Bone-patellar tendon-bone (BPTB) and semitendinosus–gracilis (STG) are the commonest grafts used for ACL reconstruction. However even after having been debated for years, there is no consensus about the ideal graft. Moreover, the literature is deficient about STG graft with preserved tibial insertion (STGPI) which preserves the proprioception. Our aim is to compare the outcome of BPTB, free STG and STGPI grafts after ACL reconstruction in professional sports persons. We compared the outcome in terms of mechanical stability, functional outcome, return to sports activity and degenerative changes.Professional sports persons aged between 16-50 years operated for ACL tear using BPTB, free STG and STGPI grafts with minimum follow-up of two years were identified from hospital records. Patients with associated knee injuries were excluded. Patients, divided in three groups according to graft used, were compared in terms of mechanical stability (arthrometric examination KT-1000 score), functional outcome (Lysholm Score), return to sports activity (Tegner score and difference in thigh circumference) and degenerative changes (KL grading).BPTB graft group was found to be better than free STG and STGPI graft groups in terms of KT-1000 score. There was no statistically significant difference among the groups in terms of Lysholm score, Tegner score, difference in thigh circumference and KL grading.BPTB graft is better than free STG and STGPI grafts in terms of knee stability. When compared for patient reported outcome, return to sports activity, osteoarthritic changes and graft failure there is no significant difference among the three types of grafts. The results of this surgery depend upon various factors including age and sex of patient, duration of injury, pre-operative knee function and type of graft5. Among the autografts, though debated since years, controversy still exists regarding the superiority of bone-patellar tendon-bone (BPTB) vs semitendinosus–gracilis (STG) graft. However, there is no ideal graft for ACL reconstruction1.Allograft, as compared to autograft is considered to achieve poorer functional outcome which is not desirable in high demand patients like sports persons6. Though the preservation of insertion of semitendinosus and gracilis tendons during graft harvesting for ACL reconstruction has been reported to preserve the proprioception of the knee joint7. There is a paucity in the literature about the functional outcome of this graft for ACL reconstruction as compared to other grafts.Also, it has been reported that a proprioception deficient knee, because of ACL injury, cannot recover fully after reconstruction surgeryOur study is a post-hoc analysis where we compared the mid to long term outcome of BPTB vs free STG vs semitendinosus–gracilis with preserved tibial insertion (STGPI) grafts after ACL reconstruction in professional athletes.We compared the three types of grafts in terms of objective mechanical stability, functional outcome, return to sports activity, degenerative changes and graft failure.The present study is a post-hoc analysis of a retrospective cohort. The professional sports persons aged between 16-50 years operated at our institute from 2011-2016 for ACL tear using BPTB, free STG and STGPI grafts with a minimum follow-up of two years were identified from our hospital record. We excluded the patients with multi-ligament knee injuries / associated meniscus injuries. The patients were contacted on phone and called for follow-up in our outpatient department.After calculating the sample size of 90, 102 patients divided in three groups were enrolled in the present study with 34 patients in each group. Group I for BPTB graft, Group II for free STG graft and Group III for STGPI graft. First 34 patients reported in each group were enrolled after taking written consent. All the patients were assessed for mechanical stability (arthrometric examination KT-1000 score), functional outcome (Lysholm Score) return to sports activity (Tegner score and difference in thigh circumference) and degenerative changes (KL grading).All the patients were treated using standard techniques for ACL reconstruction. Our post-operative rehabilitation protocol was similar in all patients. Clearance from institutional ethical board was taken before starting the study.ANOVA test was used to compare the three groups for KT-1000 score, Lysholm score, Tegner score and difference in thigh circumference. Post Hoc Tukey HSD test was used to compare Group I with Group II, Group I with Group III and Group II with Group III for KT-1000 score, Lysholm score, Tegner score and difference in thigh circumference.A total of 102 patients were enrolled in this study. There were four graft failures; one in Group I, two in Group II and one in Group III. The difference was not significant among groups (ANOVA test). We analysed 98 patients at final follow-up with mean follow-up of 28.79, 33.79 and 29.54 months in Group I, Group II and Group III, respectively. Mean age in Group I, Group II and Group III was 24.0 +/5.0, 23.21 +/- 7.5 and 23.0 +/- 5.7 years, respectively. Three groups were compared and found to be matched for age . There wIn Group I, Group II and Group III mean KT-1000 score was 1.68, 2.26 and 2.24, respectively, mean Lysholm score was 98.30, 97.30 and 97.10, respectively and mean thigh circumference difference was 1.40, 1.90 and 1.60, respectively. Results of KT-1000 score, Lysholm score, Tegner score, difference in thigh circumference and KL grading are shown in 9 however it has more donor site morbidity11. Allograft is reported to have significantly lesser stability than autograft and is considered only for multi ligament injury where autograft is insufficient13.ACL deficient knee leads to significant morbidity in a high demand patients. The purpose of ACL reconstruction is to resume the stability of the knee to pre injury level without significant complications. Despite being debated for many years, there is still controversy about the optimal graft choice for ACL reconstruction. BPTB graft is considered to have lesser laxity, high durability and larger graft diameter as compared to STG graft14. Loss of proprioception after ACL reconstruction is considered to be one of the reasons for this suboptimal outcome17. Also, it is reported that the fixation at the tibial side is the weakest point in the whole construct of ACL reconstruction when free graft is used19. The problem of loss of proprioception and fixation at tibial side can be solved by using STG graft with preserved tibial insertion. When graft is not stripped from its tibial side the nerve supply of the tendon and so its proprioception is not disrupted. However, there is insufficient literature comparing this preserved tibial insertion graft to free graft for ACL reconstruction.Though the mechanical stability of reconstructed ACL is reported to range from 85% - 90% this does not correlate with functional outcome21. If the graft is loaded before sufficient healing there is decrease in tension and loosening of the graft leading to decreased stability. Since the healing is better, there are less chances of graft loosening and knee instability with BPTB graft as compared to STG graft. This has also been reported by previous clinical studies9. However recent meta-analysis failed to find any significant difference between these two types of grafts24. Xie et al in their meta-analysis of seven studies compared the BPTB graft with four stranded hamstring graft22. The authors did not find any statistically significant difference between two groups when compared for knee stability using KT-1000 score and Lachman test. However authors reported that stability was better in BPTB group when assessed by pivot shift test. Li et al in their meta-analysis of nine studies (738 patients) found similar results23. When compared for knee stability using Lachman test there was no significant difference between BPTB and hamstring graft but BPTB was found to be significantly better than hamstring when compared by pivot shift test. Similar results were found by Magnussen et al in their meta-analysis of seven studies when knee stability was compared by using Lachman test24. The authors did not find any significant difference between BPTB and hamstring grafts. However, unlike the two above mentioned studies, the authors found that the results were comparable between BPTB and hamstring graft even when compared for pivot shift test. In our study we assessed knee stability on KT-1000 score and found that KT-1000 score is significantly better in BPTB graft as compared to free STG and STGPI grafts. Our study is different than previous studies as we also compared BPTB graft with STGPI graft. We used KT-1000 score to assess knee stability. We believe that KT-1000 score is no less reliable than other tests to assess knee stability. Though it is believed that Lachman test and Pivot test indicates anterior stability and rotational stability respectively, the relationship between Pivot test and stability needs a clarification23. Also, Abulhasan et al in their systematic review of 34 articles evaluated different knee stability measures and concluded that there is no gold standard technique to assess knee stability25. Kilinc et al compared the accuracy of the Lachman and Anterior Drawer Tests to evaluate the knee examination with the KT-1000 arthrometer in 40 ACL reconstructed knees26. The authors concluded that KT-1000 score is as reliable as Lachman and Anterior Drawer tests.BPTB graft heals more rapidly as compared to STG graft because in case of BPTB graft healing takes place at bone-bone interface whereas in STG graft healing takes place at soft tissue-bone interfaceet al22 in 2015 and Li et al23 in 2012, in their meta-analysis, analysed the patient reported outcome using IKCD and did not find any difference between BPTB and hamstring grafts after ACL reconstruction. Similarly Magnussen et al in 2011 in their meta-analysis analysed patient reported outcome after ACL reconstruction using Lysholm score, IKCD and Cincinnati scores and find no difference between BPTB and hamstring grafts24. Though IKDC score is well accepted to provide a full evaluation of the post-operative ACL knee reconstruction outcome, Lysholm score, which we used in our study, is also believed to be an acceptable psychometric parameter as patient-administered score after ACL reconstruction27.In our study we analysed the patient reported outcome using Lysholm score. We did not find any statistically significant difference among the three groups. Xie 24. The authors found that return to preinjury activity level is favoured by BPTB group as compared to STG group though the functional outcome was same in both groups.Tegner score is a standardised method of grading work and sporting activity. In our study we did not find any statistically significant difference among the groups when assessed for return to sports activity using Tegner score and difference in thigh circumference from normal limb. Our results are different than previously reported results by Magnussen in their meta-analysisAssociated meniscus injury may lead to degenerative changes after ACL reconstruction. In our study we excluded the patients having associated meniscus injuries. We used KL grading to see osteoarthritic changes after ACL reconstruction and found no significant difference among the tree types of grafts we used.24 we did not find any difference between graft failure rates of BPTB and free STG group. In addition we also compared the failure rates of STGPI grafts with BPTB and free STG groups and we did not find any difference among the groups.Similar to the reported results in previous meta-analysisWe acknowledge the drawbacks of our study. Our study is a post-hoc analysis of retrospective cohort and we believe a better designed prospective study with a bigger sample size can be more valuable.After ACL reconstruction the BPTB graft is better than free STG and STGPI grafts in terms of knee stability. When compared for patient reported outcome, return to sports activity, osteoarthritic changes and graft failure there is no significant difference among the three types of grafts at mid to long term follow-up. However, we suggest further a better designed study on this topic.


