Recurrent contracted sockets are complex situations where previous surgerieshave failed, disabling the wear of an ocular prosthesis. A combined methodof surgery and long-term fixation using custom-made, three-dimensional (3D)printed conformers is evaluated.n = 3), fireworks (n = 3), trauma(n = 2) and enucleation and radiotherapy at childhooddue to optic nerve glioma (n = 1) with three averageprevious socket surgeries (range 2–6). Treatment consisted of a buccalmucosal graft and personalized 3D-printed conformer designed to be fixatedto the periosteum and tarsal plates for minimal 2 months. Primary outcomewas the retention of an ocular prosthesis. Secondary outcome was the needfor additional surgeries.Retrospective case series of nine patients with recurrent excessive socketcontraction and inability to wear a prosthesis, caused by chemical burns. Eight cases were able to wear an ocularprosthesis after 2 months. Three cases initially treated for only the upperor only the lower fornix needed subsequent surgery for the opposite fornixfor functional reasons. Two cases had later surgery for cosmetic improvementof upper eyelid position. Despite pre-existing lid abnormalities , cosmetic outcome was judged highly acceptablein six cases because of symmetric contour and volume, and reasonablyacceptable in the remaining two.Buccal mucosal transplant fixated with a personalized 3D-designed conformerenables retention of a well-fitted ocular prosthesis in previously failedsocket surgeries. Initial treatment of both upper and lower fornices isrecommended to avoid subsequent surgeries for functional reasons. A dermis fat graft (DFG) may also be used to increase the lining of thesocket. In a subset of patients these standard procedures unfortunately fail, andsevere recurrent contraction of the socket results in the inability to wear anyocular prosthesis. This situation is referred to as stage 5 contracted socket asdefined by Krishna, where “there is recurrence of contraction of the socket after repeated trialof reconstruction”.The goal after ocular evisceration or enucleation is to fit an ocular prosthesis thatresembles a natural eye as closely as possible. In order to fit an ocularprosthesis, deep eyelid fornices with sufficient conjunctival lining are mandatory.Shortness of conjunctival lining with shallow or absent fornices can occur invarious conditions, including previous external beam radiation, multiple previousextrusions of an orbital implant, immunologic diseases such as mucous membranepemphigoid or Stevens Johnson syndrome, or after chemical or thermal trauma.Standard procedures for fornix reconstruction include fornix-deepening sutures withor without the use of buccal mucosal transplant.4Mucosal tissue is prone to contraction, but this may be reduced by mechanical restraint. Accordingly, we hypothesized that two main problems have to be overcome: tolet the graft attach to a vital surface, and to prevent the graft from contracting.Therefore, the mucosal transplant has to be both attached to a fresh wound bed andfixated in the correct position for a long time.One of the possible underlying problems for inadequate wound healing and contractionis ischemia with insufficient vascularization. In the wound healing process,myofibroblasts play a role as modulator in granulation formation and tissuecontraction. Standard, non-custom-made conformers are available and might be a reasonablealternative. 3D design offers the advantage to modify the conformer, by addingfixation holes for fornix-deepening sutures and a central wing-shaped horizontalextension (a “lip”) for fixation to the superior and inferior tarsal plates. Thiswill enable firm fixation and avoids luxation. Another advantage is that theanterior part of the conformer  can be mirrored from the healthy eye, enabling a well-formed contour thatwill be translated to the socket during the 2 months fixation. After removal, thisconformer contour can directly be translated to the definite prosthesis. In thisstudy we describe our experience with nine patients were this method of personalized3D-printed conformer with long-term mucosal transplant fixation was applied.As previously described by this group, a customized three-dimensionally (3D) printedconformer can be designed to have the optimal dimensions to fit in the operatedsocket and to serve as a pressure device for the transplanted mucosa.This retrospective case series includes nine patients who were unable to hold anocular prosthesis due to a recurrent contracted socket after previously failedsurgeries, and who were thus graded as being Krishna stage 5. Patients came toour tertiary referral center at the Amsterdam University Medical Center betweenJanuary 2016 and October 2019. Patient demographics are shown in  In short, at consultation a facial 3D photograph was made, using eitherthe handheld 3D scanner Artec Eva  or a static 3D scanningsetup . The captured data waspost-processed using the manufacturers corresponding software , exported as a file with .obj extensionand loaded into 3D modeling software . The ocularist designed the conformer as an overlay in the 3Dsoftware as if it was an adequately sized prosthesis: the anterior curvaturedefined from the eyelid contour of the fellow eye was used to define theanterior part of the conformer, the posterior part followed this curve  without interfering with the Stensen’s duct. The mouth mucosa wasinfiltrated with a solution of xylometozaline 1% or 2% with adrenalin(1:200.000). The mucosa was dissected from the buccinator muscle. If needed,local coagulation was performed. The wound was either left open for spontaneousgranulation or closed using interrupted 5-0 absorbable braided polyglactinsutures, submucosal tissue was trimmed from the graft and perforating holes weremade to both extend the size of the graft as well as to enable drainage of wounddebris. In the cases where there was hardly any mucosal tissue left in thesocket, this means the mouth mucosa should widely cover the conformer up to theextension rim. In case there was insufficient mucosa to create deep fornices orcover the conformer, a second graft was taken from the lip or other cheek. Thegraft was soaked in gentamycin solution. Gloves were changed and cleaninstruments were used for the socket part: the socket was centrally opened witha horizontal cut from the caruncle to the lateral canthus, and the availableconjunctiva was undermined superiorly and inferiorly. Scarred tissues werereleased until there was a free subconjunctival movement of the instrument intothe forniceal space up to the orbital rim. At this moment we determined whichsize of the conformer should be used: if the eyelids could not be closed easilyany disturbing tissue strand was released, or  the conformer size was exchanged to one with a smaller height. Afterdetermining the size, the conformer was placed apart. The prepared mucosal graftwas then introduced in the socket, orientating the graft so that the largestdimensions were at the deepest part of the superior and inferior fornices. Inorder to achieve this, the mucosal graft was often flipped over 45° making adiamond shape. Next, the medial and lateral mucosal flaps (about 5 mm from theedge) were fixed (with double-armed 6-0 absorbable sutures extending through theskin) in the deeper medial and lateral canthal area in order to create aboutmedial and lateral fornices. Subsequently, 6-0 absorbable running sutures wereapplied to connect the donor buccal mucosa to the recipient socket conjunctivasuperiorly and inferiorly. Care was taken to avoid knots in the socket andtherefore the sutures were extended through the skin. The large mucosal drapingwas then pushed in the correct position by gently fitting the conformer and thuscreating a “conjunctival” and “palpebral” part of the conjunctiva as in normalanatomy. Double-armed non-absorbable 3-0 monofilament sutures were transferredfrom the holes in the conformer through the forniceal folds, picking up theperiosteum, and extending through the skin where the knot was fixed on asilicone tube (fornix-deepening sutures). Two fornix-deepening sutures were madeat the superolateral and superomedial fornix, and two fornix deepening suturesat the inferior fornix. Finally, three central tarsal fixation sutures(non-absorbable 3-0 monofilament) were introduced from the superior tarsalplate, through the central hole in the horizontal extension lip and subsequentlythrough the inferior tarsal plate (tarsorrhaphy sutures).  PostoperNine patients, aged from 27 to 70 years old, with recurrent contractions aftermultiple surgeries not able to wear an ocular prosthesis were treated using thispersonalized socket reconstruction technique. Mean follow up was 20 months (range7–36 months).In eight out of nine patients sufficient fornices were created, which enabled theretention of an ocular prosthesis . The onlIn patients 1, 2 and 3, initially only the inferior fornix was constructed since wejudged the superior fornix to be adequate preoperatively. All three patients neededa subsequent reconstruction of the superior fornix since the altered position of theinferior fornix reshaped the socket and prohibited a good fit of the prosthesis. Twopatients underwent additional surgery for cosmetic reasons .All patients already had preoperative loss of levator function and scarred andabnormal position of the lid margin with entropion, misdirected or absent eyelashes, which remained after the socket surgery. For one case, after 23 months ofstable socket, it was decided to perform a correction of the superior lid entropion(lid-split with addition of mucosa and a new temporary conformer) resulting in anacceptable situation. The other patients did not want additional entropion surgery.Case 2 had upper eyelid retraction for which we performed a levator disinsertionprocedure after 22 months, also with addition of mucosa and again a temporaryconformer with good result. Despite the remaining lash abnormalities andlagophthalmos we judged the cosmetic outcome highly acceptable in six out of theeight cases who had symmetric eyelid contour and normal volume or only slightsuperior sulcus volume deficiency. The cosmetic outcome was reasonably acceptable inone patient with moderate volume loss and pre-existing shortened and irregularhorizontal palpebral fissure, and in one patient with pre-existing large volume lossand microblepharon, hence with good contour. All patients were satisfied being ableto wear a prosthesis.Commonly there was a lot of discharge from the socket in the first postoperativeweeks, for which regular superficial cleaning was advised. Approximately 2 monthsafter surgery the non-absorbable sutures  were removed. The conformer was thenreplaced by a cosmetic prosthesis. In the one case that did not succeed, theconformer luxated from the inferior fornix after 1 month which resulted inobliteration of the lower fornix, and later the superior fornix as well. As aresult, a prosthesis could not be fitted.In this paper we describe a successful method for treating severely recurrentcontracted sockets with the use of buccal mucosal transplant in combination withlong-term fixation of a personalized 3D-designed and printed conformer. In allcases, standard methods to reconstruct the fornices had failed. In this phase, angiogenesis occurs and extracellular matrix is formed.In all eight successful cases, the transplanted mucosa was retained with goodvascularization. We experienced that a well-vascularized part of the socket wasobtained after the release or excision of the anterior scarred tissue. Thisvascularized tissue should still be able to serve as a nourishing bed for the freemucosal graft, provided that the graft stays in firm contact with the vascularizedbed until the healing process has passed the proliferative phase of wound healing,which takes place between 4 and 21 days after creation of the wound. Cyclosporin eye drops are safe and easy to use during a long period. Mildsocket contraction seems to benefit from intra-operative 0.02% mitomycin applicationor four weekly 5FU injections in