Treponema pallidum, has seen a resurgence over the past years. T. pallidum is capable of early dissemination and immune evasion, and the disease continues to be a global healthcare burden. The purpose of this study was to design a multi-epitope immunogen through an immunoinformatics-based approach. Multi-epitope immunogens constitute carefully selected epitopes belonging to conserved and essential bacterial proteins. Several physico-chemical characteristics, such as antigenicity, allergenicity, and stability, were determined. Further, molecular docking and dynamics simulations were performed, ensuring binding affinity and stability between the immunogen and TLR-2. An in silico cloning was performed using the pET-28a(+) vector and codon adaptation for E. coli. Finally, an in silico immune simulation was performed. The in silico predictions obtained in this work indicate that this construct would be capable of inducing the requisite immune response to elicit protection against T. pallidum. Through this methodology we have designed a promising potential vaccine candidate for syphilis, namely Tpme-VAC/LGCM-2022. However, it is necessary to validate these findings in in vitro and in vivo assays.Syphilis, a sexually transmitted infection caused by the spirochete  Treponema pallidum subspecies pallidum, which belongs to the genus Treponema. Other pathogens from the genus cause various other non-venereal infections, such as endemic syphilis , yaws , and pinta (T. carateum) [T. pallidum subsp. pallidum is a spiral-shaped, slow-growing, obligate human pathogen with a genome size of approximately 1.14 Mb, a protein count of 967, and a GC% content of 52.8%. Long term culture methods for cultivating T. pallidum have only recently been developed, and require co-culture with Sf1Ep cottontail rabbit epithelial cells [Treponema pallidum subsp. pallidum can also cause congenital syphilis in infants through vertical transmission, known as mother-to-child transmission (MTCT). The infection occurs during gestation and can lead to fatal infections in neonates [Syphilis is a sexually transmitted infection (STI) caused by arateum) . T. pallal cells . Treponeneonates .Syphilis continues to be a worldwide healthcare burden. Even though the infection is easily identifiable and treatable, it is endemic in developing countries and on the rise within selected populations, specifically, men who have sex with men (MSM) and female sex workers (FSW), particularly within the HIV-positive population in developed countries . AccordiCurrently, Africa, the Americas, and the Western Pacific are the regions most afflicted by incident cases . Of thosWhile syphilis can still be treated with penicillin with no occurrence of penicillin-resistant strains, there have been cases of macrolide-resistant strains, such as Azithromycin . The disT. pallidum vaccine could be viable, but the immunization procedure demonstrated then was untenable for human application [T. pallidum [Proof-of-principle for successful syphilis vaccination in the rabbit model was established in 1973, indicating development of a pallidum . In thisModern computational biology techniques, such as reverse vaccinology (RV) and immunoinformatics, have proven to be powerful tools for reducing resources and minimising the time typically spent in developing vaccines . The usaT. pallidum genomes identified potential vaccine targets in the core genome of T. pallidum sequences. Since these are core proteins, which are present in all T. pallidum strains, and were predicted to be surface-exposed, they are likely to have high expression levels across multiple strains and high immunogenic potential [In order to search for and determine epitopes capable of eliciting an immune response from the host, 15 proteins, previously determined to be potential vaccine targets through reverse vaccinology, were selected from the work of Jaiswal et al. 2017 . Their aotential . In addiotential . Three ootential ,17. The For the cytotoxic T lymphocyte (CTL) epitope prediction, in an effort to improve the confidence in the selected epitopes, two different platforms were used, both of which were used to predict nine amino-acid residue long sequences. The Immune Epitope Database and Analysis Resource (IEDB-AR) is robust and has multiple epitope prediction tools . The majWe also applied a tool to assess antigenic processing and transportation. This tool, NETCTL-1.2, uses the ANN and SMM methods to perform predictions. All of the allele supertypes  , which wTo predict MHC-II epitopes for binding to Helper T Lymphocytes (HTL), we used two high-quality predictors  to identWe have used the ABCpred tool, an ANN-based tool for the prediction of linear B-cell epitopes, using the default parameters .We run an in-house python script to determine shared epitopes between the two MHC-I and MHC-II prediction methods and select the predicted epitopes with high confidence between the prediction methods. Finally, we run the script to select overlapping epitopes between MHC-I and B epitopes and MHC-II and B epitopes, that is, epitopes capable of inducing cellular and humoral immune responses. In this case, the window was 2–9 residues for CTL epitopes and 2–15 residues for HTL epitopes. Only the epitopes that showed overlaps in the two cases were selected for further analysis. In order to filter down the number of epitopes and carefully define the final structure, further filtering steps were performed: MHC-II epitopes were filtered based on IC-50, keeping only epitopes with IC50 of up to 50 nM . MHC-I eTo determine the final sequence of the chimeric protein, the final epitopes were merged using appropriate linker peptides, the purpose of which is to assist in protein folding and processing. CTL epitopes were linked by AAY linkers, and MHC-II epitopes were joined by GPGPG linkers . The selThe final chimeric protein structure was subjected to several analyses to answer important questions regarding its induction of immune response, allergic and toxic potential, and physico-chemical properties. First, VaxiJen was used to assess the antigenic capacity of the amino acid sequence through the automatic cross-covariance method, evaluating the physico-chemical properties of the protein and predicting its immunogenicity without performing alignments . EpitopeThe molecular mass, theoretical pI, extinction coefficient, aliphatic index, grand average of hydropathicity (GRAVY), estimated half-life for three model organisms , and instability index of the final protein sequence were evaluated through the ProtParam tool . The solThe PSIPRED prediction tool was used to determine the secondary structure of the final protein. This tool makes use of a complex ANN and Position-Specific Scoring Matrix (PSSM) based approach to predict the structure and generate pictures . The RapWe used Phyre2 intensive model, RaptorX, and I-TASSER servers to select the best structure for tertiary structure prediction. The Phyre2 intensive method consists of multiple alignments of the sequence of interest with homologous sequences, followed by secondary structure prediction with PSIPRED. Information from these two steps was combined to determine a hidden Markov model. A search for this model was performed in an HMM database of proteins with known structures, and the model with the best score was used to determine modelling and correct errors . RaptorXConformational epitopes are indispensable in stimulating immune responses. The refined structure was submitted to ElliPro to predict these discontinuous epitopes .+ program [iG) of the final selected complex [The Toll-like receptor 2 was identified as a vital receptor in detecting T. pallidum infection and assembling an effective immune response against the pathogen . We retr program . The PDB complex .We performed the molecular dynamics simulation using the Gromacs v5.0 program  to enhanE. coli expression system. For this purpose, the JCat tool was used for reverse translation. From that cDNA sequence, the codon optimisation for E. coli k12 was performed, returning the Codon Adaptation Index (CAI), which must have a score higher than 0.8, and the GC content should be between 30–70% [® software  accessed on 1 October 2021, where the sequence of the chimeric protein was inserted in the pET-28a(+) plasmid with the help of Blpi and BamHI restriction enzymes.In silico cloning was performed to verify the capacity of cloning and expression of the protein in an appropriate expression vector. For this, the codon usage of our peptide sequence was adapted according to the codon usage of the n 30–70% . SimulatWe used the C-ImmSim server to run an immunological simulation to enhance the description of the immune response outlined by the chimeric protein . The in Epitopes were predicted using the IEDB-AR database, yielding epitopes that can be recognized by MHC-I alleles with high frequency in the global population for all 18 proteins under analysis. We also submitted these proteins to the NETCTL 1.2 server to improve the confidence of the