Lamellar keratoplasty is fast becoming the most popular form of corneal transplantation. The adoption of Descemet membrane endothelial keratoplasty (DMEK) in the management of Fuchs endothelial dystrophy and pseudophakic bullous keratopathy is partly responsible for this shift in the paradigm of management of corneal pathology. The learning curve of DMEK, however, has been proven to be much steeper than previous endothelial keratoplasty procedures. To ease the procedure, experts have proposed multiple innovative techniques from tissue preparation to graft unfolding to aid the more novice surgeon. Here, we collate and share tips and tricks from our collective experiences to support the learning curve and outcomes in DMEK for both the novice and more experienced corneal transplant surgeons. The most common causes of endothelial failure are Fuchs endothelial dystrophy (FED) and pseudophakic bullous keratopathy (PBK) following intraocular surgery . These rThe first step towards successful DMEK surgery is to master the donor tissue preparation technique . Althoug2, then it can be used for transplantation. The tissue is fixed on a vacuum block with the endothelium facing up  to remove traces of storage media . The coracing up .Using a corneal punch blade (9.5 mm), the endothelium is superficially trephined by gentle tapping on the top of the endothelium. Strong tapping or full thickness punches can end with the endothelium margins incarcerated in the corneal stroma increasing the preparation time. The cut margins are visualized using trypan blue stain . The marThe separated periphery is then grasped using the sharp acute forceps at the superior end and is peeled towards the inferior end . The entThis technique involves the use of only 1 trephine . The corDM is peeled from TM  by gentlThe successfully peeled endothelium is then replaced back on the corneal stroma. The diameter of the punch is selected as required for the patient. The punch is placed on the graft and, before punching, the stripped and stained endothelium should be visible through the center of the punch . If the Once the graft is cut, the cornea scleral rim is removed and the stripped area should be visible and possible to grasp. The forceps should not be pressed together too strongly. Higher grasping force could break the grasping point leading to a loss of tissue and need for regrasping potentially leading to higher loss of EC. The suction of the punch should be kept on throughout the procedure. If the surgeon has experience in DMEK stripping, a suction-free peeling could be considered. Having a mobile tissue to strip is more difficult to manage but it gives more freedom in the management of tensions and vectorial forces. The stripping is then completed with a longitudinal movement trying to avoid damages and tears .During the peeling, high tension on the graft should be avoided to minimize the risk of ruptures. Sometimes however, stripping movement of the cornea could occur due to tension forces. In this case, additional toothed forceps could aid in keeping the corneal stroma in position. In the first cases, fast peelings are discouraged because they can create tight grafts . Slow peOnce fully stripped, the tissue is placed on the corneal stroma and drops of preservative medium are placed on top of it. At this point, the patient should be called to theatre to start the surgery.Graft diameter can vary as the size of the defective area changes. Corneal endothelial cell density (ECD) is higher in the periphery compared to the central cornea, especially beyond 9.00 mm , 28. DelAlthough the use of a large DMEK graft is desirable in order to deliver more endothelial cells, the size of the graft must be carefully customized by measuring the white-to-white distance in cases that are not straightforward, such as Asian populations, high hyperopic eyes, and narrow anterior chambers, where smaller grafts are preferred. On the contrary, myopic and buphthalmic eyes can benefit from grafts larger than 9.5 mm . In our We suggest performing DMEK surgery under topical anesthesia (TA) using Minims Proxymetacaine hydrochloride 0.5% w/v eye drop solution  combined with peribulbar anesthesia (PA) with lidocaine 2% and bupivacaine 0.5% in a 3 : 2 ratio. We routinely use a 24-gauge needle and a trans-eyelid approach: the needle is inserted at a right angle to the skin at the lower orbital margin and advances 1.0–2.0 cm along the orbital floor at the temporal third of the lower eyelid with the eye in the neutral position of gaze, approximately 20 min before the surgery .In cases where PA cannot be used, DMEK surgery can be safely performed under TA . Indeed,Sub-Tenon's capsule injection of local anesthesia is another method to achieve adequate local anesthesia for anterior segment surgery. Since any bleeding at the surgical site can track through the wounds and lead to fibrin formation in the anterior chamber (AC), the injection should be performed with caution to avoid large episcleral and conjunctival vessels.Many surgeons perform DMEK with a peripheral iridotomy (PI) either prior to  or durinFrom early on, the DMEK technique has been carried out with small 3.0 mm, superior, 50% scleral depth, limbal, tunneled, self-sealing, sutureless main incision and three auxiliary paracenteses . The tecDescemetorhexis, the scoring and stripping of Descemet's membrane, is usually performed with a reversed Sinskey hook or a scraper or more rarely with a cystotome in a circular fashion. The usual diameter of descemetorhexis ranges from 8.5 to 9.5, depending on the size of the graft. The descemetorhexis can be performed under air or with the help of ophthalmic viscosurgical devices (OVD) which many will find easier when starting , 48. DesDescemetorhexis under OVD has advantages such as a more stable anterior chamber, reduction of flare, and iris fluctuation . If you It is important to completely remove OVD before inserting the graft, as it can interfere with graft adherence . To doubAt this point, a 10/0 nylon suture may be placed on the main incision. Performing this step before the insertion of the graft will facilitate faster suturing at the end of the surgery, avoiding major complications like expulsion of the graft from the AC or loss of air .Staining the graft before its insertion into the AC is an important step for a successful and safe surgery. It is important to obtain a thoroughly stained graft, as it will aid with proper visualization during unfolding. The traditional dye used is 0.06% trypan blue  for 1-2 minutes , dependiGraft unfolding is the most variable step in DMEK surgery. DMEK graft, when peeled and submerged in BSS, will spontaneously roll outwards, exposing the endothelium. This requires unfolding manipulation once the tissue is injected into the AC. At the end of the surgery, DM should be well attached to the stroma. If DM spontaneously rolls inwards, complete unrolling is required to allow it to roll in the correct manner, before it can be attached using a standard AC air or sulfur hexafluoride (SF6) gas as tamponade . PatientThe presence of coexisting ocular pathologies, such as glaucoma tubes, anterior synechiae, iris malformations, and anterior chamber intraocular lenses, increases the risk of intra- and postoperative complications including AC bleeding, bubble dislocation into the vitreous cavity, and graft detachment. When first learning the DMEK procedure, such cases with higher complication risks should be avoided , 54. An The surgeon must take care not to dislocate the DMEK graft in the vitreous chamber . In postIt is better to keep the AC shallow but not completely flat . For morThe correct orientation of the graft must be repeatedly verified during surgery and especially before attaching the graft to the posterior corneal stroma. The direct observation of the Moutsouris sign or the F mark is usually quick and helpful . When thCaution is advised when adding fluid to the AC during the unfolding process, as increased AC pressure may cause the graft to be flushed out of a corneal incision when entering the AC with an instrument. Even just minimal graft dislocation into a corneal wound will make surgery more complex and will result in endothelial cell loss in the affected graft areas. We therefore suggest placing a tight suture on the main incision and only then proceed to increasing AC pressure using BSS via the side ports. When the tissue is completely unscrolled and centered, air can be inserted under it to attach the graft to the recipient stroma , 63.After air is injected in the anterior chamber, the centration of the graft can still be gently corrected using forceps  to pull the graft into the desired position. To increase graft mobility, we suggest performing this procedure with no more than a 50% AC air fill. Despite the fact that this procedure may result in a small loss of endothelial cells, it will improve the centration of the graft. Improved centration again will result in faster corneal clearing and a lesser risk of peripheral graft overlap with recipient endothelium, thus reducing the likelihood of graft detachment .At the end of the surgery, the vertical incision can be used to fill the AC with air, aiming to create a 90% air or gas fill. If available, intraoperative Optical Coherence Tomography (OCT) may facilitate all surgical steps by increasing the visualization of the graft and its orientation . If the Tips and tricks can help surgeons new to DMEK to improve their outcomes and facilitate the uptake of DMEK surgery. A well-prepared DMEK graft and different surgical techniques improve the desired surgical outcome. The tips and tricks described in this article could be beneficial for new and experienced corneal surgeons.


