Pharmaceutical companies are neglecting CHIKV vaccine development as it is a lengthy as well as expensive procedure. To deal with the cost and time factor of the drug design phase, Chemo-informatics and immuno-informatics are of immense benefit. To develop cost effective and reliable therapeutic solutions, a detailed insight of human body immune response against viral agents is imperative. Advancements of immune-informatics tools have been instantly utilized by researchers to predict possible antigenic epitopes from CHIKV proteins  for peptide based vaccine development . CHIKV sIn this study, computational approach was used to predict conserved epitope by performing multiple sequence analysis and epitope prediction tools. Once B and T cell epitopes were predicted their antigenicity, solvent accessibility, flexibility and disulphide bond potential was analyzed. Conservation of the predicted epitopes among CHIKV strains from different regions of world was analyzed to pick those epitopes that are specific to all. Conserved epitopes with high antigenicity score were selected to build their protein models using peptide building tools and docked with human HL-B7 allele to analyze enough binding specificity to induce immune response.In epitope-based vaccine development a highly conserved epitope is expected to deliver a broader protection across different strains. Moreover, CHIKV being an RNA virus is more vulnerable to mutation due to a lack of proof reading activity of RNA polymerase. So, for reliable vaccine candidate, highly conserved epitope will ensure an effective and long-lasting immunity . Online In recent studies novel antigenic epitopes of some essential and vital proteins revealed that can victoriously elicit response of immune system therefore becoming great peptide vaccines targets and protecting host from virus attack. So, the current research was conducted to predict antigenic epitopes of structural polyprotein of CHIKV. We carry out sequence, structure, and conservation analysis as well as homology modeling of structural polyprotein of CHIKV. This research discloses T and B cell epitopes which are antigenic and conserved among the CHIKV isolates of 17 different countries. These epitopes are capable to induce a particular immunologic response. We hope and rely that few of the antigenic epitopes proposed and investigated in this work might present a preliminary set of peptides for future vaccine development against CHIKV for control and prevention of this devastating epidemic.Additional file 1: Figure S1. A) PSIPRED analysis of CHIKV structural polyprotein representing helix (tea-pink), stand (yellow) and coil region; B) Representation of asymmetric heterotrimeric complex showing A chain (blue), B chains (green) and F chains (red) of three dimensional (3D) structure of CHIKV structural polyprotein. C) cartoon representation of CHIKV structural polyprotein. Figure S2. Multiple sequence alignment illustrating the sequences consensus and conserveness of CHIKV strains form 23 countries. Table S1. Predicted disulfide bonds within GP. Table S2. Emini surface accessibility prediction results.


The major difference in the chemical composition of Y-TZP ceramics, as compared with conventional porcelain, led researchers to develop alternative solutions for achieving durable and long term bonding with the zirconia surface.The study aims to evaluate the effects of glow discharge treatment on the bonding between cement and zirconia.The zirconia rings and rods were prepared with the Zirconia Y-TZP powder and TZ-3YSB-E (Tosoh-Zirconia) through auto-mix to investigate the glow discharge and thermo-cycling. An orientation Teflon mold was used to centralize each rod into the zirconia ring, and aided as a cementation jig during the cementation procedure.Cohesive failure (2/3 or more of luting agent remained on the zirconia surface) has been majorly observed with RelyX Ultimate, while adhesion failure (less than 1/3 of the luting agent remained on the zirconia surface) has been primarily observed in Ketac-Cem. Mixed failure was observed among the three specimen including Rely X Unicem 2, Multilink Auto-mix and Ceramir.The glow discharge surface treatment procedure had a major impact on bond strength to zirconia. One of the most promising non-metal-based materials is Yttria-Tetragonal Zirconia Polycrystaline (Y-TZP) ceramics. With the recent demands for maximal esthetics, Y-TZP ceramics are used in every aspect of modern dentistry. Y-TZP ceramics are proven to be a strong and reliable material for dental practitioners, researchers and manufacturers -4. DentaSeveral techniques have been introduced for improving the strength of a bond related to luting agents along with zirconia surfaces. Basic knowledge of ceramic compositions is required for the application of surface conditioning systems. Amorphuos glass matrix along with the crystalline filler are used for traditional dental ceramics. Unlike Y-TZP ceramics, the intaglio surface of silica-based ceramics are easily roughened by hydrofluoric acid and treated with silane-coupling agent 3-MPS); creating a strong siloxane bond , 3. Lack-MPS; creSeveral bond enhancement techniques are significantly considered with respect to cementation and zirconia surfaces. These techniques include chemical priming -11, PhosBy using a