chosen epitopes. These epitopes had the size of nine amino acid residues.MHC-II epitopes were predicted through the IEDB-AR and netMHCII 2.3 tools. IEDB-AR epitopes were predicted to bind to the most common MHC-II alleles, according to the IEDB-AR database. These epitopes had a size of 15 amino acid residues.The protein sequences were submitted to the ABCpred tool, and 16-mer epitopes, with scores higher than 0.51, were predicted for all 18 proteins according to the ability to interact with B lymphocyte receptors.High confidence epitopes were selected based on their overlap between the two methods for each category, MHC-I and MHC-II, for each protein at a time. Finally, epitopes capable of inducing both humoral and cellular responses were selected according to the overlap between each category and B epitopes. The number of overlapping MHC-I and B epitopes was 729. The number of overlapping MHC-II and B epitopes was 521. These epitopes were subjected to the next screening methods. After the IC-50 screening, 112 MHC-II/B epitopes fit the new threshold. When the immunogenicity screening was applied, 269 MHC-I/B epitopes had scores higher than 0.1. The remaining epitopes were subjected to a search for overlapping MHC-I and MHC-II epitopes, keeping 37 MHC-I epitopes and presenting sequences overlapping with 32 MHC-II epitopes. These epitopes were used to construct two different chimeric immunogens, which were then compared in regards to their overall population coverage and physico-chemical properties. The chimeric protein that was selected was composed of 11 MHC-I epitopes and 15 MHC-II epitopes belonging to ten different proteins . The finThe Treponema Pallidum Multi Epitope-Vaccine/Laboratory of Cellular and Molecular Genetics-2022 (Tpme-VAC/LGCM-2022) is composed of the following sequences: the cholera enterotoxin B subunit (ctxB) followed by the linker peptide EAAAK. Then, CTL epitopes linked by AAY linker peptides, followed by HTL epitopes linked by GPGPG linker peptides A. The chThe result of PSIPRED showed that, among the 558 residues of the sequence, there was an arrangement of 48% ɑ-helices, 15% β-strands, and 37% coil formation .Three different prediction methods were applied to find the model with the best structural quality. The highest quality model was constructed by the RaptorX server, the Ramachandran plot of this model showing 92.9% of residues in most favored regions . After sThe chimeric protein sequence was determined as a probable antigen according to the VaxiJen tool, with a score of 0.6852. The IFNepitope tool, using the SVM method, predicted 230 epitopes with positive and negative scores. Among these, 69 epitopes had a score greater than 1, considered more capable of inducing the production of this cytokine . The finE. coli. The protein’s aliphatic index, which is associated with stability in the face of temperature changes, was 83.89, the high indexes indicating greater stability. The grand average of hydropathicity (GRAVY) is 0.398, with positive values indicating hydrophobicity. According to Protein-Sol, the predicted scaled solubility was 0.378, a score that is lower than the solubility threshold, which is 0.45, related to solubility in E. coli, scores above it having higher solubility than the average. The pI according to this predictor was 9.520. According to the AllerTOP and ToxinPred tools, the chimeric protein sequence of the chimeric protein has shown no prospect of being allergic or toxic to humans.According to the ProtParam web tool, the theoretical molecular mass of the protein is 56,596.15 (56.59 Kd), and its isoelectric point (pI) is 9.03, indicating activity in a basic environment. The Instability Index (II), which is related to the stability of the protein, is 29.92, characterizing it as stable. Its estimated half-life in in vitro mammalian reticulocytes is 30 h, >20 h in in vivo yeast, and >10 h in in vivo iG of the complex to be −20.6 kcal/mol. A negative ΔiG value corresponds to hydrophobic interfaces, or positive protein affinity.Docking results were evaluated according to binding energy, number of members in a cluster, number of hydrogen bonds, and hydrophobic interactions. The binding energy between the chimeric protein with the TLR-2 receptor was −65.97, comprising ten hydrogen bonds. Three of the Tpme-VAC/LGCM-2022 residues involved in hydrogen bonds  belong to the adjuvant, while the remainder of the residues  belong to the selected epitopes. These bonds were formed between the vaccine construct and the extracellular portion of the TLR-2 chain. Moreover, 21 residues were involved in hydrophobic interactions, of which 8  belonged to the adjuvant, while the remainder  belong to the selected epitopes . PDBePISThe stability of the interaction between the best-docked complex was evaluated by molecular dynamics using GROMACS 5.0. In MD simulation protocol, the energy minimized structure was carried out in phases: equilibration under a constant number of particles, volume, and temperature (NVT) at 300 K, and a constant number of particles, pressure, and temperature (NPT) at 1 bar, during which the protein atoms and the solvent molecules were allowed to equilibrate around the protein molecule for 1 ns. The system was analyzed using root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF). The pressure plot indicated a fluctuation around 0.5 bar within the 1000 ps stabilization phase A. The teThe interaction was analyzed by RMSD, which reflects the complex’s structural stability. The RMSD plot shows a fluctuation ranging from 0.13 nm to 1.6 nm after an 80 ns time interval. This mild fluctuation indicates the stability of the complex during the tested time interval. To reflect the fluctuation of amino acid side chains, RMSF was analyzed. The RMSF plot shows mild fluctuations of RMSF values around 0.5, indicating uninterrupted interactions between receptor and ligand, and higher peaks with RMSF values around 2.0, indicating highly flexible loop regions in the complex C,D.E. coli k12 codon adaptation of the protein sequence, we obtained the reverse translated sequence for the protein. The GC content of the sequence was 56.45%, within the optimum range of 30–70%. The CAI index was 1.0 and within the allowed range. Using the SnapGene tool, we created BlpI and BamHI restriction sites to insert our Tpme-VAC/LGCM-2022 sequence into the pET-28a(+) plasmid vector. The complete length of the insert was 1680 bp  are generally regarded as promising candidates for vaccine targets in syphilis research [T. pallidum OMPome [The immunoinformatics strategy adopted in this study aimed to provide a multi-epitope immunogen consisting of epitopes selected from the core genome of the pathogen, thus ensuring its coverage of various strains and the scope of the displayed antigens. These epitopes were derived from eighteen research . Among tm OMPome .+ T-cell response, as well as the presence of macrophages and NK cells, whilst clearance of secondary legions is associated with a higher abundance of CD8+ T-cells. The induction of delayed-type hypersensitivity (DTH) and Th1-mediated opsonophagocitosis, which is induced mainly by CD4+ T-cells, is thought to be the main mechanism of clearance in syphilitic lesions. While this evidence points strongly to the importance of cell-mediated immune response in the response to T. pallidum infection, there is also evidence pointing that the humoral immune response is also essential. T. pallidum opsonophagocitosis has been shown to be dependent on the presence of Immune Patient Serum, highlighting the importance of the production of opsonic antibodies in the response to syphilis [During syphilis infection, the resolution of primary and secondary syphilitic lesions has generally been associated with cellular infiltrates of predominantly T-cells and macrophages. Clearance of primary lesions in humans is associated with a strong CD4syphilis .Aiming to design an immunogen capable of eliciting the desired response against the bacterium, epitopes were selected for their ability to induce CTL, HTL, and B-cell responses in various HLA supertypes, to stimulate both cellular and humoral immunity in a broad range of the global population. This careful selection of epitopes also allowed for the exclusion of potentially deleterious sequences for the construct, resulting in a multi-epitope immunogen that is non-allergenic, non-toxic, and non-homologous to any host proteins. One disadvantage of this approach is that, in contrast with whole pathogen vaccines, multi-epitope immunogens lack some antigenic determinants, requiring the addition of an enhancer as an adjuvant with strong antigenic properties . The choE. coli K12 expression model and cloned into an E. coli expression plasmid. Through modelling, molecular docking, and molecular dynamics simulations, the multi-epitope immunogen was determined to be able to form a strong, considerably stable binding to the TLR-2. TLR-2 is one of the main Toll-like receptors involved in T. pallidum detection by the host, indicating