Several interfacial surface treatments of zirconia surfaces have been proposed to improve adhesion to ceramic veneering. However, information regarding the durability and effect of such treatments on the bond strength following such treatments is lacking.This study aimed to evaluate the shear bond strength between veneering ceramic and zirconia core after different interfacial surface treatments.2O3), group II was coated by bioglass, group III was coated with ZirLiner, and group IV was subjected to wash firing (sprinkle technique). A cylinder (4 mm in diameter and 3 mm in height) of veneering ceramic was fired on top of the zirconia core. Shear bond strength (SBS) between zirconia core and veneering ceramic was evaluated by using a universal testing machine. The data was collected and statistically analysed using One-Way ANOVA followed by multiple pairwise comparisons using Bonferroni adjusted significance level. The failure modes were assessed using a stereomicroscope for each group.Fifty-two discs (8 mm in diameter and 3 mm in height) were fabricated from zirconia blanks using a microtome cutting machine. Zirconia discs were divided into four groups (n = 13). Group I was subjected to air-borne abrasion using (AlThe highest mean bond strength was recorded in group III (17.98 ± 2.51 MPa), followed by group II (15.10 ± 4.53 MPa), then group I 14.65 ± 2.97 MPa. The lowest mean bond strength was recorded in group IV (13.28 ± 3.55 MPa).Surface treatments had an effect on the zirconia-veneer shear bond strength. Liner coating revealed the highest shear bond strength values, significantly higher in comparison to wash firing (sprinkle technique) . Due to the high expectations and demands for aesthetics in routine dental treatment, the use of ceramic restorations has significantly expanded. The patient’s expectation to receive highly aesthetic restorations with good mechanical qualities has increased due to ongoing technological and procedural advancements .Due to its solid polycrystalline structure, zirconia is opaque, which affects its aesthetic properties. Hence, a veneering ceramic must be used to conceal the zirconia substrate in the aesthetic zone. Zirconia-based bilayer restorations consist of an aesthetic veneering ceramic and a durable zirconia infrastructure, both of which are biocompatible , 3.2O3), liner application, wash firing, and glass coatings [The bond between zirconia and the veneering ceramic depends on several factors, including the chemical bonding, mechanical clamping due to the difference in thermal expansion coefficient, wetting behaviour, and the glass transition temperature differences . Thus, tcoatings –8.2O3 particle roughening is the technique that is most frequently utilised. It enhances wettability and surface roughness while forming undercut zones, boosting the surface energy [Ale energy , 10. Dese energy , 11.Another technique is to add a liner between the veneering ceramic and zirconia to increase the materials’ adherence by adjusting for the difference in their coefficients of thermal expansion (CTE). However, the findings were disputable. Some authors claimed that it might reduce the degree of adhesion, while others claimed that it would strengthen the binding; several claimed that it would have no effect –16.Wash firing with nanofluorapatite veneering ceramic, called the sprinkle technique, was suggested by the manufacturer to enhance the in-depth shade and bond strength. In this technique, shade and glaze are mixed with the respective liquids to the desired consistency and applied to form a covering layer on the entire framework. After that, the nanofluorapatite veneering ceramic dentin powder is sprinkled on the restoration using a dry brush .(−6)K(−1)) comparable to that of zirconia (11.67 × 10(−6) K(−1)) and that of fluoroapatite veneer ceramic (9.85 ± 0.25 × 10(−6) K(−1)). This technique makes zirconia superficially more rough, consequently enhancing bonding [Bioglass coating is a technique where a glass mixture is utilised to cover the zirconia surface before applying the veneering ceramic, to increase the strength of the zirconia-ceramic interfacial bond. This glassy layer has a CTE  between veneering ceramics and zirconium core after different interfacial surface treatments.Power analysis was performed using a statistical software program  . Sample Sample preparation and examination was done at the Conservative Dentistry Department laboratory at the Faculty of Dentistry, Alexandria University. Fifty-two discs (8 mm in diameter and 3 mm in height) fabricated from zirconia blanks were randomly divided into 4 groups: group I was the air-borne particle abrasion group, group II was the bioglass coating group, group III was the Zirliner coating, and group IV was the wash firing (sprinkle technique) group. The materials used in the current study are listed in Table Zirconia discs were cut using a microtome cutting machine , where an automatic linear blade was used with a speed 2350 rpm and feed rate 13.2 mm/min. The zirconia discs were sintered at 1500 °C as instructed by the manufacturer in a programmable furnace  and cleaned in an ultrasonic bath  filled with ethanol (99.5%) for 5 min.2O3 particles  at 8 bar pressure.Group I discs were air-particle abraded using 110 µ AlThe working surface of each disc was abraded by placing the nozzle perpendicular to the disc surface, at a distance of 10 mm between the nozzle and the surface of the disc for 10 s. The distance was standardized by placing each disc in a custom-made cylindrical acrylic mold, at a depth of 10 mm. The sandblasting nozzle was held perpendicular to the disc with its tip flush with the opening in the cylinder as shown in Fig. After that, the zirconia surfaces were cleaned using 96% ethanol in an ultrasonic bath for 10 min to remove any loose particles caused by air-abrasion.For group II, the discs were coated with bioglass coating . Each zirconia disc was coated using a brush to produce a uniform bioglass-coated layer, then allowed to dry for 2 h and fired at 1150 °C for 16 min in a furnace .group III, discs were coated with a liner  following to the manufacturer’s instructions. The powder and liquid were mixed and applied to form a uniform thickness on the entire framework, then fired at 960 °C in the furnace .For group IV, discs were prepared using the wash firing (sprinkle) technique. In this technique, glazing material  was mixed with the respective IPS liquids to the desired consistency and applied to form a covering layer on the entire framework. After that, the nanofluorapatite veneering ceramic  dentin powder was sprinkled on the restoration using a dry brush. The wash firing was performed on a honey-comb firing tray and fired at 750 °C in the furnace .For ceramic veneering, a teflon mold was constructed to adjust the dimensions of the veneering material (4 mm in diameter and 3 mm in height). Each zirconia disc was seated in the mold, then the nanofluorapatite veneering ceramic  was applied on the treated core surface till the desired height then fired in the furnace at 750 °C . The disAn additional zirconia disc from each surface treatment group prior to ceramic veneering was fabricated for scanning electron microscope (SEM)  analysis. Each disc was coated with gold sputter coating in a machine before SEM examination. After gold coating, images were captured at magnifications of (500x and 1000x) with an accelerating voltage of 20 KV to examine surface morphology of each disc.A universal testing machine  was used for SBS testing to debond the zirconia discs from the veneering ceramics in accordance with ISO 17095:2013  at a cro2), where area of the disc equals πr2. To calculate the SBS in megapascals (MPa), the resultant values were multiplied by 0.09807.The load in kilograms, at which the eroding ceramics detached from the zirconia surface, was displayed on a digital monitor. The SBS was calculated as fracture load (kg) divided by disc surface area  by using a software . When there was no failure between the zirconia and veneering ceramic, the failure modes were observed as “adhesive”. “Cohesive” failures referred to those that occurred within the veneering ceramic, and “mixed” failures some ceramic was present, but zirconia was also exposed. Stereomicroscopic images of the zirconia fractured samples demonstrating each failure mode is shown in Fig. P value ≤ 0.05. Data were analysed using a statistical software .Normality was checked using descriptive statistics, plots and normality tests. Force and SBS showed normal distribution. Mean, standard deviation (SD), median, interquartile range (IQR) and range were calculated. Comparison between the four study groups was done using One-Way ANOVA followed by multiple pairwise comparisons using Bonferroni adjusted significance level. Significance was set at SEM analysis of the treated discs showed some variations in surface topography among the different groups. In group I, zirconia crystal structures were observed in the air-abraded surface with a uniform distribution of pores and pits due to the effect of airborne particle abrasion. In group II, bioglass coated surfaces showed the formation of glass clusters in mesh-like pattern. In group III, small areas uncovered by the liner were observed with a uniform distribution. Group IV showed wash firing resulting in large areas uncovered by sprinkle particles Fig. Quantitative data were described using range (minimum and maximum), mean, SD and median. The mean SBS in MPa are presented in Table Bonferroni adjusted post-hoc tests revealed that there was no statistically significant difference in the bond strength values between any of the groups except between group III ( liner coating) and group IV (wash firing) as shown in Table The robust bonding between the ceramic veneer and zirconia core is essential for the predictable success of zirconia-based ceramic restorations. Mechanical laboratory studies are frequently the foundation for clinical advice on materials, processes, or methodologies. The shear bond test, which was also used to assess the bond strength between core and veneer in various ceramic systems, is one of the most used methods for determining bond strength. SBS test is used in this study because of ease of preparation of the samples and simple test protocol.The surface finish of the core, which can affect mechanical retention, the emergence of flaws and structural defects at the core-veneer interface, the wetting properties, the volumetric shrinkage of the veneer, and the residual stresses caused by a mismatch in the CTE are just a few of the many factors that can affect the strength of the bond between the core and veneer. To produce a good interfacial bond strength, the core and veneering ceramic’s coefficients of thermal expansion must nearly match.Zirconia core material underwent several surface treatments in this investigation to improve its adhesion to ceramic veneering. The null hypothesis, which claimed that there would be no significant difference in SBS between veneering ceramics and zirconium cores after different interfacial surface treatments, was rejected in light of this study’s findings.A previous study by Al-Dohan et al. reported SBS in range of 22–31 MPa for commercially