The flow field of aqueous humor correlates to the stiffness of iris pigment epithelium (IPE) which acts as a wall of posterior chamber. We focus on the variations of IPE stiffness in a rat ocular hypertension (OHT) model, so as to prepare for exploring the mechanism of duration of OHT. Episcleral venous cauterization (EVC) was applied on one eye of male adult Sprague-Dawley rats to induce chronic high intraocular pressure. According to the duration of OHT , rats were randomly divided into Gw0, Gw1, Gw2, Gw4, and Gw8. Atomic force microscope (AFM) analysis was applied to test IPE stiffness in three regions: iris root, mid-periphery, and pupillary-margin in each group. Histological changes of IPE were also examined in Gw4 and Gw8. There was an overall growing tendency of IPE stiffness in EVC eye. IPE in EVC eye was significantly stiffer than fellow eye in Gw2, Gw4, and Gw8 . IPE in EVC eye in pupillary-margin was significantly stiffer than iris root in Gw4 and Gw8 (p<0.05). In EVC eye, IPE becomes thinner and IPE cell density decreases. IPE stiffness increases gradually with the duration of chronic high intraocular pressure. Persistent high intraocular pressure (IOP) has been regarded as the main clinical manifestation of glaucoma which is the first leading cause of irreparable blindness worldwide . PhysiolAccording to the theory of fluid dynamics, the flow field is directly related to stiffness of the surrounding tissues and a number of biomechanical researches have also demonstrated this point –12. So wIn fact, dating back to 1985, Shimizu and Futa  found a Many researches have shown that pathogenesis of glaucoma is closely related to the duration of high IOP –18. Our In this study, we are mainly trying to verify the first half of our hypothesis: IPE stiffness may change under chronic high intraocular pressure. First of all, IOP elevation was induced by episcleral venous cauterization (EVC) on rat eyes. Subsequently, at different time points , the IPE stiffness was measured by atomic force microscope (AFM) indentation tests, which has powerful ability to probe mechanical properties of ocular tissues including the stiffness of iris and trabecular meshwork in glaucomatous donors , 21. HisAll experiments were complied with relevant laws and institutional regulations. Male adult Sprague-Dawley rats  were obtained from the Experimental Animal Department of the Capital Medical University and given at least 48 hours of adaptation time. They were housed libitum and maintained in an air-conditioned room in a 12-h light/12-h dark cycle. All surgeries were performed under systemic anesthesia, and all efforts were made to minimize suffering.A total of 40 rats were involved in this experiment. According to different duration of OHT, they were divided into 5 groups, denoted by Gw0 which was executed before OHT induction and Gw1, Gw2, Gw4, and Gw8 which were executed at 1st, 2nd, 4th, and 8th weekend after OHT induction, respectively. μL of 5-fluorouracil  with a concentration of 25mg/mL was also performed by a 29G needle on the EVC eyes. A week after the first injection of 5-Fu, a subconjunctival injection of 100μL of 5-Fu was performed again . Sham-operation eyes (SHO eyes) were sham-operated by receiving conjunctival incision and injection of 5-Fu at the same time with EVC eyes but without cauterization. All kinds of Blank-control eyes  received no treatment.In this study, the method of OHT induction was based mainly on the research of Mittag . Rats weThe IOP of the rats was measured with a TonoLab Rebound Tonometer  at presurgery, half a week, and 1, 2, 3, 4, 6, and 8 week(s) after OHT induction as long as the rat had not been executed. In order to avoid the effect of circadian rhythm, IOP measurements were arranged between 10 AM and 12AM on the awakened rats. If the range of the IOP among three times of measurements was less than 3mmHg, the median would be regarded as an effective value.μm thin sections along the sagittal direction of the eye and processed with Masson trichrome stain. A total of 20 histological cross-sections per iris were performed. Histological cross-sections were examined by a ZEISS Axio Lab A1 light microscope .Presurgery and at 4th and 8th weekend after OHT induction, the rats prepared for histology were executed by excessive amounts of pentobarbital. A pair of eye balls was completely removed as soon as possible. The eyes were fixed with eye fixation fluid for about 24h. Then the eyes were dehydrated by graded ethanol and embedded by paraffin. The resin was cut into 3Rats were executed by injection of excess pentobarbital. Then both of the eyes were removed immediately. Each of them was put into a Petri dish (60mm×15mm) full of phosphate buffered saline (PBS). The eyeball was cut open through the sclera with micro scalpel for ophthalmic surgery. Lens and vitreous were removed. The whole circle of limbus was cut open precisely by an iris forceps, with the iris separated from the eyeball wall naturally. Then a pipette was used to suck out all of the PBS in the culture dish, keeping the iris flat. Residual PBS was dried completely by napkin without touching the iris. Adhesive tape (Scotch®) was cut in the middle to make a small hole. It was used to fix the iris on the dish with the pigment epithelium facing up. A sector field of the specimen was exposed for indentation. Finally, the dish was filled with PBS to keep the tissue alive. A performed iris sample is shown in r) was 10μm. Loading speed was 2μm/s.The iris sample in the PBS was placed under an AFM . We adopted a spherical probe  stuck to the tip of the cantilever; and its radius  prior to the first test on each experiment day. During tissue indentation, piezo-actuator translation (Z) and the cantilever deflection (d) were measured primordially. Then they were used to calculate the indentation force (F) and w, where F = k∗ d, w = Z − d. The relation between F and w could be described by Hertz equation:ν is Poisson's ratio; it was assumed to be 0.5 according to previous studies  is the correction coefficient. In this experiment, the petri dish is the substrate, and h is the thickness of iris which is about 50μm, r=10μm, and δ=1μm. So χ in this research is about 0.063. And the correction coefficient is about 1.06. If the curve of F-δ is not fitted by the corrected Hertz model but the original Hertz model, the relative error of elastic modulus of IPE is about 6%. This relative error is less than the variable coefficient of the elastic modulus of IPE which varies from 20% to 60% in all kinds of eyes. Furthermore, we are concerned with the variation trend of the IPE stiffness with chronic high intraocular pressure. The relative error from the calculation of the elastic modulus of IPE can be neglected when it is far less than its variable coefficient of variation. Therefore using the original Hertz model is viable in our research.Iris is a lamellar structure mainly composed of IPE and stroma . Our preF-w in It is also necessary to point out that the indentation of IPE in this research was performed in liquid phase. So jump-to-contact phenomenon which is mainly due to the van der Waals forces between the sample and the formation of a water meniscus when working in air operation at room conditions did not appear in the indentation curve of IPE stiffness of blank-control eyes is 100~200Pa, which is basically the same as trabecular meshwork of rat measured by AFM . We needIn this study, IPE stiffness of EVC eyes shows a growing trend with the persistence of OHT. At 8th week after OHT induction, IPE of EVC eyes in Gw8 is obviously stiffer than BC-II eyes (about 4-fold) and BC-I eyes (about 4-fold). It has also been reported that iris  and trabHowever, increased IPE stiffness may affect the flow field in turn. Previous work has verified that AH flow in posterior chamber is a noncontinuous and pulsatile flow , which mEight-week duration of OHT for rats corresponds to about 6 years for human. The stiffness of IPE of blank-control eyes in each group has not changed significantly during this period. Previous clinical studies have shown that glaucoma is a disease that usually develops in the old rats. The rats in this research are in the young age, so the basically unchanged IPE stiffness of blank-control eyes during 8-week period fits our expectations. There also have been many researches about the age-related changes in the stiffness of ocular tissues such as cornea –39, scleAs a primary study, there are also some limitations in this study. First, it is important to note that the rats used for histology and for indentation were not the same. This may lead to dimensional error. A method to determine the dimensions of the iris components individually is necessary in our future work. Second, it should be noted that the indentation test is measuring an effective modulus, but not a true modulus. The IPE is viscoelastic and anisotropic, so using Hertz contact model to describe indentation is a simplification. Third, we have only reported that OHT leads to the IPE stiffness increasing gradually, due to lack of microcosmic explanation. It, however, has reported that suppression of the Wnt pathway plays an important role in the increasing stiffness of human trabecular meshwork cells . Our futThe aim of this work did not take into consideration the homeostasis of the trabecular cells that also has relevance, but only the mechanistic aspects that however may play a role in the pupillary blocking force. During the duration of chronic high intraocular pressure, the stiffness of iris pigment epithelium increases gradually. Iris pigment epithelium in pupillary-margin was significantly stiffer than iris root in the eyes with 4-week and 8-week duration of chronic high intraocular pressure. The increased IPE stiffness may play a role in pupillary block and iris bombe. Further research on the effect of stiffer IPE on the flow field is necessary in the future work.