Superior biomechanical performance of tapered interference screws, compared with non-tapered screws, with reference to the anterior cruciate ligament (ACL) reconstruction process, has been reported in the literature. However, the effect of tapered interference screw’s body slope on the initial stability of ACL is poorly understood. Thus, the main goal of this study was to investigate the effect of the interference screw’s body slope on the initial stability of the reconstructed ACL.Based on the best screw-bone tunnel diameter ratios in non-tapered screws, two different tapered interference screws were designed and fabricated. The diameters of both screws were equal to bone tunnel diameter in one-third of their length from screw tip, then they were gradually increased by 1mm, in the lower slope (LSTIS), and 2 mm, in the higher slope (HSTIS) screws. To simulate the ACL reconstruction, sixteen soft tissue grafts were fixed, using HSTIS and LSTIS, in synthetic bone blocks. Through applying sub-failure cyclic incremental tensile load, graft-bone-screw construct’s stiffness and graft laxity in each cycle, also through applying subsequent step of loading graft to the failure, maximum load to failure, and graft’s mode of failure were determined. Accordingly, the performance of the fabricated interference screws was compared with each other.HSTIS provides a greater graft-bone-screw construct stiffness, and a lower graft laxity, compared to LSTIS. Moreover, transverse rupture of graft fibers for LSTIS, and necking of graft in the HSTIS group were the major types of grafts’ failure.HSTIS better replicates the intact ACL’s behavior, compared to LSTIS, by causing less damage in graft’s fibers; reducing graft laxity; and increasing fixation stability. Nonetheless, finding the optimal slope remains as an unknown and can be the subject of future studies. Anterior cruciate ligament (ACL) reconstruction is one of the most common orthopedic surgical procedures , in whicDespite all advancement made regarding the ACL reconstruction, fixation of the graft in tibial bone tunnel, in the immediate postoperative period, due to poor bone mineral density, also because of the direction of applied load, which is mostly in the direction of the bone tunnel, is still a serious challenge –16. Ten in-vitro biomechanical studies investigated the effect of interference screw’s length, diameter, material properties, and different manufacturer designs on critical clinical outcomes (such as displacement and strength of the fixed graft), as well as its mode of failure, through applying cycling loading, and loading grafts to failure [Some previous  failure , 22–28.  failure . Morris  failure . On the  failure . Mann et failure . Conside failure . At the  failure . For thi failure  and the  failure , there iThe main objective of this study was to investigate the effects of gradual increase of the diameter of tapered interference screws from equal diameter with the bone tunnel, known as the best screw-bone tunnel ratio, in one-third of their length from screw tip, where the engagement of screw and bone starts, to a 2 mm larger-, compared to a 1 mm larger than the diameter of the bone tunnel, on the stability of the reconstructed ACL fixation. It was hypothesized that by fixation of the interference screw with the higher body slope, compared to the lower one, intact ACL’s attachment to the bone tunnel can be better replicated, and thus it can provide a more stable graft fixation.Using a CNC TraubTx8 machine, prototypes of the two designed interference screws were made of CK45 steel, with a 30 mm length, a tapered body, and a flat head Fig. . The scr3 to simulate dense cancellous bone of tibial bone tunnel [Sixteen fresh bovine extensor tendons were cleared of adherent muscle fibers and surrounding soft tissues, wrapped, and stored frozen at -20 °C in sealed plastic bags for 3 weeks, in order to be used as soft tissue grafts . On the e tunnel . After pGrafts were inserted in prepared bone tunnels in two groups. Then, HSTIS and LSTIS were placed concentrically between graft strands in the direction of the bone tunnel. Three centimeters of the proximal end of the looped tendon strands were kept free outside of the bone tunnel. The looped strands then were secured in the custom-made rigs in Zwick/Roell (Amsler HCT 25–400), and bone blocks were also fixed with a custom-made fixture Fig. .Fig. 2EIn each cycle of incremental sub-failure loading, the slope of the best-fit line to load-displacement curve of the loading phase was measured, as the bone-screw-graft’s stiffness, independent of the other cycles . The conStatistical analysis was conducted with GraphPad Prism software , version 6. In all groups, nonparametric distribution of the data was found using the Kolmogorow-Smirnow test . ParametIn the HSTIS group, by increasing the peak values of the load in incremental sub-failure cyclic loading to 200 N, 250 N, and 300 N, one, two, and three specimens’ fixation failed, respectively. On the other hand, fixation failure occurred for one, three and five specimens, in cycles with peak values of 150 N, 200 N, and 250 N, respectively, in the LSTIS group. Moreover, due to technical errors, one of the grafts, out of 8 grafts, failed in the HSTIS fixation procedure, so the number of total graft samples was seven and eight, for HSTIS and LSTIS groups, respectively.P < 0.05); and 57.70 ± 8.04 N/mm vs. 42.71 ± 7.51 N/mm (P < 0.01), respectively ; and 2.49 ± 0.99 mm vs. 1.17 ± 0.56 mm (P < 0.05), respectively. Regarding energy loss, in all sub-failure cyclic load levels, more energy was dissipated in LSTIS group, compared to the HSTIS group ; and 151.00 ± 52.46 mJ vs. 93.04 ± 43.25 mJ (P < 0.05), respectively.An increase in the stiffness of the graft-bone-screw construct was observed in successive cyclic loading in each group Fig. a. At theP < 0.1) (Table In loading grafts to failure (step c), even though no significant difference was observed between maximum loads at failure, and the stiffness of graft-bone-screw construct, between HSTIS and LSTIS groups Table , but a n1) Table a, neckin1) Table b, and ne1) Table c. AnotheThere are still some concerns, regarding the ACL reconstruction using interference screws, such as the risk of early graft fixation failure, slippage, and laceration. Considering that an intact ACL experiences a gradual increase in stiffness as it gets closer to the point of insertion into the bone , it was To compare the capability of HSTIS and LSTIS in improving the initial stability of the reconstructed anterior cruciate ligament, stiffness of each graft-bone-screw construct was measured through applying a sub-failure incremental cyclic loading. Results of this work proved superiority of the HSTIS group, in terms of the graft-bone-screw stiffness, compared to the LSTIS group, especially in cyclic loading with the peak values of 100, 150 N Fig. a. Stiffnp < 0.05)  Fig. b. This oBy comparing the graft laxity parameter in the HSTIS group Fig. .b with tAnother cause of graft fixation failure in ACL reconstruction surgery through using interference screws, which can be observed clinically, is the graft laceration. In this study, in order to investigate the effect of the body slope of the screws on the graft damage, graft mode of failure for each sample was recorded. It was found that the graft and screw engagement in region A was the weakest site in all constructs Fig. . HoweverBased on the evidence provided in terms of lower stiffness, greater graft’s fibers damages, higher graft laxity, and displacement in the LSTIS group, compared with the HSTIS group Table  and 5, iin-vitro tests’ results can give us information about the initial stability, but they are unable to evaluate the mechanical behavior of the bone-graft-interference screw construct after graft healing and remodeling processes, which can alter graft tissue’s mechanical properties [The following points should be taken into consideration while one is trying to interpret the results of this work. First, operties . Secondloperties , 38, insoperties . HoweverIn the ACL reconstruction surgery, by using the bio-mimicked tapered interference screw, the risk of early graft fixation failure can be reduced through controlling the contact area of screw and graft, and thus by adjusting pressure distribution at the screw-graft and graft-bone interfaces, by means of screw body slope, and consequently by adjusting the screw-bone tunnel diameters ratio gradient along the interference screw. Based on this study, it can be concluded that in the area near the load exertion site, region A Fig. , engagem