available core-veneer ceramic systems . Another2, Al2O3, Y2O3 and other oxides in the liner coating coating may enable for greater chemical interaction between the zirconia and the ceramic [The highest shear bond strength was observed in group III ( liner coating group). Zirconia and ceramic are bonded together through a variety of mechanisms, including chemical and mechanical interlocking.The HFO ceramic , 7 whichIn accordance with results of the current study, López Mollá et al. evaluated the SBS of zirconia with a ZirLiner coating and a fluoroapatite-pressed ceramic and found a strength of 12.70 MPa, which is comparable to the findings of the present work (17.98 ± 2.51 MPa) . ContrarGroup II (bioglass coating group) had lower shear strength values than the group III (liner coating group). This finding may be supported by a finding of a previous study by Rivera et al., which concluded that glass coatings enhanced bond strength between zirconia and fluorapatite veneering ceramic, improving chemical and mechanical interlocking .2SiO5 and zirconium aluminium oxide were produced using the sol-gel dipping coating approach using a silica and aluminosilicate sol-gel [Group I showed SBS even lower than those of groups II and III, yet it is worth noting that there was no significant difference between the SBS values of these 3 groups. In a previous study by Bagheri et al., novel phases such Al sol-gel . This su sol-gel –35.The lowest SBS values were shown in group IV. Findings of the present study concur with those of a previous where samples that fractured cohesively within a veneer had lower SBS values than samples that fractured cohesively within the core or had a mixed cohesive/adhesive fracture pattern . In the It is worth noting that, in the current study, the failure mode of the majority of the discs showed mixed cohesive/adhesive failure at a high percentage, significantly presented in groups II and III (69.2%). That is with the exception of group IV, which displayed a cohesive mode of failure within the weak veneering ceramic (15.4%) .Adhesive failure occurred at a low percentage .According to the results of the current study, on comparing between groups there was no statistically significant difference of the bond strength values between them except between groups III and IV. These investigations showed that various surface treatments improve the bonding between the zirconia core and veneering ceramic.Because the test findings are influenced by the disc design, the type of mechanical testing, and the various materials utilised, it is difficult to compare different research. To the authors’ best knowledge, no previous studies examined and compared the effect of these surface treatments, specifically bioglass coating and wash firing (sprinkle technique), on SBS between zirconia and ceramic veneering using the same standardised mechanical testing methods.Among of the limitations of the current study is that the disc design does not represent the clinical configuration of a zirconia ceramic restoration. Nevertheless, the design used in the study allowed for the SBS testing procedure under standardized conditions. Another limitation is that the in vitro nature of the study does not reflect the conditions of the oral cavity. Moreover, the bond strength was only evaluated using the shear bond strength test, while the mouth exposes restorations to other forces. Hence, additional studies mimicking the conditions of the oral cavity should be conducted with the aim of improving the bond strength between the zirconium core and ceramic veneering.Zirconia-veneer shear bond strength was significantly affected by different interfacial surface treatments.The highest shear bond strength was found in the liner coating group, followed by the bioglass coating and air-borne particle abrasion groups. There was no significant difference between these 3 groups.The lowest shear bond strength was shown in the wash firing group, significantly lower than the liner coating group.


Filoviridae family and has been classified as a risk group 4 pathogen. To this day, there are no approved effective vaccinations or medications available to prevent or treat MARV infections. Reverse vaccinology-based approach was formulated to prioritize B and T cell epitopes utilizing a numerous immunoinformatics tools. Potential epitopes were systematically screened based on various parameters needed for an ideal vaccine such as allergenicity, solubility, and toxicity. The most suitable epitopes capable of inducing immune response were shortlisted. Epitopes with population coverage of 100% and fulfilling set parameters were selected for docking with human leukocyte antigen molecules, and binding affinity of each peptide was analyzed. Finally, 4 CTL and HTL each while 6 B cell 16-mers were used for designing multiepitope subunit (MSV) and mRNA vaccine joined via suitable linkers. Immune simulations were used to validate the constructed vaccine's capacity to induce a robust immune response whereas molecular dynamics simulations were used to confirm epitope-HLA complex stability. Based on these parameter's studies, both the vaccines constructed in this study offer a promising choice against MARV but require further experimental verification. This study provides a rationale point to begin with the development of an efficient vaccine against Marburg virus; however, the findings need further experimental validation to confirm the computational finding of this study.A hemorrhagic fever caused by the Marburg virus (MARV) belongs to the  Filoviridae family and the genus Marburg virus [Marburg virus (MARV) causes hemorrhagic fever in humans and belongs to the rg virus . It is crg virus , 3. MARVrg virus . MARV gerg virus . Seven srg virus . MARV isnd of August 2021 from multiple patients who died [The virus can spread between people via body fluids via unprotected intercourse and open wounds. It can cause similar symptoms as in Ebola virus fever and can cause bleeding (hemorrhage) and fever symptoms. In 1967, the MARV was first described . RegularAcinetobacter baumannii. Likewise, to this, the effectiveness of theoretically predicted B cell epitopes in detection against Trypanosoma vivax was also validated in wet lab experimentation. For instance, similar strategies have been used against a number of human fatal viruses, such as the Crimean-Congo hemorrhagic fever virus, Ebola, and Mokola rabies virus [Staphylococcus aureus, Shigella spp., and Ebola virus vaccines have all been developed using immunoinformatics method [There is no way to treat the virus after infection, and early treatment of symptoms like dehydration greatly increases the odds of survival . Ebola aes virus –16. The s method , 18. In s method .In this scientific investigation, predictions of linear B cell and T cell epitopes obtained from MARV proteins were carried out and investigated as viable possibilities for constructing two vaccine candidates, i.e., multiepitope subunit vaccine (MSV) and mRNA vaccine (MRV). Using in silico molecular docking methods, specific T cell epitopes were evaluated for physiochemical properties and interaction with their respective HLAs. The physical and chemical characteristics of MSV were also computed; then, secondary and tertiary structure modeling was done. The revised 3D model's affinity for binding toll-like receptors was assessed using a variety of immunoinformatics methods. This study may open the door to the creation of dynamic and effective vaccines that include a special association of several MARV protein-derived antigenic peptides that play various functions throughout the lytic stage of MARV infection.https://www.uniprot.org/) [Three proteins  of MARV were downloaded in FASTA format from the Universal Protein Knowledgebase (ot.org/) . The proot.org/) . Accordiot.org/) , 23. TheOnline servers were utilized for identification of T cell epitopes. CTL epitopes were identified by NetCTL 1.2 server at a default threshold of 0.75. The CTL predictions using NetCTL 1.2 demonstrate essential information regarding different parameters by using a weight matrix. NetCTL 1.2 has an over 0.72 sensitivity among the five percent of top-scoring short peptides in a large standardized computations including 216 HIV epitopes covering all 12 confirmed HLA super types.HTL epitopes for the MARV proteins were predicted by IEDB MHC-II considering the reference set of seven HLAs using default parameters to enable the prediction of epitopes across the largest feasible population. Since HLA alleles vary greatly in their susceptibility and sensitivity among populations, it is impossible to establish a causal relationship between the prevalence of MARV and any particular HLA allele. Each HTL epitope is given an IC50 value by the IEDB MHC-II module, which is inversely related to binding affinity of the peptide to MHC-II receptor. High binding affinity is indicated by an IC50 score of smaller than 50 nM, moderate binding affinity is shown by a score of less than 500 nM, and low binding affinity is indicated by a score of less than 5000 nM for an epitope to the MHC-II receptor. The less percentile ranks the stronger binding affinity for MHC. Numerous important parameters were considered to identify HTL epitopes fulfilling criterion that will be further used for mRNA vaccine designing. Furthermore, to select the most suitable CTL and HTL epitopes of the predicted pool, multiple parameters were analyzed for individual epitopes derived from the selected proteins , 24–26.High-throughput epitope screening and computational epitope prediction are two key approaches in immunoinformatics for identifying potential epitopes for vaccine development. In general, a combination of both HTL and CTL epitopes can be included in a vaccine to provide both humoral and cell-mediated immune protection. HTL epitopes are recognized by helper T cells and are crucial for generating antibody responses, while CTL epitopes are recognized by cytotoxic T cells and are critical for destroying infected cells. However, the selection of epitopes ultimately depends on the specific pathogen and the immune response that is needed to provide protection against it. Multiple parameters required for an ideal vaccine were considered to select the most suitable T cell epitopes.VaxiJen server predicted the antigenic capacity of both CTL and HTL epitopes . InsteadThe epitope should have the capacity to elicit a strong and specific immune reaction in the host. Immunogenicity is the capacity of an epitope to provoke an adaptive immune reaction. The IEDB MHC-I immunogenicity module was utilized to predict the immunogenic potential of MARV-derived CTL epitopes. Epitopes scoring higher than zero were classified as immunogenic and studied further .The antigenic and immunogenic epitopes were assessed for solubility and toxicity using CamSol Intrinsic and ToxinPred servers, respectively . The epiThe epitopes included in the vaccine should bind with high affinity to the MHC molecules, which present the epitopes to T cells. MHC class I alleles from the shortlisted CTL epitopes were identified using the IEDB server's consensus algorithm and IEDB MHC-I binding tool .HTL epitopes satisfying the above-described constraints were subjected