A new era is starting in the history of both the European Society for Gynaecological Endoscopy (ESGE) and Facts, Views and Vision. This partnership will create a publishing platform to provide scientific facts, to debate on controversial or well established practices, and to propose future treatments and research. It will bring together the experience built by the journal and the society over the years to achieve this task.The official journal of the ESGE, Facts, Views and Vision (FV&V) opens its doors to the clinicians, academics and researchers to publish original articles, guidelines, commentaries and opinion articles. It is impossible to think of an endoscopic journal without the use of video technology, hence video articles will be a regular feature in the new journal.In this issue of FV&V, we have a combination of these features. The British Society for Gynaecological Endoscopy (BSGE) Guideline on Laparoscopy in Pregnancy by Ball et al. cover a very relevant topic for the gynaecological endoscopist surgeons. Abdominal and pelvic surgery during pregnancy is a challenging task and decisions are usually made after hard thinking. The guideline was commissioned by the BSGE and devised using the methodology of Royal College of Obstetricians and Gynaecologists for evidence based guidelines. They concluded that only surgeons who have adequate laparoscopic skills and who perform complex laparoscopic surgery regularly should undertake laparoscopy in pregnant women. There is no doubt that the guideline will be an important resource for clinicians in years to come.Also, the FV&V journal will facilitate structured training programmes for surgical procedures. Such an example is in the article by Rusch et al. which proposes a standardised educational programme for robot assisted gynaecological surgery. The authors, are all experts in this field, nominated by the Society of European Robotic Gynaecological Surgery followed a Delphi process to develop recommendations on a stepwise training and assessment programme. This consensus document is likely to be a main reference point for training centres and trainees seeking to achieve competence in robotic gynaecological surgery.Endometriosis is frequently described as an enigmatic condition. Philippe Koninckx and his colleagues, who have many years of experience between them in this field, eloquently describe the variability amongst women with this condition and highlight the limitations of traditional statistical analysis in detecting the hidden subgroups. They propose paying special attention to the extremes of an analysis and individual data.As such, the new FV&V is an ambitious open access journal which promises to deliver multifaceted benefits. Its first issue, hereby, is an initial step to fulfilling this task. Whilst this issue includes input from the European societies, we expect to provide material from further afield, through a worldwide network of gynaecological endoscopists.I hope you enjoy reading and utilising the material in this issue and look forward to delivering the next one.ESGE EditorErtan Saridogan


It causes an infection in the upper respiratory tract mainly trachea which results in significant economic losses in the poultry industry worldwide. Vaccination against ILTV produced latent infected carriers' birds, which become a source of virus transmission to nonvaccinated flocks. Thus this study aimed to design safe multiepitopes vaccine against glycoprotein B of ILT virus using immunoinformatic tools. Forty-four sequences of complete envelope glycoprotein B were retrieved from GenBank of National Center for Biotechnology Information (NCBI) and aligned for conservancy by multiple sequence alignment (MSA). Immune Epitope Database (IEDB) analysis resources were used to predict and analyze candidate epitopes that could act as a promising peptide vaccine. For B cell epitopes, thirty-one linear epitopes were predicted using Bepipred. However eight epitopes were found to be on both surface and antigenic epitopes using Emini surface accessibility and antigenicity, respectively. Three epitopes  were proposed as B cell epitopes. For T cells several epitopes were interacted with MHC class I with high affinity and specificity, but the best recognized epitopes were 118YVFNVTLYY126, 335VSYKNSYHF343, and 622YLLYEDYTF630. MHC-II binding epitopes, 301FLTDEQFTI309,277FLEIANYQV285, and 743IASFLSNPF751, were proposed as promising epitopes due to their high affinity for MHC-II molecules. Moreover the docked ligand epitopes from MHC-1 molecule exhibited high binding affinity with the receptors; BF chicken alleles (BF2 2101 and 0401) expressed by the lower global energy of the molecules. In this study nine epitopes were predicted as promising vaccine candidate against ILTV. In vivo and in vitro studies are required to support the effectiveness of these predicted epitopes as a multipeptide vaccine through clinical trials.Infectious laryngotracheitis virus (ILTV) is a The virltovirus . The disltovirus . High moltovirus . The milltovirus , 6.Vaccination against the viral diseases is very important for protection, due to the lack of appropriate antiviral drugs, high cost, and time consuming of development of new antiviral drugs. Different types of vaccines are available for ILTV such as vaccines produced in chicken-embryo-origin (CEO), tissue-culture-origin (TCO), and recombinant vaccines . Howeverhttp://www.ncbi.nlm.nih.gov/protein) in August 2017. The strains of the virus were isolated from chicken from different geographic regions. Complete sequences of all gene subtypes were selected for various immune-bioinformatics analysis. Retrieved strains and their accession numbers and geographical regions are listed in Forty-four envelope glycoprotein B (GB) sequences of virulent isolates of ILTV were retrieved from GenBank of National Center for Biotechnology Information (NCBI) (http://phylogeny.lirmm.fr/phylo_cgi/index.cgi) [Phylogenetic tree of the retrieved sequences of glycoprotein B of ILTV was created using phylogeny.fr online software (dex.cgi) .The retrieved sequences of ILTV glycoprotein B (GB) were subjected to multiple sequence alignments (MSA) to obtain the conserve regions. This was performed using BioEdit software version 7.2.5 with the aid of ClustalW as applied in the BioEdit program to construct the alignment .http://www.iedb.org/). Epitope analysis resources were used to predict B and T cell epitopes [The reference sequence (YP_182356.1) of ILT virus glycoprotein B (GB) was submitted to different prediction tools at the Immune Epitope Database analysis resource (epitopes . Predicthttp://tools.iedb.org/bcell/). BepiPred linear epitope prediction from Immune Epitope Database (http://tools.iedb.org/bcell/result/) was used to predict linear B cell epitopes with default threshold -.012 [http://tools.immuneepitope.org/bcell/) with default threshold 1.027 [Identification of the surface accessibility, hydrophobicity, flexibility, and antigenicity was performed by analyzing candidate epitopes using several B cell prediction methods from Immune Epitope Database  Cytotoxic T-Lymphocyte Epitopes Prediction and Interaction with MHC- I. The major histocompatibility complex-1 (MHC class I) binding prediction tool (http://tools.iedb.org/mhci/) was used to predict Cytotoxic T cell epitopes [epitopes . Analysiepitopes , 27. Pep(2) Prediction of T Cell Epitopes and Interaction with MHC Class II. The MHC class II binding prediction tool (http://tools.iedb.org/mhcii/) was used to predict T cell epitopes. IC50 for strong binding peptides was set less than 1000 to determine the interaction potentials of T cell epitopes and MHC class II allele . Human MHC class I and II alleles were used in this study due to the difficulty to determine MHC B complex alleles in poultry. NN-align method was also used with IC50 less than or equal to 1000 nM [ 1000 nM . Peptidehttp://raptorx.uchicago.edu/StructurePrediction/predict/) was used for this purpose. The 3D structure was then treated with Chimera software 1.8 to display the position of proposed epitopes [Homology modeling was used for constructing the three-dimensional (3D) structure of the reference sequence of ILTV glycoprotein B. Raptor X structure prediction server (epitopes –32.∗2101 & BF2∗0401) were retrieved from the NCBI database/  and submitted to Raptor X server (http://raptorx.uchicago.edu/) for homology modeling. Chimera software was used to display 3D structure of BF alleles [Protein sequence and PDB ID of chicken alleles  to predict the linear structures from amino acid sequences [The homology modeling of the MHCI predicted peptides was performed with PEP FOLD3 (equences –35.∗2101 & BF2∗0401) and the proposed peptides from MHCI. Chicken BF alleles were set as receptors and the proposed peptides were set as ligands. Molecular docking technique of 3D structure of BF alleles and 3D modeled epitopes was performed using PatchDock online autodock tools; an automatic server for molecular docking (https://bioinfo3d.cs.tau.ac.il/PatchDock/) by submitting PDB of ligands and receptors after homology modeling by Raptor X server and PEP FOLD3 [Molecular docking was performed according to peptide-binding groove affinity, between chicken BF alleles  to trigger immune response , 39. It The use of bioinformatics analyses is an applicable method for predicting and designing new multiepitope vaccines against animals' infectious diseases as well as chickens , 41, 42.Alignment of all retrieved sequences using ClustalW through BioEdit software showed high conservancy between the aligned sequences. As shown in http://www.phylogeny.fr). The evolutionary divergence analysis of the enveloped glycoprotein B of the different strains of ILTV is presented in Phylogenetic tree was created using  revealed varying threshold for different scales see . Thirty-∗2101 from the B21 haplotype is highly expressed, leading to strong genetic links with infectious pathogens. In addition, BF2∗2101 from the B21 haplotype has principal role in provoking resistance to Marek's disease caused by an oncogenic herpesvirus [CD8+ and CD4+ T cells have principal role in stimulation of immune response as well as antigen mediated clonal expression of B cell . Severalpesvirus , to whic118YVFNVTLYY126 which interacted and linked with 16 human alleles, followed by 335VSYKNSYHF343 and 622YLLYEDYTF630 as they linked with 9 human MHCI alleles. The 3D structure of the proposed epitopes is shown in MHC-1 binding prediction tool using IEDB database predicted sixteen epitopes that interacted with the cytotoxic T cell as they strongly linked with multiple alleles. As shown in 301FLTDEQFTI309 exhibited high affinity to MHCII alleles due to great binding with 76 MHC-II alleles followed by 277FLEIANYQV285 and 743IASFLSNPF751 as they linked with 67 human alleles  ≤ 1000. The peptide (core)  alleles .FLTDEQFTI), which achieved the highest linkages with 76 alleles from MHC class II, was linked with 3 alleles only from MHCI. While the second proposed epitopes (FLEIANYQV and IASFLSNPF) that bound to 67 MHCII alleles were associated with 2 and 6 alleles from MHC1, respectively. However, of these top epitopes, only four of top MHC1 epitopes and two of best epitopes from MHCII were not linked to any alleles from MHCII and MHCI, respectively.Twelve epitopes from top five proposed MHC class I were associated with at least to 15 alleles from MHC class II epitopes see . It was ∗2101; BF2∗0401) were selected. The docked epitopes  using peptide-binding groove affinity were used to evaluate the ability of predicted epitopes to bind with chicken BF alleles/receptors to chicken alleles BF2 (BF2∗2101 and BF2∗0401). Results indicated that the docked epitopes achieved strong binding affinity to Chicken BF2 alleles based on global energy and attractive VDW in kcal/mol unit. The lowest binding energy  was selected to predict probable CTL epitopes. Docked ligand epitopes  with BF2 2101 alleles (receptor) showed higher binding affinity which expressed by the lower global energy . However, BF 2 0401 allele as a receptor produced less binding affinity with docked ligands . These results indicated that the binding affinity of ligands is higher with the receptor BF2∗2101 allele compared with the other allele (BF2 0401) which produced less binding affinity. In addition, the docked molecules showed different groove binding site for both BF alleles. The top ranked CTL proposed epitopes were selected for molecular docking to predict and symbolize the image of real CTL epitopes interaction with chicken alleles. For this purpose, two types of chicken BF alleles (BF2Smart computational techniques which provide tremendous predictive and analytical information facilitate the prediction of novel epitopes that may act as a powerful vaccine through immunoinformatic technology.This is the first in silico study to design peptide vaccine against avian ILTV through humoral and cell mediated immune responses. The expected epitopes in this study could help in prevention of latent infection caused by the use of attenuated vaccines and developing more effective and trustable prophylactic and therapeutic vaccines than conventional methods.In this study new epitopes were proposed as promising multiepitopes vaccine for ILTV. CTL epitopes were selected as vaccine candidates due to their high binding affinity with different alleles. The result should be supported by designing the peptide vaccine in the lab and through clinical trials.