chemical priming, a chemical bond can be created with the use of modern primers with metacrylate and organophosphate functionalities , 7 or biMonobond Plus (Ivoclar Vivadent) is a primer that contains 3-Methacryloxypropyltrimethoxysilane (3-MPS). It is known to improve the bond strength to zirconia ceramics , 18. Airet al., [et al., [As per the Intellectual Property Rights (IPR), other manufacturers have developed new phosphate monomers capable of improving the bond to zirconia regarding the structure of MDP-monomer. These include Selective Infiltration Etching (SIE), Glaze-on, and Gluma desensitizer. According to Aboushelib et al., , SIE is [et al.,  has show[et al., .However, this study has used glow discharge plasma surface treatment on the bonding between cement and zirconia. Glow discharge plasma surface treatment is an approach, which is used for modifying the physical characteristics without affecting material bulk. This treatment has been previously studied on the shear strength bond , support2in vitro design of the study consists of specimen preparation and testing of the hypothesis.The 2.11 has presented the formation of cement according to the bonding groups. It has been apparent that RelyX Ultimate, Rely X Unicem 2, and Multilink Auto-mix were bonded through self-etching/self-adhesive groups. However, Ceramir and Ketac-Cem were bonded through acid based reaction. Table 2 presents the specimen distribution for the separation force test with and without glow discharge. A total of “10 glow discharge samples” have been included in group A while “10 No glow discharge samples” are included based on force testing in group B. The zirconia rings were seated in place with a Teflon orientation/cementation jig and all excess cement was removed for groups A and B (Table 2). All dual-cure resins were polymerized with a Triad 2000 machine  for 60 seconds on each top and bottom surfaces. Then specimens were stored wet at 37 °C for 24 hours. Bonded specimens were placed in a Delrin holder with clearance for the rod inside the holder. The rods were pushed out of the rings with a hardened steel compression rod with a round end, by using a universal testing machine (Instron Model 4202) at a crosshead speed of 0.5 mm / minA simulation system has been used comprising of custom-made zirconia rings and cylindrical rods. The zirconia rings and rods were prepared with the Zirconia Y-TZP powder and TZ-3YSB-E (Tosoh-Zirconia) through auto-mix to test the glow discharge and thermo-cycling. Three hundred custom-made zirconia rings were prepared with an outside diameter of 12 mm, height of 6 mm, and a centered hole of 6 mm diameter. Table Zirconia rings were made by pressing 3 grams zirconia powder  with the use of a straight dye set and load of 5000 pounds or 22250N. A putty index has been assembled to enable both the pressing pins provide uniform density independently.1).The specimens were first partially fired at 1100°C and then fully sintered in a Vita ZYrcomat furnace  at 1530°C, with a holding time of 2 hours. Heating rate has been set at 10°C per minute, as per the manufacturer’s recommendation. After sintering, dimensions of the rings were 12.5 ± 0.1 mm in outer diameter, 5.5 ± 0.1 mm in height and 5.9 ± 0.1 mm in a diameter , and at a rate of 4.2 gram per minute. The zirconia rings and the titanium rods were pretreated for bonding according to the manufacturer’s instructions.An orientation Teflon mold was used to centralize each rod into the zirconia ring, and aided as a cementation jig during the cementation procedure. All cements were mixed as per the instructions, and were applied to the external surface of the titanium rods and the internal surface of the zirconia rings. Peak separation loads were recorded and separation force was measured.The bond strength was computed as follows:Bond strength (MPa) = Load (KN × 1000)/ surface areaWhere:Load = Load at separation in KN,Surface area = 2∏ rh,r = Radius of specimen (mm),h = Height of specimen (mm).2.22A and 2B).Plasma is considered as an ionized gas, which have neutral and charged particles that are known as molecules, electrons, radicals, ions, and atoms. Chamber of glow discharge was evacuated to an optimal pressure by using a vacuum pump . Low pressure was maintained by an air inlet. A high frequency voltage  was applied between 2 electrodes, and was also adjusted until stable glow discharge plasma was generated. The schematic and actual glow discharge apparatuses are displayed in Figs. .Specimens were further treated and inspected under SEM  with an acceleration voltage of 15 kV.2.4Descriptive analyses were recorded as means, standard deviations and coefficients of variance. Furthermore, paired sample t-test has been used to analyze the comparison between adhesive and cohesive groups.33. As per the cement specimen, cohesive failure has been majorly observed with RelyX Ultimate (n = 12) as compared to 5 specimens of adhesive failure. In addition, adhesion failure has been majorly observed with Ketac-Cem (n = 20) as compared to cohesive failure (n = 0). This indicates that cohesive group was more materialized towards Ketac-Cem cement. Moreover, cohesive failure was also observed with RelyX Unicem 2, and Multilink Auto-mix (n = 8) as compared to adhesion failure (n = 4). This shows that both cements are suitable for