to allergenicity prediction using Algpred2.0 server . AlgpredPopulation coverage is a significant consideration in in silico vaccine design, since it helps to ensure that the vaccine provides broad protection against a pathogen, and is effective in a diverse range of individuals. The chosen T cell epitopes in the mRNA vaccine construct should efficiently cover highly populated countries across the globe. The chosen T cell epitopes were examined (country as well as region-wise) using default parameters set in IEDB population coverage tool to assure that the proposed vaccination will cover the majority of the global population . The aboLinear B cell epitopes are a useful component of vaccine design, as they are easy to identify and design and highly immunogenic and can help to generate a diverse range of antibodies that target different regions of the pathogen. The kernel approach is a novel technique used by the BCPred service to estimate linear B cell epitopes . The AllMolecular docking of epitopes with HLAs is a crucial step in computational vaccine design, as it enables the estimation of how epitopes will bind to HLA molecules and can help to identify the most promising candidates for inclusion in a vaccine. Selected T cell epitopes were docked in silico with corresponding human leukocyte antigens using the CABS-dock platform. The server does not need a predetermined peptide/epitope 3D structure; thus, all that was required was a PDB file of the receptor and the epitope sequences. The RMSD (root-mean-square deviation) score of the epitope/peptides was computed by CABS-dock. The ligand-RMSD score indicates the quality of the docking pose. The RMSD score is directly related to the quality of ligand-receptor binding, and a smaller RMSD score indicates better key binding contacts, indicating a good docking position. High-quality predictions have RMSD scores of less than 3, whereas moderate-quality predictions have RMSD scores between 3 and 5.5. The respective HLAs were retrieved from RCSB. Furthermore, HawkDock server was employed to estimate the binding free energy for the best conformation determined by CABS-dock server , 36.In this study, two vaccine candidates were devised to make use of the shortlisted T and B cell epitopes.A computationally designed mRNA vaccine consists of several components that are designed and optimized using bioinformatics and computational approaches. By optimizing the antigen sequence, noncoding regions, delivery system, adjuvants, and other components, it is possible to create a highly effective and specific vaccine against a target pathogen or disease. The presence of five components is considered essential of an ideal mRNA vaccine. (1) It should have 5 prime and 3 prime UTR regions, (2) a Kozak sequence should be there, (3) the vaccine structure should also have efficient B and T cell epitopes, (4) also, suitable linkers should be used to combine the epitopes, and (5) a stop codon at the end that tells the translation machinery where to stop. The AUG codon has to be included within the Kozak sequence , althougThe tissue plasminogen activator (tPA) (UniProt ID: P00750) secretory signal sequence that has been reported to be helpful in increasing antigen presentation and release of translated epitopes was included in the vaccine sequence . Since mSubunit vaccines are made up of antigenic sections of a pathogen that are used to initiate immune response in host. To construct the final vaccine, the anticipated T cell and linear B cell epitopes were joined sequentially. To increase immunological response, a vaccine sequence was created adding mammalian beta-defensin (a 45-mer peptide) as an adjuvant. Interactions of adjuvants with toll-like receptors (TLRs) polarize CTL responses and cause a strong immune response. TLR1, TLR2, and TLR4 receptors can all be triggered by beta-defensin adjuvant. PADRE sequence was added along with the adjuvant to help overwhelm the issues created by extremely polymorphous HLA alleles. The adjuvant and PADRE sequences were linked using EAAAK linkers. The CTL, HTL, and LBL epitopes were conjugated using AAY, PMGLP, and GGGGS linkers, correspondingly.Predicting different physicochemical properties of a vaccine is an important step in vaccine design, as it can help to optimize the stability, solubility, bioavailability, and immunogenicity of the vaccine. By optimizing these properties, it is possible to improve the effectiveness of the vaccine and increase the likelihood of a successful immune response. VaxiJen, Algpred2.0, and ProtParam servers were utilized for the calculation of the physiochemical properties  of the multiepitope's subunit vaccine.The SOPMA predicted the secondary structure of the MSV, whereas for tertiary structure prediction, Robetta server was employed. Predicting the 3D structure of a vaccine is an important step in computational vaccine design, as it is directly connected to the stability, epitope accessibility, immunogenicity, and design of multiepitope vaccines. By optimizing the 3D structure of a vaccine, it is possible to improve its effectiveness and increase the likelihood of a successful immune response. The protein structure modeling technique used by the Robetta server includes the identification of a template model. If in case a template protein with suitable identity and similarity is identified by utilizing BLAST, PSI-BLAST, 3D-Jury, or FFAS03, the server uses the comparative modeling approach for modeling. The predicted structure was further refined using the GalaxyRefine webserver.Z-score). For instance, if the Z-score falls within the range determined for experimentally established protein models, the submitted protein model is most expected to have no errors. The region of the input structure, if there are problems, can be visualized on the result page of the ProSA-web server.The validation of 3D models of vaccines is an important step in the computational design of vaccines. By ensuring the accuracy and reliability of the predicted structure, researchers can optimize the design of the vaccine for improved immunogenicity, efficacy, and safety and can support the regulatory approval of the vaccine. In this study, ERRAT, ProSA-web, and Ramachandran plot by Procheck were used to validate the quality of vaccine's 3D model generated and refined by Robetta and GalaxyRefine webservers, respectively. ERRAT assesses the non-bonded interactions within a given 3D structure. ProSA-web is commonly used to authenticate a protein 3D model and assign a quality score  of an epitope is directly related to binding potential to MHC-I receptors. The selected most suitable epitope multiple parameters were considered, high COMB score, immunogenicity score higher than 0, antigenicity above threshold of 0.4, and solubility above -1; and for an epitope to be classified as nontoxic, it should have a score less than 0. Four CTL epitopes that fulfilled the criterion mentioned above were selected for construction of vaccine sequence. Furthermore, IEDB MHC-I binding module predicted corresponding HLA molecules having considerable binding affinity for the selected CTL epitopes. The selected CTL epitopes with HLAs and scores are provided in HTL epitopes are bind to MHC-II receptors and activate helper T cells of host's immune system. IEDB MHC-II predicted HTL epitopes that corresponds to a reference set of 7 human leukocyte antigens or shortly as HLAs. Epitopes that have lowest adjusted percentile rank indicate higher binding affinity of that epitope to the MHC-II receptor. Multiple parameters with respective thresholds were considered to select HTL epitopes suitable for vaccine construction. Epitopes with low adjusted percentile score, antigenicity above 0.4, immunogenicity greater than 0, solubility score greater than -1, allergenicity score lower than 0.3, epitope having the ability to induce interferon-gamma response, and toxicity score lower than 0 were selected for vaccine construction. Overall, five HTL epitopes were selected based on the aforementioned criterion. The selected HTL epitopes with corresponding scores are provided in The population coverage analysis was carried out using IEDB population coverage tools. The complete set of HLAs available in the IEDB database was used to evaluate and select T cell epitopes covering most of the population worldwide. The overall results suggest the selected T cell epitopes covering 100% of the population worldwide; furthermore, region-wise population coverage had similar results . MoreoveLinear B cell epitopes are crucial for an ideal vaccine; in our study, we predicted linear B cell epitopes using BCPreds. Linear B cell epitopes with high scores are directly related to strong binding to respective receptor present on B cell surface. Screening of the epitopes was based on multiple scores, high binding score, antigenicity above threshold of 0.4, and allergenicity above the threshold level of <0.3. Six epitopes were selected to be included in the final vaccine construct. The selected epitopes with respective scores are presented in ∗08:01 with PDB ID: 7NUI, HLA-B∗58:01 with PDB ID: 5VWH, HLA-A∗01:01 with PDB ID: 4NQX, HLA-DRB1∗07:01 with PDB ID: 6BIJ, and HLA-DRB1∗15:01 with PDB ID: 1BX2, and prepared for docking. The total binding free energy for HLA-B∗08:01-TTEERTFSL was reported to be -53.55 kcal/mol, -49.48 kcal/mol for HLA-B∗58:01-LTNLGILLL, -57.15 kcal/mol for HLA-B∗58:01-LTNRELLLL, and -63.51 kcal/mol for HLA-A∗01:01-ISPNLLGIY. The docked complexes (CTL) are shown in Figures ∗15:01-AQHLVYFRRKRSILW425-439 , -77.18 kcal/mol for HLA-DRB1∗07:01-SEWLLLEVTSAIHIS123-137, -46.04 kcal/mol for HLA-DRB1∗15:01-NRELLLLMARKMLPN103-117, and-71.46 kcal/mol for HLA-DRB1∗15:01-TLENLGHILSYLHRS150-164. The docking pattern and BFE are given in Figures The list of HLAs with corresponding PDB IDs is presented in Molecular simulation of complex revealed stable dynamics calculated as root-mean-square deviation (RMSD). Each complex shown in An adjuvant multiepitope subunit vaccine must be cloned, expressed, purified in vitro, and injected to the host subcutaneouslyA self-adjuvant mRNA vaccine must be produced in vitro, delivered to the host via an appropriate delivery system, i.e., nonviral lipid nanoparticles, administrated intramuscularly, and expressed in vivoIn this study, two vaccine candidates were constructed using the selected set of 4 CTL, 4 HTL, and 6 LBL peptides:A cluster of epitopes joined together through AAY, PMGLP, and GGGGS linkers was used to construct the vaccine candidate. The final included CTL, HTL, and B cell epitopes are presented in Tables 2The development of a multiepitope vaccine design that is safer for human usage has evolved with the use of computational models in vaccine design. The MSV construct comprised of beta-defensin (adjuvant), 4 CTL, 4HTL, and 6 LBL epitopes joined together using suitable linkers. To boost immune response, beta-defensin was added at the N terminal of the vaccine construct using EAAAK linker. Another sequence, PADRE, is placed between adjuvant and epitopes using the GGGGS linker. Furthermore, AAY, PMGLP, and GGGGS linkers were added to join CTL, HTL, and LBL epitopes together. These linkers stop the self-folding of the epitopes and allow the vaccine