Almost 25% of all human cancers are located in the gastrointestinal tract (GIT), making it the dominant cancer-affected site. The reason for this could be constant GIT exposure to organ damage and chronic inflammation . DespiteThe most important risk factor for BE and EAC is gastroesophageal reflux disease (GERD). The majority of GERD patients present with heartburn and effortless regurgitation. GERD is one of the most common indications for visiting primary care physicians and gastroenterologists and the most common GIT-related diagnosis worldwide -7. The pMultifactorial pathophysiology and risk factors for GERD are still the subject of many investigations ,5-7. A rCroatian Medical Journal (Having all this in mind, we were delighted to read the article by Markoš et al, published in the current issue of the  Journal . The aut Journal .


TM technique (Arthrex-tech group); continuous loop graft using the Kessler anastomosis technique (Kessler-tech group); and continuous loop graft using a Double-Z anastomosis technique (Double Z-tech group). Each group of eight specimens underwent cyclic loading followed by a load-to-failure test. The M–technique yielded a smaller graft diameter (8.4 ± 0.5 mm) compared to the statistically equivalent diameters of the three continuous loop techniques . The continuous loop grafts formed by the Double Z-Technique showed outstanding performance among the tested techniques in terms of ultimate failure load (982 ± 121 N) and cyclic elongation (3.7 ± 1.0 mm). There was no significant difference between the four groups in cyclic stiffness. Of the assessed techniques, the Arthrex technique resulted in the lowest ultimate elongation (2.0 ± 0.7 mm), followed by the Double Z-tech (4.5 ± 1.8 mm), the M-tech (5.2 ± 3.9 mm), and the Kessler-tech (5.3 ± 2.4 mm). The Arthrex-tech group (5.98 ± 0.38 min) displayed the shortest graft preparation time, followed by the M-Tech (7.94 ± 0.58 min), Kessler-tech (9.03 ± 0.39 min) and Double Z-Tech (13.29 ± 1.14 min). Double Z-Tech can improve the construct of continuous loop tendon graft with regard to mechanical performance.We introduce a new approach for a continuous loop tendon-graft preparation, benchmarking it against established graft preparation techniques widely used in conjunction with non-adjustable interference screw fixation. A four-strand bovine tendon graft was prepared using the following graft preparation techniques: standard graft using the baseball stitch technique (M-tech group); continuous loop graft using the GraftLink Therapeutic strategies of ACL ruptures have received extensive attention from scholars and with the development of sports medicine and arthroscopic techniques, arthroscopic anterior cruciate ligament reconstruction (ACLR) has become the most common surgical strategy for the treatment of ACL rupture3.Untreated ACL rupture can be associated with the occurrence of knee osteoarthritis and knee instability5. Fixation with these techniques is usually achieved with suspensory or interference screw fixation with suspensory fixation being preferred by many surgeons6. Whereas conventional cortical fixation devices provide fixed length suspension, adjustable loop fixation7 introduced the possibility of individual graft length adjustment and maximization of graft-bone contact8. Depending on the surgeon’s preference, adjustable loop fixation devices can be used for femoral-only as well as for femoral and tibial graft fixation. In the latter case, the tendon explant is folded twice over the two adjustable loop fixation devices and the two tendon ends are connected to form a continuous double-loop tendon graft. This graft configuration is in contrast to the tendon-graft configuration used for more conventional fixation techniques such as interference screw tibial fixation where the two tendon ends are “baseball- stitched” and most commonly folded twice to create a tendon graft folded in M-configuration. Whereas adjustable loop fixation devices may provide sufficient fixation strength and rigidity the biomechanical behavior of continuous double-loop tendon grafts has not been reported.Currently, mainstream ACLR techniques utilize autogenous lower limb tendon grafts  and bone patellar tendon bone (BPTB) grafts with tendon grafts being the most common7  to a conventional M-configuration technique (M-Tech) used in tibial interference screw fixation. Meanwhile, we also explored modified graft suturing techniques (Kessler-tech and Double Z-Tech) designed to improve the construct with regard to mechanical performance in isolated biomechanical testing. It was hypothesized that continuous loop tendon grafts were superior to the M-Tech graft in mechanical properties. The second hypothesis was that a graft using Kessler-tech or Double Z-Tech can improve the construct of continuous loop tendon graft with regard to mechanical performance.In this biomechanical study, we compared a frequently applied hamstring graft preparation technique used for tibial adjustable length suspensory graft fixation9. Specimens were preserved at −20 °C and defrosted at room temperature for 1 hour before graft preparation. During graft preparation and biomechanical testing, phosphate buffered saline  was used to keep the tendon hydrated per 10 minutes. Excess soft tissues were removed from the surfaces of tendons before graft preparation. The tendons were cut in half along their long axis for the 4-folded grafts to have a diameter of approximately 8 mm.This study was performed in accordance with the National Institutes of Health guidelines for the use of experimental animals, and all animal protocols were approved by the Institutional Animal Care and Use Committee of University of Zurich. The Ethics Committee of Canton of Zurich approved the study protocol, including any relevant details. Testing was performed using 16 bovine calf tendons purchased from a local slaughterhouse, with structural and biomechanical properties similar to those of human hamstringtendonsTM graft preparation technique (Arthrex-tech), continuous loop graft using Kessler technique (Kessler-tech), and continuous loop graft using Double-Z technique group (Double Z-tech).The 32 tendon specimens were randomly divided into four groups: 4-strand graft using baseball stitching at both tendon ends to form a double-folded tendon graft in M-configuration (M-tech), continuous loop graft using the GraftLinkAs shown in Fig. Continuous loop grafts were prepared by inserting the tendon into two looped size 5 polyethylene loading sutures  twice to form a double-loop continuous tendon graft Fig. . The fre10. Briefly, the graft was quadrupled symmetrically over two looped size 5 polyethylene loading sutures, and the free ends were whip-stitched and then sutured with a buried-knot technique7 Technique was aided by use of guides for the continuous loop GraftLink ue7 Fig. .11. The suture was hereby inserted longitudinally into the tendon core for a length of 10 mm. Then the suture was passed to the side of the tendon and reinserted into its core and brought back to the end of the tendon  technique was selected to connect the free tendon ends. One stitch location was near the loading suture at the outside the circular contraction sutures (group A); one stitch location was under that of the circular contraction suture (Group B) and in the middle of the two circular sutures (Group C). Each locations type contains 6 samples and was tested according to the following protocol.13. The preconditioning protocol was aimed to simulate the loading regime the graft undergoes during surgical insertion. Then the tendon grafts were ramp loaded until failure at a machine speed of 20 mm/min14. Table Before mechanical testing, the diameter of the graft was measured using a graft sizing device . The tendon grafts were then placed into a universal testing machine  suspended with size 5 suture on both sides Fig. . After aForce-displacement data was recorded using TestXpert 10 software  and processed in MATLAB . Ultimate failure load (N) and ultimate elongation (mm) were defined as maximum force achieved and respective elongation with elongation zeroed at the end of preconditioning. Cyclic elongation (mm) describes total graft elongation during the 5000 test cycles. Cyclic stiffness (N/mm) was measured by averaging the slope of the linear curve connecting minimum and maximum force-position of each cycle Fig. .Sample testing was monitored by Dr. Chen Zhang and Tobias Götschi alternately and documented using a digital camera . Four modes of sample failure were documented: Baseball stitch failure, loading ropes cut through tendon, continuous loop anastomosis  rupture, and continuous loop anastomosis loosening and subsequent structural failure during cyclic testing.Time required for the preparation of the graft using either of the techniques was recorded.α was 0.05.Statistical results were generated by using a standard statistical software . Measurement data were expressed as the mean ± SD. Kolmogorov-Smirnov test was used to test if the values were adequately fit by Gaussian distributions. The measurement data were statistically analyzed using one-way analysis of variance for multiple group comparisons and the Dunnett t test for 2 group comparisons. The test level of The Ethics Committee of Canton of Zurich approved the study protocol.After the baseline biomechanical assessments, we found most of the stiches loosened and failed due to the pulling force of the loading loop when the graft securing sutures were located near the loading suture (6 of 6 in group A) and under the circular contraction sutures (5 of 6 in group B). No visible gap nor anastomosis loose was observed during cyclic testing in group C. Stitch location which was in the middle of the two circular sutures was thus chosen for the experiments in this study.Each group of 8 graft specimens was mechanically tested and analyzed Table , Fig. 5.Each group of 8 graft specimens was mechanically tested and analyzed Table , Fig. 5.tK/M = 2.673, p = 0.0203; tZ/M = 2.668, p = 0.0205), whereas the diameter of Arthrex-tech (A) group (8.94 ± 0.56 mm) was not significantly different . However, there was no significant difference among the three continuous loop graft groups .Analysis indicated that M-tech (M) group has the smaller diameter (8.43 ± 0.53 mm) than the three continuous loop graft groups Fig. . The meatZ/M = 2.542, p = 0.0259; tZ/A = 3.794, p = 0.0022; tZ/K = 3.196, p = 0.0077) .Optimized continuous loop grafts of Double Z –tech group (982 ± 121 N) had significantly higher ultimate failure load than conventional hamstring graft (777 ± 175 N) and continues loop graft using Arthrex-tech (762.2 ± 103 N) and Kessler-tech (712 ± 187 N) (77) Fig. . FailuretZ/M = 5.561, p = 0.0001; tZ/A = 2.838, p = 0.014; tZ/K = 3.899, p = 0.0021). The Arthrex-tech group also showed significantly better performance than M-tech group and Kessler-tech group . While cyclic elongation of the Kessler-tech group was the largest among the continuous loop groups, it was not statistically different to the conventional hamstring graft. No difference was observed between Kessler-tech group and M-tech group .The continuous loop grafts showed outstanding performance than hamstring graft in terms of cyclic elongation Fig. . The DoutA/M = 2.265, p = 0.0413; tA/K = 3.721, p = 0.0026; tA/Z = 3.612, p = 0.0032). No difference was found between Kessler-tech group, Double Z-tech group and M-tech group .The ultimate elongation of continuous loop grafts was not inferior to conventional hamstring graft preparation Fig. . The ArtF = 1.416, p = 0.2428). The measured cyclic stiffness of Arthrex-tech group was 2219 ± 862 N/mm, the Kessler-tech group was 1758 ± 1051 N/mm, the Double Z-tech group was 1927 ± 1436 N/mm, and the M-tech group was 1405 ± 433 N/mm , with one of the four grafts failing completely at the 2200th cycle due to continuous loop anastomosis loose and structural failure. The remaining 7 samples completed the cyclic testing protocol failed as Kessler anastomosis rupture during ramp loading. Of the eight tested grafts in the Double Z-tech group, one was excluded due to data output failure, five failed in the form of Double Z anastomosis rupture and two from loading rope cut through. No visible gap nor anastomosis loose was observed during cyclic testing in Double