adhesive surfaces and proves the applicability of glow discharge treatment for the adhesion surfaces. Table 4 has shown the comparison between adhesive and cohesive failure with respect to cement specimen used in this study. From the findings, it has been clearly observed that there is a significant difference between the use of cement specimens with respect to adhesive and cohesive surfaces.Failure pattern of specimens displayed a wide range of qualities, including cohesive failure (2/3 or more of luting agent was remained on zirconia surface), adhesive failure (less than 1/3 of the luting agent was remained on zirconia surface), and mixed (adhesive/cohesive failure). The count of the failure modes for the specimens is displayed in Table 3-6). The observations for failure modes observed during the study are presented in the following figures.Selected SEM photographs of each group are displayed in Figs. , regardless of the surface treatment. It appears that a chemical bond forms between the zirconia metal oxides and the phosphate-ester group of MDP. The bond eventually improves the bonding effectiveness. Such results are similar to those of several studies , 29-32. et al., . CeramirRelyX Unicem 2 and Multilink Automix cements contained Bis-GMA (bisphenol A-glycidyl methacrylate), UDMA (urethane dimethacrylate) or TEGDMA (triethylene glycol dimethacrylate) that have 2 C = C groups at the end of chain. Bonding agent containing MDP  has one C = C group. It was hypothesized that the polymerization of MDP-monomer forms a linear polymer; while Bis-GMA and UDMA formed a cross-linked polymer. Cross-linking had a major effect on mechanical properties of polymer , which eStudies reviewing different surface treatment techniques on zirconia have presented a contradiction with the findings. It has been reported that surface treatment usually enhances the bonding to zirconia -11, 27. It is known that high bond strength can get influenced by the application method of the priming mixture. These systems are thus, perceived sensitive to the technique because the operator tends to have a strong influence on the bonding quality of luting system . In contThe study evaluated the effects of glow discharge plasma surface treatment on the cements that bond to the zirconia. The treatment of glow discharge plasma is a common approach that has been in use for various services in dental health. It has been analyzed that the research lacks proper information about the impact of glow discharge surface treatment on cement bonding to zirconia. It is important to utilize these modes of management with high care as they may lead to the unlikely outcomes, including contamination. Outcomes of this assessment indicated that the glow discharge treatment significantly enhances the retention of zirconia to titanium rods. Thus, the results of the study will be effective in which application area of dentistry.


Skin engraftment of intraoral defects is known to be inconsistent due to mobility of the oral structure, uneven wounds, and accumulation of saliva under the skin graft. To improve the success rate of oral skin graft, we proposed a novel and simple dressing technique for intraoral skin graft to control saliva accumulation, in comparison with the conventional bolster dressing.n = 21) and modified bolster group (n = 10). In the modified bolster group, a polyvinyl alcohol sponge was designed to fit the skin graft and a suction drain was inserted with tagging suture to apply continuous suction. We analyzed the success rate, the size of the skin grafts and clinical variables of each method.We retrospectively reviewed 31 patients reconstructed with skin graft in their intraoral defect. The patients were divided into two groups; conventional bolster group (p = 0.015). The duration of bolster dressing and the time to start an oral diet were shorter in the modified bolster group.The overall success rate of oral skin graft was not different between the two groups (90.0 and 90.5%). However, partial necrosis in the engrafted skin was observed frequently in the control group (57.1% versus 20.0%). The relative engrafted area was significantly higher in the modified bolster group  contains supplementary material, which is available to authorized users. Intraoral defects can be reconstructed with various methods depending on different factors, including subsite and extent of defect, patient factors, surgical skills and institutional resources. Skin graft is a useful method for repairing wound, particularly for superficial defects that could not be closed primarily. It is a simple and less time-consuming procedure compared to regional and free flaps. Transplanted skin graft can protect the wound bed from further trauma and provide an important barrier to infection . To achiDifferent from other organs, an intraoral wound has some disadvantages in skin engraftment due to constant mobility of the oral structure, uneven wound bed, and accumulation of saliva between grafted skin and wound bed . To overHowever, few trials have been conducted to control undesirable accumulation of saliva under intraoral skin grafts. Saliva aids intraoral wound healing by providing a humid environment and it contains many growth factors . HoweverClinicalTrials.gov NCT02546895). From 2014 to 2017, a total of 31 patients who had undergone