small comparative studies.10 As we have not used this, andas our experience is currently limited to these cases, we cannot give clearrecommendations on which anti-inflammatory treatment to use for severecontraction.To decrease the inflammatory response, we prescribed steroid eyedropspost-operatively. We also empirically started cyclosporin drops simultaneously, ascyclosporin inhibits the release of pro-inflammatory cytokines. Therefore care has to be taken that the fornices are “frozen” in their properposition. Our fixation of the buccal mucosal graft with the custom-made conformerkept the graft attached to the underlying fresh vascular bed, and simultaneouslyprevented it from mechanical contraction.Looking at the sizes of the postoperative conformers, we were able to preserve thefornices and have thus prevented severe contraction which was seen after theirprevious surgeries. We assume the long-term fixation of the adequately sizedconformer added to this result. In vitro studies have shown the contraction ofmucosal tissue to be around 50% after 28 days. Mechanical restraint reduced thiscontraction to 22%.13 Other surgeons have used ahard palate composite graft with buccal mucosa. The stiff hard palate is generallyless vulnerable to contraction. Choi et al. described additional fornix surgery in three out of four cases duringfollow-up due to fornix contraction and Lee et al. described that five of 13 cases needed additional surgery after initialsocket reconstruction. Using the conformer however, a hard palate graft is notnecessary. Standard, non-custom-made conformers are available as a reasonablealternative, but do not offer the advantages of optimal size, the ability to adaptthe fixation holes exactly where needed, the possibility to fixate upper and lowereyelids to the conformer so that fornices are well formed, and the easytransformation to the cosmetic ocular prosthesis which is based on the 3D-printedconformer. Obviously, the costs for producing 3D-printed objects depend on access toa 3D-printer which is by far the most expensive part of the process. If such aprinter is available in the clinic, or the ocularist comes to an agreement with acompany to share a printer, it is also possible to add the conformers to an alreadyplanned print session intended for other objects. In this way, the costs are limitedto the design time, and the print material. The resin we used is about €257 apackage for 1000 ml, and as the average conformer is 0.8 ml we can theoreticallyprint 1250 conformers out of one package, leading to an average cost of 20 cents perconformer. If a printer needs to be purchased solely for the purpose of designingthese conformers the total costs might not outweigh the benefits.Other examples to prevent contraction are the use of a silicone expander and siliconefixative after fornix reconstruction. It should be evaluated whether these scaffolds will be able to result inviable mucosal tissue in recurrent contracted cases, since it may need a certainamount of healthy pre-operative mucosal tissue in the recipient.To avoid disturbing sufficient tissue, three cases were initially only treated forone fornix because there was a moderate opposite fornix. We noted the socketreshaped after the procedure, ultimately needing a second surgery also on thisopposite fornix. This made us adapt the procedure to preferably one large graft thatreconstructs both upper and lower fornices, enabling a better formation and fixationof the complete socket. It has to be noted that for this procedure (so preferablyaddressing both fornices), the harvested buccal mucosal grafts should be as large aspossible. The size of this graft can be enlarged by perforating holes and stretchingthe graft, like with skin transplants. Additionally, as the mucosa willre-epithelialize intraorally, it is possible to repeat this procedure multipletimes. Alternatives for buccal mucosa can be nasal mucosa, amniotic membrane with orwithout mitomycin, and synthetic options such as polymers or collagens serving asscaffolds for self-regenerating progenitor conjunctival epithelial and goblet cells.A strength of our study is that it presents a relatively simple innovative way to acomplex problem: to fixate a mucosal graft using a custom-made conformer, which canbe worn during a relatively long period and ultimately benefits the socket lining.In all but one case our treatment goal was reached, at least for the relatively longfollow-up time of 6–36 months. A limitation is the small number of cases. Anotherlimitation is that we did not repair the  entropionusing this method. Our primary goal in our series was the retainment of aprosthesis. Although not all patients wished for additional improvement, we mightconsider correcting entropion as a secondary procedure or perhaps adjust the surgeryto a one-step procedure including a lid-split procedure, or the use of a palatumcomponent during the primary surgery.In conclusion, we described nine cases for whom custom-made, 3D-printed conformershave aided fixation of mucosal transplants in contracted sockets where previoussurgeries had failed, enabling the retention of a well-fitted ocular prosthesis ineight cases. We advise initial treatment of both upper and lower fornices to avoidsubsequent surgeries. In some cases, extra lid surgery is desirable for cosmetic,but not essential for functional reasons.


The aim of this experimental study was to analyze the impact of applying different configurations of the transpedicular fixation system on selected mechanical parameters of the thoracolumbar spine under conditions of its instability (after simulated fracture). Five study groups were tested: physiological, with compression fracture of the vertebra, with two-segment fixation, with three-segment fixation, and with four-segment fixation. Each of the analyzed study groups was subjected to axial compression, flexion, and extension. Based on the conducted experimental tests, the mechanical parameters, i.e., stiffness coefficient and dissipation energy, were determined for all groups under consideration. The stiffness value of two-segment fixation is significantly lower than the physiological value (during flexion and extension). The use of long-segment fixation considered in two configurations (three- and four-segment fixation) may result in excessive stiffness of the system due to the high stiffness values achieved . The use of long-segment fixator design shows better results than short-segment fixation. Considering both biomechanical and clinical aspects, three-segment fixation seems to be a compromise solution as it saves the patient from more extensive stiffening of the spinal motion segments. According to statistics, fractures of the thoracolumbar spine Th11-Th12-L1) account for almost 90% of all vertebral fractures and, moreover, approximately 50% of fractures are compression fractures of the Th12 or L1 vertebra 1-Th12-L1. The thoIn the case of vertebral fractures, surgical treatment of back injuries with implants has become standard clinical practice. However, it is often a major clinical problem and leads to many failures , 5. ThisHowever, there are discrepancies in the available literature regarding the behavior of the spinal segment subjected to loads under the conditions of discontinuity of the anterior column structure (compression fractures) and after its fixation –14. AccoTherefore, experimental research may help to better understand the behavior of the thoracolumbar spine stabilized with implants. The aim of this experimental study was to analyze the impact of applying different configurations of the transpedicular fixation system on selected mechanical parameters of the thoracolumbar spine under conditions of its instability (after simulated compression fracture).Experimental tests were carried out on specimens collected from eight domestic swine aged 6–10 months and weighing 90–110 kg. Isolated specimens of the thoracolumbar spine (Th7-L5) were cleaned of the surrounding soft tissues, leaving intact intervertebral discs, ligaments, and articular joints as well as partially ribs and muscles. The test material was stored in double foil packaging at -20°C until testing. The specimens were thawed at room temperature for several hours prior to testing.In order to determine the impact of using different configurations of transpedicular fixation on selected mechanical parameters, five study groups were tested . First, Next, transpedicular fixation was performed in each of the damaged thoracolumbar segments in three configurations: two-segment fixation (S2—1 level above and 1 level below the compression fracture), three-segment fixation (S3—2 levels above and 1 level below the compression fracture), and four-segment fixation (S4—2 levels above and 2 levels below the compression fracture). These three types of fixation design were selected for the analysis as they are most frequently used in clinical practice in the surgical treatment of compression fractures.The experiment used the SOCORE transpedicular fixation system by NovaSpine, inserting polyaxial pedicle screws into the spine specimens (30° range of motion of the head relative to the screw). The vertebral bodies of the thoracic spine were implanted with screws with a diameter of 5 mm and a length of 35 mm, while the vertebral bodies of the lumbar spine were implanted with screws with a diameter of 6 mm and a length of 40 mm. Pedicle screws were connected with rods, 5.5 mm in diameter and of variable length depending on the length of the stabilized segment. In order to verify correct implantation of the pedicle screws in the vertebral bodies , each of the specimens of the thoracolumbar spine was diagnosed by X-ray in the sagittal and coronal planes.The specimens were loaded with forces reflecting normal life activities. The loading was carried out using an MTS 858 MiniBionix testing machine. The prepared specimens were mounted in a purpose-built test rig fitted with two grips (upper and lower), in which vertebral bodies Th7 and L5 were clamped with eight cylindrically arranged screws .In the first stage, each of the analyzed study groups was subjected to pure axial compression with the force ranging from 150 to 650 N , 17. TheBased on the conducted experimental tests, the mechanical parameters, i.e., stiffness coefficient and dissipation energy, were determined for all five study groups. Assessment of the stiffness of the different transpedicular fixation configurations allowed to determine which of the variants best reflected the physiological stability of the spine. By measuring the stiffness, it was checked which type of stabilization had a better effect on restoring the physiological stiffness and thus indirectly on restoring the physiological stability of the spine column. Stability was defined in the context of the system stiffness assessment.k) was determined from the force-displacement loading curve obtained for the test specimens. The axial stiffness coefficient was determined in the force range from 350 N to 550 N for the 20th load cycle because in this section, the curve slope was linear and the increase in displacement was directly proportional to the applied load.In the case of compression load, the axial stiffness coefficient (kM) was determined from the torque-deflection angle loading curve in the deflection angle ranging from 1.5° to 3.5° for the 20th load cycle because in this section, the curve slope was linear and the increase in the deflection angle was directly proportional to the applied load.In the case of loads simulating flexion and extension, the bending stiffness coefficient  is determined, among others, by the viscoelastic nature of the annulus fibrous , 22. Thep < 0.05.All the results obtained from experimental testing were statistically analyzed using Statistica 13.1 software . The results for the five analyzed study groups were presented in the form of means with standard deviations. The parameters describing the effect of the type of fixation on the behavior of the spinal structures were analyzed using the Kruskal–Wallis