its capacity to activate the receptor. To enhance our understanding of the protein’s immunogenic capacity, an immune simulation was performed. The results indicated it to be a putative inducer of both the innate and adaptive immune systems, with wide differentiation of B-cell population, antibody production, and high activation of Th1 Helper T-cells. Both types of responses are vital to the clearance of active treponemes from syphilis wounds and limit the bacterium’s ability to spread within the host [The chimeric protein’s physico-chemical properties were determined to be within the acceptable parameters for recombinant protein production and application as a sub-unit vaccine. The sequence was optimized for expression in the the host . The immTreponema pallidum proteins, was constructed using an immunoinformatics-based approach. Several criteria were applied to select epitopes capable of inducing strong humoral and cellular responses while assessing the protein’s allergenicity and toxicity to construct a safe immunogen. The designed immunogen has suitable structural, physiochemical, and immunological properties that can successfully elicit humoral and cellular immune responses against T. pallidum. Finally, the in silico immune simulation enhanced our understanding of the capacity of the immunogen to elicit an immune response, highlighting that it is capable of inducing both humoral and cellular immune responses. This feature is vital in the development of a syphilis vaccine, as both the clearance of active treponemes in infectious sites and prevention of its ability to spread within the host are key for effective syphilis prevention. Therefore, a promising immunogen candidate for T. pallidum was designed, notwithstanding, to ensure immunologic efficiency and memory development. Experimental validation must, however, be performed in vitro and in vivo. Further validation of Tpme-VAC/LGCM-2022 may be performed through the recombinant synthesis of the immunogen construct in a vector expression system (pET-28a(+), for expression in E. coli). Once synthesized and purified, the construct can then be used in in vitro and in vivo immunization assays in the rabbit model before progressing to human studies.In this study, a novel multi-epitope immunogen (Tpme-VAC/LGCM-2022), comprising high-ranked epitopes from eighteen


Vascular grafts are widely used for vascular surgeries, to bypass a diseased artery or function as a vascular access for hemodialysis. Bioengineered or tissue-engineered vascular grafts have long been envisioned to take the place of bioinert synthetic grafts and even vein grafts under certain clinical circumstances. However, host responses to a graft device induce adverse remodeling, to varied degrees depending on the graft property and host’s developmental and health conditions. This in turn leads to invention or failure. Herein, we have mapped out the relationship between the design constraints and outcomes for vascular grafts, by analyzing impairment factors involved in the adverse graft remodeling. Strategies to tackle these impairment factors and counteract adverse healing are then summarized by outlining the research landscape of graft innovations in three dimensions—cell technology, scaffold technology and graft translation. Such a comprehensive view of cell and scaffold technological innovations in the translational context may benefit the future advancements in vascular grafts. From this perspective, we conclude the review with recommendations for future design endeavors. Vascular grafts are widely used for vascular surgeries. Their uses occur in a variety of forms, including vascular access for hemodialysis, as well as surgical bypass for diseased coronary artery, cerebral artery, or peripheral artery in the abdomen  or in the leg , routing blood flow around an area of blockage or narrowing. A vascular graft can be a native artery or vein, or fabricated from synthetic or natural biomaterials. The current gold standard for a graft is autologous blood vessels such as a saphenous vein, which has better clinical outcomes than biomaterials-based grafts , even thThus, bioengineered or tissue-engineered vascular grafts (TEVGs) have long been envisioned to take the place of synthetic grafts and even vein grafts under certain clinical circumstances. Selective TEVGs have gradually progressed into clinical trials, for both adult patients  and pediTo this end, this review starts with analyzing healing- or regeneration-impairment factors, which are tackled by recent graft innovations. To withstand or override the influences of impairment factors, pro-healing and/or pro-regenerative environments could be precisely defined and built into a graft through structural optimizations, signal molecule incorporations, and integration of bioactive materials, cells or cell products. As illustrated in In clinic, graft outcomes in the treatment of vascular diseases are often determined by the size and location of the devices as well as the patients’ developmental and health conditions, besides the graft type . Herein, we examined these graft design constraints and associated clinical outcomes, by analyzing relevant impairment factors such as flow-graft interactions, cellular and molecular environments around grafts, and graft bioinertness.Abnormal blood flow in the graft has long been known as a key impairment factor that alters graft healing and overall patency . ComputaThe graft diameter is key to reach a hemodynamic balance. Bypass grafts with unfavorable diameter ratios to the original vessel can result in competitive flow, ultimately leading to graft failure. Competitive flows occur when a partially stenotic blood vessel is bypassed with a graft, but still retains some of its throughput, thereby competing with the newly grafted path and causing irregular flow through it . A high The roles of graft compliance and length in determining the graft patency are also well known . CompliaThe graft failure in the cases of coronary bypass, dialysis access, or stent graft have been variably associated to age, diabetes mellitus, hypertension or other health conditions . Sex mayBesides comorbid diseases, the patency of a biological graft, compared to an inert synthetic graft, might be more influenced by cellular and molecular environments associated with older age. Older patient’s arteries tend to be more easily calcified making them more prone to abnormal shear stress, inflammation and other endothelial dysfunction . Due to Graft bioinertness, or lack of biointegration, is another major impairment factor for long-term graft patency. It is mainly related to commercial grafts such as those made of PTFE or Dacron, but could be variedly applicable to different TEVGs, particularly prior to the completion of vascular tissue regeneration. Previous review articles have elaborated the main issues with the graft inertness, which include thrombosis, inflammation and intimal hyperplasia of a graft. Infection can also be a serious issue , in partTo tackle above-mentioned factors involved in adverse graft remodeling, we summarize a three-thronged approach being taken in this field. A focused literature review of recent publications is taken to map out the research landscape of graft innovations in three dimensions, namely innovations in cell technology, scaffold technology and translation . Each rein situ or in vivo cellularization technique into the realm of cell technology.The contribution of cell technology to counteract adverse remodeling is to enhance graft acceptance and integration with native vessels, including attenuating chronic inflammation, reducing thrombogenicity, and promoting fast healing and vascular regeneration. The cell technology includes the technology to isolate, culture and use different types of cells for vascular graft applications, the technology to recruit cells into grafts, and the technology to produce cell products crucial for regeneration. Herein, a brief overview is taken on each cell type used for vascular grafting, highlighting recent innovations in tissue bank, immune-tolerant cells, and bioreactors. Recent cell technology has been expanded to include the use of cell secretomes, while on the other end adding a range of Autologous matured vascular cells, such as SMCs, endothelial cells and fibroblasts , were emin vitro, and consequently progenitor seeded-grafts produced more organized elastin when implanted in vivo as jugular replacements in lambs  have the potential of generating patient-specific SMCs, endothelial cells, and fibroblasts for TEVGs . The avain vitro often requires an incubation time ranging from days to months in a dish or in a sophisticated bioreactor. Bioreactor designs vary from a rotary bioreactor for stem cell differentiation  body as a bioreactor, added a novel option into the cell technology for vascular grafts. This approach starts with placing a tube composed of Teflon  Shortening the production time for cell sheet self-assembly method ; 2) loadBiopolymers used for the graft