skin graft reconstruction for oral cavity defect were reviewed. Of 31 patients, 21 used conventional bolster dressing (without continuous suction) and 10 used our modified bolster dressing. Clinical data including patient age, gender, pathology, primary tumor site, clinical and pathological stages, and the sizes of defect and graft were collected. Gross photographs of the intraoral wound and the medical record were used to analyze successful engraftment of the skin graft. We analyzed the success rate and size of skin graft, duration of bolster dressing, and the time start of an oral diet from operation day.We retrospectively reviewed prospectively enrolled head and neck cancer patients in our institution. All patients submitted written informed consent for use of their clinical and biological data. The study protocol was approved by our Institutional Review Board  with interrupted sutures at the graft edge. The surface of the skin graft was processed with the pie-crust technique. A polyvinyl alcohol sponge  was designed to fit to skin graft size and applied directly with tie-over sutures to compress the skin graft.After the same procedure of applying a skin graft with the pie-crusted technique in the conventional bolster method, a sterile, absorbable gelatin sponge USP  was put on the skin graft. A Merocel® was designed to fit the skin graft size and a 100 cc Jackson-Pratt (JP) drain  was inserted inside the Merocel® sponge with tagging sutures Fig. -b. Then,The suction line of the JP apparatus was directly connected to the suction device, without using the collection bulb. Although sometimes we used the wall suction device (when the patient was in his bed), the main suction device, exerting the negative pressure on the sponge, was a portable suction device . This was the vacuum device for the negative pressure dressing (a vacuum assisted closure therapy system).We adjusted the JP drain pressure in the range of 100 to 180 mmHg to effectively remove the accumulated saliva in each patient Fig. . Daily aGraft status was evaluated based on graft healing and size by visual inspection at the time of wound healing (at postoperative 1-4 months). In this study, we regarded it as successful engraftment if there was no definite dehiscence or detachment of the skin graft from the wound bed. When there was necrotic debris or exudates at a portion of the graft site which consequently led to an incomplete healing of the skin graft, we regarded it as a partial engraftment. If there was no remnant skin graft in the bed, we classified it as a graft failure. Representative cases of successful engraftment and partial success are shown in Fig. p = 0.045). Nine out of 21 patients in the conventional bolster group and 9 out of 10 patients in the modified bolster group had clinical T1 tumors. Eleven out of 21 patients in the conventional bolster group and 1 out of 10 in the modified bolster group had clinical T2 tumors. However, the area (size) of skin graft was not different between the two groups (p = 0.31). All pathological diagnoses were squamous cell carcinoma except one (intermediate grade mucoepidermoid carcinoma). Individual clinical characteristics of all patients in two groups are listed in Table A total of 31 patients with a median age of 62 years (range 29–79) who were reconstructed with skin graft after resection of oral cavity cancer were reviewed in this study. Of these, 21 patients used the conventional bolster method and 10 patients used our modified bolster method. There was no statistical difference between two groups in age, sex, site of primary cancer and graft type   (90.0 and 90.5%). However, partial necrosis  in the engrafted skin was observed frequently in the control group (57.1% versus 20.0% in the modified bolster group) Fig. . As a re15) Fig. . These fp = 0.028)  of bolster (Merocel®) placement was significantly shorter in the modified bolster group (5.6 days) than in the conventional bolster group (6.5 days) (28) Fig. . Althougp = 0.002)  earlier than in the conventional bolster group  Fig. . While pIn the modified bolster group, successful engraftments were achieved in all patients except three  has been introduced to facilitate skin graft adherence , 18–20. Our modification is simple and effective in removing saliva accumulation under skin graft. Two or three suction holes in the JP drain are buried in the Merocel®. Therefore, saliva or secretions around the oral cavity wound can be sucked out through the drain. In our study, we applied a continuous suction pressure ranging from 100 to180 mmHg. Though this pressure seems to be quite strong, it does not produce direct pressure on the oral cavity wound because the sponge (Merocel®) with a suction drain was placed in an open space (not negative pressure). Also, when we apply suction pressure via a JP drain, the sponge collapses around the holes in the drain and reduces the actual suction pressure on the wound and the skin graft. If saliva was sticky, we elevated the drain pressure up to 180 mmHg. Thus, the important role of suction drain is to effectively remove the absorbed secretions in the sponge, drying it to absorb other secretions. Furthermore, when starting an oral diet with continuous suction, no patient complained about the high suction pressure.We experienced malfunctions of continuous