nonparametric test with Dunn's multiple comparison post hoc test. The Kruskal-Wallis test is a nonparametric alternative to the one-way analysis of variance, allowing to compare the distributions of more than two independent groups, whose size is at least three in each case. Dunn's test is a post hoc pairwise test for multiple comparisons of mean rank sums. The statistical analyses were performed at a significant level of k) for the group of physiological specimens averaged 224.0 ± 51.3 N/mm. As expected, the fracture disturbed the transmission of loads through the spinal column and led to instability, as indicated by an approximately 20% decrease in the mean value of the axial stiffness coefficient (179.7 ± 52.0 N/mm) compared to the value obtained for the physiological system.The axial stiffness coefficient  caused a significant increase in the stiffness coefficient compared to the values obtained for physiological specimens. These values were greater by approximately 22% (274.5 ± 68.5 N/mm) and approximately 29% (289.9 ± 65.3 N/mm), respectively. The increase in the mean k coefficient between S3 and S4 fixation was insignificant and amounted to approximately 5%. The demonstrated values of stiffness between the examined fixation systems showed no statistically significant differences (p > 0.05)  .kM) for the group of physiological specimens averaged 14.4 ± 3.4 Nm/rad. It was shown that the fracture caused instability, as reflected by the mean stiffness coefficient lower by approximately 28% compared to the value obtained for the physiological system. The use of S2 fixation resulted in decreased stiffness compared to the value obtained for physiological specimens. The mean stiffness coefficient was almost 41% lower than that obtained for physiological specimens . There were also statistically significant differences between S2 and S3 fixation (p = 0.009) as well as between S2 and S4 fixation (p = 0.04).In addition, statistically significant differences were demonstrated between the values obtained for the specimens with the Th12 vertebral fracture and the specimens treated with S3 fixation .During extension, the stiffness coefficient fracture . The usep = 0.003).Analysis of the results of dissipation energy obtained for the axial compression showed that the mean damping energy of the examined spine specimens was 125.2 ± 23.2 mJ . The greDuring flexion, the mean damping energy of the examined physiological spine specimens was equal to the value obtained for the specimens with a fracture. The smallest loss of dissipation energy was obtained for the group of specimens treated with S2 fixation. This value was lower by approximately 32% compared to the value obtained for physiological specimens. It was also noticed that dissipation energy for the specimens with S3 fixation was slightly higher (by approx. 3.5 mJ) than the value obtained for the group with S2 fixation. The highest values of dissipation energy were obtained in the case of S4 fixation. No statistically significant differences were found between the analyzed study groups.It was shown that during extension, the mean damping energy of the examined physiological spine specimens was practically equal to the value obtained for the specimens with a fracture. The values obtained for all three fixation configurations ranged from 23.5 to 26.2 mJ. The lowest loss of dissipation energy was obtained for the group of S4 specimens, and this value was smaller by approximately 48% compared to the value obtained for physiological specimens. No statistically significant differences were found between the analyzed study groups.The analysis of the obtained results showed a relationship between the stiffness coefficient and dissipation energy obtained for a given load case. An increase in the stiffness coefficient caused a decrease in the dissipation energy. The use of a more rigid system for long-segment fixation dampened less energy due to the fact that the acting load was transmitted mainly by the fixator and not by the system of intervertebral discs and ligaments with hyperelastic properties. A decrease in the stiffness coefficient increased dissipation energy, which in turn was probably related to the fact that the system needed more energy to resist the force while maintaining the original properties.The high values of the standard deviation presented in It was also observed that in the case of Th12 vertebra fracture, the stiffness coefficient decreased for the compressive and bending loads and was significantly lower that the stiffness obtained for the physiological system. However, during extension, stiffness was similar to the physiological group and the group of vertebral fractures. This demonstrates that articular processes transmitted a significant part of the load during extension and fractures of the vertebra were not able to significantly disrupt the load transfer mechanism.Based on the conducted experimental tests, it was shown that during flexion and extension, the stiffness coefficient obtained for S2 fixation was significantly lower than the physiological value, which in practice negates the usefulness of this solution because the fixator does not perform its function. This can be explained by the fact that fixation of the S2 segment is not able to completely prevent displacements in the area of the damaged vertebra, which is also indicated by the authors of other research studies , 14, 23.The analysis of the obtained results showed that S4 fixation can give better results than S2 fixation. It was noticed that during flexion and extension, the values of the stiffness coefficient obtained for S4 fixation were closest to the values obtained for the physiological system. However, the use of S4 fixation resulted in significantly longer time of surgery. On the other hand, the S3 configuration allows for a less extensive fixation of the spine compared to S4. From the functional point of view, the shorter the fixation time, the better for the patient. Moreover, to obtain good clinical effects of treatment, it is crucial to reduce kyphosis and restore the correct sagittal position of the spine, which can be ensured by S3 fixation relative to S2 fixation .McDonnell et al.  and BoleElmasry et al.  and McDoSun et al.  reportedS2 fixation can reflect the physiological stiffness in the case of compression because the obtained value is the closest to that obtained for the physiological caseDuring flexion and extension, the stiffness value of S2 fixation is significantly lower than the physiological value, which in practice negates the usefulness of this solution because the fixator does not perform its functionDuring both compression and flexion, the use of long-segment fixation considered in two configurations (S3 and S4) may result in excessive stiffness of the system due to the high stiffness values achieved During loading simulating extension, the greatest excessive stiffness may be caused by S3 fixation due to the highest stiffness coefficient while S4 fixation works best in the process reflecting the physiological stiffnessThe obtained results allowed to assess which fixation configuration can best restore the physiological stiffness. The test results showed the following:Summarizing the results obtained from experimental testing, it can be said that fixation of the Th12 vertebra fracture over two segments does not provide adequate stability to the spinal column during flexion and extension. On the other hand, the use of long-segment fixation for this type of fracture may lead to excessive stiffness of the considered spinal segment. Consequently, this may contribute to increased mobility of the segments immediately adjacent to the implantation site, raising the risk of increasing curvature of thoracic kyphosis, which may result in the breakage or loosening of transpedicular screws , 28. BreAnimal-derived preparations are commonly used as research models to replace human preparations  in experimental research. Due to the limited availability of human preparations, scientists are constantly looking for a representative animal model that reflects the biomechanics and anatomy of the human spine. Therefore, in experimental studies, the porcine spine was used as a physiologically correct preparation, unchanged under the influence of deformation.The lumbar spine of porcine is characterized by a high anatomical and structural similarity to the lumbar spine of humans, mainly in terms of geometric parameters of the vertebrae , 32. BusIt was also proved that the heights of human and porcine vertebrae in individual sections of the spine were similar to each other. By comparing the width and height of the spinal canal in individual vertebrae between the human and porcine spine, it can be concluded that porcine vertebrae can be a representative model when testing fixation system techniques. In particular, it is noted that the width of the spinal canal of porcine vertebrae indicates remarkable similarity to the same parameter in human vertebrae , 34. TheDespite the fact that differences in the curvature of individual sections of the spine are observed when comparing the dimensions of the spinal canal in individual vertebrae between the human and porcine spine, the literature shows that porcine vertebrae can be a representative model when testing fixation system techniques , 33.Additionally, while developing the testing protocol used in this study, it was observed that tests performed on such long segments of the spine (Th7-L5) sometimes led to their buckling during loading with compressive force. It has been noticed that the danger of loss of stability is not due to simulated trauma but due to the lack of active support of the muscular system. Therefore, in order to eliminate the undesirable phenomenon of buckling, the costovertebral joints  and muscles surrounding the isolated specimens were not removed. Moreover, the limitation of the number of load cycles was also applied for each tested case in order to minimize the risk of loss of stability, which could consequently (in some cases of the applied stabilization) lead to a second loss of stability.Our findings can be used in studies using human cadavers and subsequently in clinical research related to the introduction of new implant solutions for the fixation of thoracolumbar spine injuries. Due to the anatomical similarity , it can be suggested that the simulated type of the fracture and its influence on the obtained mechanical parameters after stabilization also occur in the case of the human spine. The cognitive aspects also include the fact that the obtained mechanical parameters can be used to implement complex numerical models of the spine.It should be emphasized that experimental studies on animal models are more accurate than numerical simulation. Experimental studies reflect the variability of material properties to a greater extent, especially when it comes to spine structure. The spine is a complex structure with nonlinear material properties, especially the intervertebral disc, which has viscoelastic properties. Often, to optimize the numerical simulations, simplifications are introduced in the model. The simplifications mainly relate to among others the material parameters or boundary conditions. This is a limitation of the numerical analysis carried out.The use of long-segment fixator design shows better results than short-segment fixationShort-segment fixator design does not provide sufficient stability, especially in the case of flexion. The use of this type of fixator configuration may not provide adequate stiffness, thereby resulting in excessive mobility of the transitional zoneThe use of S3 fixation allows to optimize the biomechanical and clinical conditions in a situation where it is impossible to insert a screw into a fractured vertebraThe experimental results of the biomechanical study allow us to draw the following conclusions:


In recent years, scientists have focused on research to replace petroleum-based components plastics, in an eco-friendly and cost-effective manner, with plant-derived biopolymers offering suitable mechanical properties. Moreover, due to high environmental pollution, global warming, and the foreseen shortage of oil supplies, the quest for the formulation of biobased, non-toxic, biocompatible, and biodegradable polymer films is still emerging. Several biopolymers from varied natural resources such as starch, cellulose, gums, agar, milk, cereal, and legume proteins have been used as eco-friendly packaging materials for the substitute of non-biodegradable petroleum-based plastic-based packaging materials. Among all biopolymers, starch is an edible carbohydrate complex, composed of a linear polymer, amylose, and amylopectin. They have usually been considered as a favorite choice of material for food packaging applications due to their excellent forming ability, low cost, and environmental compatibility. Although the film prepared from bio-polymer materials improves the shelf life of commodities by protecting them against interior and exterior factors, suitable barrier properties are impossible to attain with single polymeric packaging material. Therefore, the properties of edible films can be modified based on the hydrophobic–hydrophilic qualities of biomolecules. Certain chemical modifications of starch have been performed; however, the chemical residues may impart toxicity in the food commodity. Therefore, in such cases, several plant-derived polymeric combinations could be used as an effective binary blend of the polymer to improve the mechanical and barrier properties of packaging film. Recently, scientists have shown their great interest in underutilized plant-derived mucilage to synthesize biodegradable packaging material with desirable properties. Mucilage has a great potential to produce a stable polymeric network that confines starch granules that delay the release of amylose, improving the mechanical property of films. Therefore, the proposed review article is emphasized on the utilization of a blend of source and plant-derived mucilage for the synthesis of biodegradable packaging film. Herein, the synthesis process, characterization, mechanical properties, functional properties, and application of starch and mucilage-based film are discussed in detail. Over the past few years, non-degradable plastic-based materials have created a major challenge for the whole world due to their excessive use in the industrial and domestic sectors and global production which exceeds 400 Mt/year . SynthetGenerally, biopolymer (starch–mucilage)-based edible film can be synthesized by two methods: the casting and extrusion techniques, also known as wet and dry methods . MoreovePlantago psyllium starch and seed mucilage by Krystyjan et al. [Glycerol is most commonly used to dissolve or disperse the starch and mucilage polymers; this process is known as solubilization. The resulting solution is poured into a specified mold or glass plate during the casting process. The drying process allows the solvent to evaporate, resulting in a starch–mucilage film that binds to the mold . For then et al. . In thein et al. .Opuntia ficus-indica mucilage (10% W/W) with waxy maize and acetylated or normal rice starches (70% W/W) and glycerol (20% W/W). addition of Opuntia ficus-indica mucilage improved the functional and mechanical properties of tubular and extruded films [W/V) blended with polyvinyl alcohol (PVOH) (4% W/V). Therein, glycerol (25% W/W) was used as a plasticizer. The result of a study proved that films prepared blended with starch and PVOH are highly suitable and manageable for food packaging applications. This is because the incorporation of PVOH into potato starch enhanced the water absorption capacity, and improved mechanical and functional properties and gas permeability of the film. However, biodegradable films produced from potato starch and PVOH blends had a homogeneous appearance, with no obvious bubbles or phase separation, were transparent, and were easy to unmold. Satisfactory compatibility was observed during the processing in all the blended formulations. Moreover, bindings of hydrogen bonds between polysaccharide chains of starch–mucilage and glycerol were analyzed by Fourier-transform infrared spectroscopy (FTIR) analysis of films prepared from the potato husk starch and pear peel mucilage [Consequently, plasticizers and cohesive matrix can be used for the development of easily peeled starch–mucilage edible films with an excellent uniform microstructure, thermal stability, barrier properties, and mechanical properties. The starch–mucilage pre-fabricated film can be used in several industrial applications, mainly in food applications. Here, the film can act as a good barrier, protector, and reduce the loss of water from food products . Therefoed films . Becauseed films . Furthered films . Howevered films , and filglycerol % W/W wasmucilage . Furthermucilage ,32. As amucilage .Nowadays, physical and chemical modifications have been suggested as ways to improve the physicochemical and mechanical properties of starch–mucilage-based films, which seem to be very effective . The maj−1 [Plantago psyllium starch–mucilage by FTIR spectroscopy. In their study, a high number of changes was observed in the spectrum of the film prepared from the starch only, compared to film prepared from the starch–mucilage film. Broadband spectra were recognized at about 3200–3300 cm−1 in O-H stretching. Furthermore, asymmetric vibrations of the ring appeared at around 1100 cm−1. Spectra at 1080–960 cm−1 corresponded to C-O groups with stretching vibrations. Spectra of all the prepared film samples presented bands at 3200 cm−1 and 2880–2900 cm−1 which confirmed the C-H group, as well as CH2 group, were confirmed bands at 1245, 1405–1465, 2855, and 2916–2936 cm−1. Similarly, in chia-seed mucilage nanocomposites, films with starch nanocrystals were produced [−1 linked to the OH (hemicellulosic) group. Characteristic peaks of protein structures were observed at 1417, 1545, and 1643 cm−1. Spectra were deconvoluted at the range between 1200 and 900 cm−1. Moreover, the observed peak at 1033 cm−1 was due to hemicellulosic compound glycosidic linkage. Peaks of aliphatic CH wagging were detected around 922 cm−1. The OH groups in starch and glycerol, which are added to the mucilage film, are likely to create new functional groups with hydrogen bonds in the mucilage structure; therefore, the OH stretching band will become wider as the H bands increase due to the formation of new intermolecular hydrogen bonds. Likewise, Wang et al. [Dioscorea opposita Thunb. mucilage (DOM) and starch. In their study, there were peaks at 1250 cm−1 when the effects of pure starch and DOM films are combined. Peaks at 1250 cm−1, 923 cm−1, and 525 cm−1 were seen in the spectra of Dioscorea opposita Thunb. mucilage films and starch only when the effects of pure mucilage, starch, and DOM films were combined. In another study, psyllium gum and modified starch composite films were prepared by Askari et al. [−1 (C-H stretching and C-O stretching of the anhydroglucose ring), 1593 cm−1 (O-H blending of water absorbed), 2924 cm−1 (C-H stretching), and 3000–3600 cm−1 (O-H stretching). Moreover, increases in the percentage of Psyllium gum enhanced the intensity of the broad-band at 3300 cm−1 (O-H stretching of starch). This might be due to Psyllium gum having more hydroxyl groups, which could lead to more hydrogen bonding.Fourier-transform infrared spectroscopy (FTIR) is a technique used to determine the functional groups and chemical structure of the film. Generally, it is used in the wavelength range between 4000 and 400 cm−1 . In this−1  characteg et al.  producedDioscorea opposita Thunb. mucilage and starch [Scanning electron microscopy is a part of an electron microscope, used to evaluate the surface morphology of starch–mucilage films. Electrons are bombarded on the prepared sample through electron beams resulting in the reflection of electrons towards the object from the sample . Generald starch , and alsd starch . Likewisd starch . Scannind starch  investigPlantago psyllium starch mucilage films, an endothermic effect was observed at low temperature with the relation of water polysaccharides [W/W) and mucilage (0.1% W/W) and film based on potato starch (H2) (3% W/W) and mucilage (0.2% W/W) contained approximately 15% less water than film based on single potato starch (H0). The highest effect of exothermic with comparable magnitude was observed close to 330 °C. The thermal characteristics of S0 are different from those of S1 and S2 as determined by DSC. Its thermogram showed two exothermic peaks with maximum temperatures of 304 °C and 237 °C (which might be linked with decomposition), but S1 had just one peak at 291 °C and 317 °C which was connected with film composite degradation. This may be due to the addition of mucilage to the solution reducing decomposition, making the films more thermally stable. Furthermore, the addition of mucilage decreased the water content in the system based on the endothermic peak at around 70 °C, which corresponds to the dehydration of some film samples due to mucilage filling the gaps between starch molecules that were interacting with each other through hydrogen bonds. On the other hand, thermogravimetry analysis (TGA) is used for the measuring of mass variation of starch–mucilage films by temperature [Dioscorea opposita Thunb. mucilage and starch by Wang et al. [Dioscorea opposita Thunb. mucilage films without apparent peaks in TG.Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) can be used to determine the behavior of physical and chemical changes during the thermal processing, and the difference between the distinct formulation of starch–mucilage films, respectively . Araujo charides . Five fiperature . Edible g et al. . Around Dioscorea opposita Thunb. mucilage and starch films (0.79 to 4.26), Plantago psyllium seed mucilage and starch (0.56–1.38 MPa), chia-seed mucilage and starch nanocrystals films (6.7–7.5 MPa), nopal mucilage, and rice starch (2.80–3.96 MPa) were observed [Opuntia ficus indica mucilage (0.64–3.75 MPa), mango kernel starch and guar and xanthan gums (3.57–10.24 MPa), banana starch and peel fibers (8.9–11.1 MPa), and potato starch and coconut fiber nanocrystals (4.09–8.20 MPa) were observed as shown in Generally, the tensile strength of the film is the most important mechanical property of starch–mucilage films. Therefore, the casting method is widely used to obtain a high mechanical parameter of starch–mucilage films. Good tensile strength and low elongation are significant to provide the better structural protection of food products during the transportation and storage of products . Becauseobserved ,39,51. Astarch 0.–1.38 MPaDioscorea opposita Thunb. mucilage and starch can be used as an edible coating or edible films due to excellent water solubility—41.11% to 62.74% [Plantago psyllium seed mucilage and starch (16.76–22.85%), okra mucilage and corn starch (11.53–89.82%), chia-seed mucilage and starch nanocrystals (80–86.1%), nopal mucilage and Rice starch (18.42–22.59%), prickly pear peel mucilage and potato husk starch (39.67–54.43%) were observed, as compared to mango kernel starch and guar and xanthan gums (36.26–44.63%), potato starch and zedo gums (25.02–43.67%), and cassava starch and hydroxyethyl cellulose (28.73–93.26%) [Plantago psyllium seed mucilage and starch, films were highly soluble in water as compared to alone starch-based films [Dioscorea opposita Thunb film ranged from 51.85 ± 2.56 to 63.08 ± 1.07 g mm/m2 d kPa [WVTR (water vapor transmission rate) and water solubility are key properties and parameters evaluated during the characterization of films that help us to understand how the film behaves and interacts with water. It can be also measured by a water vapor transmission tester . The reso 62.74% . In addiarch 11.5–89.82%, 18.42–22.%, prickl–93.26%) ,61,62. Tch 39.67–.43% were–93.26%) . In the ed films . Moreove36.26–44.