OBJECTIVES/GOALS: Face transplant can offer functional and aesthetic restoration to patients who have exhausted reconstructive options. Ethical issues in face transplant still abound, including that of patient selection. The goal of this study was to assess ethicists’ viewpoints on face transplant. METHODS/STUDY POPULATION: A large-scale online survey of attendees of the International Conference on Clinical Ethics Consultation (N = 401) was performed to assess ethicists’ opinions on issues in face transplant. Questions were asked regarding the risk-benefit ratio of immunosuppression, permissibility of face transplant for more recipient subpopulations (including children and blind patients), donor-recipient age, gender, and ethnicity mismatches, and ethics committee make-up. RESULTS/ANTICIPATED RESULTS: Among 84 respondents, 84% agreed it is permissible to perform a face transplant on an adult with no medical contraindications. The majority of respondents agreed that it is permissible to perform a face transplant on a child or blind recipient. An issue of continued concern was risk of immunosuppression. Respondents had a high threshold of permissibility for ethnic mismatches between donor and recipient, and 43% reported it is permissible to have a gender mismatch. A 10 year age difference between donor and recipient was the most commonly accepted. Questions regarding the ideal composition of a face transplant ethics committee demonstrated consensus on the roles that should be represented. DISCUSSION/SIGNIFICANCE OF IMPACT: This study provides insight into ethicists’ viewpoints on face transplant, which demonstrates a high level of permissibility towards the procedure. This may be due to the early success of face transplants and the shifting ethical issues in the field to practical aspects of the procedure. This research also provides guidance to programs regarding questions of donor and recipient selection, ethics committee composition, and offers insight into strengthening the ethical framework of the field.


The evolvement of the newly started subsection IJMS molecular oncology is discussed. The breadth and depth of the journal articles is alluded to. A bright future for this subsection is anticipated, developing into a top tier cancer journal. International Journal of Molecular Sciences, subsection Molecular Oncology. This is an incredible success considering that the first article was published in 2018. The scope of the subsection is posted as follows on the Journal website:This editorial commemorates the publication of more than 1000 articles in the International Journal of Molecular Sciences (IJMS) aims to rapidly publish contributions on all aspects of the most recent research on human cancers. This section lies at the interface between medicinal chemistry and oncology research. We encourage the submission of high-quality manuscripts that provide novel mechanistic insights and details of the molecular signatures of oncogenic transformation. The scope of this section includes, but is not limited to, the following:Biological processes like gene activity and metabolisms in the development and progression of cancersMechanisms of metastasis (vascular and lymphatic dissemination and seeding)Molecular pathology and immunology in tumor developmentBiomarkers in diagnostic processesNovel therapies like drug therapy, radiotherapy, gene therapy, hormonal therapy, and immunotherapyCancer vaccines”“This section of the It easily can be claimed that these anticipations have successfully been met when assessing the journal’s current track record. Thus far, 564 original articles and 420 reviews have been published, and 133 Special Issues are open or have been completed. The publications cover a broad range of various aspects of molecular oncology. Addressed are different tumor types such as prostate, lung, breast, gastric, colon, skin, leukemia and others. Molecular signaling responsible for conferring cancer phenotypes, examples of which are related to the cell cycle , Wnt 8]8], p53 , Myc 1010, cytokRecent topics of cancer research are well represented. Novel diagnostics and biomarkers ,17, the IJMS Molecular Oncology in the cancer community, making it widely accepted as a top tier journal for the publication of the most recent cancer research of the highest quality.In summary, the journal aims have all been successfully fulfilled during the short time it has been active and the future seems bright. The challenge lying ahead is to spread the excellence of


Sogatella furcifera Horvath, commonly known as the white-backed planthoppers (WBPH), is an important pest in East Asian rice fields. Fungal endosymbiosis is widespread among planthoppers in the infraorder Fulgoromorpha and suborder Auchenorrhyncha. We successfully obtained complete mitogenome of five WBPH fungal endosymbionts, belonging to the Ophiocordycipitaceae family, from next-generation sequencing (NGS) reads obtained from S. furcifera samples. These five mitogenomes range in length from 55,390 bp to 55,406 bp, which is shorter than the mitogenome of the fungal endosymbiont found in Ricania speculum, black planthoppers. Twenty-eight protein-coding genes (PCGs), 12 tRNAs, and 2 rRNAs were found in the mitogenomes. Two single-nucleotide polymorphisms, two insertions, and three deletions were identified among the five mitogenomes, which were fewer in number than those of four species of Ophiocordycipitaceae, Ophiocordyceps sinensis, Hirsutella thompsonii, Hirsutella rhossiliensis, and Tolypocladium inflatum. Noticeably short lengths (up to 18 bp) of simple sequence repeats were identified in the five WBPH fungal endosymbiont mitogenomes. Phylogenetic analysis based on conserved PCGs across 25 Ophiocordycipitaceae mitogenomes revealed that the five mitogenomes were clustered with that of R. speculum, forming an independent clade. In addition to providing the full mitogenome sequences, obtaining complete mitogenomes of WBPH endosymbionts can provide insights into their phylogenetic positions without needing to isolate the mtDNA from the host. This advantage is of value to future studies involving fungal endosymbiont mitogenomes. Sogatella furcifera Horvath commonly known as the white-backed planthopper (WBPH) is a planthopper belonging to the infraorder Fulgoromorpha [Sogatella furcifera has already been registered in the National Species List of Korea [S. furcifera has been sequenced successfully [Cardinium bacterial endosymbiont of S. furcifera was also completed from the same raw reads generated by the whole genome project [Wolbachia, which alters host reproductions by parthenogenesis, feminization, male-killing, and induction of cytoplasmic incompatibility in arthropods [Cardinium endosymbiont [romorpha  and suboromorpha . It has romorpha –6. In paromorpha . Sogatelof Korea  indicatiof Korea . Becauseessfully , 11. The project . Anotherthropods , also casymbiont .Ricania japonica [Nilaparvata lugens, a brown planthopper [Ricania speculum, a black planthopper [Besides these bacterial endosymbionts, fungal endosymbiont has been identified using PCR method in planthopper, japonica . This yejaponica . In addinthopper , 17 whicnthopper . Howevernthopper . This minthopper . This renthopper , 19–37.R. speculum [R. speculum, forming an independent clade. Once additional planthopper fungal endosymbiont mitogenomes become available, their phylogenetic relationships as well as evolutionary histories based on their complete mitogenomes will become clearer.Here, we reported the first complete mitogenomes of fungal WBPH endosymbiont from five WBPH samples isolated in Korea and China. The five mitogenomes display 55,390 to 55,406 bp in length, shorter than that of speculum . The numAll four WBPH samples were captured at two places in Korea . One indDe novo assembly, with confirmation, was accomplished with Velvet v1.2.10 [http://geis.infoboss.co.kr/) like the previous studies of mitogenomes [ v1.2.10  after fi v1.2.10 . After o v1.2.10 , and all v1.2.10 ) against v1.2.10 . The cirogenomes , 43–91.R. speculum fungal endosymbiont (NC_049089) [Geneious Prime® 2020.2.4  was used for mitogenome annotation with referring