construction kept diversifying the graft repertoire. For instance, the recent addition of shape memory polymer as a self-enclosable external support to a graft reduced stenosis . CurrentBy and large, a long-term, dynamic, mechanistic view is taken into the design parameters and optimizations for vascular grafts, while continuous advancements in fabrication techniques and materials allow diverse choices and better controls over design parameters. A central finding in the graft optimization is that dynamically evolving mechanical properties can dictate the levels of inflammation and stress shielding of cells, and in turn affect mechanobiologically-mediated ECM production, remodeling and degradation . TherefoEvents immediately occurring upon vascular grafting in the body involve endothelial injury platelet activation, and acute inflammation. Properly regulating these early events is crucial to the fate of a graft, either promoting regeneration or preventing failure from thrombosis or intimal hyperplasia . Talacuain vivo  growth factors, (b) genes, (c) peptides, and (d) antioxidants. For in situ vascular regeneration could benefit from an exquisite graft design orchestrating the functions of multiple signaling molecules.One of the most adopted growth factors is VEGF . DespiteThe gene incorporation into a graft can also effectively enhance vascular regeneration. Genes such as miRNAs and vectors encoding VEGF receptor-2 ligands  were useThe antioxidant approach could address both regenerative need for a functional endothelium and therapeutic need for inhibiting neointimal growth or calcification, which is related to oxidative stress-caused cellular damage . A naturEssential to the future development is continual efforts to balance the mechanistic understandings underlying adverse graft remodeling in clinic with the innovations in grafting technologies. Translational innovations are as important as innovations in cell technology and scaffold technology. Assessment tests and preclinical models for vascular grafts are to be properly matched to the targeted clinical application , in termin vitro device tests also include biological function assessment. As TEVG improvements focus on the graft development by attending to the main causes of graft failure—thrombus formation and intimal hyperplasia, in vitro thrombogenicity tests as well as proliferation and migration tests of vascular cells have been widely employed.Related to tubular vascular implants, FDA guidance documents have specified testing criteria for a number of physical and mechanical properties of grafts, including porosity, burst strength, compliance, elastic modulus, water permeability, compliance, and kink diameter . VasculaThe goal of TEVG regeneration studies is to translate new findings to human clinical therapy, which relies on animal models to generate enormous data useful to the translation. Ideally, the selection of animal model considers the animal’s analog close to human patients in terms of similarities in anatomical, structural, pathological and/or functional aspects. Immunodeficient animal models further allow long-term accommodation of humanized vascular grafts . To evalA closer analog to clinical conditions allows one to better predict graft healing and regeneration in clinic. A huge discrepancy exists between endothelialization outcomes in animal models and those occurring in clinic . DespiteOverall, the design of future preclinical studies would better address the clinical relevance, for example, dialysis access  and mesoClinical trials are a true success measure and a determinant to the commercial value of any graft innovation. Prior trials have been performed on three types of grafts: Biological grafts , bioreactor-manufactured grafts, and readily implantable grafts. Though trial results are encouraging and enlightening for future TEVG strategies, the overall success is still limited.Biological grafts, referring to acellular blood vessels from human or animal  origin, have undergone clinical trials for decades. Bovine carotid artery grafts, such as those commercialized as Artegraft™, were used as arteriovenous fistula in patients undergoing chronic hemodialysis. These grafts outperformed PTFE grafts, though incrementally . LindseyBioreactor-manufactured grafts produce TEVG by culturing human autologous cells and/or allogeneic cells with or without a scaffold in a bioreactor with controlled physical, mechanical and/or biological environments. Clinical trials have illuminated promises and challenges with current methods. in vivo degradation, remodeling, regeneration and maturation. One recent approach is embedding a biotube in subcutaneous spaces of a patient for months, using patient’s body to fabricate a graft. This has been used successfully as a pulmonary artery substitute in pediatric patients (More readily implantable grafts are desirable in clinic, as they require minimal or no need of cell culture time, relying on patients . Anotherpatients . Researcin vitro studies and animal models. A combined approach employing both experimental and computational tools could significantly expedite the determination of optimal design parameters in a disease- or host-specific condition. From a perspective of clinical translation and graft commercialization, the considerations about costs and graft processing logistics become more and more important and are also highlighted in other recent reviews and studies. To address scientific questions to the point of clinical relevance, it is critical to highlight and increase the use of appropriate cell models, such as 3D cell models in diseased conditions, appropriate animal models, such as senescent animals, diseased animals, animals with slow vascular regenerative rates. Additionally, to more quantitatively and systematically understand the mechanisms, it would be helpful to standardize protocols such as sampling times across studies to obtain critical mechanobiological modeling inputs such as material degradation, inflammation, growth/remodeling/regeneration parameters.In conclusion, we reviewed the studies in the context of three innovations, i.e., cell technology, scaffold technology, and translational stages. Regenerative medicine has demonstrated great promises in the TEVG context. Future endeavors require the technological innovations to be coupled with translational innovations, in order to tackle the adverse remodeling of vascular grafts Also, it is expected that the adaptation and integration of bioengineered or tissue-engineered vascular grafts to human patients are heterogeneous because of cells’ varied ability to adapt to new microenvironments. A comparable case of graft heterogeneity in clinic is the use of vein grafts. Some veins are poor remodelers due to lack of adaptation, which had a 13-fold increased risk of failure at 2 years compared with “robust remodelers” . One facWe acknowledge several limitations of this review study, which include the lack of in-depth analyses and justifications of graft design, fabrication, biomaterials, and molecular mechanisms underlying graft failure and integration.


Shape memory elastomers have revolutionised the world since their introduction in the 20th century. The ability to tailor chemical structures to produce a family of materials in wide-ranging forms with versatile properties has propelled them to be ubiquitous. Recent challenges in the end-of-life management of polymeric materials should prompt us to ask, ‘what innovations in polymeric materials can make a strong case for their use as efficient materials?’ The development of smart elastomers that can acquire, convey, or process a stimulus  and reply by creating a useful effect, specifically a reversible change in shape, is one such innovation. Here, we present a brief overview of shape memory elastomers (SMEs) and thereafter a review of recent advances in their development. We discuss the complex processing of structure-property relations and how they differ for a range of stimuli-responsive SMEs, self-healing SMEs, thermoplastic SMEs, and antibacterial and antifouling SMEs. Following innovations in SEMs, the SMEs are forecast to have significant potential in biotechnology based on their tailorable physical properties that are suited to a range of different external stimuli. Smart materials, also known as intelligent or responsive materials, are a useful name for a broad group of different chemical substances. A common characteristic is that one or more properties may change significantly, even reversibly, in response to the specific stimulus input of preset form and extent over short or appropriate timescales and return to its original shape as the stimulus is taken away. The stimulus could be strain, stress, temperature, pressure, electric and magnetic fields, irradiation, or chemicals. Examples of smart materials include coatings that transform colour owing to the presence of chemicals, materials that have a shape memory at specific temperatures and paint that self-heals when it is scratched ,4,5,6,7.Shape memory polymers (SMPs) can memorise an original shape and can be tailor-made to some temporary shapes, whereas they naturally return to their original permanent shape from the temporary deformations upon simultaneous disclosure to various (even multiple) stimuli without additional mechanical effort. Typically, SMPs exhibit at least two phases: the first is a stable form that stabilises the SMP and is responsible for the retention of the original shape and the second that responds to the external trigger ,23,24,25Tg) –pyrimidinone motifs (UPyMA). After bulk damage, a great healing performance of two parts of a specimen was obtained after thermal treatment as shown in Chen et al.  presenteFurthermore, the indexes for quantifying the self-healing abilities were presented in Buaksuntear et al. (2022) . GeneralSMPs also include thermoplastic and thermoset  polymeric materials. Thermoplastic elastomer or TPE is classified as a category of thermoplastic polymer, although it is sometimes referred to as thermoplastic rubber because it is similar in its characteristics and performance to rubber ,54,88. TStyrene block copolymers (SBCs) are a type of thermoplastic elastomers, which include polystyrene–block–polyisoprene–block–polystyrene (SIS), polystyrene–block–polybutadien–block–polystyrene (SBS), and polystyrene–block–poly(ethylene–co–butylene)–block–polystyrene (SEBS) block copolymers. SBCs are commercially available and the required elastomer should be useful in many applications due to its shape memory effect capability such as under changing conditions . The required SEBS triblock copolymer was developed to induce intrinsic shape memory behaviour. The mechanism of SBCs’ shape memory effect is presented in b–amide) multiblock copolymers (PEBAs) are a type of thermoplastic elastomer (TPEs) due to their tuned characteristics with soft polyether (SSs) and crystalline aliphatic–amide (HSs). In 2018, Shibasaki and co-workers utilised monodisperse telechelic aromatic N–methylbenzamide-based molecules (MABx–x) with H2N–PEG–NH2 in high-molecular-weight multiblock poly(N–methylbenzamide)–b–poly–(ethylene glycol) polymers [poly(MABx–x–b–PEG)] using solution polycondensation to make moisture-responsive materials that contain hydrophilic PEG domains. The results revealed three thermal transitions: the glass transition (Tg) and melting (Tm) temperatures of the PEG segment and the Tg of the MABx–x segment. The shape memory behaviour was only obtained for the multiblock copolymer films with both crystalline PEG and aggregated monodisperse MABx–x phases. The co-crystalline structure was necessary for the stability of the temporary shape; the copolymer film returned to its original shape above the Tm of the PEG phase. After immersion of the film in water for 5 days, it swelled by decreasing the density. The film also exhibited moisture-responsive behaviour after exposing its surface to the humid air [Poly represents a two-phase separated structure that consists of soft and hard segments. Hu et al. (2021) reported that the hard segment via hydrogen bonding played an important role in shape recovery. However, the hydrogen bonding was always weak causing a decrease in the shape recovery effect of the TPU. Therefore, they presented a new type of diol chain extender with anthracene groups incorporated into the hard segment of the TPU to improve the shape recovery behaviour. The results seen in In another example, the shape memory behaviour of ethylene propylene diene monomer (EPDM)/polypropylene (PP) thermoplastic vulcanisates (TPVs) was developed by magnesium acrylate (MgMA). The MgMA reacted both to the PP and EPDM, which increased the compatibilisation of the EPDM/PP blend. The PP phase provided the temporary shape and the EPDM phase induced the recovery to the original shape. The interfacial compatibilisation between the EPDM and PP phases played an important role in the stress passage between the two phases. An appropriate amount of MgMA could be used to increase the crystallinity of the PP phase, which consequently contributed to the shape fixing of the TPV .Nowadays, the antibacterial and antifouling properties of a material are very interesting as elastomers can be degraded by environmental agents, such as mechanical stress, heating/cooling, chemicals, and hydrolysis under working conditions, causing the polymer to be vulnerable to bacterial contaminations or infections, which are a severe threat to humans. Antibacterial refers to anything that destroys bacteria or suppresses their growth or their ability to reproduce, whereas antifouling systems can be determined as the surface treatment on a substrate to prevent contamination by unwanted organisms ,90,91.ε–caprolactone) diol (PCL), 1, 4–butanediol (BD), and SRGO dispersion in DMAc were used with the desired amount of xylene. After that, TDI was slowly added to the mixture at room temperature. A tough HPU/SRGO SME represented the shape memory effects under sunlight and exhibited a tremendous enhancement of the mechanical properties. The nanocomposites also showed microbial inhibitory effects against Escherichia coli, Staphylococcus aureus, and Candida albicans. The HPU only possessed the shape memory property, whereas the SRGO had great energy absorbing ability and good thermal conductivity, which absorbed and then transferred energy to the HPU matrix efficiently. Therefore, the HPU/SRGO showed a great healing performance and the nanocomposites revealed fast self-healing behaviour [The hyperbranched polyurethane/sulfur nanoparticles decorated with reduced graphene oxide (HPU/SRGO) were prepared; poly(ehaviour .E. coli (Gram-negative bacterium) and S. aureus (Gram-positive bacterium). Pictures of the bacterial suspensions co-cultured with the cryogels are presented in E. coli and S. aureus due to the quaternary ammonium groups of QCS that were delivered to the bacterial cell membrane [In another example, Yao et al. (2022) presented shape memory and antibacterial cryogels for clinical application. A shape memory cryogel was obtained via mixing quaternized chitosan (QCS) and mesoporous bioactive glass (MBG) to obtain QCS/MBG cryogels. The results revealed that the mechanical properties depend on the degree of the interconnected macroporous structure. membrane .Petroleum is a fossil fuel that is usually used in transportation and industry; however, it generates global warming. Regarding the UN’s Intergovernmental Panel on Climate Change, petroleum use must cease by the end of the 21st century. Thus, the development of environmentally friendly intelligent materials from renewable sources is urgent.2 during petroleum consumption; however, it also has an appropriate glass transition temperature for SMEs. Polyfuranyl PLAs and polymaleimidyl linkers were reacted to form recyclable shape–memory elastomers. Four-arm hydroxyl-terminated poly(lactic acid) (4HP) and four-arm furanyl-terminated poly(lactic acid) (4FP) were synthesised by the linkers tris amine (TMEA) and hexamethylene dimaleimide (HDM). X-ray diffraction (XRD) and dynamic mechanical analysis (DMA) showed that the transition in the shape memory behaviour was induced by the crystallinity of the PLA moiety. The thermal properties of the crosslinked PLA samples were investigated, and the macromonomer 4HP represented the construction and destruction of a crystalline domain. The 4FP-TMEA also established a reversible characteristic in the PLA-based elastomer. These results revealed that recycling the elastomer is possible based on the Diels–Alder reaction observed in this system. These elastomers were also improved in their mechanical properties for disposable products [Poly(lactic acid) (PLA) can be applied to decrease the production of petroleum resources and the emission of COproducts .In this mini review, we have explored recent developments in processing approaches and the resulting achievable structures, behaviours, and properties of shape memory elastomers (SMEs) suitable for biotechnology applications. SMEs possess unique utilities in comparison to commodity polymers. SMEs are an exciting category of polymeric materials, with the potential to incorporate multi-functionality. Being elastomers, they are soft (low modulus) and can be deformed to large strains and yet recover almost completely. The shape memory aspect enables SMEs to respond to an external stimulus by producing a useful effect such as changing shape and recovering from damage . As trigger mechanisms for SMEs can be as creative as the chemical design of the SME, including temperature, pressure/stress, electromagnetic field, moisture/humidity, pH, and solvent, SMEs can be developed and optimised on an application-by-application basis, particularly for biotechnology. In some respects, SMEs can combine the robustness of polymers with the flexibility of hydrogel systems. Indeed, the good hysteresis behaviour and cycle life  of SMEs, in comparison to other shape memory polymers (SMPs) is an attractive quality for biotechnology. Moreover, the