construct to stabilize and boost the immune response. The final MSV construct was 317 amino acids in length. The MSV construct is graphically presented in To assess the antigenic potential of the MSV, VaxiJen server was used; however, to ensure the nonallergenicity of the vaccine construct, Algpred2.0 server was used. The results ensured the MSV construct is antigenic and nonallergenic. Furthermore, molecular weight, PI, half-life, aliphatic index, and GRAVY were calculated using ProtParam server. The antigenic, nonallergenic, and other physicochemical properties are presented in SOPMA server predicted that the MSV has 29.97% alpha helix, 16.09% extended strand, and 53.94% random coil. The secondary structure of the MSV is presented in Model 1 was selected from Robetta for further processing using GalaxyRefine server. The best one based on several parameters, GDT-HA (0.98), RMSD (0.30), MolProbity (1.61), clash score (12.6), poor rotamers (0.4), and R plot (98.1), was validated using several tools . ERRAT, ProSA-web, and R plot calculated by Procheck were used to estimate the quality of refined MSV model . The ProPyDock server was used for in silico molecular docking of MSV with TLR7 (PDB ID: 7CYN) and TLR8 (PDB ID: 6ZJZ), whereas PDBsum was used to explore interaction analysis of MSV-TLR complexes. The PDBsum interaction analysis revealed the 2 salt bridges and 9 hydrogen bonds, while 227 are nonbonded contacts. The complex formed ten hydrogen bonds between chain A (MSV) and chain B (TLR7) Ser550-His151, Ser575-His151, Ser577-Lys169, Ser577-Ser148, Ser577-Ser148, Asp605-Ser148, Asp605-Ser148, Asp605-His151, and Asp607-Ser148 residues. The two salt bridges were formed between Asp605-His151 and Asp607-Lys169 residues. Furthermore, 10 hydrogen bonds and 243 nonbonded contacts were reported between MSV and TLR8, Arg429-Gln-120, Ser516-Asn-159, Arg541-Met-115, Arg541-Leu-143, Arg541-Thr-147, Arg541-Met-115, Arg541-Leu-143, His566-Thr-147, Tyr567-Thr-147, and His593-Tyr-113 residues. The molecular docking and interchain residual interaction maps of MSV-TLR7 and MSV-TLR8 are provided in Figures nd and 3rd doses as compared to the first dose of the vaccine which represent real-world scenario of an ideal vaccine  represent the success of mRNA-based vaccines, and the aforementioned examples of mRNA vaccines represent an efficient subgroup of such vaccines from the first successful mRNA-based vaccination in 1990. Two vaccine candidates, a multiepitope subunit vaccine that must be synthesized outside the host body and mRNA vaccine candidate that must be synthesized inside host cell machinery, were designed in this study. TCRs recognize antigens that are visible on the surface of APCs, or antigen-presenting cells, which are joined to class I and class II MHC molecules. The cytotoxic and helper T cells, correspondingly, identify these antigens. BCRs recognize and respond to antigens on viral proteins that are accessible on their surface, releasing antibodies and commencing humoral host defenses , 59.Tropheryma whipplei [For instance, researchers are very interested in computational approaches to reveal the molecular basis and identify novel therapeutics, vaccines, or peptides for various infections , 60–62. whipplei . In addiwhipplei .In our study, we used a variety of immunoinformatics tools for prediction and evaluation of B and T cell epitopes. T and B cells epitopes are presented to respective cells of the immune system; the epitopes included in the final vaccine were parts of three major proteins of MARV. The residues of the epitopes give empirical information about how immunogenic the vaccine is and also give information about exposure of the epitope on surface of respective cells. The immunogenicity and surface exposure information is calculated from presence of residues having aromatic side chains . The resOptimal spacers were inserted into the mRNA sequence to avoid interaction between domains. It also gives stability to vaccine . At the The current framework of the creation of mRNA and MSV candidates has shown to be tremendously advantageous, notably in terms of inducing cell-mediated or adaptive immunity. In this work, immunoinformatics and computationally meta-analysis methods were used to identify possible B cell and T cell epitopes generated from antigenic MARV proteins. It was determined whether certain T cell epitopes could bind to the appropriate HLA molecules and the modelled MSV protein with TLR7 and TLR8, using peptide modeling and in silico molecular docking. The final vaccine candidate validated through various methods could instigate the immune system against MARV, and further verification of its effectiveness using in vitro and in vivo tests is necessary to confirm the vaccine's efficacy against MARV. This research may aid in the development of RNA-based vaccinations, as well as prompt early corrective measures and efficient defenses against MRV.


In December 2014, the International Committee for Monitoring AssistedReproductive Technology under the umbrella of the World Health Organizationconvened an expert meeting to re-examine, update and expand the infertilityglossary previously published in 2009. Thus, the International Glossary ofInfertility and Fertility Care was developed and published in 2017simultaneously in Fertility and Sterility and Human Reproduction. In thisarticle, we present the glossary translated into Spanish, obtained afterevaluation by Argentinian experts in the field of assisted reproductivetechnologies, reviewed by Dr. Zegers-Hochschild and approved by the board of theArgentinian Society of Reproductive Medicine (SAMeR). The translation of theglossary to Spanish will facilitate communication between professionalsresponsible for the practice of ART in Spanish-speaking communities. Moreover,it will lend support to promote better understanding as well as safer and bettercare for Spanish-speaking minorities and those experiencing cross-borderreproductive care. The International Committee Monitoring Assisted Reproductive Technologies (ICMART) is anindependent, international non-profit organization that has taken a leading role in thedevelopment, collection and dissemination of worldwide data on assisted reproductivetechnology (ART). Regional and national registries, such as the European Society ofHuman Reproduction and Embryology (ESHRE), the Society for Assisted ReproductiveTechnology (SART) and the Latin American Network of Assisted Reproduction (REDLARA),report their results to ICMART. Thus, in an effort to synchronize the reporting, ICMARTin collaboration with the World Health Organization (WHO) compiled a standard set ofdefinitions on ART published in 2006 , Dutch . Since nThe International Glossary of Infertility and Fertility Care, 2017 is ultimately theresult of a work led by ICMART together with a worldwide consortium of internationalorganizations related to reproductive health: the American Society for ReproductiveMedicine (ASRM), the European Society of Human Reproduction and Embryology (ESHRE), theInternational Federation of Fertility Societies (IFFS), March of Dimes (MOD), theAfrican Fertility Society (AFS), the Groupe Interafricain d’Etude de Recherche etd’Application sur la Fertilité (GIERAF), the Asian Pacific Initiative onReproduction (ASPIRE), the Middle East Fertility Society (MEFS), the Latin AmericanNetwork of Assisted Reproduction (REDLARA), the International Federation of Gynecologyand Obstetrics (FIGO). It was agreed with the corresponding editors to have the glossarypublished simultaneously in the journals Fertility and Sterility (The current article contains a translation into Spanish of the 283 terms and definitionsdescribed in the ICMART 2017 glossary (Zegers-We believe this Spanish version of the glossary will standardize and facilitatecommunication in the practice of ART not only in Spanish-speaking countries but also incountries where Spanish-speaking communities are visibly present. The Spanishtranslation of the glossary may be advantageous to Spanish-speaking minorities since itmay attenuate language barriers, which are well recognized to have a negative impact onthe quality of patients’ care (


P = 0.011). Within the subgroup of recipients without hepatocellular carcinoma, the inferiority of female-to-male LT became greater . Despite good graft quality, 1y-graft-failure-probability was 37.9% (23.1%–57.9%) in female-to-male LT within this subgroup while such exceptionally high probability was not shown in any other subgroups even with worse graft quality. When donor was > 40 years or macrosteatotic, graft-failure-risk was not significantly different between female-to-male LT and others (P > 0.60). These results were in agreement with the estrogen receptor immunohistochemistry evaluation of donor liver. In conclusion, we found that the inferiority of female-to-male LT was only found when donor was ≤ 40 years and non-macrosteatotic. Abrogation of the inferiority when donor was > 40 years or macrosteatotic suggests the presence of dominant contributors for post-transplant graft-failure other than graft quality/quantity and supports the role of hepatic-estrogen-signaling mismatch on graft-failure after female-to-male LT.Greater graft-failure-risk of female-to-male liver transplantation (LT) is thought to be due to acute decrease in hepatic-estrogen-signaling. Our previous research found evidence that female hepatic-estrogen-signaling decreases after 40 years or with macrosteatosis. Thus, we hypothesized that inferiority of female-to-male LT changes according to donor-age and macrosteatosis. We stratified 780 recipients of grafts from living-donors into four subgroups by donor-age and macrosteatosis and compared graft-failure-risk between female-to-male LT and other LTs within each subgroup using Cox model. In recipients with ≤ 40 years non-macrosteatotic donors, graft-failure-risk was significantly greater in female-to-male LT than others (HR 2.03 [1.18–3.49],  Recent research further validated the inferiority of female-to-male LT in living donor LT10. Our previous research further demonstrated that the inferiority of female-to-male LT is not attributed to anatomical size mismatch between females and males 11.The sex difference in graft failure risk is well-known in liver transplantation (LT). That is, post-transplant graft failure is more prevalent in male recipients with female donors, so-called female-to-male LT, compared to other donor-recipient sex combinations. Since the landmark study by Kahn et al.15. The liver is one of the target organs of estrogen, and hepatic estrogen signaling plays an important role in mitigating injury and enhancing recovery of the liver18. Hepatic estrogen signaling increases in relation to the increase in systemic estrogen secretion or hepatic estrogen receptor content19. In this regard, previous experimental and clinical studies have demonstrated that the tolerance of various liver injuries is greater in females with greater systemic estrogen secretion and hepatic estrogen receptor content compared to males21. However, abrupt decrease in hepatic estrogen receptor occurs in the transplanted female liver after exposure to the male hormonal milieu14, and consequent decrease in hepatic estrogen signaling and