to the mitogenome of _049089)  by trans_049089)  was also_049089) .Single-nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs) were identified using the “Find variations/SNP” function implemented in Geneious Prime® 2020.2.4  based on multiple sequence alignment of the five mitogenomes of WBPH fungal endosymbionts conducted by MAFFT v7.450 . Each idhttp://ssr.pe.kr/; Park et al., in preparation). Based on the conventional definition of an SSR on an organelle genome, monoSSR (1 bp) to hexaSSR (6 bp), the total length of SSRs on the mitogenome exceeds 10 bp. Owing to the different criteria of SSRs on organelle genomes [Simple sequence repeats (SSRs) were identified on the mitogenome sequence using the pipeline of the SSR database  [Five conserved PCGs, including T v7.450  and concve.info) . The modve.info) . The neive.info) . In the ve.info)  plug-in R. speculum  [R. speculum, there were slightly fewer PCGs and tRNAs found in the WBPH endosymbiont mitogenomes. There were three fewer PCGs for three reasons: the smaller number of LAGLIDADG endonucleases, the absence of one endonuclease and a GIY-YIG endonuclease, and the presence of two additional PCGs—a hypothetical protein and a LAGLIDADG/HNH endonuclease. This particular configuration of PCGs is usually identified in other fungal mitogenomes; for example, two mitogenomes of Fusarium oxysporum (GenBank accessions are MN259514 and MN259515) display two completely different PCGs in each mitogenome [R. speculum [We successfully assembled fungal endosymbiont mitogenomes from four WBPH samples isolated in Korea and China and one public dataset of NGS raw reads . This is,785 bp) . In thes,785 bp) , 108–112togenome , 56. Thespeculum ). This dspeculum .COX1 contains three introns, and COB has five introns in the Hirsutella thompsonii mitogenome [Aspergillus pseudoglaucus and Aspergillus egyptiacus are longer than the other Aspergillus mitogenomes because of the presence of many introns on major PCGs [COB, COX1, NAD1, ATP8, COX3, COX2, and NAD2  and the KR.11D isolate were identical to that of KR, while the other three WBPHs captured in other locations in Korea displayed intraspecific variations. The sample used in the WGS originated from the University of Science and Technology of China , indicating that KR 11D and KR WBPH samples obtained in Korea have migrated from the similar region to the WGS sample. However, further analyses of their complete mitogenomes or whole genomes will be needed to provide more supportive data for identifying their origins.Aspergillus flavus [Fusarium oxysporum f.sp. lactucae [There is a relatively small number of intraspecific SNPs and INDELs identified from these fungal mitogenomes in comparison to those of other fungal mitogenomes, for example, 16 to 17 SNPs (0.055% to 0.0582%) and 22 to 27 INDELs (0.075% to 0.092%) on s flavus , 53 and lactucae . They arlactucae , 45–51.Ophiocordyceps sinensis, Hirsutella thompsonii, Hirsutella rhossiliensis, and Tolypocladium inflatum, contain more than one complete fungal mitogenome (Buchnera aphidicola (Aphis gossypii) displays a low level of intraspecific variation in comparison to those of host mitogenome , and second, the whole genome of endosymbiont of Pediculus humanus capitis also shows low-level intraspecific variations in comparison to those of their whole genomes [Based on 25 available complete fungal mitogenomes in Ophiocordycipitaceae, four species, togenome . We invetogenome . There a genomes .Simple sequence repeats (SSRs) identified from organellar genomes have been utilized as molecular markers in various species such as plant species , 118–122Ophiocordyceps sinensis (up to 24 bp) [Pestalotiopsis fici (up to 45 bp) [R. speculum (NC_049089) [The length of the identified SSRs is relatively short  , as wello 45 bp) . Moreove_049089)  was 18 bOut of 191 normal SSRs, extended SSRs, and potential SSRs, 84 (43.98%) are located in the genic region , NAD1, NAD6, ATP synthase F0 subunit a, and LAGLIDADG/HNH endonuclease, displayed a relatively large number of SSRs  [Ophiocordyceps sinensis fungal mitogenome (KP835313), five PCGs, NAD5, COB, COX1, NAD1, and NAD4, containing introns are not correctly annotated. Only five conserved PCGs, ATP8, COX2, NAD2, NAD3, and NAD4L, were selected and aligned individually. Subsequently, this alignment was concatenated to construct three phylogenetic trees.We constructed bootstrapped maximum-likelihood (ML) and Bayesian inference (BI) phylogenetic trees using 26 fungal mitogenomes consisting of 5 mitogenomes used in this study, 25 mitogenomes in the Ophiocordycipitaceae family, and 1 outgroup species (inearum) . Due to R. speculum (NC_049089) [R. speculum endosymbiont and the five WBPH endosymbionts, suggesting that other fungal endosymbionts may also be independently clustered with other fungal species in the sample family, Ophiocordycipitaceae. In terms of evolution, it can be explained by the two hypotheses: (i) independent evolution once this endosymbiont entered the host insect species or (ii) independent taxonomic groups of Ophiocordycipitaceae entering into the host insect species multiple times during evolution. To determine which hypothesis is more likely, we would need more endosymbiont mitogenomes from various host insect species of infraorder Fulgoromorpha and suborder Auchenorrhyncha as well as mitogenomes from neighboring noninsect endosymbiont fungal species.Five fungal endosymbiont mitogenomes of WBPH were well clustered with another fungal symbiont mitogenome of _049089)  with higHirsutella thompsonii, Hirsutella rhossiliensis, Ophiocordyceps sinensis, and Tolypocladium inflatum, also display rigid clades covering all mitogenomes of each species with high supportive values  compared to those of other fungal mitogenomes. Nearly half of the SSRs are in the genic region, suggesting that these SSRs may be more conserved and they may affect the functionality of PCGs. Based on the phylogenetic trees of 5 conserved PCGs of 26 fungal mitogenomes, including one outgroup species, WBPH fungal endosymbiont mitogenomes were clustered with that of R. speculum with high supportive values. This suggests that these insect-hosted fungal endosymbionts have been evolved independently from the other fungal species in the Ophiocordycipitaceae family. Owing to the advantages of NGS raw reads, which can detect sequences from unknown or unexpected organisms [We successfully elucidated the five complete mitogenomes of the fungal endosymbiont of WBPH from various sources of NGS raw reads obtained from WBPH samples. These five complete mitogenomes show common and their own characteristics in comparison to the previously elucidated complete mitogenome of the symbiont . There wrganisms , 19–37,


BackgroundA wide array of diseases can lead to skin defects of the male genitalia. Although reconstructive options have been debated in the literature, no study has compared the effectiveness of a meshed split-thickness skin graft (STSG) and a sheet STSG in perineal and scrotal wound coverage. In this study, we report our experience in a tertiary trauma center.MethodologyIn this retrospective study, we included cases with a skin defect of the male genitalia, for which genital reconstruction with a skin graft was performed at our hospital from December 2017 to February 2020. This study was approved by the institutional review board. The analysis was performed at 95% confidence interval using the Statistical Package for Social Science (SPSS) version 23.0 .ResultsA total of 27 patients were included in the study. The most common indication for genital reconstruction was Fournier’s gangrene (59.3%). In 15 (55.6%) patients, a meshed skin graft was utilized to cover the defect, whereas a sheet graft was utilized in 12 (44.4%) patients. Out of the 15 patients who underwent genital reconstruction with a meshed graft, 10 (66.6%) had complete graft take. On the other hand, out of the 12 (44.4%) patients who underwent genital reconstruction using a sheet graft, five (41.6%) had complete graft take. A statistically significant relationship was found between aesthetic and functional outcomes and the type of skin graft used. The satisfaction rate was higher among meshed skin graft recipients (86.2%) compared to sheet skin graft recipients (41.7%) (p = 0.014).ConclusionsBased on our observational experience, we found that meshed STSG to cover male genital