%, potato× 10−10 (control) to 4.29 × 10−10 with 0.6% guar gum. Starch-based edible films have a low barrier capacity against water vapors due to their hydrophilic nature [In another study carried out by Krystyjan et al. , 2 cm ofc nature . Furtherc nature .Dioscorea opposita Thunb. mucilage and starch (0.21–0.34 mm), Plantagopsyllium seed mucilage and starch (0.130–0.209 mm), okra mucilage and corn starch (0.04–0.08 mm), chia-seed mucilage and starch nanocrystals (0.042–0.045 mm), nopal mucilage and rice starch (0.47–0.50 mm), and prickly pear peel mucilage and potato husk starch (0.09–0.22 mm) were observed as compared to thermoplastic starch and Opuntia ficus indica mucilage (0.125–0.150 mm), potato starch and zedo gums (0.22–0.207 mm), and cassava starch and hydroxyethyl cellulose (0.04–0.08 mm) [p < 0.05), the transparency of the films decreased, most likely due to greater polymeric chain compaction changing the refractive index and restricting light passage through the film matrix [Transparency is one of the most important aspects of packaging material, especially when product visibility is important. In such cases, the package’s degree of transparency is a critical characteristic that might influence a consumer’s choice to buy a product  a produch 0.21–0.4 mm, Pla 0.125–0.0 mm, pot0.08 mm) ,60,61,62m matrix .B. thuringiensis, S. aureus, E. coli, S. typhmurium, P. aeruginosa and S. mutans. The results of the antimicrobial assay were 18.23 ± 0.68, 18.89 ± 0.72, 19.56 ± 0.79, 17.86 ± 0.62, 18.19 ± 0.61, and 17.52 ± 0.51 mm, respectively. Moreover, several biopolymers have been investigated to be used in the development of biodegradable, edible, and antibacterial food packaging films, such as starch, mucilage, gums, cellulose, and lipids. Several researchers have studied chitosan/starch blends films. The incorporation of chitosan into corn starch increased the antimicrobial activity of films. In this context, biodegradable composite films were produced from brown rice starch (BRS) and chitosan (CH) by Hasan et al. [Staphylococcus auerues) and Gram-negative bacteria (Escherichia coli). The inhibition zones of Escherichia coli against BRS100, BRS70CH30, BRS50CH50, BRS30CH70, and CH100 were 7.33 ± 0.33, 8.00 ± 0.00, 8.00 ± 0.00, 8.00 ± 0.00, and 8.00 ± 1.00, and for Staphylococcus auerues, they were 6.67 ± 0.58, 7.67 ± 1.15, 7.00 ± 1.00, 10.33 ± 0.58, and 12.33 ± 0.58, respectively. Chitosan’s antimicrobial activity is influenced by electrostatic interaction between the positive charge of the protonated amino group from chitosan and the negative charge on the surface of the microbial cell, resulting in the formation of a membrane on the microbe’s surface that prevents the microbe from gaining nutrients [Natural gums such as mucilage are biocompatible, environmentally friendly, and capable of a wide range of chemical changes. However, due to their low water solubility and distinct flavor, their industrial applicability is limited. Composite technology can help improve solubility and stability ,70. The n et al. . In thisutrients .In addition, naturally derived biopolymers are widely accepted in the food packaging industry due to their high biodegradability . To measBiodegradable polymers such as starch are some of the most important groups of commercially accessible bio-based products at the moment. Since the 1980s, a growing number of starch polymers have been developed. Thus, simple products like pure thermoplastic starch and polyolefin/starch blends were introduced in the beginning. These products had a detrimental influence on public perception of biodegradable polymers and harmed the reputation of the firms involved due to the incomplete biodegradability of starch/polyolefin blends . HoweverCurrently, researchers are more focused on research to replace petroleum-based component plastics with plant-derived biopolymers, offering desirable properties. Additionally, due to the high impact of plastic-based materials on the environment, human health concerns for the formulation of biobased, non-toxic, biocompatible, and biodegradable polymer films are emerging. Several plant-derived biopolymers such as starch, cellulose, gums, agar, milk, cereal, and legume proteins are always the first choice for eco-friendly packaging materials as the substitute for non-biodegradable packaging materials. Starches from various sources have usually been considered as a favorite choice of material for food packaging applications, however, suitable barrier properties are impossible to attain with single polymeric packaging material. Therefore, the properties of edible films can be modified based on the hydrophobic–hydrophilic qualities of biomolecules. In certain chemical modifications of starch, however, the chemical residues may impart toxicity in the food commodity. Therefore, in such cases, several plant-derived polymeric combinations could be used as an effective binary blend of the polymer to improve the mechanical and barrier properties of packaging film. Recently, scientists have shown great interest in underutilized plant-derived mucilage to synthesize biodegradable packaging material with desirable properties. Mucilage has a great potential to produce a stable polymeric network that confines the starch granules, delaying the release of amylose and improving the mechanical property of films. Due to techno-economical challenges and lack of awareness, the utilization of biodegradable materials is still not mainstream. Therefore, more research is required to synthesize effective packaging materials with desirable properties.


Fusarium thapsinum  and Macrophomina phaseolina  cause internal damage to the stalks of the drought tolerant C4 grass, sorghum (Sorghum bicolor (L.) Moench), resulting in reduced transpiration, reduced photosynthesis, and increased lodging, severely reducing yields. Drought can magnify these losses. Two null alleles in monolignol biosynthesis of sorghum  were used to investigate the interaction of water limitation with F. thapsinum or M. phaseolina infection.As effects of global climate change intensify, the interaction of biotic and abiotic stresses increasingly threatens current agricultural practices. The secondary cell wall is a vanguard of resistance to these stresses. bmr12 plants inoculated with either of these pathogens had increased levels of salicylic acid (SA) and jasmonic acid (JA) across both watering conditions and significantly reduced lesion sizes under water limitation compared to adequate watering, which suggested that drought may prime induction of pathogen resistance. RNA-Seq analysis revealed coexpressed genes associated with pathogen infection. The defense response included phytohormone signal transduction pathways, primary and secondary cell wall biosynthetic genes, and genes encoding components of the spliceosome and proteasome.The bmr12 plants.Alterations in the composition of the secondary cell wall affect immunity by influencing phenolic composition and phytohormone signaling, leading to the action of defense pathways. Some of these pathways appear to be activated or enhanced by drought. Secondary metabolite biosynthesis and modification in SA and JA signal transduction may be involved in priming a stronger defense response in water-limited The online version contains supplementary material available at 10.1186/s12870-021-03149-5. Sorghum bicolor (L.) Moench] is heat and drought tolerant, requires low nitrogen and water inputs, and is grown as a staple food crop, biofuel feedstock and animal feed worldwide ) and to a lesser extent corn earworms ([Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae)], as compared with pith from wild-type plants, though this was affected by growth conditions of the stalks  had significantly smaller mean lesions than wild-type (156.4 ± 26.6) (p = 0.04) (Table F. thapsinum-inoculated bmr6 (98.3 ± 13.3) were not significantly smaller than similarly-treated wild-type (156.0 ± 30.4) (p = 0.09). When considering the two water treatments with the same inoculum and plant genotype, bmr12 plants under water-limitation conditions  had significantly shorter mean lesion lengths than when inoculated with respective pathogens under well-watered conditions  (p < 0.01) bmr12 plabmr12 plants also had significantly elevated levels of salicylic acid (SA), jasmonic acid (JA), gibberellin A19 (GA19), and significantly lower levels of wall-bound and soluble syringic acid   and columns (traits) to identify patterns in these correlations in tissues sampled from all days Fig. . Genes w2-transformed lesion lengths. The relative expression of the dark turquoise module is considerably higher in bmr12 samples regardless of lesion length.Genes from the lesion-related subset Table  were repbmr12 genotype in tissues sampled at day 0  and a stress-response A/B barrel domain-containing protein ortholog (Sobic.008G035400).SbCAD5, a cinnamyl alcohol dehydrogenase (Sobic.007G076000), is a candidate susceptibility gene in this module.The salmon module was positively correlated with PDB inoculation and time to bloom. The salmon module was enriched for phenylpropanoid biosynthesis, including four peroxidases, PAL, CCoAOMT, and CCR1 . No significant KEGG enrichment was detected in these modules. The light cyan module eigengene was also positively correlated with ABA and SA content, shorter lesions, and time to bloom; the dark turquoise module was also positively correlated with the phenolic and phytohormone profile of bmr12  is a priming gene positively correlated with soluble sinapic acid levels and negatively correlated with abscisic acid content.The magenta module was positively correlated with SbTCP5 (Sobic.002G198400), a transcription factor strongly positively correlated with water limitation.The white module was enriched for carbon metabolism. It contained genes coding for glutathione S-transferase (GST) (Sobic.001G318900), a GDSL esterase/lipase (Sobic.003G018800), and a catalase (Sobic.004G0115660) that were putative priming genes, indicative of a response to oxidative stress. The GST and GDSL esterase/lipase were positively correlated with sinapic acid content. The white module also contained an early response LRR-like gene (Sobic.010G217400) and Fusarium infection across the three timepoints, and chitinases Sobic.009G130100 and Sobic.006G132400 were associated with the water limitation treatment across the three timepoints.The light green module was enriched for flavonoid biosynthesis , protein processing in the ER, proteasome, and peroxisome. Several of the genes coding for flavonoid-related enzymes, including chalcone synthases 4 (Sobic.005G136300) and 6 (Sobic.005G137300/), a eugenol O-methyltransferase-like (Sobic.007G058800), and a putative flavonone 2-hydroxylase (Sobic.002G000400), were identified as putative priming genes. An early response tricin synthase (Sobic.007G218700) was strongly correlated with water limitation. The light green module also contained an RGA3-like disease resistance protein (Sobic.007G058800), also a putative priming gene. Early fungal response genes in this module included pathogenesis-related thaumatin-like protein (Sobic.001G145700) and chalcone synthase I (Sobic.005G137000). Additionally, three predicted chitinases  and four pathogenesis-related (PR) proteins  were early response genes in this module, highlighting the plant-pathogen interaction at the cell wall. These chitinases and pathogenesis-response proteins were associated with SbEREB93 (Sobic.006G168100), a PP2C-like gene (Sobic.001G462800), and a calcium-dependent protein kinase (Sobic.004G279100).The green module is enriched for plant pathogen interactions through calcium-dependent protein kinases and calmodulin-dependent signal transduction cascades , and a flavonoid 3′-monooxygenase (Sobic.004G200800) as putative priming genes.F. thapsinum inoculation. The red module was enriched for central metabolism, alpha-linolenic acid metabolism , phagosome, protein processing in the endoplasmic reticulum, phenylpropanoid biosynthesis, proteasome subunits, cysteine and methionine metabolism , and the citric acid cycle. It contained a gene coding for an acetylserotonin methyltransferase (Sobic.005G216100), which