Z-tech group.Of the eight grafts that were tested in M-tech group, one was excluded due to data output failure, five failed in the form of baseball stitch failure and two of loading rope cut through. In the Arthrex-tech group, all of 8 samples failed by whipstitch rupture. Interestingly, 3 of 8 samples non-critically damaged with a visible gap forming at the 115tA/M = 7.952, p < 0.001; tA/K = 15.52, p < 0.001; tA/Z = 17.19, P < 0.001). While graft preparation time of the Double Z-tech group was the longest among the continuous loop groups, the mean graft preparation time of continuous loop groups (9.32 ± 0.68 min) was not statistically different to the M-tech graft .The Arthrex-tech group (5.98 ± 0.38 min) displayed the shortest graft preparation time compared to the M-tech group (7.94 ± 0.58 min), Kessler-tech group (9.03 ± 0.39 min) and Double Z-tech group (13.29 ± 1.14 min)  to a conventional M-configuration technique (M-tech) used in tibial interference screw fixation. Arthrex-tech grafts outperformed the more traditional M-tech in terms of cyclic- and ultimate elongation. In this work, we also explored modified graft suturing techniques designed to improve the construct with regard to mechanical performance in isolated biomechanical testing. Using a newly introduced Double-Z graft preparation technique (Double Z-tech) we could significantly increase ultimate failure load (by 28.9% compared to the Arthrex-tech) and reduce cyclic elongation of the continuous-loop construct (by 24.7% compared to the Arthrex-tech). To the authors’ knowledge, the current investigation is the first to functionally assess and compare these graft configuration techniques.It was originally hypothesized that the diameters of continuous loop grafts and hamstring grafts would show no significant difference. However, the diameters of all three grafts were larger than hamstring graft, although there was no statistical difference between Arthrex-tech group and hamstring graft group. The reasons may be due to the continuous loop technology itself. As is known, the suture configuration ultimately determines the diameter of the entire graft. In fact, Arthrex-tech’s whip stitch suture is a side-by-side suture between the free ends of the tendon, which potentially increases the diameter of the suture configuration. However, due to the applied compression of the graft, the diameter of the tendon could be reduced by a circular contraction suture, leading to a lack of any significant differences in diameter between the Arthrex-tech group and the M-tech group. Both the Kessler-tech and the Double-tech Z group differ from the Arthrex-tech in that the suture passes through the interior of the tendon entirely, restricting compression of the tendon and consequently increasing the end diameter. Considerations for decreasing the diameter of the graft will be pursued in follow-up studies.et al.15 reported a biomechanical result of cyclic elongation using the Arthrex technique with a tendon–button suspensory device named TightRope RT . They reported a total elongation of 4.5 ± 0.7 mm after 1000 cycles (50 and 250 N). Our results using the Double Z-tech group and Arthrex-tech group are comparable - indicating that our implementation of the Arthrex technique can be considered as standard, and demonstrates that the Double Z-tech can provide superior technical performance in terms of ultimate failure load and cyclic elongation.Elongation of the ACL graft should be kept to a minimum in order to avoid postoperative laxity. Our data indicated that continuous loop grafts showed better performance in terms of cyclic elongation and ultimate elongation compared to other grafts. In this respect, the Double Z-tech group and Arthrex-tech group outperformed the Kessler-tech group and M-tech groups. Notably, cyclic elongation in Double Z-tech group was superior to the Arthrex-tech group. This aspect may be useful in minimizing the widely-discussed windshield-wiper/bungee cord phenomenon and could help to potentially avoid postoperative laxity. Petre For the purpose of making the tendon graft more compact and increasing its stability, the doubled continuous tendon loop was secured using a three-suture technique. The actual location of three stitches were realized to be extremely important to the ultimate stabilization of the graft structure. We first performed a baseline biomechanical study analyzing three different locations for the stitch end. One stitch location was near the loading suture at the outside the circular contraction sutures; one stitch location was under that of the circular contraction suture and in the middle of the two circular sutures. In these biomechanical assessments we found most of the stiches loosened and failed due to the pulling force of the loading loop when the graft securing sutures were located near the loading suture and outside the circular contraction sutures. It should be noted that locating stitches beneath the circular contraction, while stabilizing the construct, can substantially enlarge the graft diameter. Locating the stitches in the middle of the two circular sutures yielded both good biomechanical performance and smaller diameters than other configurations, and was thus chosen for the experiments described in this study.In this study, the Arthrex-tech group displayed the shortest graft preparation time while graft preparation time of the Double Z-tech group was the longest among the continuous loop groups. The reasons were due to the continuous loop technology and Double Z-tech themselves. As we introduced above, Arthrex-tech, M-tech and Kessler-tech were more mature and easy suture surgical techniques. In the sample preparation process, they were easier to implement. Because of the innovation and complexity of Double Z technology, it will take longer preparation time. However, the preparation time of these four surgical techniques were completed within 15 minutes, which was sufficient and reasonable for the preparation of the tendon for ACLR surgery.During this time, the surgeon can complete surgical procedures such as joint cleaning, ligament exploration and bone bed preparation, without affecting the implementation of the operation. Therefore, although Double Z technology has the longest preparation time, it can still meet clinical needs. Considerations for decreasing the preparation time of the grafts will be pursued in follow-up studies.in vitro study focuses only on mechanics of graft and neglects biological factors such as healing and incorporation of the graft into the surrounding bone tunnel. Additional intra-articular biomechanical experiments are planned to clarify some of these aspects.Among the limitations to the present study, is the fact that bovine tendons rather than human hamstring tendons were used as a testing model. Bovine tendons yield a homogeneous graft size that allows the suturing method effects on ultimate implant size to be more accurately compared. Further, since no tendon rupture and no differences in stiffness were observed in the present study, it can be assumed that the differences between the groups were not affected by differences in tendon properties. Of course, such uniformly high-quality tendon does not reflect actual clinical practice. Third, as we discussed above, the diameter in continuous groups were unsatisfyingly large, and additional work will be required to refine the methods in a manner that minimizes increases in graft diameter. Finally, it is important to note that this Altogether, we hypothesized that continuous loop tendon grafts were superior to the M-Tech graft in mechanical properties and the Double Z-Tech can improve the construct of continuous loop tendon graft with regard to mechanical performance. Our data indicated that continuous loop grafts showed better performance in terms of cyclic elongation and ultimate elongation compared to other grafts. In this respect, the Double Z-tech group outperformed the Kessler-tech group and M-tech groups. Our findings confirm our hypothesis and oriented a new approach for continuous loop tendon-graft preparation. Future work is warranted to exploit potential performance increases that can be achieved by improved graft design.This study shows that continuous loop tendon grafts perform equivalently to the standard M-folding technique in cyclic and ramp-to-failure testing. We introduce a novel “Double Z” technique that biomechanically outperforms other preparation techniques, albeit with the drawbacks of being more time-consuming and yielding slightly larger overall graft diameters.


In some clinical situations, the vertical length of either a prepared tooth or an implant abutment is short, while the occlusal clearance to be restored by a porcelain crown is large. Incisal thickness of the veneering porcelain should be considered to prevent mechanical failure of the crown.  The aim of this study is to evaluate the effect of two different incisal veneering porcelain thickness on the fracture resistance of the anterior all-ceramic CAD/CAM zirconia crown system as compared with the conventionally used metal ceramic crown system.  CAD/CAM zirconia all-ceramic and metal ceramic crowns were fabricated on the prepared dies with standardized dimensions and designs using standardized methods according to the manufacturer's instructions. All crowns were then adhesively luted with resin-based cement (Multilink cement system), subjected to thermal cycling and cyclic loading, and were loaded until fracture using the universal testing machine to indicate the fracture resistance for each crown material in each veneering thickness.  Statistical analysis was carried out, and the results showed that the fracture resistance of the nickel-chromium metal ceramic group was significantly higher than that of the CAD/CAM zirconia all-ceramic group. Also, the fracture resistance of crowns with 1.5 mm incisal veneering thickness was significantly higher than those with 3 mm incisal veneering thickness in both groups. Furthermore, there was no significant difference in the fracture mode of the two groups where 50% of the total specimens demonstrated Mode II (veneer chipping), while 35% demonstrated Mode I (visible crack) and only 15% demonstrated Mode III (bulk fracture).  High failure load values were demonstrated by the specimens in this study, which suggest sufficient strength of both incisal veneering thickness in both crown systems to withstand clinical applications; however, the fracture patterns still underline the requirement of a core design that support a consistent thickness of the veneering ceramic, and it is recommended to conduct long-term prospective clinical studies to confirm findings reported in the present study. Since the late 1950s, the metal ceramic crown system has remained a standard modality for rehabilitation of anterior dentition, thanks to their good mechanical properties and to somewhat satisfactory esthetic results, along with a clinically acceptable quality of their marginal and internal adaptation . LongeviIn the last 30 years, the growing patients' demand for highly esthetic and naturally appearing restorations has led to the development of new all-ceramic materials, whose mechanical characteristics have been dramatically improved to provide suitable longevity and limitation of the technical problems. Furthermore, a great advancement in dental ceramics has been achieved since the introduction of high-strength tetragonal yttria-partially stabilized zirconia (Y-TZP) which became the most interesting polycrystalline ceramic available for dentistry mainly due to the transformation-toughening mechanism , 10. HowPorcelain compatibility is a concern on veneered zirconia restorations since recent studies on all-ceramic restorations reported a significant amount of porcelain chipping (15–62%), cracking (25–50%), delamination (less than 10.7%), and large fractures (3–33%) –14. SeveSo, it is obvious that restoration of the anterior teeth with crowns that have a framework for porcelain support is further complicated by the requirement, generally placed on the veneering porcelain, to simulate a lifelike tooth appearance. This is particularly challenging for anterior restorations where a high level of translucency, especially in the incisal and middle third of the tooth, is required, while the presence of the framework in these areas is thought to be necessary to provide mechanical resistance to fracture.Therefore, this study was conducted to evaluate the effect of two different incisal veneering porcelain thicknesses on the fracture resistance of the anterior all-ceramic CAD/CAM zirconia crown system when comparing it with the conventionally used metal ceramic crown system.The materials used in this study were as follows.All-ceramic crown materialsMetal ceramic crown materialsThe description of the crown materials is shown in Universal self-curing resin-based system with the light-curing option  was used for cementation of both groups in this study.Metal die Doublident Epoxy resin material CAD/CAM Zirkonzahn ceramic systemThermal cycling machine Custom made by the load cyclic machine Universal testing machine A specially designed stainless steel metal master die was milled to simulate the maxillary central incisor crown prepared to receive metal ceramic and all-ceramic full coverage crowns . The apiTwenty negative replicas of the prepared master die were made by the duplicating addition silicon material and filled with the epoxy resin material having the same elastic modulus of dentin to get twenty positive replicas of the prepared maxillary central incisor and then the reproduced dies were smoothly polished .Group I. CAD/CAM zirconia all-ceramic crowns (coping thickness of 0.5 mm). Group II. Nickel-chromium metal ceramic crowns (coping thickness of 0.5 mm).   