comparisons of SMEs and shape memory alloys (SMAs) or metals and ceramics were described. The main advantages of SMEs are that they are a soft material, have high flexibility, are lightweight, have good processibility, and have high shape transformations and good recovery compared to the SMAs. The disadvantages of SMEs are the weak recycling process and low thermal conductivity. However, SMEs can be utilised in medical applications ,95.Current research in SMEs seems to focus on developing material processes and technologies (without an application in mind at the outset) with limited work then going on to identify applications, develop prototypes, and further enhance and optimise the processes and properties. The latter is an important challenge that needs dedicated attention. In terms of the development of SME material technologies, research has principally explored (i) new and improved processes, (ii) concepts and mechanisms, (iii) material constituents/ingredients, as well as (iv) emerging structures, properties, and multi-functionality. For biotechnology applications of SMEs, we think that specific directions for research would be very welcome.In terms of processes, with the advent of low-cost 3D printing and its significant potential in biotechnology, exploring fused-deposition moulding processes with SMEs could be an exciting avenue. This could further help the introduction of specific architectures  to enhance the properties and tailor the form of the shape change  as well as produce precision parts.Tg).Following innovations in shape memory elastomers, new concepts and mechanisms could also be explored. For instance, elastomers with dynamic bonds formed through click chemistry or supramolecular chemistry or even the use of interpenetrating networks and co–networks could produce SMEs with a wider range of properties. The nature of these dynamic bonds is inherently amenable to multi-functionalities, such as self–healing, as well as being compatible with the inherent properties of elastomers such as high-failure strains. The use of dynamic bonds and supramolecular interactions is common in nature and could further enhance the dynamic properties, hysteresis, and recovery behaviour. A range of trigger mechanisms has already been explored in the literature. Furthermore, the programming process for the temperature in the static and dynamic methods in tension mode was used to quantify SMEs in order to find the optimal mechanical point and mechanical properties around the glass transition temperature , would be useful. In addition, although most research has explored one-way SMEs (being triggered by a single stimulus) that have one temporary shape, SMEs with a memory of two or more temporary shapes that can be triggered at different levels of a single stimulus (one-way SMEs) or even two stimuli (two-way SMEs) are of particular interest for biotechnology applications. For instance, these would enable a more complex set of action patterns for potential use in biomedical devices such as artificial muscles, soft robotics and deployable structures. An example of the latter is in minimally invasive surgery, where an SME device is to be activated in its temporary, compact shape through key-hole surgery and then stimulated to relax to its original, functional shape.SMEs can be tailored by altering the ingredients and constituents. Exploring SME composites comprising property-enhancing fillers and reinforcements of nano- to macro-scale is another avenue of further growth in the future. Notably, although the use of a filler may enhance certain properties , it may be at the expense of other properties  and hence these would need evaluation. Moreover, the addition of a filler creates an interphase and interfaces—the effects and enhancements of these also require attention.Finally, with regard to research on the emerging properties of SMEs for biomedical applications, their biodegradability and biocompatiblity  would be highly advantageous, for instance, to eliminate the need for additional surgery for the removal of a device. The principal challenges would be in designing and managing this multi-functionality, particularly with regard to the choice of stimulus and execution of trigger mechanisms at will or on demand, as well as balancing other physical properties (such as stiffness), and the rate and degree of recovery/reversibility, as well as the cycle time . Imparting durability and managing temporal changes (cycle life) could also be important for biotechnology applications such as in antibacterial and antifouling SMEs. Indeed, properties such as cycle time and cycle life are rarely tested or reported in the literature.Although the field of SMEs has a lot of growing to do, with several challenges and areas for further exploration, SMEs are in good shape to meet the required specifications for products in the ever-growing and diversifying markets of healthcare and fitness, ranging from personal health monitoring devices and fitness gear and comfort fit products to soft robotics, prosthetics/orthotics, and biomedicine.


Performing biopsy for intermediate lesions with PI-RADS 3 has always been controversial. Moreover, it is difficult to differentiate prostate cancer (PCa) and benign prostatic hyperplasia (BPH) nodules in PI-RADS 3 lesions by conventional scans, especially for transition zone (TZ) lesions. The purpose of this study is sub-differentiation of transition zone (TZ) PI-RADS 3 lesions using intravoxel incoherent motion (IVIM), stretched exponential model, and diffusion kurtosis imaging (DKI) to aid the biopsy decision process.A total of 198 TZ PI-RADS 3 lesions were included. 149 lesions were BPH, while 49 lesions were PCa, including 37 non-clinical significant PCa (non-csPCa) lesions and 12 clinical significant PCa (csPCa) lesions. Binary logistic regression analysis was used to examine which parameters could predict PCa in TZ PI-RADS 3 lesions. The ROC curve was used to test diagnostic efficiency in distinguishing PCa from TZ PI-RADS 3 lesions, while one-way ANOVA analysis was used to examine which parameters were statistically significant among BPH, non-csPCa and csPCa.p<0.001) and could correctly classify 89.39% of the subjects. Parameters of fractional anisotropy (FA) (p=0.004), mean diffusion (MD) (p=0.005), mean kurtosis (MK) (p=0.015), diffusion coefficient (D) (p=0.001), and distribute diffusion coefficient (DDC) (p=0.038) were statistically significant in the model. ROC analysis showed that AUC was 0.9197 (CI 95%: 0.8736-0.9659). Sensitivity, specificity, positive predictive value and negative predictive value were 92.1%, 80.4%, 93.9% and 75.5%, respectively. FA and MK of csPCa were higher than those of non-csPCa , while MD, ADC, D, and DDC of csPCa were lower than those of non-csPCa .The logistic model was statistically significant (χ2 = 181.410, FA, MD, MK, D, and DDC can predict PCa in TZ PI-RADS 3 lesions and inform the decision-making process of whether or not to perform a biopsy. Moreover, FA, MD, MK, D, DDC, and ADC may have ability to identify csPCa and non-csPCa in TZ PI-RADS 3 lesions. The pathological and clinical features of prostate cancer (PCa) vary in different anatomical regions of the prostate. Compared to peripheral zone (PZ) cancers, transition zone (TZ) cancers have lower Gleason scores, higher tumor volumes, higher prostate-specific antigen (PSA) levels, and are often confined to the prostate . AlthougMultiparametric MRI has been increasingly used to detect and locate tumors in patients highly suspected of PCa based on clinical examination results, such as PSA level and digital rectal exam. The recently updated version 2.1 of Prostate Imaging-Reporting and Data System (PI-RADS) guidelines have standardized the imaging acquisition and interpretation, providing more detailed principles for evaluating prostate lesions to reduce interreader variability .Generally, PI-RADS 1 or 2 lesions are considered as “negative” MRI, and have a negative predictive value of more than 90% for clinically significant prostate cancer  . In addiSeveral studies have shown that csPCa in PI-RADS 3 have higher PSA density, lower prostate volume, and apparent diffusion coefficient (ADC) value compared with benign lesions in PI-RADS 3 –14. UsinIn strict compliance with the evaluation criteria of PI-RADS V2.1, the present study attempted to differentiate PCa and BPH in PI-RADS 3 lesions in TZ with advanced diffusion-weighted imaging  and to analyze which parameters have potential value in predicting csPCa.This study was approved by the ethics committee (subheading on ethics committee incompleted). A total of 637 patients with an abnormal increase in serum PSA  underwent prostate MRI examination between December 2018 and January 2021. Inclusion criteria: patients with the highest PI-RADS category 3 lesions in TZ with a diameter larger than 10 mm. Exclusion criteria (1): patients underwent prostate biopsy (n=11) within 2 weeks before prostate MRI (2); the interval between prostate MRI examination and magnetic