impairment of hepatic protection capacity has been implicated in the greater graft failure risk8.Although the exact mechanisms underlying the sex difference is not fully understood, hepatic estrogen signaling mismatch is considered to play an important role25 and the liver is without macrosteatosis 27. These findings suggested that female’s superior hepatic protection capacity decreases in relation to the decrease in hepatic estrogen signaling with poor ovarian reserve or macrosteatosis; thus, we deduced that the degree of acute post-transplant defeminization of the graft after female-to-male LT decreases in relation to donor poor ovarian reserve and macrosteatosis. In this study, we aimed to evaluate whether the inferiority of female-to-male LT changes by donor age of 40 years or graft macrosteatosis.In our recent study of healthy living liver donors, we found that the tolerance of hepatic ischemia–reperfusion injury is greater in females than in males only when the age is ≤ 40 years We screened the records of 813 recipients who underwent a first adult-to-adult living donor LT between December 2005 and April 2016 (representing the 500th–1700th LT) in our hospital. We excluded 32 recipients of grafts consisting of a partial liver other than the right lobe . We further excluded one recipient who underwent an auxiliary partial orthotopic LT. The remaining 780 recipients were included in the analysis. All data analyzed in the study were derived from our institution’s electronic medical records and LT database (prospectively collected). All recipients were followed until September 2017 or the last medical follow-up, for a maximum of 5 years. The Institutional Review Board of Samsung Medical Center approved this retrospective cohort study (SMC 2020-03-015-002) and waived the requirement for written informed consent. The study was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice Guidelines.2, macrosteatosis degree ≤ 30%30, and donor residual liver volume ≥ 30%. Individuals with any type of hepatitis, including steatohepatitis, or liver fibrosis were excluded from donation.The sex of the donor and recipient was not considered in selection of the donor. Acceptance criteria for liver donation were age of ≤ 65 y, body mass index < 35 kg/m32 as well as the study reporting that hepatic estrogen receptor content is lower in females with > 5% macrosteatosis than in females with ≤ 5% macrosteatosis16.All living donors underwent a wedge liver biopsy before liver resection, and frozen biopsy sections were stained with hematoxylin–eosin and Oil Red O. In general, the size of the biopsy section was 3 μm in thickness, 15 mm in length, and 5 mm in width, containing 15–20 portal fields. Permanent biopsy sections were prepared with tissues being fixed in 4% neutral buffered formalin solution, embedded in paraffin, and stained with hematoxylin–eosin. The pathology report was recorded based on the review process of both frozen and permanent biopsy sections , describing the average percentage of macrovesicular fatty droplets occupying the surface area of the parenchyma. For the purpose of the study, macrosteatosis was defined if ≥ 5% hepatocytes contain macrovesicular fat droplets based on a non-alcoholic fatty liver disease activity score system33. ER positive expression was defined when the Allred score is greater than or equal to 4 based on previous reserach34.To examine the mechanisms underlying the specificity of female-to-male LT and its interaction with graft macrosteatosis and donor age, the data of our previous study on immunohistochemistry for estrogen receptor performed for the liver tissues biopsied during the donor hepatectomy were analyzed while 457 patients with available tissues were overlapped between the two studies. The liver tissue was stored in an in-house bio-bank using a a Bond-max autoimmunostainer  with Bond™ Polymer refined detection, DS9800 . After deparaffinization of formalin-fixed and paraffin-embedded tissue sections in xylene for a total of 15 min, antigen retrieval was performed at 97 °C for 20 min in ER2 buffer. Briefly, antigen retrieval was performed at 97 °C for 20 min in ER2 buffer. After blocking endogenous peroxidase activity with 3% hydrogen peroxidase for 10 min, slides were incubated with mouse monoclonal estrogen receptor antibody  and rabbit monoclonal AR-V7 specific antibody  for 15 min at room temperature, at a dilution of 1:200. Normal breast tissue was used as a positive control. For ER status evaluation, the Allred score, which is the sum of two scores , was calculated35. Blood salvage and auto-transfusion were routinely used to reduce recipient exposure to allogeneic red blood cells37. Immunosuppression and hepatitis B virus prophylaxis were performed according to the standardized protocol, as described previously38.All study donors underwent computed tomography angiography and magnetic resonance cholangiopancreatography to determine the anatomy of the hepatic vascular and biliary system. Expert liver radiologists and transplant surgeons carefully discussed anatomical considerations and related surgical issues in a joint meeting prior to transplantation. All grafts consisted of segment 5 through 8, excluding the middle hepatic vein trunk. Graft implantation was performed using the piggyback technique. After the portal vein anastomosis was complete, the graft was reperfused by consecutively unclamping the hepatic vein and portal vein. Subsequently, hepatic artery anastomosis was followed by biliary anastomosis. Transfusion of allogeneic blood was strictly controlled based on a restrictive and prophylactic strategy in which each blood component was transfused separately according to its respective indication38. Furthermore, for graft failure occurred during the early postoperative period (90 days), early allograft failure simplified estimation (EASE) score, which is the indicator for quantifying the early post-operative failure risk, was calculated compared between two groups40. Regarding estrogen receptor analysis, the multivariable Cox model was used to test whether estrogen content of donor liver is associated with graft failure risk while the independent variables which were significant during the multivariable analysis for graft failure were included. Because the largest part of graft failure occurred during the early post-transplant period, we performed the analysis for the first 6 months post-transplantation. The continuous variables were described as median with interquartile range and analyzed using the Mann–Whitney U-test. The categorical variables were expressed as frequency (%) and analyzed using χ2 test or Fisher's exact test. All reported p-values were two-sided, and p < 0.05 was considered significant. Data were analyzed using SPSS 19.0 .The primary outcome was post-transplant graft failure (death or retransplantation). The secondary outcome was post-transplant overall death. Survival analysis was performed using the Cox model, and the results were described using hazard ratios (HR) with 95% confidence interval. Backward stepwise selection was performed for multivariable analysis with p < 0.05 for inclusion and p > 0.10 for removal. Subgroup analysis was performed within the recipients who did not have hepatocellular carcinoma (HCC) to remove the bias from HCC-related graft failure because our recent research demonstrated that the risk of post-transplant HCC recurrence is greater in recipients of grafts from male donorsThe indications for transplantation in the 780 recipients were as follows: HCC arising from viral hepatitis , alcoholic hepatitis (n = 16), or unknown origin (n = 18); liver failure secondary to viral hepatitis , alcoholic hepatitis (n = 51), biliary obstruction (n = 14), metabolic disease (n = 5), or unknown origin (n = 17); autoimmune hepatitis (n = 16); toxic hepatitis (n = 13); Budd-Chiari syndrome (n = 7); and liver tumors other than HCC (n = 6). There were 207 male recipients of grafts from female donors. Other 573 recipients consisted of 59 female recipients with female donors, 104 female recipients with male donors, and 410 male recipients with male donors. Clinical data of male recipients with female donors and other recipients are described in Table The median follow-up time was 35 months with interquartile range of 20 to 60 months. Within the subgroup of recipients with ≤ 40 years non-macrosteatotic donors Fig. A, graft Figure Within the subgroup of non-HCC recipients, the gap in graft failure risk between female-to-male LT and other LTs when donors were ≤ 40 years and non-macrosteatotic became greater  compared to the results for the whole cohort  despite worse graft quality18, supporting the role of hepatic sex hormonal mismatch in the inferiority of female-to-male LT. When donor was > 40 years or macrosteatotic, the inferiority of female-to-male LT was abrogated and graft failure risk was comparable between female-to-male LT and other LTs, suggesting that anatomical size mismatch  is not the reason of the inferiority of female-to-male LT, being in agreement with our recent research11. Overall, our data suggest the presence of dominant contributors for graft failure other than graft quality/quantity and supports the role of hepatic estrogen signaling mismatch in greater graft failure after female-to-male LT. Also, our data suggest the risk of female-to-male LT can be changed by modification of donor/recipient hepatic estrogen signaling.Continuing from our previous work, demonstrating the greater tolerance of hepatic ischemia-reperfusion injury in females than in males only with age ≤ 40 years and no macrosteatosis15. Hepatic estrogen signaling is positively correlated with hepatic protection capacity18. A previous study in mice demonstrated that 100% of female mice survived after 70% hepatic inflow occlusion for 45 min, whereas all male mice died within 5 d with greater initial hepatocyte injury10. In the same study, ovariectomy or estrogen antagonist administration increased hepatic ischemia–reperfusion injury, reduced regenerative capacity, and increased mortality, whereas administration of 17β-estradiol improved recovery10. In rats, translocation of estrogen receptors from the cytosol to nucleus and activation of cell signaling occurred after liver injury to stimulate the healing process43. Our recent study of healthy living liver donors has also demonstrated that the tolerance of hepatic ischemia–reperfusion injury following intermittent hepatic inflow occlusion was greater in females than in males18. Thus, it has been hypothesized that acute decrease in hepatic estrogen signaling (including decreased hepatic estrogen receptor content) of the transplanted female liver after the exposure to male hormonal milieu impairs the recovery of female grafts8. The abrogation of the inferiority of female-to-male LT when the donor was > 40 years25 or with macrosteatosis27 supports the relationship because experiencing some degree of defeminization with lower hepatic estrogen signaling prior to transplantation may mitigate the impact of male hormonal milieu after transplantation.Post-transplant liver graft failure has been known to be more prevalent in male recipients of female donors compared to other donor-recipient sex combinations