was positively correlated with water limitation and was an early response gene. The red module also contained priming genes, including a dirigent protein-like ortholog (Sobic.005G101600), which was also correlated with water limitation. This module also contained two sets of PR proteins that were directly adjacent to one another on the same chromosome . Sobic.001G400800 and Sobic.001G400900 were putative priming genes and Sobic.001G401100 and Sobic.001G401200 were early response genes. Another early response gene included in this module was a laccase (Sobic.005G215300) that was correlated with bmr12 across the three timepoints. Four LRR-like genes  were found in this module.The red, orange, steel blue, and grey60 modules were associated with The orange module was enriched for ribosomal proteins and RNA transport (translation initiation factors and components of the nuclear pore complex) and contained a putative secreted peroxidase (Sobic.007G014200) that was identified as a larger lesion gene. The steel blue module was enriched for ribosomal proteins  and ribosome biogenesis, including small nucleolar ribonucleoproteins , ubiquitin mediated proteolysis (E3 ubiquitin ligases), proteasome (proteasome subunits), and spliceosome . It contained five out of the seven constitutive lesion genes including an LG2 transcription-factor like gene (Sobic.003G363600). This module also included a peroxidase 47-like protein (Sobic.007G014200), which was not influenced by genotype but was positively correlated with soluble ferulic acid and GA53. Isoflavone reductase (Sobic.003G104350) was identified as an early response gene in this module. SbCAD4 (Sobic.002G195600) was also correlated with the bmr6 mutant, corroborating previous research identifying this gene as upregulated in bmr6 plants compared to wild-type [There were fewer modules associated with only ild-type , 36. EnrThe saddle brown module eigengene was positively correlated with the well-watered treatment. It was enriched for protein processing in the endoplasmic reticulum, plant-pathogen interactions (primarily through heat shock proteins) and the spliceosome. This was the only module correlated to watering treatment.SbGLK7 (Sobic.002G016300), which was strongly positively correlated with water limitation and bmr12 and negatively correlated with soluble malic acid, and SbMyb60 (Sobic.004G273800), which impacts phenylpropanoid biosynthesis and lignin formation [SbCAD5 (Sobic.004G071000) was strongly positively correlated with the bmr6-ref in this module, as described earlier.The midnight blue module eigengene was positively correlated with time to bloom. It was enriched for DNA replication (the MCM helicase complex), phenylpropanoid biosynthesis, and glutathione metabolism. It also contained a putative priming gene, a chalcone synthase 5 (Sobic.005G136200). The midnight blue module also contained the transcription factors ormation , 37. SbCThe purple module was positively correlated with time to bloom and with JA and SA content. Its enrichment included oxidative phosphorylation , carbon metabolism, TCA cycle, pyruvate metabolism, and phagosome (26 s proteasome subunits) , correlations to physiological traits unrelated to inoculum dominated the module-trait relationships. In tissues sampled at day 13, these physiological processes were variously represented by the sky blue, dark orange, green, royal blue, light green, white, midnight blue, and red modules and were positively correlated with time to bloom. The black, purple, light cyan, and dark turquoise modules were negatively correlated with peduncle diameter, while the brown module was positively correlated with peduncle diameter.Genotype and time to bloom were strongly correlated with several expression modules identified in tissues sampled at 0 DAI. These relationships may be representative of the initial transcript pool determining the cellular environment encountered by the fungus upon inoculation and can shape early host-pathogen interactions , F-type and V-type ATPase subunits, and NADPH dehydrogenase orthologs. Putative sulfoquinovosyltransferase Sobic.002G000600 was correlated with low average moisture and time to bloom.bmr12 at 0 DAI. This corroborates the elevated levels of JA detected in bmr12 plants at 3 DAI, though bmr12 plants did not have elevated levels of IAA nor ABA. The purple module eigengene was additionally correlated with time to bloom.The tan, black, and purple module eigengenes were negatively correlated with peduncle diameter. The black module contained several smaller lesion-associated genes, including a putative malate dehydrogenase (Sobic.001G073900). The black module, enriched for phytohormone signal transduction in JA, IAA and ABA pathways, was correlated with The yellow module was enriched for spliceosome  and RNA transport . A probable protein-phosphatase 2C (PP2C)-like gene (Sobic.004G332900) is a potential susceptibility gene assigned to this module, which was positively correlated with a longer photoperiod at planting time and negatively correlated with time to bloom.bmr12 genotype  and SbEREB107 (Sobic.007G077001). Across all days, a flowering time (FT)-like ortholog (Sobic.010G164200) within the black module was strongly correlated with bmr12. Within the magenta module, putative sulfoquinovosyltransferase Sobic.002G00060 was correlated with bmr12. The pink, pale turquoise, brown, dark red, purple, black, and dark turquoise module eigengenes maintained a consensus association with plant physiological traits, including time to bloom and peduncle diameter  and acetylserotonin O-methyltransferase I . Two yieldins   were early genes , and 4-coumarate-CoA ligase 1 (4CL1) . A glycogenin glucosyltransferase (Sobic.001G479800) and cellulose synthase (Sobic.001G021500) from the dark red module were constitutive lesion genes.The study of monolignol mutants requires attention to the impact of drought and pathogen infection on cell wall processes. The expression of many primary cell wall  and monobmr12 mutation and time to bloom, but negatively correlated with peduncle diameter. This expansin was also correlated with decreased levels of soluble syringic acid, syringaldehyde, and 4-coumaric acid, and elevated levels of soluble 4-hydroxybenzoic and sinapic acids. As previously observed, expression of the Bmr12 and Bmr6 genes  were reduced in lines with their respective mutant allele. However, expression levels of the Bmr6 locus was positively correlated with the bmr12 null allele. Notably, the expression of two CAD genes  were positively correlated with the bmr6 mutation.There were components of the cell wall that were also correlated with specific genotypes, highlighting the impact of monolignol biosynthetic mutations on diverse components of the cell wall  and SbHSF11 (Sobic.002G271100), were positively correlated with the well-watered treatment. Their predicted targets were enriched for protein processing in the ER and for plant pathogen interactions. The predicted targets of SbDOF23 , a zinc finger transcription factor, were also in the blue module and were enriched for ubiquitin mediated proteolysis and with one carbon pool by folate. In the red module, SbWRKY85 (Sobic.009G234100) is a transcription factor putatively involved in priming, and several genes from pathogenesis-related enriched categories were also early response or priming genes in this module  was associated with F. thapsinum and, in tissues sampled at 3 DAI, with M. phaseolina. SbMed14a  was positively correlated with M. phaseolina inoculation. Expression of SbMed25a  was positively correlated with lesion length but not associated with fungal inoculation. SbCyc1 , the cyclin component of Mediator complex, was negatively correlated with lesion length in tissues sampled at 3 DAI and SbMed30  was positively correlated with lesion length in tissues sampled at 3 DAI.The Mediator complex is the group of basal transcription factors that coordinate the interaction of RNA pol II with gene specific transcription factors. Originally discovered for its involvement in growth and development, the role of the Mediator complex in phenylpropanoid metabolism, immunity and abiotic stress tolerance has been of increasing interest –42. In tM. phaseolina or F. thapsinum infection.The objective of the current study was to examine the influence of cell wall modification on mechanisms of drought adaptation, and to identify potential mechanisms and pathways which promote or prevent lesion development during The cell wall is a hub of stress surveillance and response, and the monolignol biosynthesis pathway is a component of a vastly interconnected metabolic grid whose manipulation can affect a broad array of other pathways . The pribmr mutations in CAD and COMT genes, bmr6 and bmr12 (respectively), provide a valuable system to study the impact of lignin alteration on drought and disease response in sorghum [The reduction of lignin content is a key objective for cellulosic biofuel and forage production, necessitating an investigation into the robustness of reduced lignin lines to biotic and abiotic stresses. The  sorghum , 23, 44. sorghum –49, maki sorghum –53.M. phaseolina, the necrotrophic causative agent of charcoal rot and one of the most destructive plant pathogens, encodes an extraordinarily high number of secreted cell wall-degrading enzymes that likely aid in host infection [Fusarium species have also been shown to encode cell wall degrading enzymes [F. thapsinum is not available, it is likely that F. thapsinum also encodes these enzymes based on its pathology and lifestyle, like other closely-related Fusarium species [Cell wall degradation is a component of many plant-fungal interactions, and cell wall integrity maintenance is crucial to plant homeostasis and adaptation. nfection . A numbe enzymes –57. Alth species .F. thapsinum and M. phaseolina explain a relatively small amount of disease resistance, ranging from 9 to 30%, and several loci are environment specific. The current findings support the consistent observations that despite decreased lignification, bmr6 and bmr12 plants are not more susceptible to the common stalk pathogens F. thapsinum and M. phaseolina under fungal pathogen infection, nor under combined pathogen and drought stresses. Unexpectedly, bmr12 plants under water limitation had shorter lesions upon fungal inoculation than under well-watered conditions at 13 DAI , were also correlated with water limitation. The expression of tricin synthase I (Sobic.007G218700) and acetylserotonin O-methyltransferase I (Sobic.005G216100) were correlated both with water limitation and with n plants –68. In tn plants , 70. Ovebmr12 at 0 DAI were also positively correlated with F. thapsinum at 3 DAI, suggesting that these modules may have components that contribute to an earlier and more effective resistance response.Other modules that were correlated with fungal infection at 3 DAI were enriched for ribosomal proteins, protein processing in the ER, ubiquitin-mediated proteolysis, and proteasome, highlighting the dramatic role of protein turnover and the increased synthesis of defensive enzymes and other defensive proteins in the pathogen response. Modules positively correlated to bmr mutations may be able to compensate for their lignin biosynthesis impairment with promiscuous orthologs. A Zrp4-like O-methyltransferase gene  was strongly correlated to bmr12, which might explain why sinapic acid is still produced in bmr12 plants despite a hypothetical loss of ability to produce sinapoyl groups . This corroborates the elevated levels of JA observed in bmr12 plants at 3 DAI.ens C58C  and Fusa. niveum . Fusariurganisms , raisingrganisms , 74, 75.bmr12 tissues sampled at 3 DAI had elevated levels of JA and SA. JA and SA are signaling molecules with roles in defense whose pathways have been primarily understood to act antagonistically [The stically . Howeverstically , 78. Forstically , 80. In Pseudomonas syringae and the fungal pathogen Botrytis cinerea in Arabidopsis but does not currently have a reported host function [F. thapsinum than in plants inoculated with PDB, but it was not below the significance threshold of α = 0.1. Nonetheless, this trend suggests that IAA-Asp may play a role in Fusarium spp. pathogenesis on sorghum. In tissues sampled at 3 DAI, the modules positively correlated with IAA-Asp were enriched for pathways associated with protein turnover, including heat shock proteins and ubiquitin-mediated proteolysis. This result suggests that IAA-Asp may be involved in stress response by inducing heat shock proteins for protein stability. Heat shock transcription factors SbHSF4 and SbHSF11 were coexpressed with their predicted targets, which were enriched for heat shock proteins.Pathogens are adept at manipulating plant hormone signals . It has function . In the Arabidopsis with beta-aminobutyric acid (BABA), a known priming agent for disease resistance, was shown to result in altered levels of phenolic compounds including sinapic acid, primary metabolites, and the oxylipin-derived phytohormones OPDA and JA [F. thapsinum inoculation, and sinapic acid levels were elevated in bmr12 compared to wild-type tissues. However, soluble sinapic acid levels were not elevated in F. thapsinum-infected tissues.In addition to these defense and infection-related hormones, soluble phenolics have also been implicated as part of the priming response. Treatment of A and JA . In the bmr12 plants. The diverse number of pathways that have been linked to defense priming in this study and in previous studies suggests that there may be numerous and interacting ways to activate and tune these pathways, and that drought may be an environmental trigger of priming in bmr12 plants.Drought conditions may prime defense pathways in bmr12 plants impacted coexpression of genes involved in multiple pathways, including plant hormone signal transduction, RNA and protein processing and turnover, and transcription and translation. In particular, the coexpression patterns of primary and secondary cell wall biosynthetic genes and putative regulatory pathways that respond to both drought and disease point to the cell wall as the site of intricate connectivity between defense, growth, and development. Thus, drought conditions may have further primed bmr12 plants for disease resistance through the activation of defense pathways. Monolignol mutant lines are not more susceptible and may even be hardier to some diseases, depending on environmental conditions. This method of activating pathways associated with priming may also be a way to increase disease resistance in crops while providing reduced lignin lines for bioenergy or forage production.Altering cell wall structure enables the study of the resultant metabolic and transcriptomic alterations on drought and disease response. Monolignol biosynthesis enzymes have been shown to interact with components of the immune system , 85. In Seed of these genetic stocks are maintained and distributed by the USDA-ARS, Wheat, Sorghum, and Forage Research Unit, University of Nebraska, Lincoln, NE 68583–0937, and will be provided without cost to each applicant on written request. Genetic material of this release is deposited in the U.S. National Plant Germplasm System where it is available for research purposes, including development and commercialization of new varieties or cultivars. Released seed stocks are available upon request or through GRIN-Global.bmr6 and bmr12, near-isogenic to the wild-type, in the genetic background RTx430 were previously developed and are maintained by USDA-ARS, Lincoln, NE [bmr6, and bmr12), two watering conditions (well-watered and water-limited), three inocula , and three timepoints . Eight such replicates were collected. The bmr12 plants exhibited delayed bloom [bmr12 plants, five bmr6 plants, and no wild-type plants were culled from the experiment  medium for 4 days. The F. thapsinum isolate (H03-11S-9) was originally from a field in Lincoln, NE, and the M. phaseolina isolate (MP01–001) was a kind gift from G. Odvody . Plants were inoculated by producing a small wound on the peduncle, 5 cm below the base of the head, and inserting a fungus- or PDB-incubated toothpick into the wound [Toothpicks were incubated in batch culture at room temperature (22–23 °C) in potato dextrose broth (PDB) alone (the mock inoculation) or inoculated with agar discs  from a fungal culture of he wound . SamplesStatistical testing for the greenhouse pathology data was conducted in the SAS programming environment using the PROC MIXED procedure for linear mixed models . The modFrom one-half of the split peduncle, 2-cm sections were harvested either surrounding the wound (if lesion was less than 20 mm) or from the base of the lesion . Phenolics and phytohormones were assessed from a 2 cm peduncle section distal to the lesion  for further processing. RNA integrity was assessed at UNMC using an Agilent 2100 BioAnalyzer . Libraries were constructed using the QuantSeq REV 3′ 96 barcode kit  and were assayed for quality on the BioAnalyzer prior to pooling and sequencing. Library fragments were also analyzed by BioAnalyzer; quality control data (RIN and fragment size) is presented in Additional file bmr12, well-watered and water limited, mock-inoculated and F. thapsinum-inoculated samples on both 0 and 13 DAI were sequenced in Run 3. Because the runs were sequenced in batches, and not every condition was replicated in every batch, the separation between the 3 DAI samples and the 0 and 13 DAI samples is confounded with the sequencing run. This is reflected in the clustering pattern of the samples on a PCA plot . For RNA, 50–100 mg of each sample was extracted using TRIzol reagent  and purified with an RNA Clean and Concentrator kit . The purified RNA samples were quantified using 260/280 ratios and 1–10 ng were sent to the University of Nebraska Medical Center Genomics Core Facility (S. bicolor genome (v3.1) [Barcodes were removed and the 132-bp adapters trimmed with a Lexogen-recommended script   downloade (v3.1)  using kae (v3.1) . Kalliste (v3.1) .Pairwise differential expression testing was performed in DESeq2 but resulted in < 20 DE genes under each condition tested. The code for performing these comparisons is included in Additional file https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/), modified for three groups [Reads were pre-filtered to genes containing cpm > 10 and transformed with the native variance stabilizing transformation in DESeq2, as recommended by the authors of WGCNA , 95. A ce groups , 95. Mode groups –98.http://kobas.cbi.pku.edu.cn/) adjusted for FDR with BH [http://planttfdb.cbi.pku.edu.cn/) [KEGG enrichment in modules identified through WGCNA was calculated using Fisher’s exact test in KOBAS  .Phytohormone analysis was conducted at the UNL Proteomics and Metabolomics Facility following procedures described previously –104.Phenolic analysis was conducted as described previously  with modMetabolite analysis was performed in the R programming environment (3.6.1)  of samples indicating significant separation by day of sampling (A), which is confounded with RNA sequencing run, and little separation based on water treatment (B). Separated by day of run (C), the separation between treatment conditions is clarified.Additional file 9. Lexogen-recommended script for trimming raw RNA-Seq reads.Additional file 10 Shell script for using Kallisto to pseudoalign reads to the S. bicolor genome.Additional file 11. R markdown notebook containing WGCNA and downstream analysis.Additional file 12. GC/MS protocol, adapted from Palmer et al. 2008.Additional file 13. R markdown notebook containing time to bloom and metabolite analysis.Additional file 14. Pseudoalignment statistics from Kallisto v0.44.Additional file 15. Excel workbook of qPCR correlation analysis.Additional file 16. Complete Cq values for qPCR validation.


Cryptococcus neoformans var. grubii. It has high global morbidity and mortality among HIV patients and non-HIV carriers with 99% and 95%, respectively. Furthermore, the increasing prevalence of undesired toxicity profile of antifungal, multidrug-resistant organisms and the scarcity of FDA-authorized vaccines were the hallmark in the present days. This study was undertaken to design a reliable epitope-based peptide vaccine through targeting highly conserved immunodominant heat shock 70 kDa protein of Cryptococcus neoformans var. grubii that covers a considerable digit of the world population through implementing a computational vaccinology approach.  Cryptococcosis is a ubiquitous opportunistic fungal disease caused by Cryptococcus neoformans var. grubii's heat shock 70 kDa protein were retrieved from the NCBI protein database. Different prediction tools were used to analyze the aforementioned protein at the Immune Epitope Database (IEDB) to discriminate the most promising T-cell and B-cell epitopes. The proposed T-cell epitopes were subjected to the population coverage analysis tool to compute the global population's coverage. Finally, the T-cell projected epitopes were ranked based on their binding scores and modes using AutoDock Vina software. Results and Discussion. The epitopes  had shown very strong binding affinity and immunogenic properties to B-cell.  and  exhibited a very strong binding affinity to MHC-I and MHC-II, respectively, with high population coverage for each, while FYRQGAFEL has shown promising results in terms of its binding profile to MHC-II and MHC-I alleles and good strength of binding when docked with HLA-C∗12:03. In addition, there is massive global population coverage in the three coverage modes. Accordingly, our in silico vaccine is expected to be the future epitope-based peptide vaccine against Cryptococcus neoformans var. grubii that covers a significant figure of the entire world citizens. A total of 38 sequences of  Cryptococcus neoformans that causes life-threatening pneumonia and meningoencephalitis in immunocompromised patients [Cryptococcus neoformans var. grubii strains (serotype A) are more virulent, are widely distributed all over the world, and cause 99% of infections in HIV patients and 95% of the non-HIV carriers [Cryptococcosis is a ubiquitous opportunistic infection caused by patients –3. Cryptpatients –6. Commocarriers , 8.C. neoformans expresses a significant number of immune-proteomic factors that evoke host immunity that could be useful targets as diagnostic markers or vaccines [Candida [Histoplasma [Mycobacterium [Clinical and experimental evidence suggests that cell-mediated immunity by T helper CD4+ plays a crucial contribution to host defense against intracellular cryptococcosis –12. Nevevaccines –20. Intevaccines , 21–23. vaccines , Trypanovaccines , Toxoplavaccines , Candida[Candida , 28, Histoplasma , 30, andacterium  species.Cryptococcus neoformans var. grubii by implementing an emerging approach in computational vaccinology. This strategy is quite advantageous because it speeds up the process of successful identification of potential epitope-based peptide vaccine candidates and significantly downsizes the number of epitopes to be synthesized and analyzed for experimental assays [Cryptococcus neoformans. Accordingly, this is the first computational-based study to utilize heat shock 70 kDa protein as an attractive immune-proteomic factor that is able to stimulate desirable immune responses against cryptococcosis.Cryptococcosis has become more prevalent and the main cause of morbidity and mortality in the last 30 years for the eruption of the HIV epidemic . Despitel assays . TherefoThe flow chart demonstrates the overall process of peptide vaccine designing as illustrated in Supplementary Figure Cryptococcus neoformans var. grubii sequences with a length of 773 mer were retrieved in FASTA format from the National Centre for Biotechnology Information (NCBI) protein database (Accession No. XP_012053205.1) on 30th July 2018 at https://www.ncbi.nlm.nih.gov/protein . The retrieved antigen sequences were run against the NCBI Reference Protein (RefSeq) using ClustalW as implemented in BioEdit sequence alignment editor software version 7.2.5 .B-cell epitopes are determinants on the surface of pathogens that interact with B-cell receptors. B-cell epitopes can be either continuous or discontinuous. Approximately 10% of B-cell epitopes are continuous, consisting of a linear stretch of amino acid. In consequence, the majority of B-cell epitopes are a discontinuous or conformational structure , 42.http://tools.iedb.org/bcell/. The classical propensity scale methods and hidden Markov model programmed analysis resource were applied from IEDB to fulfill the following physiochemical criteria .Analysis of epitope binding affinity to B-cell was assessed by the IEDB B-cell epitope prediction tool at (1) Prediction of Linear B-Cell Epitopes. BepiPred was conducted as a linear B-cell epitope prediction method to sort out the linear conserved regions with a default threshold value of 0.249 [of 0.249 .