Each group was subdivided into two subgroups according to the incisal veneering porcelain thickness.Subgroups Ia and IIa. The incisal veneering porcelain thickness was 1.5 mm. Subgroups Ib and IIb. The incisal veneering porcelain thickness was 3 mm. The specimens were divided into two main groups , each ofCopings of group I crowns were milled out from Zirkonzahn zirconia blanks in the following steps: optical impression of the specimens, designing of the coping using modeling software “Zirkonzahn modeler,” milling of zirconia blanks, and firing of the milled zirconia copings.Ten CAD/CAM zirconia copings were veneered with Ceramco®PFZ using the layering technique. To standardize the specimen's size, two extra specimens were milled to the full anatomical contour of maxillary central incisors, and the only difference was in the incisal edge thickness. One of the specimen had 2 mm incisal edge thickness , and the other had 3.5 mm incisal edge thickness ; the thickness of both was checked using the Iwanson gauge. Two impressions were made for the full contoured crowns from duplicating addition silicon with the aid of a metal ring to reproduce two silicon molds (mold #1 and mold #2) which were then split along the long axis to be used as guidance during building of the porcelain veneering, and then all the specimens were fired according to the manufacturer's instructions Figures . Each crFor standardization of the metal coping thickness and contour with that of CAD/CAM zirconia coping, duplicating addition silicon was used to make an impression for one of the zirconia copings; then, hard stone type IV was poured, and the stone die  was trimTen resin copings were sprued, invested using phosphate-bonded investment, and then placed inside the preheating furnace where burn out was done followed by casting of the nickel-chromium alloy according to the manufacturer's instructions. After divesting the metal copings, the sprues were cut and the copings were sandblasted and then finished and ultrasonically cleaned to get rid of all investment traces. Finally, the metal copings were seated onto their corresponding epoxy resin dies and checked for thickness and proper fit.Ten nickel-chromium copings were veneered with Ceramco 3 using the layering technique, and as means of standardization, the split silicon molds that were used for fabrication of zirconia all-ceramic crowns were used again for fabrication of metal ceramic crowns with the same technique. After checking the metal ceramic crowns thickness and fit, the crowns were finally finished and glazed according to the manufacturer's instructions.Self-curing luting resin with the light-curing option (Multilink N system pack) was used for luting all of the crowns according to manufacturer's instructions, and the restoration was seated in place and fixed by finger pressure for 15 seconds and then under static load of 5 kg for 10 minutes [(1) Thermal Cycling. Thermal cycling is the laboratory model used to mimic oral temperature changes. Each specimen was placed into a specially designed tray suspended from the arm of a thermal cycling machine. The specimens were subjected to 300 thermal cycles corresponding to six months of clinical service [ service , 17; the(2) Cyclic Loading. Each specimen was embedded in an acrylic block of size 19 × 19 × 20 mm, and then cyclic loading was performed using a specially designed custom-made load cycling machine where the loading stylus ended with a beveled metal rod with a dimension of 7 mm × 2 mm to apply load at 135° angle to the long axis of the tooth at the palatal surface of the crown, 2.5 mm from the incisal edge to simulate class I occlusion relationship with the antagonist tooth [st tooth –22.Cycling loads in this study were corresponding to 6 months of clinical service. Accordingly, samples were exposed to 120,000 mechanical cycles . The loaA rubber sheet of 0.5 mm thickness was inserted between the metal stylus and the crown to prevent sharp contacts, to distribute the applied force equally and to represent the consistency of the food substance  Figures–12.(3) Fracture Resistance Test. To determine the fracture resistance of the specimens, a universal testing machine was used Analysis of numeric data included the independent sample Analysis of categorical data included the Mont Carlo exact test and Fisher's exact testAll statistical analysis was done using two-tailed tests and an alpha error of 0.05. Most of the recommendations for a clinically relevant in vitro load-to-fracture test for ceramic restorations described by Kelly  and RekoAfter thermal and load cycling, no signs of fracture were detected in the specimens of each group. Signs of fracture were demonstrated by the specimens of each group only in the fracture resistance test, and the load at which fracture occurred for each specimen was recorded in newton.P < 0.05) between the fracture resistance of subgroups Ia and Ib where t = 4.2. CAD/CAM zirconia ceramic crown specimens with 1.5 mm incisal veneering thickness showed higher mean values (1428 ± 72.2 N) than those with 3 mm thickness (1278.2 ± 33.3 N)  .P < 0.05 between the fracture resistance of subgroups IIa and IIb where t = 3.5. The mean values for specimens with 1.5 mm veneering thickness was 1940.5 ± 153.7 N which is higher than those with 3 mm veneering thickness (1667.9 ± 80.2 N)  .This finding is in accordance with Swain  who repoP < 0.05) in the fracture resistance between the two groups. The metal ceramic group showed a higher mean value (1804.2 ± 184.4) than the mean value of the CAD/CAM zirconia all-ceramic group (1353.4 ± 95.3)   Table 3P < 0.05 This finding is similar with other studies conducted by Silva et al.  and HienRegarding the failure load values in this study, both groups demonstrated failure load values around 1500 N  and thisAccording to Waltimo and Könönen study, the mean maximum incisive force of the anterior teeth was 263 N for men and 243 N for women . All of It was found that there was no significant difference in the modes of fracture demonstrated by the two groups in the two incisal veneering thicknesses since 50% of the total specimens demonstrated Mode II (veneer chipping). However, 35% demonstrated Mode I (visible crack), and only 15% demonstrated Mode III (bulk fracture)  Table 3Table 3.Veneer chipping demonstrated by 50% of total specimens ; this fiThe chip size in the current study was much greater with zirconia crowns, creating more unacceptable defects; the surfaces where the veneering porcelain were delaminated from the core appeared smooth with no residual porcelain detected, and these observations were similar to previous studies , 43.Visible crack demonstrated by 35% of total specimens ; this fiBulk fracture demonstrated by 15% of total specimens and only by the CAD/CAM zirconia all-ceramic specimens , and thiObservations in this study and other studies revealed that fracture or chipping of veneering porcelains can be either a fracture of the porcelain itself or a fracture originating from the interfaces between the coping and porcelain . A strucPoor wettability of feldspathic porcelain is also another material specific factor for veneering chipping and is influenced by some processing parameters such as the roughness of the core surface and the atmosphere in which the feldspathic veneer was fused on the dental zirconia core. Such factors have a huge effect on the bond strength between zirconia and the overlying porcelain . Coping Regarding the fracture pattern, in the present study, the fracture pattern was extending from the lingual to the labial surface, and this finding is in consistent with Geminiani et al.  and Kim P=0.494), no interaction between the tested crown material and the modes of fracture (P=0.634), and also no interaction between the veneering thickness and the modes of fracture (P=0.737).The results revealed that there was no interaction between the tested crown material and the veneering thickness  investigation of the initiation and propagation direction of the cracks and failures would have been beneficial in studying the association between the defect at the loading point and the crack or fracture lines. Use of finite element analysis (FEA) also would be helpful in investigations of stress distribution to evaluate the mechanical behavior of restorations.The current study  suggests


To evaluate the influence of baseline parameters on the occurrence of stent-graft surface movement after endovascular aneurysm repair (EVAR) and to investigate its association with migration and stent-graft-related endoleaks (srEL).In this retrospective, cross-sectional study, three-dimensional surface models of the stent-graft, delimited by landmarks using custom-built software, were derived from the pre-discharge and last follow-up computed tomography angiography (CTA). Stent-graft surface movement in the proximal anchoring zone between these examinations was considered significant at a threshold of 9 mm. The Cox proportional hazards model was used to determine baseline variables associated with the occurrence of stent-graft surface movement. The association between migration and srEL with stent-graft surface movement was tested with the chi-square and the Fisher exact test, respectively.p = 0.017), proximal neck diameter , infrarenal aortic neck angulation , and proximal neck length  were significantly associated with the occurrence of stent-graft surface movement. Migration and srEL occurred in 17 (31.5%) and 5 (9.3%) patients, with and 11 (8.3%) and 2 (1.5%) without stent-graft surface movement .Stent-graft surface movement was observed in 54 (28.9%) of 187 patients. Multivariate analysis revealed that age  contains supplementary material, which is available to authorized users. The robustness of the overlapping area between the aortic wall and the stent-graft is a determining factor for the long-term durability of endovascular aortic repair (EVAR) , 2. StenThe primary objective of this study was to evaluate the influence of baseline clinical and morphological parameters on the probability of stent-graft surface movement in patients undergoing EVAR. The second aim was to investigate the association of stent-graft surface movement with migration and stent-graft-related endoleaks (type 1 and 3), representing the leading causes of rupture after EVAR , 21.This was a retrospective single-center, cross-sectional study of patients with abdominal aortic aneurysm (AAA) who were followed after elective EVAR. The institutional review board (No. 1843/2018) approved the study protocol and waived written, informed consent.Our institutional database was screened for patients who underwent elective endovascular AAA repair between May 2002 and July 2014 and who met the following inclusion criteria: (1) EVAR with bifurcated stent-grafts without intra-procedure