and hepatic estrogen signaling is considered to play an important role in this sex difference46, along with the significant multicollinearity between donor sex and donor characteristics like weight and hight as indicated previously11. Also, biasing effects of HCC-related graft failure might have contributed because the impact of donor gender on HCC-unrelated graft failure and HCC-related graft failure is different based on our and other data38. In other words, analyzing the interaction of donor/recipient sex with donor age and macrosteatosis, instead of simply incorporating those variables in multivariable model, on HCC-related/unrelated graft failure may help improve research quality and derive more consistent results regarding the impact of female-to-male donation. This is important because there is a possibility that donor selection process may ultimately improve post-transplant clinical outcome if donor sex is appropriately taken into account while the greater graft failure risk in female-to-male LT has been being reported repeatedly. This is particularly relevant in living donor LT because multiple donation candidates can be obtained in some cases44. On the other hand, our data suggested that female-to-male LT is not inferior in all cases; thus, variables interacting with donor sex (e.g. donor age and macrosteatosis in the current study) needs to be further elucidated to minimize the risk subgroup. Also, our data suggest that pre/post-transplant hepatic estrogen conditioning to prevent acute defeminization of the graft helps maintain the hepatic protection capacity and prevent graft failure. More scientific data are warranted to incorporate donor sex and hepatic estrogen signaling into LT process.The current study suggests that the insignificance of female-to-male LT in few previous articles were attributable to the lack of consideration of hepatic estrogen signaling or pretransplant liver graft defeminization according to reproductive aging and macrosteatosisIn our analysis, we observed that the significant difference in graft failure between female-to-male LT and other LTs was only found with non-macrosteatotic donors ≤ 40 years. The absence of a correlation between EASE score and female-to-male sex match further supports that the physiopathologic mechanism underlying the inferiority of the female-to-male LT is not dependent on graft quality. An unexpected finding was a significant protective effect of recurrent ascites in the subgroup ‘female-to-male LT'. Despite the initial counterintuitive nature of this finding, potential confounding factors and complex underlying mechanisms need to be considered. The presence of recurrent ascites may trigger closer monitoring, early interventions, and more intensive management strategies, leading to improved graft outcomes. These interventions may include adjustments in immunosuppressive regimens, fluid balance optimization, and close surveillance for complications. An interesting finding was the association between female-to-male LT and sepsis-induced graft failure. Because the number of event was too small with the lack of statistical power, the current study cannot draw a conclusion for this finding warranting further investigation.45.Despite the retrospective design, our study presented several strengths for generation of robust data. First, this study included a homogeneous living donor liver transplant population. All recipients received right hemi-liver graft without variation. Most recipients underwent elective surgery and were in stable condition without acute deterioration. Accordingly, thorough perioperative anesthetic and surgical care could be performed strictly based on the institutional standardized protocols. Second, only three patients were lost to follow-up, and graft failure was defined as a clear objective outcome; thus, the data regarding the time-to-graft failure were highly reliable. Another advantage of this study was the routine use of Oil Red O staining to examine liver parenchyma. Conventional hematoxylin and eosin staining alone tends to underestimate the degree of steatosis46. Thus, it is unclear that the effects of donor age and macrosteatosis are observed in the same way in deceased donor LT because more various factors, aside from donor age and macrosteatosis, may affect hepatic estrogen signaling.This study has several limitations as well. First, due to the retrospective nature of the study design, we were unable to exclude the possibility of bias from unmeasured variables, although we performed adjustment of all established contributors by subgroup analysis and multivariable analysis. Second, mechanisms underlying the sex differences in post-transplant graft failure as well as its change according to donor age and macrosteatosis have not yet been confirmed although the involvement of hepatic estrogen signaling can be assumed based on results of previous and the current studies. Third, in contrast to living donors, deceased donors are frequently attended with multiorgan dysfunction, unstable hemodynamics, vasoactive drug use, lack of sufficient hepatic inflow, and prolonged graft ischemia timeIn the current study, we found that the greater graft failure risk of female-to-male LT was abrogated when the donor was > 40 years or with macrosteatosis, which is known to be related to the decrease in hepatic estrogen signaling. Therefore, our findings shed light on the importance of hepatic estrogen signaling mismatch between donor and recipient to post-transplant graft failure and suggest that the strategies to avoid abrupt decrease in hepatic estrogen signaling prevent graft failure in female-to-male LT.Supplementary Figure 1.Supplementary Figure 2.Supplementary Table 1.


We describe a bilateral craniodorsal dislocation of the hip joint in a free-ranging young roe buck and the associated pathological changes in the ossa coxae and femora of the animal. The highly symmetrical dislocation, which is considered to have developed secondary to hip dysplasia, caused the formation of two false acetabula that each consist of several, partially fused bone portions. The femora exhibit symmetrical outgrowths that extend from the greater trochanter along the intertrochanteric crest to the lesser trochanter. Formation of these outgrowths is attributed to abnormal traction at muscle attachment sites due to the displacement of the femora. On radiographic examination, both femora show signs of avascular necrosis in their head regions and of fatty marrow necrosis in their shafts, which is attributed to the damage of the arterial blood supply of the femora that was associated with the dislocation. The fact that, according to the hunter who shot the buck, the animal’s locomotion was inconspicuous suggests that the false hip joints functioned quite well, thereby demonstrating a remarkable capacity of the musculoskeletal system for functional recovery. The coxofemoral (hip) joint is formed by the acetabulum of the hip bone (os coxae) and the head of the femur (caput ossis femoris). In mammals, the acetabulum is deepened by a well-developed ring of fibrocartilage, the acetabular lip (labrum acetabulare), which is attached to its bony rim (margo acetabuli) . The hipThe term dislocation describes a condition in which the contact between bones that normally articulate has been completely lost , 3. The Hip dysplasia is a congenital, bilaterally occurring condition characterized by a lack of conformity between the femoral head and the acetabulum , 8. A coHip dysplasia is a well-known predisposing factor for hip dislocation, which can develop spontaneously due to the inherent instability of the joint or following a mechanical impact that in a normally developed joint would not lead to a displacement of the femoral head from the acetabulum . In domePan troglodytes) [Capreolus capreolus) that had caused the formation of highly symmetrical pathological changes in both ossa coxae and the proximal portions of both femora.Compared to domesticated animals, hip dislocation has only rarely been reported from deer , 14 and lodytes) , and infWe studied the hip bones and femora of a young roe buck that had been taken in 1999 in the course of regular hunting operations in the state-owned forest district Königsforst near Cologne . The European roe deer is listed as a huntable species under both the German federal hunting law and the state hunting law and not subject to further species protection. Therefore, no additional permits were required for obtaining the specimen under study. The largely macerated and dried hip bones and femora of the roe buck were originally submitted to the Research Station of Wildlife Biology of the federal state of North Rhine-Westphalia  by the state forest officer in charge of the area, and from there forwarded to the first author for examination. According to observations prior to culling, locomotion of the roe buck was inconspicuous. During evisceration, the buck’s ossa coxae were split in the symphyseal region and the dorsal portion of the wing of the ilium  was cut off bilaterally. No further skeletal material from the animal was available.For comparison with the pathological bone specimens, we also studied the ossa coxae  and femora of an adult female roe deer (collection number of the skeleton: 3945) from the osteological reference collection at the Center for Baltic and Scandinavian Archeology . Henceforth, these bones are referred to as “controls”.The bones analyzed in this study originated from animals killed during legal hunting operations for population management. As the individuals had not been killed for the purpose of this study, ethical approval was not required.The hip bones and femora of the male and the female roe deer were photographed with a digital camera (Canon EOS 80D) and radiographed using a digital X-ray apparatus . In addition, the pathological femora of the buck were scanned using an in-vivo microCT-scanner  operated at 90 kV and 200 μA (FOV 73 × 73 mm). The acquired images were processed using Adobe Photoshop. Following image acquisition, the left femur of the buck was transversely sectioned in its distal portion and the object located in the medullary cavity was removed. A transverse slice from this object was subsequently analyzed by multipoint laser-induced breakdown spectroscopy (LIBS) using a Keyence EA-300 elemental analyzer attached to a Keyence VHX-7000 microscope.The ossa coxae of the roe buck show a number of highly symmetrical pathological changes Figs . Both acThe most conspicuous pathological change in both ossa coxae is the presence of a newly formed joint socket  that is located craniodorsal to the acetabulum in the region of the spina ischiadica Figs . The falThe proximal portions of the pathological and the control femora are shown in In both pathological femora, an elongated flattened (laminar) outgrowth extends from the caudal margin of the greater trochanter along the intertrochanteric crest to the lesser trochanter . The surThe surface of the intertrochanteric fossa of both pathological femora is uneven with bony projections (exostoses) that are more pronounced in the right femur . BilaterRadiographic examination showed the presence of an elongated (freely movable) object of low