suction in some patients due to obstruction of the drain. The main cause of drain obstruction was local collapse of the sponge around small holes in the JP drain. In such cases, repositioning the drain line or lowering the power used for the continuous suction was needed.The duration of the continuous suction with Merocel® was decided based on the status of skin graft adherence by regular examination of skin graft through the gap between the Merocel® sponge and the wound bed. If there was no definite dehiscence or detachment in skin graft, we removed Merocel® and continuous suction. When removing the dressing, the soft gelatin layer (Gelfoam®) could play a role as a good physical buffer to prevent the skin graft from accidental detachment. With our method, we could reduce the average duration of bolster to 5 days from the operation day.In our experience, applying continuous suction was harder for tongue lesions than for floor of the mouth lesions. This might be because the tongue is more mobile. On the floor of the mouth, saliva accumulation is more prominent than at a tongue lesion. Considering this, our technique could be more effective particularly in the floor of the mouth area. Moreover, in patients with oral cavity cancer, there would be a higher chance of saliva accumulation because of decreased swallowing function (before or after surgery). This will likely have a negative influence on successful engraftment.In summary, the modified bolster technique can help the skin graft adapt to the intra oral wound bed by removing saliva and exudates. Removing exudates prevents skin graft detachment from the wound bed, which can result in necrosis of the skin graft. In terms of patient convenience, no patient had complained about maintaining the oral suction line during the post-operative period.As we mentioned before, the modified bolster technique can advance the starting time of an oral diet, which can eventually reduce the duration of hospital stay. Furthermore, allowing the patient to try an oral diet earlier can increase patient comfort.One of the limitations of this study is the small number of patients of each group, especially in the modified bolster group, so that it is difficult to generalize the results. Another limitation is that this study has a selection bias because of the retrospective nature of the data collection. We expect a further prospective study with a large number of patients to be performed to support our preliminary results.This study suggests the technical feasibility of using our modified method to remove exudate and saliva under an intraoral skin graft. Further trials are needed to verify our method. Furthermore, detailed technical refinement is also needed to control saliva or exudates more effectively. Though this study has some limitations, these results suggest a new technical modification to the conventional method. This might trigger future focus on the effect of saliva control on the success of intra oral skin graft.Additional file 1:Table S1. Clinical characteristics of patients with the conventional bolster dressing (controls). Table S2. Comparison of baseline variables between the modified bolster dressing group and the control group. (DOCX 25 kb)


Concerns have been raised that the transplants performed in the local context at the time of procedures reported in this article  may haveDetails as to the donor sources and methods of obtaining informed consent from donors were not reported in , and theIn addition, the authors did not provide documentation when requested by the journal to confirm that the study had institutional ethics approval, and they did not respond to inquiries about the availability of underlying data supporting this study.PLOS ONE Editors retract this article.Owing to the lack of documentation to demonstrate this study had prospective ethical approval, insufficient reporting, unresolved concerns around the source of transplanted organs and whether they included organs from prisoners, and in compliance with international ethical standards for organ/tissue donation and transplantation, the The first and third author notified the journal that all authors agree with the retraction. The other authors either could not be reached or did not respond directly.


Concerns have been raised that the transplants performed in the local context at the time of procedures reported in this article  may haveDetails as to the donor sources and methods of obtaining informed consent from donors were not reported in , and wheIn addition, the authors did not provide documentation when requested by the journal to confirm that the study had institutional ethics approval, and the authors did not respond to inquiries about the availability of underlying data supporting this study.PLOS ONE Editors retract this article.Owing to the lack of documentation to demonstrate that this study had prospective ethical approval, insufficient reporting, unresolved concerns around the source of transplanted organs and whether they included organs from prisoners, and in compliance with international ethical standards for organ/tissue donation and transplantation, the The first author and corresponding author notified the journal that all authors disagree with the retraction. The other authors either could not be reached or did not respond directly.