implantation of proximal adjunctive sealing devices and (2) available standardized CTA, (a) pre-procedure, within 3 months prior to intervention, (b) pre-discharge within 1 week after intervention, and (c) after a time interval of at least 6 months.All patient data were anonymized and de-identified prior to analysis. One hundred eighty-seven patients 14 female) with a mean age of 73.2 ± 8.0  years were available for final analysis. Details of patient selection and patient characteristics are given in Fig. 4 female n = 122) or a dual-source  scanner. The institutional standard protocol included an arterial phase, ranging from the celiac trunk to the groin solely for pre-interventional imaging, followed by a late phase limited to the extent of the stent-graft in case of postinterventional imaging. Acquisition parameters are given in Table CTAs were performed in the caudo-cranial direction using a 16-slice , sex, and body mass index (BMI). Baseline morphological parameters included anatomical variables measured in the pre-procedural CTA scans, device-dependent variables collected from the pre-interventional CTA and/or the stent-graft procedure, and early postinterventional variables assessed on the pre-discharge CTA. Measurements were performed with the syngo.via imaging software .2) [Anatomical variables determined in accordance with the Society for Vascular Surgery standards for EVAR  included2) . This vaThe presence of a thrombus with > 2 mm thickness at the circumference of the proximal anchoring zone was evaluated and classified as follows: (1) no thrombus, (2) thrombus < 25%, (3) thrombus 25–50%, or (4) thrombus ≥ 50%. The presence of calcifications was categorized according to the same classification. On the basis of gathered anatomical variables, compliance requirements with the manufacturer’s instructions for use (IFU) were analyzed, and rated as within or outside the IFU.n = 36, 19.3%), Excluder , Zenith , Endurant , Anaconda , Aorfix , Powerlink , and Treovance . Device-dependent variables included (1) proximal oversizing factor, (2) distal oversizing factors, (3) proximal fixation level , (4) implantation side of the modular limb (right/left), and (5) presence of any active fixation mechanism .The followed devices were implanted: Talent  the presence of a type 2 endoleak (T2EL) and (2) the renal artery to stent-graft distance (RSD). RSD was measured along a semi-automatically drawn CLL, as described by Bastos Goncalves et al . RSD wast test. Non-normally distributed data were described by medians and interquartile ranges (IQRs). Possible differences between groups were tested with the Wilcoxon-Mann-Whitney U test. Dichotomous variables were described in absolute numbers and percentages, and possible differences between groups were tested by the chi-square test or the Fisher exact test, as appropriate. The univariate Cox proportional hazards model, with calculation of hazard ratios with 95% confidence intervals, was used to investigate the influence of baseline clinical and morphological parameters on the probability of stent-graft surface movement-free survival. A multivariate Cox proportional hazards model was conducted by using a backward selection of parameters, with a limit of p < 0.1 required to enter and to stay in the model. The proportional hazards assumption was tested for each variable individually using the time-dependent covariate method [J statistics. The Kaplan-Meier life table method was used to determine freedom from stent-graft surface movement, migration, and stent-graft-related endoleaks. For these survival analyses, all observations were censored at the time of the patient’s last CTA. All tests were two-sided; significance was assumed at p < 0.05. All statistical analyses were performed using SPSS for Windows .Normally distributed, continuous data were presented as the mean ± standard deviation. Potential differences between groups were compared using the e method . No relep < 0.001) more frequently in patients with , compared to those without , stent-graft surface movement  more frequently in patients with , compared to those without , stent-graft surface movement  of 187 patients during a mean MSCTA follow-up of 33.5 ± 25.4 (median and IQR) months. Overall, cumulative freedom from stent-graft surface movement rates after 1, 3, and 5 years were 94.6%, 78.8%, and 55.3%, respectively , proximal neck diameter , and infrarenal aortic neck angulation  were significantly associated with a higher incidence of stent-graft surface movement. Proximal neck length  proved to be significantly associated with a decreased incidence of stent-graft surface movement in the multivariate analysis (Table There were four (1.7%) type 1a endoleaks caused by stent-graft migration that resulted in rupture in two patients, 45 and 42 months after treatment. Successful redo-EVAR was performed by implantation of an aorto-mono-iliac device implanted in one patient, and a bifurcated endoprosthesis in the second patient. The two other type 1a endoleaks were treated by implantation of proximal extensions . Furthermore, two patients with a left-sided modular limb suffered from type 1b endoleaks , which were treated by extension of the iliac limb into the external iliac artery after coil occlusion of the internal iliac artery. One type 3 endoleak, caused by disconnection of the left modular limb, was over-stented. Baseline clinical and morphological parameters of the entire cohort, separated for patients with and without stent-graft surface movement, are given in Table p = 0.410), 0.607 , 0.633 , and 0.530 , respectively.For identification of patients with stent-graft surface movement, the optimal cut-off points for age, neck diameter, neck length, and infrarenal neck angulation were 72.3 years, 2.65 cm, 4.15 cm, and 39.5° with areas under the curve of 0.538  previously reported 8% previo–34, whicDifferent anatomical aspects of proximal neck morphology were found to be associated with the incidence of migration. In this context, the aortic neck diameter was found to be a risk factor for stent-graft migration , 4, 35, Another mechanism with a negative influence on wall compliance and aneurysm morphology is atherosclerotic disease progression , which pDifferent approaches were made to streamline the surveillance protocol proposed by the Society of Vascular Surgery , designeOur study has several limitations. First, data were evaluated in a retrospective manner and we had to exclude 50 patients with early re-interventions for stent-graft failure, rendering the assessment of stent-graft surface movement impossible. Since we have no information on possible late complications of these patients, we cannot completely rule out a possible selection bias. Second, inclusion of second-generation devices and rudimentary planning in the early period of our study may have negatively affected the outcome in certain patients. Third, comparing the pre-discharge and the last CTA follow-up resulted in a heterogeneous time interval for measurements. As a consequence, the study design does not allow a conclusion about the time point for the occurrence of stent-graft surface movement and migration, nor on its chronological sequence. Fourth, due to the relatively small sample size and heterogeneity by means of implanted devices, we are not able to draw a reasonable statement on the performance of different devices.Older patients, those with larger aneurysm neck diameters, and those with increased infrarenal aortic neck angulation experienced higher rates of stent-graft surface movement. Consideration of these risk factors may help to identify patients who benefit from possible use of adjunctive, prophylactic sealing systems and a regular CTA surveillance or even open repair as a primary intervention. Detection of stent-graft surface movement during CTA surveillance may allow for planning of timely prophylactic re-interventions to prevent stent-graft migration and stent-graft-related endoleaks.ESM 1(DOCX 18 kb)


Closure of skin defects after scalp surgery may be accomplished by grafting; either split- or full-thickness. Both methods are used in Denmark, and the optimal approach on scalp defects without exposed bone is not known. This study aimed to investigate if the two methods were equal regarding graft take as primary outcome and as secondary outcomes complications and number of outpatient visits/ number of days from surgery until the last outpatient visit for the recipient site , hypothesizing that they were. The present retrospective single-center case series reported our experience using the two types of skin grafts after scalp surgery in the inclusion period from 1.1.2014 to 30.09.2015. Data were analyzed according to graft type with a full-thickness skin graft (FTSG-group) or a split-thickness skin graft (STSG-group). p>0.05).In the inclusion period, 106 patients had surgery (28 with a FTSG and 78 with a STSG). Irrespectively of which skin graft that was used, we found no statistically significant difference regarding percentage of adherence, complications or number of outpatient visits and time from operation until last outpatient visit regarding the recipient site (Our findings supported that use of either FTSG or STSG in scalp lesions were equal choices. FTSGs are usually harvested from the groin, the upper inner arm and the supraclavicular fossa or, for scalp surgery, the adjacent scalp.5Scalp surgery for skin tumors often produces defects larger than what can be closed directly and therefore, reconstructive surgery is necessary. Multiple methods of reconstruction are available, including free flaps, local advancement flaps and skin grafting.5STSGs are usually harvested from the upper anterior or lateral thighs, but almost any body region can be used if necessary. After harvest at the donor site, the graft is applied to the defect and a dressing is placed over to secure contact with the wound bed and prevent bleeding and formation of hematoma by providing slight compression. The dressing is held in place using either sutures or staples, and remains until the patient arrives for the first outpatient visit, typically on the fourth-seventh day after surgery.5 Limited donor areas for FTSG can be a reason for using STSG. There are a number of advantages and disadvantages with the two types of grafts. The usage of a FTSG leaves the donor area to be closed by suturing, stapling or in rare cases with a split-thickness skin graft.6-9For scalp-surgery, including region-parietalis, region-occipitalis and the postauricular area, surgeons use either a STSG or a FTSG as opposed to the facial area or the hand where a FTSG is most commonly used due to cosmetic and functional reasons.6–9 An inadequate wound bed is a major factor affecting especially FTSG take, which with its added thickness places higher demands regarding nourishment and revascularisation.10 STSGs have better initial take, larger donor site availability, and, in the scalp, contraction can be useful to decrease the size of the scar /alopecia.10FTSGs heal with a better cosmetic and functional result, give better coverage and have less secondary contraction, but require substantial nourishment, which leaves them vulnerable to insufficient vascularization.10A STSG is harvested from the donor area with a dermatome  and the donor area is left for re-epithelialization. A reason for using this type of graft for skin cancer patients is that they often present with recurring skin cancer, requiring more surgery and therefore it could be argued to leave their full skin graft reserves for tumors located in the face. In this study we did not consider the cosmetic aspect of what graft to use.10The scalp is an area where the cosmetic and functional result is often of not as great importance as other areas, e.g. the face or hands. The possibility to mesh (to make holes in) this type of graft makes it able to cover larger areas than the size of the graft first harvested. Skin graft contraction occur in two stages; primary and secondary contraction. Primary contraction, which is greater in a FTSG, refers to the immediate reduction in size after harvest and secondary contraction, which is greater in a STSG, refers to wound bed contraction.10 Both types of grafts leave a difference in skin level, which is slightly more in STSG, and this graft is also