resonance imaging-Transrectal ultrasound (MRI-TRUS) biopsy >1 month (n=2) (3); pathology was not obtained by MRI-TRUS fusion biopsy (n=14) (4); the lesions were located at the base of the prostate so that biopsy specimens could not be obtained (n=2). Finally, 145 patients with 198 PI-RADS category 3 lesions located in TZ were included in the present study. Among 198 PI-RADS 3 lesions in TZ, 149 lesions were pathologically confirmed as BPH, while 49 lesions as PCa, among which 37 were non-clinically significant prostate cancer  and 12 were csPCa Figure 1b values  were used. IVIM were obtained with following parameters: repetition time msec/echo time msec, 3500–4400/87.5; section thickness, 3.6 mm; intersection gap, 0.5 mm; field of view, 28 cm×28 cm; matrix, 128×128; and 11 b values  were used. The details of MRI scan sequences and parameters are shown in All the enrolled patients underwent prostate MRI examination at 3.0 T MRI (GE Discovery MR 750W) with an 8-channel phased-array coil. Conventional prostate MR images, including axial T1-weighted imaging, axial T2-weighted imaging, coronal T2-weighted imaging, sagittal T2-weighted imaging, diffusion-weighted imaging, and dynamic contrast-enhanced imaging, were obtained according to the scanning protocol recommended by PI-RADS version 2.1. DKI was obtained with the following parameters: repetition time msec/echo time msec, 3800–4600/87.4; section thickness, 3 mm; intersection gap, 0.5 mm; field of view, 28 cm×28 cm; matrix, 128×128; 15 directions and 3 f) are fit for a biexponential equation:IVIM model and its parameters of diffusion coefficient (D), pseudo-diffusion coefficient (D*), and perfusion fraction  and heterogeneity index (α) are fit by the following equation:b represents the signal intensity at a specified b value, S0 is the signal intensity based on b = 0 s/mm2. DDC is a measure of the rate of signal decay with various b values, representing mean intravoxel diffusion rates. The heterogeneity index (α) is the water molecular diffusion heterogeneity and related to intravoxel water diffusion heterogeneity . A higher α value indicates low intravoxel diffusion heterogeneity, which approaches pure monoexponential decay. Conversely, an alpha = 0 indicates a higher degree of multiexponential signal decay  and mean kurtosis (MK) are obtained. The parameter MK represents the apparent diffusional kurtosis, and MD is the diffusion coefficient that is corrected to account for the observed non-Gaussian behavior.Where SAccording to PI-RADS version 2.1 (3), we screened the PI-RADS 3 lesions strictly located in TZ Table 2.2 were randomly drawn at the maximum 3 slices of each PI-RADS 3 lesion in TZ. The average value was recorded and used for data analysis. Detail showed in Acquisition of quantitative data was carried out on GE AW 4.6 workstation. The two radiologists  depicted every region of interest (ROI) separately on related parameter maps of IVIM, stretched exponential model, and DKI. Nine ROIs with area of 50 mmThe BK transperineal MRI/TRUS fusion biopsy system  was used for all biopsies. Three target biopsy cores were taken from each target lesion before the systematic biopsies Figure 3All the pathological samples were separately evaluated by two pathologists  who were blinded to MR imaging results. In case of deviation in the results, an agreement was reached. The criteria for pathological evaluation were based on ISUP recommendations in 2014 . The patp-value< 0.05 was considered statistically significant. All analyses were performed with SPSS .One-way ANOVA was used to test whether the clinical characteristics of patients were statistically significant. In order to examine the association between parameters of DKI, IVIM, and stretched exponential model with the probability of PCa in PI-RADS 3 lesions, binary logistic regression analysis was applied. After binary logistic regression analysis, the receiver operating characteristic (ROC) curve of combination parameters, which were statistically significant in binary logistic regression analysis, were analyzed to test the diagnostic efficiency of detecting PCa in PI-RADS 3 lesions. One-way ANOVA was used to analyze whether parameters of DKI, IVIM, and stretched exponential model was helpful in differentiating BPH, PCa, and csPCa in PI-RADS 3 lesions. A p >0.05), respectively. The average T-PSA of BPH patients and PCa patients was 6.17 ng/ml and 8.21 ng/ml (p >0.05), respectively. The detailed clinical characteristics are shown in Among 198 PI-RADS category 3 lesions, 75.25% (149/198) lesions were pathologically confirmed as BPH Figure 4f of IVIM, DDC, and α of the stretched exponential model were included in the statistical analysis. Finally, the logistic model was statistically significant, χ2 = 181.410, p<0.001. The model could correctly classify 89.39% of the subjects. Among the nine independent variables included in the model, only FA (p=0.004), MD (p=0.005), MK (p=0.015), D (p=0.001), DDC (p=0.038) were statistically significant. Odds ratio (CI 95%) were 3.165 (2.169-8.645), 0.158 (0.008-0.427), 2.886 (1.365-7.491), 0.185 (0.019-0.382), 0.221 (0.053-0.917), respectively , MK, MD of DKI, ADC, D, D*, ectively Figure 6-3mm/s, 0.123×10-3mm/s, 0.063×10-3mm/s, 0.173×10-3mm/s and 0.330×10-3mm/s, 0.328×10-3mm/s, 0.243×10-3mm/s, and 0.434×10-3mm/s, respectively.Results of one-way ANOVA analysis showed that parameters FA, MD, MK, ADC, D, DDC were statistically significant Table 5.Our study included 198 PI-RADS category 3 lesions located in TZ, where the detection rate of non-csPCa was 18.69%, and the detection rate of csPCa was 6.06%. In our study population, 23.45% of patients were non-csPCa, and thus required active surveillance at the time of initial biopsy according to NCCN guidelines. In addition, we detected 8.28% of csPCa requiring active treatment. Hansen et al.  found th-3mm/s, 0.662×10-3mm/s, 0.989×10-3mm/s) were significantly lower than that of BPH , which is consistent with the results of previous studies . Usually, lesions with these manifestations are assessed as PI-RADS category 4 or 5. However, in PI-RADS 3 lesions, detection of PCa using conventional multiparameter prostate MRI can be challenging. Our results showed that combining quantitative parameters FA, MD, and MK of DKI, parameter D of IVIM, and parameter DDC of the stretched exponential model could effectively identify PCa from PI-RADS 3 lesions. D value, DDC value and MD value of PCa (0.583×10 studies –25. D, D studies . In our  studies , 26–28.  studies , 15. How2 and ADC value of<1000 mm2/sec yielded an AUC of 0.91 for csPCa. Moreover, Hermie et al. (For TZ PI-RADS 3 lesions, our diagnostic model, built with parameters FA, MK, MD, D, and DDC, showed that AUC for detection of PCa was 0.9197. Sensitivity, specificity, positive predictive value and negative predictive value were 92.1%, 80.4%, 93.9% and 75.5%, respectively. Felker et al.  examinede et al.  showed te et al.  examinedActive treatment is needed for csPCa . Screenif had no statistical significance among BPH, non-csPCa, and csPCa. On the contrary, Beyhan et al. (f of PCa in TZ was significantly lower than that of BPH. In addition, D* of PCa in TZ was also significantly higher than that of BPH, which might be because their study population was smaller than ours. Moreover, Oto et al. (f cannot distinguish BPH, PCa, and csPCa. Our results showed that α wast no statistical significance, which is consistent with the previous studies (Our results revealed that D* and n et al.  studied o et al.  have fou studies , 37. On  studies  have fou studies . These cThe present research has some limitations. First, in our study, the diameter of the lesion was larger than 10 mm. Therefore, we do not know whether our results are applicable to lesions with a diameter that is less than 10 mm. Second, our sample size was small, especially csPCa, which only included 12 patients. Third, this was a single-center study, and the study results may be biased. In order to make the study results more robust and generalizable, multi-center and large sample studies may be needed to confirm our current results.In the summary, the relevant parameters of IVIM, stretched exponential model and DKI were used to establish the model, in which FA, MD, MK, D, and DDC can predict PCa in TZ PI-RADS 3 lesions and inform the decision-making process of whether or not to perform a biopsy. Moreover, FA, MD, MK, D, DDC, and ADC could differentiate csPCa and non-csPCa in TZ PI-RADS 3 lesions.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethics committee of the second hospital of Dalian Medical university. The patients/participants provided their written informed consent to participate in this study.K-PZ, H-BH, CB: these authors contributed equally to this work and share first authorship. JB: this author is the corresponding author. Q-YL: this author is the co-corresponding author and contributed equally to this work and share corresponding authorship. All authors contributed to the article and approved the submitted version.