skin defects is safe with satisfactory cosmetic outcomes. Further prospective randomized studies are needed. Numerous diseases can affect the male genitalia leading to significant dysfunction, profound skin defect, and aesthetic disfigurement . These dPatient selection and study designIn this single-center retrospective study, we included all patients who underwent male genital reconstruction with a skin graft at our hospital between December 2017 and February 2020. Patients who had perineal or scrotal skin loss regardless of the etiology were included in the study. Patients’ age, comorbidities, etiology of perineal and scrotal skin loss, type of skin graft applied, site of skin loss, graft uptake, length of hospital stay, aesthetic and functional outcomes, and postoperative complications were obtained from the medical records.Ethical considerationAll patients signed an informed consent to allow the use of their preoperative and postoperative images for publication. This study was approved by the institutional review board of King Saud Medical City, Riyadh, Saudi Arabia (Ref. No. 10/6924/IRB).Description of surgical techniqueAll patients were operated on under endotracheal intubation and general anesthesia. A warming mattress was used in all patients to avoid hypothermia. Preoperatively, patients were placed in the lithotomy position. The lower abdomen, genital region, and bilateral thighs were shaved. Intravenous broad-spectrum antibiotics were administered. Debridement constituted excising devitalized tissue as well as skin edges. The viability of the testicles, spermatic cord, and the extent of penile damage was confirmed by exploration. STSG was harvested from one or bilateral thighs depending on the size of the defect. The graft was harvested using mineral oil on the thigh’s anterior aspect, and a 0.015-inch thickness Zimmer dermatome  was used. Subsequently, Sofra-tulle  dressing was applied over the graft donor site. After two STSGs were harvested from the thigh, one was kept as unmeshed while the other was meshed (1:1). The unmeshed sheet graft was sutured circumferentially around the penile shaft. Vicryl 3/0 was used to suture the graft along the base of the penile shaft subcoronal tissue. On the other hand, the meshed graft was sutured with Vicryl 3/0 around the scrotum to cover the defect. A urinary catheter was placed before applying the dressing. The Sofra-tulle dressing was applied on the recipient sites, and the penile shaft was immobilized under erectile conditions using a sponge for optimal take of the graft and to ensure less mobility. For all cases, the penile shaft was stretched at the graft application time and dressing, as wrinkles within the graft can cause poor graft take. Patients were then commenced on intravenous augmentin, ciprofloxacin, and metronidazole considering the potential for developing a polymicrobial infection. Broad-spectrum intravenous antibiotics were administered for the first 24 hours and then switched to oral augmentin for five days. Graft immobility for at least five days is the most potent factor contributing to the extent of graft take. Patients were usually discharged home after initial operative management on days six to eight.Statistical analysisData were checked for errors before analysis. Data analysis was performed at 95% confidence interval using the Statistical Package for Social Science (SPSS) version 23.0 . Categorical variables were presented as frequencies and percentages. Continuous variables were presented as mean and standard deviation. The relationship between the type of skin graft and age and length of hospital stay were assessed by the Mann-Whitney U-test. Relationship with the type of skin graft and comorbidities, etiology, site of skin loss, outcomes, aesthetic and functional outcomes, and complications were assessed by the chi-square test. A p-value of 0.05 was used to determine statistical significance.Demographics and etiologyA total of 27 males were retrospectively included in the study who suffered scrotal and/or perineal skin loss. The mean age was 35.26 ± 13.35 years. Of the 27 patients, 18 (66.6%) had comorbidities, and the most common comorbidity was diabetes mellitus in nine (33.3%) patients. In total, three (11.1%) patients were obese. Others had arterial hypertension, autoimmune disease, and hyperlipidemia. The most common cause of scrotal and perineal skin loss was Fournier’s gangrene observed in 16 (59.3%) patients. The most common site of skin loss was the “penis, scrotum, and perineum” (40.7%). Table Meshed split-thickness skin graftOut of 15 patients in whom meshed graft was used, 10 (66.6%) had complete graft take. Overall, three (20%) patients had graft loss mounting to 10%, one had graft loss of more than 30% and one had complete graft loss. These cases were managed conservatively and healed by secondary intention. None of the patients in this group required a second surgery. In total, nine (60%) patients were discharged after the first postoperative dressing on days three to four. The remaining six patients were discharged after the second postoperative dressing on days five to six.Sheet skin graftOut of 12 (44.4%) patients in whom sheet graft was used, only 5 (41.6%) had complete graft take. Two patients had graft loss of more than 25%, while five had more than 50% graft loss. In these five patients in whom 50% of the graft was lost, a second surgery was done and a sheet graft was applied; the remaining two patients in whom 25% graft was lost were managed conservatively. Five patients in this group were discharged on day five after the second dressing, two patients on day seven, and five patients in whom the second surgery was done were discharged on day ten after the first surgery. The average length of stay in the meshed group was 4.4 days (range: 3-7 days), while in the sheet group, it was 5.2 days (range: 3-9 days). Both groups were followed up in the outpatient department and showed similar aesthetic and functional outcomes.No statistically significant relationship was found between the type of skin graft and the age of the patients (p = 0.903). In addition, no statistically significant relationship was found between the type of skin graft and the length of hospital stay p = 0.354) . The only statistically significant relationship was between aesthetic and functional outcomes and the type of skin graft used. The satisfaction rate was higher among meshed skin graft recipients (86.2%) compared to sheet skin graft recipients (41.7%)  (Table Perineal wound defects are challenging to manage. The main goal of reconstruction is wound coverage, preservation of voiding, sexual function, and a good cosmetic outcome ,8. The wAlthough FTSGs are thought to provide a superior aesthetic result, there are some disadvantages such as limited donor sites that provide high-quality skin and donor site comorbidity. STSGs have several advantages over FTSGs including good graft take in cases of wound contamination such as trauma, avulsion injuries, burns, and hidradenitis ,10. SkinLimitationsThis study was limited by the small number of patients. This might be attributed to the low incidence of perineal and scrotal skin losses. The assessment of functional outcome was subjective, and the International Index of Erectile Function Questionnaire was not used. To date, there is no standard method of assessment for graft take; thus, our graft take assessment was done based on the surgeon’s clinical judgment and expertise. There is an inherent need for further clinical studies to include a larger population size, for improved validity, as well as to utilize validated tools in the assessment of function, clinical, and cosmetic outcomes. In addition, prospective cohort studies are needed to confirm the results of this study.In the context of male genital skin defects, a single-step reconstructive procedure using a meshed skin graft to cover the skin defect has proven to be the superior option compared to a sheet graft for achieving good aesthetic and functional outcomes. Based on our experience, a meshed skin graft to cover male genital skin defects is safe and demonstrates satisfactory cosmetic outcomes. Further, we propose making male genital reconstruction using a meshed graft the standard of care offered as initial surgical management. However, further prospective studies are needed to confirm these findings.