more prone to dryness afterwards, since sebaceous glands are not transferred with the graft.10This contraction can work in advantage for the patient over the long term by decreasing the grafted area over time. A relative contraindication exists for the use of a STSG over joints or where contractures must be avoided. However, it remains a necessary tool for larger defects, for example burn-wounds.There are no recommendations regarding which method is the most optimal to use on the scalp, and both are used in Denmark. The present study reported our experience with patients receiving the two types of skin grafts after scalp-surgery with excision of a skin tumor. The hypothesis was that the two methods were equal regarding graft take and complications.The present study is a retrospective single-center case series including patients undergoing full-thickness or split-thickness skin grafting after scalp surgery for skin tumors during the period of 01.01.2014 and 30.09.2015. Patients were identified through the local hospital database using treatment codes for excision of a tumor on the head or neck, full thickness skin graft and split thickness skin graft. The study complies with the declaration of Helsinki. The primary outcome was percentage of skin graft adherence. Secondary outcomes were complications and number of outpatient visits/number of days from surgery until the last outpatient visit for the recipient site . Results were analyzed according to FTSGs and STSGs. Inclusion criteria were surgery for a skin tumor in region-parietalis, region-occipitalis, the postauricular- or scalp-area leaving a defect that was either covered with a split-thickness or a full-thickness skin graft. If the patient had more than one surgery during the period, we only included the initial one, and in case of simultaneous tumors, the largest was included. A diversity of tumors was represented among the patients screened for inclusion. The diagnosis was not considered to influence what type of skin graft was used, nor the course of healing after excision of the tumor. We did not include region-frontalis in this study since this in many cases could be located in the face area rather than on the scalp, and a FTSG is almost always used in the facial area, if not a local flap.Exclusion criteria were missing description of the primary lesion, missing follow-up to last planned outpatient visit or if the patient had a chronic wound after previous surgery in the area. Data on demographics, site of pathology, skin tumor diagnosis, ulceration or infection before surgery, longest excision diameter , donor site location, length of hospital stay, complications, number of outpatient visits, time from operation until the last outpatient visit at the plastic surgery department, previous radiation therapy to the recipient area or chemotherapy within a month prior to surgery were obtained from hospital files. p value <0.05 was considered significant. All statistics were done using SPSS software . Infection before surgery was defined as cases where antibiotics were prescribed preoperatively. Data were analyzed according to grafting with a full-thickness skin graft (FTSG-group) or a split-thickness skin graft (STSG-group). Data are presented as number of patients and median (ranges). Fisher´s exact test and Chi-Square test were used for categorical data, Mann-Whitney U for ordinal or continuous data, and 95% confidence intervals as appropriate. A The medical records of 106 patients were reviewed. There were 28 patients receiving a full-thickness (FTSG-group) and 78 a split-thickness skin graft (STSG-group). Patient demographics were shown in p=0.001). There was a significant difference between the two groups regarding excision to galea fascia or pericranium. In the FTSG-group, 16 (57.1%) of the patients had an excision to the galea fascia and 12 (42.9%) to the pericranium as opposed to 24 (31.6%) and 52 (68.4%) patients in the STSG-group. For 84 (79.2%) of the patients, two of the following materials were used as inner layers in the dressing; euflavine, jelonet and nitrofuranzoin. Preoperative data, surgical information and tumor diagnosis are shown in , 22 (78.6%) of the patients had a graft take of 90-100% at the date of unpacking of the dressing, 4 (14.3%) of 60-90%, 1 (3.6%) of 30-60% and 1 (3.6%) of <30%. Two patients (7.1%) had full necrosis of the skin graft with one of them undergoing a second skin grafting and the other one was left to secondary healing  of the patients had a graft take of 90-100%, 6 (7.7%) of 60-90%, 4 (5.1%) of 30-60% and 4 (5.1%) of <30%. Two (2.6%) of the patients had a full necrosis of the skin graft with 1 (1.3%) of them undergoing a second skin grafting and 1 (1.3%) left to secondary healing. Eight (10.3%) of the patients were treated with antibiotics due to postoperative infection. Two patients (7.1%) were treated with antibiotics due to postoperative infection. One patient developed a hematoma. In the STSG-groupp=0.79). At the donor site, one patient in the FTSG-group had wound dehiscence. One (3.6%) in the FTSG-group had infection of the donor site treated with antibiotics as opposed to 4 (5.1%) in the STSG-group, who were treated with Flamazine or Biatain Ag dressing. Two (2.6%) patients developed a hematoma and 5 (6.4%) a seroma. There was no significant difference between the two groups regarding graft adherence and surgical complications. Overall complication rate was 5 (17.9%) in the FTSG-group and 18 (23.1%) in the STSG-group  developed a hematoma and one had partial necrosis of the donor site. There was no statistically significant difference in donor site complications between the two groups. Duration of hospital stay had a median of 0 (0-2) days in the FTSG-group and 0 (0-4) in the STSG-group (p=0.10). Duration of re-hospitalization had a median of 0 (0-0) days in the FTSG-group and 0 (0-11) in the STSG-group (p=0.55). The number of outpatient visits was 1 (1-14) and 2 (1-14) in the FTSG-group and the STSG-group, respectively (p=0.43). Time from surgery until the last outpatient visit for wound care was 7 days (4-191) in the FTSG-group and 10.5 days (4-330) in the STSG-group (p=0.97).In the STSG-groupIn the present study there were no significant statistical difference regarding preoperative data, adherence of the skin graft, complications or overall complication ratio. There was a significant difference concerning length and depth of the excision and also regarding the time from surgery until the last outpatient visit of the donor site, but not for the recipient site. We found a significant difference with the longest excision diameter in the split-thickness group and a larger percentage with deeper excision, to the pericranium, in the split-thickness skin graft group. This is most likely a matter of confounding by indication: larger size defects call for larger skin grafts and STSG would more often be chosen in such cases. Deeper defects can be at risk of more healing problems due to a less vascularized wound bed, and in such cases most surgeons would probably chose STSG over FTSG. The pericranium is though well vascularized, and no real contraindication for using a STSG.The time from surgery until the last outpatient visit at the plastic surgery department could be seen as a proxy for the healing progress. We found no difference regarding the recipient site, but a significant difference regarding the donor site with a longer time for a split skin thickness graft. There were though missing data from over 50% of the patients in each group and most donor sites got controlled by the patients general practitioner. et al.11 found in their analysis of 31 patients with STSG and 16 with FTSG for coverage after radial forearm free flap harvest, no significant difference in time to healing at the donor site. Al Shlash et al.12 found in their study with 85 burn patients who received STSG (56 cases) and FTSG (29 cases) no significant advantage regarding graft failure, graft contraction, hyperpigmentation, altered sensation, infection rate and hospital stay.Wellington In the process of deciding what graft to use potential complications, cosmetics of the donor and recipient site and patient satisfaction should all be considered, where the last part was not analyzed in this study. Hereafter, practical matters such as draping of the patient during surgery could also be determinant for the chosen graft and at last, with no difference in complications between the two groups, recommendations of which skin graft to use could also be based on the economical aspect; the costs of the surgery. Our main limitation is the retrospective nature of our study, leaving it vulnerable to confounding by indication, i.e. the surgeons could have been choosing STSG for complicated cases. If that was the case, STSG could be superior to FTSG. However, this cannot be determined from the present study. Furthermore, most patients were followed up at their General Practitioner concerning their donor site, from where we do not have data. However, we expect these cases to be rather uncomplicated, otherwise they should have been referred back to the department. Finally, due to the small number of patients in our case series, rare complications might not have been seen. Cosmetics and patient satisfaction could be analyzed retrospectively, although, given the limitations of a retrospective study, only a randomized controlled study will be able to examine if the two procedures are really equal. In conclusion we could not find any significant difference regarding graft take with the use of either FTSG or STSG in scalp defects, which is reassuring. The authors declare no conflict of interest.


Croatian Medical Journal (CMJ), I took over the task of maintaining the reputation of a journal, which although it is one of the best general medical journals in an EU member country, is still a small medical journal at the scientific periphery. It is difficult for such a journal to compete with the likes of the Lancet, British Medical Journal (BMJ), or Journal of American Medical Association (JAMA), although we try to uphold the same high standards of reporting research and publication integrity. By working in the CMJ as a full-time employee for two years I have become familiar with all the aspects of journal publishing process, and therefore I know that we should be realistic. The CMJ's goal from the beginning has not been to publish the most influential studies, but to allow scientists who have good ideas but come from countries with lower investment in science to publish their work in an internationally indexed journal. And even more than that, our goal has been to teach younger, but also more experienced scientists, how to set up research well, write good scientific articles, and give them a springboard for later publication in more prestigious journals (CMJ, continued their series of quality publications in other well-known journals from their field of interest (By assuming the role of the editor in chief of the journals -3. Thereinterest -3.However, despite the author-helpful policy and diamond open access, maintaining the citation impact indicators and attracting quality manuscripts remain to be challenging tasks. There are many factors affecting the authors' choice of a journal ,5. AlthoIn the next year, we will continue publishing Knowledge Landscapes, a series of texts on the challenges the digital society presents to medicine, which has been a standard column for the past two years. We also plan a new column about biostatistics. This column will deal with new statistical concepts and dilemmas that researchers face when setting up research, and will help them apply biostatistical knowledge in their work. Also, we will try to add new thematic issues to the standard ones, such as International Society for Applied Biological Sciences thematic issue scheduled for June 2019.Finally, it will be hard to surpass the excellent previous editors in chief, but I see the editor’s job to be honest and rewarding, so I am sure when I leave the journal it will be ranked even higher and be more influential. The main goal in next period will be to increase the journals' impact factor and to attract more quality articles, both from Croatian and foreign scientists. I hope that in achieving this goal, I will have your help.