radiodensity in the medullary cavity of both pathologic femora . In the The case described here represents an example of bilateral craniodorsal hip dislocation and associated pathological changes in both hip bones and femora of a free-ranging roe buck. To the best of our knowledge, no such case of highly symmetrical pathological changes in the ossa coxae and femora has previously been reported in roe deer or other cervid species. The bilateral nature of the lesions strongly favors the interpretation that the condition developed secondary to congenital hip dysplasia. The bilateral craniodorsal dislocation of the hip joint had led to the formation of a false acetabulum in both ossa coxae.viz. C1 when the femoral head articulates with a false acetabulum, and C2 when there is no false acetabulum. Applying these criteria to the present (non-human) case, the condition in the roe deer would be classified as a bilateral C1 case. Applying the 5-grade classification scheme of canine hip dysplasia by the FCI , the condition would be classified as the most severe grade E [In humans, various attempts have been made to classify congenital hip disease  according to its severity. In adults, Hartofilakidis et al.  distingu grade E .The marked osteophytosis along the acetabular rim precluded detailed measurements of the width and depth of the true acetabula in the roe buck. However, visual comparison with the control specimens suggests their abnormal shallowness, and it is concluded that this was the predisposing factor for the bilateral joint dislocation.The dislocation must have been a long-standing condition, since bilaterally a deep false acetabulum had formed that consisted of several, partially fused portions. Bilateral formation of a false acetabulum is considered the consequence of a constant irritation of the periosteum of the hip bone by the femoral head and the formation of larger quantities of new bone in response to this stimulus . It has Displacement of the femoral head from the true acetabulum must have been associated with severe damage to the acetabular lip and the ligaments of the hip, injury to the joint capsule with loss of synovial fluid, and lesions in surrounding soft tissues , 7.viz., osteophyte formation around the acetabular margin and eburnation of the femoral head, correspond to those typically seen in the case of DJD [Some of the pathological changes seen in the ossa coxae and femora, e of DJD , 18, 19.It is suggested that the bilateral hip joint dislocation in the roe buck was associated with severe damage to the arterial vessels supplying the femora, causing traumatic osteonecrosis in the femoral head and shaft due to a disrupted blood supply. Evidence for avascular necrosis in the femoral heads is the co-occurrence of sclerotic and lucent bone lesions, which are considered characteristic for osteonecrosis . The lucThe two loose objects present in the marrow cavities of the pathological femora were diagnosed as dried and condensed remnants and/or breakdown products of fatty marrow, thus indicating a case of “mummified marrow necrosis” . BecauseIn conclusion, the pathological changes present in the ossa coxae and proximal femora of the roe buck are diagnosed as the result of a bilateral craniodorsal hip joint dislocation that most likely developed secondary to congenital hip dysplasia. The long-standing condition had caused the formation of two large false acetabula, each of which is composed of several, partially fused bone. Formation of a laminar bony outgrowth in the proximal region of both femora is attributed to abnormal muscle traction resulting from the hip joint dislocation. According to our interpretation, the dislocation was associated with severe damage to the arterial vessels supplying the femora, causing osteonecrosis in the femoral head and shaft.A limitation of the study is the fact that only macerated dry bones were available for study. However, in our view a reliable diagnosis of the pathological condition in the roe buck is nevertheless considered possible, based on the detailed analysis of the lesions present in its hip bones and femora. The observation that locomotion of the buck was inconspicuous suggests that the false hip joints functioned quite well, thereby demonstrating a remarkable capacity of the musculoskeletal system for functional recovery. Survival of the buck may have been favored by the fact that large predators were not present in the area.S1 Fig(a) Left lateral view, (b) Dorsal view. (c) Radiograph . 1: Tuber coxae. 2: Tuber sacrale. 3: Spina ischiadica. 4: Tuber ischiadicum. 5: Margo acetabuli. 6: Fossa acetabuli. 7: Symphysis pubica. 8: Foramen obturatum. 9: Arcus ischiadicus.(TIF)Click here for additional data file.


Renal graft biopsies were performed in 43 of the kidney transplant recipients (62.3%) with allograft dysfunction. Antibody-mediated rejection (ABMR) was detected in 29/43 (67.4%) of the patients. The incidence of ABMR was similar among patients with C1q-binding and non-C1q-binding DSA . Graft loss occurred in 30/69 (43.5%) of the patients at a median time of 82.5 months (IQR 45–135) from DSA detection. C1q-binding DSA was present in more patients who experienced graft loss . Higher MFI values and inferior clinical outcomes occurred in most of the kidney transplant recipients with C1q-binding dnDSA. Complement activation by HLA antibodies is a key component of immune-mediated graft injury. We examined the clinical outcomes of kidney transplant recipients with complement-fixing de novo donor-specific antibodies (dnDSA) who were followed in our center. The C1q-binding ability was retrospectively assessed in 69 patients with dnDSA and mean fluorescence intensity (MFI) values > 2000 out of the 1325 kidney transplant recipients who were screened for DSA between 2015 and 2019. Luminex IgG single antigen beads (SAB)and C1q-SAB assays (One Lambda) were used. C1q-binding dnDSA was identified in 32/69 (46.4%) of the patients. Significantly higher MFI values were observed in C1q-positive DSA (18,978 versus 5840,  Progress in antibody detection techniques and immunosuppressive therapy has led to a continuous decline in early antibody-mediated rejections (ABMR) and an improvement in short-term graft survival rates in recent decades ,2. DespiThe interaction between circulating alloantibodies and human leucocyte antigens (HLA) on the renal graft endothelium is a major pathogenetic mechanism of alloimmune injury ,7,8,9,10Complement fixation is essential for the pathogenesis of antibody-mediated rejection. The interaction between C1q and anti-HLA antibodies bound to the renal graft endothelium results in the activation of the classical pathway, leading to cleavage of the complement molecules C3 and C5. The breakdown products, C3a and C5a, serve as potent chemotactic factors, attracting inflammatory cells to the site, which target the graft endothelium. Furthermore, C5b production results in the formation of the membrane attack complex (MAC), which causes cell lysis and tissue injury ,29,30,31Identifying which dnDSA may exert harmful effects on renal grafts remains a pivotal issue in the transplant community. Immunological risk stratification currently relies primarily on the determination of the mean fluorescence intensity (MFI) values of the HLA antibodies or the performance of a renal graft biopsy, which carries an associated risk of complications . The aimFor this retrospective analysis, we initially identified 116 subjects with dnDSA out of a total of 1325 kidney transplant recipients who were screened for DSA at our histocompatibility laboratory between 2015 and 2019. The C1q binding capacity was retrospectively assessed in 69 patients with dnDSA and MFI values > 2000. All of the included patients had received a kidney graft at our renal transplant center between 1989 and 2018. Kidney transplant recipients with pre-existing antibodies before transplantation, as well as patients with dnDSA and MFI values < 2000, were excluded from the study. Renal graft biopsies were performed in 43 out of the 69 kidney transplant recipients upon clinical indication, specifically new-onset proteinuria and/or increase in serum creatinine levels. Rejection episodes were classified based on the current Banff classification system. A database of the 69 kidney transplant recipients, including demographic characteristics, immunological data , immunosuppressive therapy regimens, histological data, and renal graft function indices, was employed for the analysis. Graft loss was considered when the patient progressed to end-stage kidney disease (ESKD). ® platform and commercially available SAB kits  according to the manufacturers’ instructions. EDTA treatment of the sera was performed as previously published in all samples in order to prevent the prozone effect [All of the patients were routinely tested for the presence of circulating DSA using serum samples obtained at the time of transplantation and after the transplantation annually or at the time of biopsy. The presence of HLA Class I and II IgG DSA was determined using a Luminexe effect ,42. NormThe same banked sera of the patients found to be positive for DSA using the conventional IgG-SAB assay were further tested for complement-binding ability of antibodies. Detection of the antibodies capable of fixing to the complement was performed using a C1qScreen assay (One Lambda) according to the manufacturer’s instructions. The cutoff for a positive reaction was set at a normalized MFI value of 500 or greater ,18,43,44p-values < 0.05 were considered statistically significant. Statistical analysis was performed using IBM SPSS Statistics Version 23.Continuous variables were expressed as means and standard deviations or medians and interquartile ranges (IQR), depending on the normality of the data distribution. Categorical values were presented as absolute values and percentages. Means and proportions were compared using the two independent samples t-test or Mann–Whitney U test and chi-square test, as appropriate. p = 0.005), while proteinuria did not differ significantly . Sixty-nine kidney transplant recipients with dnDSA and MFI > 2000 comprised the study cohort. Their clinical and demographic characteristics are shown in p: 0.004) in our study. Moreover, subjects with C1q-positive dnDSA had significantly higher MFI values than those with C1q-negative dnDSA . The majority of DSAs with MFIs greater than an arbitrary threshold of 6000 were found to be capable of fixing the C1q complement factor (p < 0.001).Among the 69 kidney transplant recipients with dnDSA and MFI > 2000, 11 had Class I IgG (15.9%), 48 had HLA Class II IgG (69.5%), and 10 had both Class I and Class II IgG (14.4%) HLA dnDSA. The HLA-DQ antibodies were more frequently represented (69.5%) and were observed in most patients with HLA Class II dnDSA (89.5%). Most C1q-positive kidney transplant recipients had HLA Class II antibodies (p = 0.046). Among the 43 kidney transplant recipients who underwent a graft biopsy, 29 (67.4%) were diagnosed with ABMR within a median time of 19 months (IQR 68) after dnDSA presentation. The incidence of ABMR was similar between the patients with C1q-binding and non-C1q-binding DSA . Graft loss occurred in 30 (43%) out of 69 kidney