They are the causative agents of an infectious disease called dengue, an important global public health problem with significant social-economic impact. Thus, the development of safe and effective dengue vaccines is a priority according to the World Health Organization. Only one anti-dengue vaccine has already been licensed in endemic countries and two formulations are under phase III clinical trials. In this study, we aimed to compare the main anti-dengue virus vaccines, DENGVAXIA®, LAV-TDV, and TAK-003, regarding their antigens and potential to protect. We studied the conservation of both, B and T cell epitopes involved in immunological control of DENV infection along with vaccine viruses and viral isolates. In addition, we assessed the population coverage of epitope sets contained in each vaccine formulation with regard to different human populations. As main results, we found that all three vaccines contain the main B cell epitopes involved in viral neutralization. Similarly, LAV-TDV and TAK-003 contain most of T cell epitopes involved in immunological protection, a finding not observed in DENGVAXIA®, which explains main limitations of the only licensed dengue vaccine. In summary, the levels of presence and absence of epitopes that are target for protective immune response in the three main anti-dengue virus vaccines are shown in this study. Our results suggest that investing in vaccines that contain the majority of epitopes involved in protective immunity  is an important issue to be considered.The four serotypes of Dengue virus (DENV1-4) are arboviruses ( There are four distinct serotypes: DENV-1, DENV-2, DENV-3, and DENV-4 (Dengue virus (DENV) is an arbovirus , pre-membrane protein (pre-M), and the envelope glycoprotein (E). In addition, the genomic RNA also encodes seven non-structural (NS) proteins: NS1; NS2A; NS2B; NS3; NS4A, NS4B, and NS5, which participate both in the replication of the viral genome and in the assembly of the new viral particles , 7.Controlling dengue by suppressing mosquito vectors is only effective if applied before the circulation of viruses in a certain region. Due to epidemiological complexity of the disease, once it is established, agencies responsible for disease prevention face significant technical, scientific and operational difficulties in controlling dengue . Thus, aBoth, antibodies and T lymphocytes play important roles in fighting DENV . Antibod® from Sanofi Pasteur; LAV-TDV from NIAID/Butantan, which is under phase III of the clinical trial; and TAK-003 from Takeda, which concluded phase III of the clinical trial. Dengvaxia® is a live, attenuated and tetravalent recombinant vaccine. Its antigens consist of chimeric viruses in which genomic sequences encoding the pre-membrane (prM) protein and the envelope glycoprotein (E) of the 17D strain of Yellow fever virus (YFV) were replaced by those of each of the four serotypes of DENV. LAV-TDV is a live, attenuated and tetravalent vaccine solely based on DENV. The viruses were attenuated by deletions in their genomes and one of them consists of a chimera of a DENV2 and a DENV4. And TAK-003 is also a live attenuated tetravalent vaccine that is based on DENV2 (16681) PDK 53, which was used as a backbone in the constructs of the chimeric vaccine viruses of DENV1, DENV3 and DENV4 . The search filters used at ClinicalTrials.gov were as follows: Condition or disease: “dengue”; type of study: “interventional clinical trials”; study results: “with result”; Eligibility criteria: “child (birth–17)”, “adult (18–64)”, “elderly (65+)”; Gender: “all”; Study phase: I, II, III and IV. Pubmed searches were performed using keywords: dengue, vaccine, clinical trial. We selected three vaccine formulations that were either licensed or were under phase III of the clinical trial or had finished phase III of the clinical trial: DENGVAXIA® from Sanofi Pasteur; LAV-TDV from NIAID/Butantan Institute and TAK-003, from Takeda.We performed a search for anti-dengue vaccine formulations both, in clinical trial records  and papers obtained from PUBMED (https://www.ncbi.nlm.nih.gov/nucleotide/). The assembly was performed using the software ApE-A plasmid Editor, and the translation from DNA to protein sequence was performed using the Translate tool (https://web.expasy.org/translate/). Then, the sequences were used to compose a dataset in FASTA format  platform (Flavivirus previously described (The DENV 1-4 polyprotein sequences dataset was built by applying the same filters for escribed . Sequenchttps://www.iedb.org/) for B cell epitopes validated in neutralization assays using the following filters: “any epitope”, “dengue1-4”, “B-cell positive essays”, “any MHC restriction”, “human host”, “infectious diseases” and “only positive B-cell assays with neutralization essays”. We retrieved 50 B cell discontinuous epitopes from IEDB, namely, 20 from DENV1, 13 from DENV2, 13 from DENV3, and 4 from DENV4  (https://www.ncbi.nlm.nih.gov/pubmed/) using the following search criteria: i) studies evaluating the survival or viral load control in challenge experiments, either using human or humanized animal models ; ii) infection of human host by DENV without presentation of pathological signs; iii) identification of important epitopes in the context of an immune response induced by anti-dengue vaccines known to be protective. We constrained our search to studies that contained epitope binding to HLA (human leukocyte antigen) and analysis of the proinflammatory cytokine production profile by CD4+ T cells or cytotoxicity mediated by CD8+ T cells. We also selected only studies in which at least two different methods were used to validate the secretion of pro-inflammatory cytokines or cytotoxic activity. The keywords used were: CD4+ T lymphocyte, CD8+ T lymphocyte, epitopes, protection, immunity, Dengue virus.Additionally, we carried out a bibliography search for the studies about the protective anti-dengue response induced by T lymphocyte epitopes that were experimentally characterized. The data was retrieved from Pubmed database  was performed as previously described , as previously described  was used to perform the epitope mapping. The 3D model protein was retrieved from the protein database (https://www.rcsb.org/).Epitopes conserved in vaccines were mapped in a 3D envelope glycoprotein model, as previously described . The PyMStatistical analysis was performed to compare the results of the epitope conservation scores that were present in at least one of the three vaccines. Scores were defined based on the levels of conservation of epitopes present in vaccines in circulating viruses. This method is shown in details in ® vaccine, the sequences used for genetic recombination were extracted from GenBank, according to the accession numbers in Chimeric yellow fever virus-DENV (CYD) were inserted, and the four constructs were assembled  according to the accession numbers in TAK-003 is a tetravalent vaccine. Its antigens are based on the genetic backbone of the DENV-2 PDK-53-V variant, as previously described . In orde® . Both epitopes are located in the envelope glycoprotein in the LAV-TDV vaccine. It is important to highlight that all of these conserved epitopes are discontinuous. In addition, most of them are located in the envelope glycoprotein and only one of them is located in prM. Moreover, our analyses showed that circulating DENV2 contain the highest number B cell epitopes targeted by neutralizing antibodies . A score-based scale associated with conservation percentages of epitopes obtained from circulating viruses  in children and adolescents from 2 to 16 years old , 27968 FINEP/RTR/PRPq/REDE COVID-19 (9-UFOB). AB is supported by a grant from Instituto Serrapilheira/Serra-1708-15285. JA is supported by grants from Consórcio Multifinalitário do Oeste da Bahia and 27968 FINEP/RTR/PRPq/ REDE COVID-19. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


H, and gelatinization properties has a stronger correlation. This study provides practical information for improving the nutritional benefits of waxy and non-waxy proso millet in food applications.Resistant starch (RS) is widely used in the food industry because of its ability to regulate and protect the small intestine, but their distinct effects on the structural and functional properties of waxy and non-waxy proso millet starches are not completely understood. The crystalline structure and physicochemical properties of waxy and non-waxy proso millets’ starch samples were analyzed after heat-moisture treatment (HMT). The analysis revealed significant differences between the RS of waxy and non-waxy proso millets. The crystal type of proso millets’ starch changed from type A to type B + V. The relative crystallinity of the RS of waxy proso millet was better than that of non-waxy proso millet. The gelatinization temperature and thermal stability of RS significantly increased, and the pasting temperature (PTM) of the RS of waxy proso millet was the highest. The water solubility and swelling power of the RS in proso millet decreased, and the viscoelasticity improved. The correlation between the short-range ordered structure of RS and Δ Panicum miliaceum L.), commonly known as yellow rice, is one of the oldest crops in the world. It was domesticated in China for the first time about 10,000 years ago [Proso millet , a renewable and abundant polysaccharide, and is widely used in food production as a stabilizer, water-retaining agent. and thickener . HoweverRecently, the physicochemical properties and other aspects of starch of waxy proso millet were reported in detail by Chao et al. . BesidesWaxy proso millet (Shanmi1), non-waxy proso millet (Chimi2), and pea (Xiwan1) were harvested in 2020 at the Northwest A&F University’s experimental site , Yu’lin Institute of Agricultural Sciences, Shaanxi Province, China. In the experiment, management was conducted according to local production practices and crop requirements. The potato was bought from the supermarket. Pea and