(2) Prediction of Surface Accessibility. The Emini surface accessibility test was implemented to discriminate the surface conserved epitopes with a default cut-off value of 1.000 [of 1.000 .(3) Prediction of Antigenicity. The Kolaskar and Tongaonkar antigenicity method was used to differentiate the immunogenic sites with a default cut-off value of 1.024 [of 1.024 .(4) Prediction of Beta-Turn. The Chou and Fasman beta-turn prediction tool was used to predict beta-turn sites with a default cut-off value of 0.950 [of 0.950 .(5) Prediction of Hydrophilicity. The Parker hydrophilicity prediction tool was utilized to distinguish hydrophilic residues with a default cut-off value of 1.949 [of 1.949 .http://tools.iedb.org/ellipro/. ElliPro implements three algorithms evaluating the approximation of the protein shape as an ellipsoid, calculation of residue protrusion index (PI), and clustering of neighboring residues based on their PI values. The minimum score and maximum distance (angstrom) were calibrated in the default mode with a score of 0.5 and 6, respectively [The reference sequence of heat shock 70 kDa protein was subjected to the Swiss model in order to get the 3D structure . Based oectively .CD8+ T cytotoxic cells, which recognize peptides displayed by MHC-I moleculesCD4+ T helper cells, which recognize epitopes in association with MHC-II moleculesT-cells identify antigens as a short peptide segment in association with MHC molecules on antigen-presenting cells. There are two categories of T-cells:T-cell epitopes only recognize linear peptides. MHC-I binding predictions are now very strong and have wide allelic coverage by integration with predictions of proteasomal cleavage and TAP binding sites .http://tools.iedb.org/mhci/. The functional cleft of MHC-I molecules is closed and can only accommodate short peptides ranging from 9 to 11 amino acids; all epitope lengths were set to the optimum length of 9 mers [50) with a default threshold value of 500 nM [50 value indicates greater binding affinity and vice versa. Based on a rough protocol, all conserved epitopes with an IC50 score of less than 50 nM have high affinity, less than 500 nM intermediate affinity, and less than 5000 nM low affinity [50 are selected for further analysis whereas epitopes with IC50 greater than 500 were omitted.The analysis of epitopes binding to MHC-I molecules was assessed by the IEDB MHC-I prediction tool (version 2013-02-22) at f 9 mers , 52. Artf 500 nM . It is gf 500 nM . Therefoaffinity . Conservhttp://tools.iedb.org/mhcii/. Unlike MHC-I, MHC-II has a flexible pocket with a series of polymorphic pockets and plateaus that interact with several side chains of the peptide core sequence; this provides the specificity of the MHC-peptide interaction and can accommodate peptides of varying lengths, typically 12 to 26 mer. The consensus sequence of the peptides is set to be 9 mer [50) with a default threshold value of 100 nM [50 were selected for further mapping analysis whereas epitopes with IC50 greater than 100 were dismissed [The analysis of MHC-II selected candidates were assessed by the IEDB MHC-II prediction tool at be 9 mer . For thef 100 nM . Finallyismissed .http://tools.iedb.org/population/ [To ensure the universal coverage within heterogeneous populations, it is crucial to calculate global population coverage for the chosen epitopes since the HLAs are among the most polymorphic proteins and vary among different geographical regions around the world  and becaulation/ , 60.The main purpose of vaccination is to induce an immune response after injecting the vaccine into the body. Therefore, it is essential to define the physiochemical parameters associated with the vaccine. The physicochemical properties of vaccine construct were analyzed using BioEdit sequence alignment editor software version 7.2.5  and ExPAThe reference sequence of heat shock 70 kDa protein was submitted to Raptor X template-based tertiary structure prediction in order to get the 3D structure . After wMolecular docking was performed using AutoDock Vina software  to prediIn terms of continuous B-cell epitope prediction analysis, the default threshold score of heat shock 70 kDa protein to B-cell was given to be 0.249 in BepiPred Linear Epitope Prediction, and only 298 linear epitopes were predicted. In Emini surface accessibility prediction, the default threshold score of the surface accessibility test was found to be 1.000; 211 epitopes were potentially at the surface by passing the default threshold. In Kolaskar and Tongaonkar antigenicity prediction, the default threshold score of antigenicity was set to be 1.024, fifteen immunogenic epitopes passed the test, and out of all, only eight have passed beta-turn and hydrophilicity prediction tools. Hence, fifteen linear conserved surface antigenic epitopes were passed . HoweverWe found that 213 conserved epitopes interacted with different MHC-1 alleles. Among the core epitopes, YVYDTRGKL was noticed to be the dominant binder as judged by its interaction with 9 alleles , followed by LTFYRQGAF, RATPSLVSF, and FTQLVAAYL that interact with 7 alleles for each . It is followed by FYRQGAFEL and FFGGKVLNF which are believed to bind with 10 alleles for each , >20 hours in yeast (in vivo), and >10 hours in Escherichia coli (in vivo). The instability index was computed to be 35.99, which indicates the thermostability. The aliphatic index and the GRAVY value of the vaccine were determined 85.80 and -0.416, respectively. The high aliphatic index shows that the vaccine is stable in a wide range of temperatures, and the negative GRAVY value indicates vaccine hydrophilicity and has better interaction with the surrounding water molecules. The amino acid composition is shown in Supplementary Figure The protein length was found to be 773 amino acids. MW and pI parameters were calculated as 85.69 kDa and 5.12, respectively. The pI value indicates that the protein is acidic in nature. The total numbers of negatively and positively charged residues were 124 and 98, correspondingly. The extinction coefficient of vaccine at 280 nm was measured 67520 MThe population coverage test was performed to compute the world coverage of epitopes that bind to separate MHC-I alleles, MHC-II alleles for each, and combined MHC-I and MHC-II and to sort out the most predominant promising epitopes for each coverage mode through the IEDB population coverage analysis tool.Three epitopes had exhibited the highest coverage percentage  in the isolated MHC-I mode. The maximum population coverage percentage over these epitopes was found to be 60.93% for YVYDTRGKL . In the Three epitopes had exhibited the highest coverage percentage . The most abundant coverage percentage of these epitopes in the world was granted to FFGGKVLNF with a percentage of 98.20%. For the top three coverage epitopes, the coverage percentage together was found to be 99.77% .Cryptococcus neoformans var. grubii and the sequential location of FYRQGAFEL were a promising MHC-I and MHC-II epitope, with massive population coverage, within the 3D structure of heat shock 70 kDa protein , seven discontinuous B-cell epitopes, and six immunogenic MHC-I and MHC-II epitopes  that are proposed to be used in epitope-based vaccine designing.C. neoformans cell burden from the CNS of rabbits and pulmonary fungal growth clearance in infected mice [Candida, Hsp60 in Histoplasma, and Hsp70 in Schistosoma [in silico and in vivo integrative approach on Hsp70 of Trypanosoma cruzi and found that four immunodominant epitopes  were assayed for their recognition by CTL of HLA-A∗02:01 and T. cruzi-infected transgenic B6-A2/Kb mice. Of those, TLQPVERVL and RIPKVMQLV were also recognized by CTL of HLA-A∗02:01 Chagas disease patients, indicating that these peptides are processed and displayed as MHC-I epitopes during the natural history of T. cruzi infection. Khalil's group [Cryptococcus neoformans var. grubii using an immunoinformatic approach and found three potential MHC-I and MHC-II epitopes for each  and  correspondingly and four promising B-cell epitopes . Nooney et al. [C. albicans which is conserved with the corresponding protein in C. neoformans. The study concluded that mycograb and amphotericin B can act in a synergistic fashion against multiple Candida species and C. neoformans clinical isolates. Khan and coworkers [Cryptococcus neoformans' immunogenic proteins and utilizing the promising antigenic epitopes in peptide vaccine designing. A therapeutic vaccine is able to prevent reactivation and is effective in the setting of established cryptococcosis [The importance of Hsp70 family proteins as stand-alone immune response modulators is a widely held view since it prolongs the survival rate of the animal model by decreasing ted mice –23, 68. istosoma –29. Maraistosoma  conductey et al.  reportedoworkers  identificoccosis , 73, 74.C. neoformans field. For example, killed vaccines have generally been ineffective and some have enhanced infection. Live vaccines using attenuated mutants have been shown to induce stronger, longer-lasting immune responses in those immunocompetent [Haemophilus influenza type B, and Streptococcus pneumonia has shown the safety and effectiveness of this approach [C. neoformans in mice which represent a potential vaccine candidate. Patients with suppressed T-cell responses will undoubtedly suffer from reduced memory responses and disease relapses, rendering conventional vaccine strategies useless. Hence, implementing novel combined T-cell and B-cell vaccines that have the potential to mediate protective immunity against C. neoformans would improve the quality of life of immunocompromised patients [An overview of a cryptococcal vaccine has already been provided in a perspective review article by Ueno and coworkers , extensiompetent –78. Howeapproach . Severalapproach , 73. Upaapproach  found thpatients  and provpatients . NonetheG binding value, the interaction between epitope and HLA can also be studied by analyzing the intermolecular interaction between them. The 2D interaction analysis revealed that more hydrogen bonds were present in FYRQGAFEL–HLA-C∗12:03 than in FYRQGAFEL–HLA-DRB1∗01:01 complexes. However, the ΔG binding value for FYRQGAFEL–HLA-C∗12:03 has shown a more negative value. This is maybe due to the inequality of hydrogen bonds and the contribution of other intermolecular interactions, which depend on the atom distances and angles  that might possess the therapeutic and prophylactic potentials to combat lethal cryptococcosis in immunocompromised patients. Accordingly, this in silico vaccine is expected to be the future epitope-based peptide vaccine with potential immunogenicity that is able to stimulate desirable immune responses against all strains of Cryptococcus neoformans var. grubii with massive global population coverage. Therefore, we recommend assessing its in vitro and in vivo antigenic potential through experimental validation.Cryptococcosis is a serious global problem concerning morbidity and mortality in immunocompromised individuals. Unfortunately, the unavailability of vaccines and the failure of antifungals against cryptococcosis have led to affecting many precious lives in various regions of the world. This