Apostictopterus and Barca (Lepidoptera: Hesperiidae), remain ambiguous. We sequenced and annotated the two mitogenomes of Apostictopterus fuliginosus and Barca bicolor and inferred the phylogenetic positions of the two genera within the Hesperiidae based on the available mitogenomes. The lengths of the two circular mitogenomes of A. fuliginosus and B. bicolor are 15,417 and 15,574 base pairs (bp), respectively. These two mitogenomes show similar AT skew, GC skew, codon usage and nucleotide bias of AT: the GC skew of the two species is negative, and the AT skew of A. fuliginosus is negative, while the AT skew of B. bicolor is slightly positive. The largest intergenic spacer is located at the same position between trnQ and ND2 in A. fuliginosus (73 bp) and B. bicolor (72 bp). Thirteen protein-coding genes (PCGs) start with ATN codons except for COI, which starts with CGA. The control regions of both mitogenomes possess a long tandem repeat, which is 30 bp long in A. fuliginosus, and 18 bp in B. bicolor. Bayesian inference and maximum likelihood methods were employed to infer the phylogenetic relationships, which suggested that A. fuliginosus and B. bicolor belong in the subfamily Hesperiinae.The systematic positions of two hesperiid genera,  Despite considerable efforts in recent years5, the higher-level phylogenetic relationships within the family Hesperiidae are still unsatisfactorily resolved. The taxonomic affinities of many genera are not conclusive, even at the subfamily level6, including Apostictopterus and Barca.Skipper butterflies (Lepidoptera: Hesperiidae) include approximately 4,000 species in 567 genera worldwideApostictopterus and Barca have been controversial. They were assigned to the Heteropterus group of the subfamily Hesperiinae close to the Astictopterus group in Evans’s classification7, while Chou8 assigned Apostictopterus to the tribe Astictopterini and Barca to the tribe Heteropterini. Since Higgins9, the Heteropterus group of Evans has widely been regarded as Heteropterinae at the subfamily level. In previous studies10, these two genera were both treated as members of the subfamily Heteropterinae. However, on the basis of morphological evidence, Warren et al.6 were more likely to place them in Hesperiinae.The taxonomic positions of the two monotypic genera 14. Insect mitochondrial genomes (mitogenomes) are typically compact circular molecules of 15–18 kb containing 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), and two ribosomal RNAs (rRNAs)16. In addition, the mitogenome mostly contains a control region (an AT-rich region due to a high A + T content) that has a longer sequence than the other regions and embraces essential regulatory elements for transcription and replication20. However, this region cannot be well sequenced by high-throughput sequencing techniques, as the depth of coverage is strongly positively correlated with the GC content21.The difficulty of morphologically based phylogenetic systematics has been shown, whereas molecular phylogeny has been contributing to the development of a more stable classification. Since mitochondria are characterised by maternal inheritance, a rapid evolutionary rate, and little or no genetic recombination, they have been extensively used in the field of genetics and evolutionary biology21 had obtained promising results on the genus-level relationships of swallowtail butterflies using mitogenomes. Thus far, 30 complete or nearly complete mitogenomes of skippers have been sequenced. In this study, we sequenced two additional complete mitogenomes of A. fuliginosus and B. bicolor and then elucidated the composition of the genomes. Finally, we inferred the phylogenetic relationships from the 27 available mitogenomes within the Hesperiidae26. We did not use three mitogenomes. Polytremis jigongi and Polytremis nascens showed very low homology to the other species. There are two mitogenomes of Daimio tethys that are basically in line, so we randomly selected the one from Korea based on a computation-efficient strategy.Mitogenomes are data rich and relatively accessible source of information. CondamineA. fuliginosus and B. bicolor are 15,417 bp and 15,574 bp , 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNA: lrRNA and srRNA), and an AT-rich region. These assembly units are identical to those of the other skippers, and the encoding protein genes’ ORF direction is the same as in most skippers. Both mitogenomes have 15 intergenic regions. The maximum intervals of A. fuliginosus and B. bicolor, both between trnQ with ND2, are 73 bp and 72 bp, respectively. Only a few genes  are from the N strand, and the remaining 23 genes (nine PCGs and 14 tRNAs) are from the J strand. The nucleotide composition of A. fuliginosus is A (40.1%), T (40.6%), C (11.8%), and G (7.4%); the AT nucleotide content is as high as 80.7%. In B. bicolor, the composition is A (40.0%), T (39.4%), C (12.9%), and G (7.7%); the AT nucleotide content is as high as 79.4%. In these two mitogenomes, the GC skew of two mitogenomes and the AT skew of A. fuliginosus are negatively biased, while the AT skew of B. bicolor has a slightly positive bias  and 3,731 (B. bicolor) amino acids, which account for 72.6% and 71.9% of A. fuliginosus and B. bicolor, respectively. All PCGs in both mitogenomes start with typical ATN codons, except for COI, which is initiated by CGA, as is common in Lepidoptera. Stop codons in the PCGs include two types: TAA or T. Though incomplete stop codons always appear in lepidopteran mitogenomic PCGs, translation will not be affected at all because the codons will be automatically filled by added As during the transcription process27. We calculated the relative synonymous codon usage (RSCU) of the PCGs in the two mitogenomes  encoding the small and large ribosomal subunits are located between trnL(CUN) and trnV and between trnV and the AT-rich region. The lrRNA and srRNA lengths are 1,415 and 774 bp, respectively, in A. fuliginosus, and are 1,419 and 773 bp in B. bicolor.The two rRNA genes , which lacks the DHU arm both in A. fuliginosus and B. bicolor  has been demonstrated29. In addition, the number of bases in the dihydrouridine loop ranges from 4 to 8 bp, which is not uniform because the DHU stem is highly variable30.Both A. fuliginosus and eight in B. bicolor, with sizes ranging from 1 to 8 bp. The maximum overlap of the two mitogenomes are located between trnW and trnC 55 in A. fuliginosus and two dinucleotide repeats (TA)36 and (AT)54 in B. bicolor. Furthermore, we found a long tandem repeat of 30 bp (AAATAAAAAATTAAAATAATTATTTTAATT) in A. fuliginosus and a tandem repeat length of 18 bp (TAAAAAAATAATTATTTT) in B. bicolor. There was also a structure in the AT-rich region of both species with the poly-T stretch in a position close to the srRNA. Several microsatellite-like A/T sequences following the motif ATTTA in the control region were found in A. fuliginosus and B. bicolor, which were also discovered in the other skipper mitogenomes33. Moreover, our predicted results showed that there are two stem-loop structures in A. fuliginosus and three stem-loop structures in B. bicolor ; here, we present the results based on the PRT dataset as a basis for subsequent analyses. 16 best-fitting partitioning schemes , is recovered as monophyletic with weak support. Hesperiinae is obtained as monophyletic.Similar topologies were inferred from phylogenetic analyses with MrBayes and IQ-TREE Fig. . Six majA. fuliginosus and B. bicolor formed a strongly supported subclade (Clade A); this subclade branches after Heteropterinae and is followed by Hesperiinae with high support. Our results do not agree with placing them in the subfamily Heteropterinae10. We thus tentatively assign these two genera to the subfamily Hesperiinae. Previous studies have inferred a close relationship among Heteroptinae, Trapezitinae and Hesperiinae, but the sister relationships were uncertain6, and none of these studies sampled Apostictopterus and Barca. In this study, we were unable to include Trapezitinae to test for close relationships with Hesperiinae along with Apostictopterus and Barca, as no mitogenome is yet available. Hence, more samples in Trapezitinae are needed to confirm this hypothesis and clarify their systematic positions.In the phylogenetic tree, The phylogenetic analyses based on four datasets  using two methods revealed very similar topologies except for the phylogenetic position of Eudaminae and Pyrginae. In the BI and ML analyses from different datasets, the topologies were largely congruent except for three strategies with little discrepancy. As many studies have concluded, the mitogenome can provide robust and stable phylogenetic analyses. The result from the PCGR dataset showed that Eudaminae branched after Euschemoninae in the BI analyses. In the ML analyses, however, the topologies based on the PCGC and PCGD datasets revealed that Eudaminae nested within Pyrginae  following the manufacturer’s instructions.The adult specimen of et al.23 except for SF2, SF10, SF18, SF22 and SF27, which are newly designed  via the following protocol: initial denaturation for 2 min at 94 °C, followed by 35 cycles of denaturation for 30 s at 94 °C, annealing for 45 s at 40–50 °C, and extension for 1 min at 72 °C, and a final extension step at 72 °C for 10 min. For the AT-rich region, we used KOD high-fidelity thermostable DNA polymerase  to improve the accuracy of the amplification and employed the following PCR conditions: initial denaturation of 2 min at 94 °C, followed by 35 cycles of 10 s at 98 °C, annealing for 45 s at 42 °C, and extension for 1 min at 68 °C, and a final extension at 72 °C for 10 min.36. PCGs were identified by finding the ORFs on the NCBI website (https://www.ncbi.nlm.nih.gov/orffinder/) with the invertebrate mitochondrial genetic codes. The tRNAs and rRNAs were identified using the MITOS Web Server (http://mitos.bioinf.uni-leipzig.de/index.py)37. Moreover, to confirm the accuracy of the boundaries of different genes, 37 genes were aligned using ClustalW in MEGA v7.0.238 and manual inspection. The nucleotide composition statistics and relative synonymous codon usage (RSCU) were calculated using MEGA v7.0.2. The AT skew and GC skew39 values used for measuring the deviation of the base were calculated by the following formulas: AT skew = (A − T)/(A + T); GC skew = (G − C)/(G + C). The circular maps were drawn by CGView Server 40. The tandem repeats of the control region were identified with the Tandem Repeats Finder on-line server (http://tandem.bu.edu/trf/trf.html)15. Stem loop structures of the AT-rich region were predicted by DNAMAN. The two complete mitogenomes were deposited in GenBank with accession numbers MH985707 and MH985708.We assembled and proof-read the sequences using the software Geneious v7.1.441 with the G-INS-i strategy. Each of the two rRNAs was aligned separately using the Q-INS-i strategy through the MAFFT V7 online alignment server 42. We removed gaps and ambiguous sites from the 13 PCGs by using the Gblocks V0.9143 online server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) with default settings.We downloaded 33 available lepidopteran mitogenomes from GenBank, including 27 Hesperiidae, three Papilionidae and three Geometridae. The species used in this study are listed in Table rd codon removed; 2) PCGC: the 13 complete PCGs; 3) PRT: the 13 complete PCGs, two rRNAs and 22 tRNAs; and 4) PCGR: two rRNAs and 13 PCGs with the 3rd codon removed. We employed PartitionFinder V2.1.144 to identify the best partitioning strategies under the Bayesian information criterion (BIC). Maximum likelihood (ML) analyses were performed on the IQ-TREE web online server (http://iqtree.cibiv.univie.ac.at/)45 with 1000 ultrafast bootstraps (UFBS) to estimate the branch support. The best-fit models produced by ModelFinder46 implemented in IQ-tree. The Bayesian inference (BI) analyses were performed using MrBayes V3.2.6 on the CIPRES Science Gateway 3.347. We used reversible-jump MCMC to allow sampling across all substitution rate models instead of specifying one substitution model, as suggested by PartitionFinder in BI analysis. Four Markov chains (one cold and three heated chains) were run simultaneously for 1 × 107 generations with sampling every 1,000 generations. We examined the average standard change of the split frequencies in Tracer V1.748 to determine the values falling below 0.01. We discarded the first 25% of the sampled trees as burn-in. The remaining trees were then used to calculate the posterior probabilities (PP) under the majority rule consensus.To compare the phylogenetic signal information of the different dataset combinations, four datasets were used: 1) PCGD: the 13 complete PCGs with the 3Supplementary information