potato served as controls in this study.w/v) NaOH suspension and incubated at room temperature for 12 h. The suspension was first filtered through a 100-mesh sieve and then through a 200-mesh sieve and centrifuged thrice at 4000× g for 10 min. The top yellowish layer was discarded, and the precipitate was dissolved in distilled water. Then, the suspension was neutralized with HCl (1 mol/L). After neutralization, precipitates were washed thrice with distilled water. Finally, the starch was dried at 40 °C for 24 h, and the dried starch was ground into powder and passed through a 100-mesh sieve. The starch was put into self-sealing plastic bags and numbered for later use. The starches from pea and potato were separated according to the method of Joshi [Starch was extracted from proso millet following the alkaline soaking method . The seeof Joshi  and Wu eof Joshi .w/v) emulsion of NS was prepared from four samples. The sample of 10% (w/v) emulsion was heated in an high-pressure sterilization at 121 °C for 30 min and placed at 4 °C overnight; this step was repeated twice. Then, the obtained sample was dried at 60 °C and sieved through an 80-mesh sieve to obtain the RS.The four RS  was prepared from proso millet, pea, and potato following the method by Gao et al. . A 10%  at an acceleration potential of 5 kV . Dried sColor characteristics of NS and RS were measured using a colorimeter . L* value represents the lightness , a* , and b* . The parameters of L*, a*, and b* were measured following the method by Wang et al. . The totThe amylose content of the NS and RS was determined by the iodine-binding method by Kim et al. . About 1The crystalline structure of starch was analyzed using XRD  according to the method described by Liu et al. . The dif−1 and 1022/995 cm−1 were calculated from the obtained original spectra.The short-range ordered structure of NS and RS samples was analyzed by ATR-FTIR  spectroscopy . The values of 1045/1022 cmWater solubility and swelling power of starch were determined through the method of Wang et al. . The 0.0To), peak (Tp), and completion (Tc) temperatures and the gelatinization enthalpy (ΔH) of NS and RS was calculated using the universal Analysis 2000.The differential scanning calorimeter  was used to determine the starch thermal properties. Approximately 3 g of NS and RS was mixed with 9 µL of distilled water in an aluminum DSC pan, and the mixture was allowed to sit for 12 h at 4 °C . An emptApproximately 3 g of the sample from NS and RS with 14% water content were weighed and mixed evenly with 25 mL of distilled water. A rapid viscosity analyzer  was used to determine the gelatinization and pasting characteristics of the NS and RS. The peak viscosity (PV), breakdown viscosity (BD), final viscosity (FV), setback viscosity (SB), and gelatinization temperature (PTM) and peak time (PT) of starch were analyzed using the program parameters set according by Li et al. .The DHR-1 rheometer  was used to determine the dynamic rheological properties of NS and RS. Here, the parallel metal plate of rheometer diameter was set at 40 mm and the gap at 1000 μm. About 0.4 g of starch was mixed with 5 mL of distilled water in a 10 mL centrifuge tube and gelatinized in a boiling water bath for 10 min. The mixture was loaded onto the rheometer plate, and the edges of the gap were covered with a layer of dimethylpolysiloxane (50 cP viscosity) to reduce evaporation. The experimental conditions were modified as described by Kong et al. .p < 0.05.All measurements were performed in triplicate and data are represented as the mean ± standard deviation. All data were analyzed using variance analysis . Differences between samples were considered statistically significant at Granule size and the morphology of NS and RS of proso millet, pea, and potato were observed by SEM . No signThe particle size of starch affects the physicochemical properties, such as swelling power and rheological properties, as well as the functional properties of food . The parp < 0.05). The L* values of starches from the four NS samples ranged from 92.75 to 95.42 and from the four RS samples ranged from 81.86 to 89.11. The RS was light yellow compared with NS; the b* value of RS increased significantly, while the ΔE* reduced considerably upon treatment, probably because HMT induced chemical and physical changes in the thermally unstable substances of food, destroying the food color [Results of color analysis of the eight samples are shown in od color .The level of amylose content will have a certain impact on cooking quality and eating quality. The amylose content of NS and RS of proso millet, pea, and potato is shown in In general, the amylose content of RS was significantly higher than that of NS, indicating that RS has high temperature resistance and strong stretchability. Waxy proso millet (Shanmi1) RS had the lowest amylose content; the RS of non-waxy proso millet (Chimi2) was significantly higher than potato but lower than the pea. The amylose content of starch has an impact on food digestion. According to Riley’s research, starch with low amylose content gets easily digested . Our finXRD analyzed the structure characteristics of starch granules. Three crystalline types, A, B, and C, were identified in the plant starch-based on XRD peaks . The XRDThe relative crystallinity of NS of waxy proso millet (Shanmi1), non-waxy proso millet (Chimi2), pea, and potato was 30.42, 28.64, 28.34, and 29.96%, respectively. The differences in relative crystallinity between different varieties of NS may be related to crop variety and amylose and amylopectin content . These f−1  were the highest, with higher crystallinity. However, the ΔH values and the crystallinity of pea starch were low, and the amylose content increased compared with proso millet and potato starch. These observations indicate that the thermal properties of starch are mainly related to crop species, crystalline type, and amylose content [The ectively . The Tp To, Tp, and Tc of RS were significantly higher, while the ΔH was lower than NS in proso millet, pea, and potato  and loss moduli (G”) were used to determine the elasticity and viscosity of starch. The G’ and G” curves of the starches are shown in G’ of NS was greater than G”, and both G’ and G” increased with the increase in frequency, indicating better elastic properties than the viscous properties and the dependency of G’ and G” on frequency. When the frequency was greater than 16 Hz, the G’ of RS of potato was the largest, and that of pea was the smallest, indicating greater potato elasticity and softer pea gel. The G” values of NS of potato and pea were higher than that of proso millet, and indicated that their viscosity was higher, being consistent with pasting properties results. The G’ of RS of non-waxy proso millet (Chimi2) and pea was higher, indicating improved starch stability. The G’ of RS in waxy proso millet (Shanmi1) was the lowest, indicating low elasticity due to low amylose content [G’ is directly related to the cross-linking density in the gel network. More rigid gels have limited application in food processing and may cause uneven mixing of processed materials as they require greater flow force [G’ and G” values of RS in non-waxy proso millet (Chimi2) and pea increased compared with NS, probably because the crystalline structure of starch particles got destroyed by high pressure and high temperature. Moreover, the crystalline structure of RS was stable after HMT, and the flexibility of the starch gel improved [Storage moduli ( content . Besidesow force . The G’ improved . The RS −1, To, Tp, Tc, and ΔH (−1 in RS and negatively correlated with relative crystallinity, swelling power, and ΔE*. The thermal performance of NS was negatively related to the gelatinization properties, while that of RS was closely related. However, the correlation between the short-range ordered structure of RS and ΔH, and gelatinization properties has a stronger correlation compared with NS. This indicates that the short-range ordered structure of RS promotes the gelatinization of starch, so that the functional properties of RS are not easily damaged in food processing.Furthermore, a heat map was constructed to analyze the relationship between the amylose content of NS and RS and relative crystallinity, swelling power, water-solubility, thermal properties, and gelatinization properties. Amylose content positively correlated with FV and SB in NS and negatively correlated with relative crystallinity, swelling power, 1022/995 cm, and ΔH . MeanwhiG’ and G” values of RS in non-waxy proso millet increased. Therefore, RS has strong gelatinization ability and good thermal stability. These findings suggest that HMT rearranges the proso millet starch particles to form a more ordered and stable crystal structure than pea and potato. Therefore, in today’s pursuit of food diversification, the resistant starch of broomcorn millet can be used as a new type of food processing material, and has great potential for development in the food industry due to its strong structural stability. The study’s findings provide a theoretical basis for applying proso millet starch as an additive and in the production of new foods.This study analyzed the crystal structure and physicochemical properties of the RS of waxy and non-waxy proso millet. Differences in the effects of HMT on different properties between NS and RS were analyzed. HMT resulted in the rearrangement of starch particles and a larger size of grains. The A-type crystalline morphology and short-range ordered structure of proso millet starch changed after HMT. Further, the A-type structure of waxy and non-waxy proso millet starch transformed into B + V type, which was more stable than peas and potatoes. HMT increased the amylose content, relative crystallinity, gelatinization temperature, and gelatinization time of starch from the different plant sources. Meanwhile, water-solubility, swelling power, and gelatinization enthalpy reduced. The