Journal of Educational Evaluation for Health Professions (JEEHP) was included in Embase, the invaluable biomedical literature database maintained by Elsevier, which provides over 32 million records published in over 8,300 indexed peer-reviewed journals. This represents another opportunity for the journal to be disseminated and searched by biomedical researchers from all over the world. Although JEEHP has been indexed in MEDLINE since March 2016, it was included in Embase in May 2018. The rigorous review process for being indexed in Embase provides further evidence of the journal’s high quality and brand.We are proud to announce that in May 2018, Fundación Educacion Médica, Medical Education, Medical Teacher, and South-East Asian Journal of Medical Education. The World Federation for Medical Education Conference 2019 will be held at the Grand Walkerhill Seoul in Seoul, Korea from April 7 to 10, 2019 (http://www.wfme2019.org/). I hope to invite the authors who present at this conference to publish their results in JEEHP.Although JEEHP has been an affiliated journal of the World Federation for Medical Education since September 2016 [In April 2016, we adopted an open data policy ; howeverIn January 2018, the third version of the ‘Principles of transparency and best practice in scholarly publishing’ was announced by the Committee on Publication Ethics, the Directory of Open Access Journals, the Open Access Scholarly Publishers Association, and the World Association of Medical Editors. It consists of 16 items that should be announced and adhered to by scholarly journal editors and publishers . A complThis year, I invited a number of reviewers to review submissions to JEEHP. Some of them accepted the review request and provided invaluable comments that contributed to the better presentation of study results. Without their help, it would not be possible to publish this small, but unique, journal. I understand very well that they were all very busy, but they nonetheless shared their time to contribute to advances in educational evaluation for health professions. I appreciate them from the bottom of my heart. The reviewers for the 2018 issue are listed below:Abdolghani Abdollahimohammad, Katharina Brandl, Su-Jin Chae, Lap Ki Chan, Cheol-Woon Chung, Derek Clewley, Fabrizi Consorti, Lionel Di Marco, Kyung (Chris) T. Han, Geum-Hee Jeong, Karen Huhn, Yera Hur, Oscar Jerez, Sun Hee Kang, Nayoung Kim, Yong Sung Kim, Na Jin Kim, Young-Min Kim, Sue Kim, A Ra Cho, Seock-Ho Kim, Kwang Hwan Kim, Sun Kim, Oh Young Kwon, Young Hwan Lee, Eun Young Lim, Nesreen El Mekawy, Younjae Oh, Cesar A. Orsini, Robin Parish, Janghee Park, Mee Young Park, Quan Pham, Amelia Richardson, Dong Gi Seo, Ji-Hyun Seo, Ravi Shankar, Luiz Troncon, Julie Youm, Hon Yuen, Pete Yunyongying, Gagani Athauda, Fabrizio Consorti, and Gregory RoseWe published 3 articles on computerized adaptive testing in this year’s issue introduction piece of SimulCAT, a software program developed for conducting CAT simulation studies ; post-hoIn the New Year, I hope to receive and publish more manuscripts with complete adherence to the journal’s style and format with interesting topics for instructors in the medical and health fields.


As we approach the end of our 22nd year as the pioneering and preeminent Open Access journal in the field of organic chemistry and natural products, time has come to formally announce what has been the de facto reality of the journal for the past few years, the expansion of the range of topics we cover.Originally dedicated exclusively to organic synthesis and natural products chemistry, the evolution of these fields into multidisciplinary areas where the lines between chemistry, biology and materials are blurred, has led us to publish over the years increasing numbers of papers that did not fit the classical definition on the journal’s masthead. Molecules , which henceforth will include (but will not be limited to):Organic chemistryMedicinal chemistryNatural productsInorganic chemistryPhysical chemistryMaterials scienceNanoscienceCatalysisChemical biologyAnalytical chemistrySupramolecular chemistryTheoretical chemistryGreen chemistryPhotochemistryMoreover, we have not been alone in this evolution, and as several other MDPI journals have also refocused their own fields of coverage over the years, we felt that this left some interdisciplinary topics without a suitable forum. To remedy all this we are now formally expanding the scope of As always, we encourage any authors who feel that their work does not fit into one of these boxes, to think of us as a venue for publishing their results. This refocus in no way changes our commitment to provide rigorous peer review and enable rapid publication of cutting-edge research to educate and inspire the scientific community worldwide. As our main objective is the full diffusion of experimental details and theoretical results, there will still be no restriction on the length of the papers, we will continue to insist on the publication of all experimental results and to encourage collaboration, we will continue to alert readers of the availability of samples and encourage the deposit and diffusion of chemical samples through the international non-profit organization Molecular Diversity Preservation International (MDPI). On behalf of the publisher and our Editorial Staff, I look forward to your thoughts and comments as we expand our Editorial Board to bring on experts in these new fields and especially, to receiving and publishing your papers.