transplant recipients at a median time of 82.5 months (IQR 90) from DSA detection. Biopsy-proven ABMR was the cause of renal graft loss in 15 (51.7%) of the patients. However, in eight  of the kidney transplant recipients who lost their renal graft, no renal graft biopsy was performed. The kidney transplant recipients with C1q-binding DSA experienced graft loss more often than those with non-C1q-binding DSA . Higher levels of DSA (MFI > 6000) were observed in patients who were diagnosed with ABMR (p: 0.079) or experienced graft loss (p: 0.131) (n = 30), class II dnDSA was detected in 86.7% (p: 0.64) of them, mostly HLA-DQ (80%). Comparing the histological features, no statistically significant difference was observed between the patients with C1q-fixing and non-C1q-fixing DSA (p: 0.09), moderate interstitial fibrosis/tubular atrophy (25–50%) was present in 86.4% of the patients (p: 0.22), and transplant glomerulopathy was present in 40.9% of the patients (p: 0.85). Renal graft biopsies were performed in 43 patients (62.3%) in a median time of 12 months (IQR 57) after DSA identification. The patients with C1q-binding DSA underwent a graft biopsy more often than those with C1q-negative DSA  . Among txing DSA . ChronicLate-onset or chronic ABMR associated with the presence of dnDSA remains one of the major causes of chronic allograft injury and reduced graft survival ,46. Prev® assays in risk stratification for ABMR or allograft dysfunction and loss [® assays to better stratify ABMR risk compared to conventional Luminex® assays [Only a limited number of studies have addressed the diagnostic and prognostic value of complement-binding Luminexand loss ,50. Someand loss ,51. Rece® assays . Notably® assays . Several® assays  were the® assays  reported® assays ,54,55 ha® assays  have pubMost clinical studies have investigated the role of pre-existing C1q-binding DSA in kidney transplantation ,56,57. NThe presence of dnDSA post-transplantation is a well-established independent risk factor for ABMR and graft loss. The question of whether C1q-binding DSA may result in an inferior kidney allograft outcome is still unclear. According to our results, there was no association between the presence of C1q-positive dnDSA and ABMR. This could be partly explained by the small sample size of our cohort and our decision for logistical reasons to only include patients with dnDSA and MFI values > 2000 in the analysis. Another limitation of our study was that no protocol graft biopsies were performed after dnDSA detection, so it was not possible to identify the occurrence of subclinical rejection.Complement activation is strongly implicated in the pathogenesis of ABMR. Studies regarding the clinical impact of C1q-binding HLA antibodies provide the basis for clinical trials investigating the effect of complement inhibitors, specifically eculizumab or C1 esterase inhibitor, in the management or prevention of ABMR . InteresIn conclusion, the distinction between clinically relevant and non-significant dnDSA is still a major concern for transplant care. Our findings indicate that patients with C1q-positive dnDSA exhibit higher MFI values and are possibly at a greater risk of renal graft loss. In contrast with most previously published studies, no association was found between the presence of antibody-mediated rejection and the ability of newly developed DSA to fix complements. However, not all kidney transplant recipients in our cohort underwent a renal graft biopsy. The pathogenicity of dnDSA may be currently best evaluated by a renal graft biopsy until emerging non-invasive biomarkers for rejection, such as donor-derived cell-free DNA , become


To report late central graft detachment after repeat Descemet membrane endothelial keratoplasty (DMEK) without visual reduction.2. Given the lack of corneal edema, visual reduction or subjective visual complaint, the graft detachment was followed-up for up to 20-months post repeat-DMEK with no further intervention, where the central cornea remained clear.A 71-year-old patient with Fuchs’ endothelial corneal dystrophy received a DMEK in his left eye. At 11 month post-operatively, a subtotal graft detachment was noted. Due to increasing corneal edema with vision loss, the first DMEK was removed and a repeat-DMEK was performed. At four months post repeat-DMEK, the graft was fully adherent to the posterior stroma. There was no significant corneal edema, and the best corrected visual acuity was 20/25. At 16-months after repeat-DMEK, a central graft detachment was noted, but there was no concurrent corneal edema or any loss of visual acuity. The mean density of the central endothelial cells was measured at 842 cells/mmTo our knowledge, this is the first report of a central repeat-DMEK graft detachment that occurred 16 months after surgery despite initial attachment. Interestingly, there was no concurrent corneal edema or vision reduction. We describe a potential mechanism for clear central cornea in the presence of a central graft detachment after repeat-DMEK. •Isolated central graft detachment can occur after repeat-DMEK.•Central graft detachment can occur without loss of visual acuity or corneal edema.•Endothelial cell density should be measured before repeat-DMEK.•Stromal polishing may be performed during repeat-DMEK before graft insertion.•Central graft detachment with good visual acuity after repeat-DMEK can be tolerated. This case report presents a case of a late central graft detachment occurring after repeat-DMEK in an eye that previously underwent DMEK for FECD.Graft detachments are typically observed in the early postoperative period, which highlights the importance of close follow-up examinations following the surgery. To our knowledge, a graft detachment occurring numerous months after surgery has not been reported before, particularly a detachment that 2A 71-year-old White male presented to our department with blurry vision in his left eye. He had previously undergone DMEK in this eye 11 months ago for corneal decompensation due to FECD. Other previous ocular history was unremarkable except for cataract surgery on both eyes more than 10 years ago. His systemic comorbidities included myasthenia gravis, rheumatoid arthritis, and obstructive sleep apnea syndrome.The left eye showed a best-corrected visual acuity (BCVA) of 20/200, and the slit-lamp examination revealed a diffuse corneal edema involving the optical axis. Optical coherence tomography (OCT) demonstrated a subtotal DMEK graft detachment A and a cThe repeat-DMEK was performed with no intraoperative complications and the new graft was completely attached to the stromal surface under 20% SF6 gas tamponade. Both the slit-lamp and the anterior-segment OCT confirmed a fully adherent graft position A at 2-daAt 1- and 4 months after surgery, the graft was still fully attached to the host stroma and the BCVA had improved to 20/25. The central corneal thickness was 489 μm.When the patient followed up at 16-months postoperatively, however, a central graft detachment was observed B. A broa2. The HRT3 Rostock Cornea Module , which is widely used to analyze the depth and composition of corneal layers, confirmed the location of the endothelial cells in the central posterior corneal stroma. Given the clinically stable situation with no visual impairment, we decided to continue observation with no further surgical intervention.Given the good BCVA and the lack of any subjective visual complaints, we decided against a surgical intervention and followed-up in 4 months. At 20-months after repeat-DMEK, the magnitude of central graft detachment remained stable with consistent diameter and height of the detachment C, and th3,only occurred in the central cornea with no signs of concurrent corneal edema or loss of visual acuity.DMEK graft detachment generally occurs in the early postoperative period and is often accompanied by vision loss due to increasing corneal edema in the area of detachment.It is unclear which exact mechanism led to late graft detachment that only involves the central cornea. In our case, a subtotal graft detachment was first observed at 11-months after the initial DMEK. As the patient had failed to follow-up between 1 and 11 months after the initial DMEK, it is unknown for how long the graft had remained detached in this eye as shown in Immediately after repeat-DMEK, the migrated endothelial cells on central posterior stroma, together with peripheral endothelial cells from the second DMEK graft, then presumably began to dehydrate the edematous cornea. This is reflected in the gradual improvement of BCVA and corneal clarity at 4-months post repeat-DMEK, which is in alignment with a report by Lazaridis et al. who observed a recovery of corneal clarity at 3-months following DMEK.In such a case, one would expect the gradual fluid “pouch” above the area of graft detachment to continue to increase and ultimately cause a total graft detachment. Interestingly, however, the size of graft detachment remained unaltered. One possible mechanism may be that the healthy endothelial cells residing in central repeat-DMEK graft also transport a similar amount of fluid from the accumulating fluid pouch, thereby establishing a certain balance in the amount of fluid that is being transported 1) from the central corneal stroma into fluid pouch and 2) from the fluid pouch into the anterior chamber .Fig. 4IlSurely, a re-bubbling may be a possible option to treat such a graft detachment. However, a re-bubbling in the setting of such a central graft detachment could apply pressure on the central fluid accumulation and lead to a further increase in graft detachment. A third DMEK may also be a possibility. However, the central corneal clarity as well as the lack of visual loss or complaints dissuaded us to perform a third corneal transplantation.In conclusion, late central graft detachment can occur after repeat-DMEK, possibly due to endothelial cells that migrate from the first DMEK tissue onto the host posterior stroma. This suggests that stromal polishing may be useful when performing a repeat endothelial keratoplasty to remove all residual migrated endothelial cells. Central graft detachments with no associated corneal edema or visual loss can be observed with no surgical intervention. Future studies are necessary to confirm the mechanism of late repeat-DMEK graft detachment described herein.Written informed consent for publishing personal information and case details was obtained from the patient. A copy of the written consent is available upon reasonable request.No funding or grant support.Maximilian Friedrich: Methodology, Investigation, Data curation, Writing – original draft, Visualization. Hyeck-Soo Son: Methodology, Data curation, Writing – review & editing, Visualization. Ramin Khoramnia: Resources, Writing – review & editing, Supervision. Gerd Uwe Auffarth: Resources, Writing – review & editing, Supervision. Victor Aristide Augustin: Conceptualization, Methodology, Investigation, Resources, Writing – review & editing, Supervision, Project administration.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ramin Khoramnia – Heidelberg Engineering – Lecture feeThe following authors have no financial disclosures:M.F.; H.-S.S.; G.U.A.; V.A.A.