Stiffness is a fundamental property of living tissues, which may be modified by pathologies or traumatic events but also by nutritional, pharmacological and exercise interventions. This review aimed to understand if specific forms of exercise are able to determine specific forms of tissue stiffness adaptations. A literature search was performed on PubMed, Scopus and Web of Science databases to identify manuscripts addressing adaptations of tissue stiffness as a consequence of long-term exercise. Muscular, connective, peripheral nerve and arterial stiffness were considered for the purpose of this review. Resistance training, aerobic training, plyometric training and stretching were retrieved as exercise modalities responsible for tissue stiffness adaptations. Differences were observed related to each specific modality. When exercise was applied to pathological cohorts (i.e. tendinopathy or hypertension), stiffness changed towards a physiological condition. Exercise interventions are able to determine tissue stiffness adaptations. These should be considered for specific exercise prescriptions. Future studies should concentrate on identifying the effects of exercise on the stiffness of specific tissues in a broader spectrum of pathological populations, in which a tendency for increased stiffness is observed. DiffTissue stiffness has been recently discovered to possess a role in orienting cell division, maintaining tissue boundaries, directing cell migration or driving differentiation , 6. ThisSince strain and stress may vary considerably in vivo, measuring tissue stiffness is particularly challenging. To date different methods are employed to measure tissue stiffness, ranging from myometers which are mainly employed for musculoskeletal purposes for evaluating myofascial stiffness, a condition frequently associated with pain , to ultrAll the above-mentioned disorders and the methods developed to measure stiffness imply a modification from a physiological to a pathological condition. Nevertheless, there are different therapeutic strategies employed to reduce tissue dysfunction and reverse stiffness adaptations. These may involve both pharmacological and non-pharmacological interventions, for example, beta-blockers or ACE inhibitors  and nutrAnother important implication regarding tissue stiffness is shear displacement of connective tissue. This latter wraps muscle fibres and muscle fascicles and during muscle contraction the rate of force development changes in relation to geometrical modifications of the connective tissue . It has Despite exercise interventions being currently used to effectively treat pathological conditions or to improve health outcomes, there is still a lack of knowledge about the physiological adaptations induced by different exercise regimes for specific tissue stiffness modifications and therefore which should be the appropriate exercise modalities adopted. Therefore, the current review aims to clarify aspects related to changes in tissue stiffness as a consequence of long-term physical exercise. In addition, different forms of exercise will be taken into account to identify specific exercise-induced tissue stiffness adaptations. Combined forms of exercises or acute protocols will not be discussed since these study designs would not allow the defining of a specific form of tissue stiffness adaptation.This article was conducted to narratively review studies that addressed changes in tissue stiffness as a consequence of long-term physical exercise. To retrieve eligible articles, a manual search was conducted on the following database: PubMed, Scopus and Web of Science. The search was conducted between the 1st of September 2021 and the 31st of October 2021. Articles published up to the 1st of September 2021 were included, with no limitation on the beginning of the search period. The following search strategy was adopted to identify eligible articles: (stiffness) AND ((aerobic) OR (resistance training) OR (stretching)) AND ((muscul*) OR (vascular) OR (nerv*) OR (tendon) OR (fascia) OR (connective)). This search was extended utilizing the bibliography within the recruited texts. Articles published in any language were considered. To summarize the findings of this review, we divided the review into subsections, each of which elucidated adaptations to a specific form of exercise . Main outcomes are reported in Table The effects of different exercise modalities have been classified according to the different targeted tissues, which are classified as muscular, tendinous (for the connective tissue), blood vessels and peripheral nerves. A synthesis of the studies can be found in Table Studies on muscle stiffness presented large variability in terms of protocols and assessment modalities. In addition, some studies also evaluated the effects of muscle tendon unit (MTU) properties. These differ in paradigms, since if MTU stiffness properties are assessed through ultrasonography, these may reflect aspects related to the muscular tissue , while eDespite consistent evidence regarding muscle adaptations to exercise being to date available, information pertaining to muscle stiffness after longitudinal resistance training (RT) protocols is scarce. A general overview highlights conflicting information, which also differs regarding exercise paradigms .Within the single analysis of the retrieved protocols, two studies have found no significant effects on different targeted muscles, using ultrasound SW elastography (SWE) , 32. TheAn eccentric hamstring training was tested by Uysal et al. in physically inactive individuals . The autA different exercise regime was administered by Santos et al. in order to assess maximal concentric or eccentric training effects on vastus lateralis muscle characteristics . SedentaDue to the contradictory results, the effects of RT on muscle are still unclear. Common points of all studies are that the relative intensity was greater or equal to 65%RM, while differences regarding contraction mode, volume, training frequency, duration of intervention and stiffness assessment techniques were present.Limited evidence exists regarding muscle stiffness adaptations to plyometric training –41. The There is concordance between studies despite different exercise regimes that plyometric training can increase the stiffness of the targeted muscles. However, further investigations are needed to confirm such findings.Only one study assessed the effect on muscle stiffness after a specific form of aerobic training (AT). This work was carried out during a mountain ultra-marathon (330 km) competition in Italy . ParticiThe lack of additional records regarding AT on muscle stiffness suggests that more studies with a longitudinal design are required to underline adequate conclusions about muscle stiffness responses.Many investigations evaluated changes in muscle stiffness after acute bouts of stretching. The overall results of these studies indicate that a reduction in stiffness is present following the stretching interventions, especially if performed at high intensity ; howeverHowever, few longitudinal interventions are present addressing outcomes related to stretching and stiffness. All the retrieved studies applied passive stretching interventions –52, exceIn detail, Blazevich et al.  applied From the analysed manuscripts, all stretching protocols pertaining to the plantar flexor muscles elicited a decrease in the levels of evaluated stiffness.The interventions of Marshall et al.   in an intensity driven direction . Such moIncreased tendon stiffness seems also linked to the duration of the interventions . HoweverWhen comparing measures of tendon stiffness across populations, it seems that independently of the type of sport, tendon stiffness is increased in athletes compared to controls , 67. IncOutcomes relating to RT and tendon pathology provide different results. Studies evaluating patellar tendinopathy found a general decrease in tendon stiffness following RT programs, performing either heavy loads or eccentric exercise , 72. TheOnly one study took into account the possible relation between tendon stiffness in runners and parameters regarding oxygen consumption, following RT . The stuThe retrieved studies regarding tendon stiffness indicate that RT increases stiffness in healthy subjects. Despite limited evidence available in pathological populations, exercise interventions have the potential to induce a stiffness adaptation towards a physiological condition.Few studies evaluated the effects of plyometric training on tendon stiffness , 74, 75.It is to be noted that the majority of studies also taking into account muscular aspects highlight an increase in muscle stiffness following this form of exercise, which may explain the incremental trend observed. Muscular aspects have been discussed in the Muscular Tissue section above.Similarly to adaptations of the muscular tissue, many stretching studies evaluate tendon tissue properties following acute bouts of exercise. It is not the aim of this review to evaluate protocols of such nature, also considering the enormous heterogeneity in the adopted protocols. Regarding longitudinal adaptations, three studies by Konrad and Tilp have applied a 4 × 30 s static stretching protocol , a balliLongitudinal interventions taking into account tissue stiffness following stretching in physiology or pathology are warranted since no conclusive evidence can be drawn from the currently included studies, which were all performed by the same research group.As discussed in the Muscular Tissue section above, different assessment techniques were adopted to evaluate MTU properties, which may reflect different intrinsic characteristics of specific tissues. In order to include information regarding such specific anatomical areas, all studies regarding MTU have been discussed below.Only three studies –81 evaluNo evidence was retrieved regarding adaptations of the MTU to RT performed with concentric contractions or other specific means of delivery. No clear effect regarding MTU stiffness properties following RT performed through eccentric contractions was present.Effects of MTU stiffness properties have also been described regarding stretching protocols. All the investigations analysed adaptations of muscles of the lower limb, in particular the plantar flexor muscles , 82–85 aThe overall results indicate, despite the limited number of manuscripts, that passive stretching decreases the stiffness of the MTU. Overall, the contradictory results pertaining to the muscular tissue suggest that stretching could act on muscle properties mainly at the level of the MTU rather than the muscle fibres.Studies taking into account stiffness of blood vessels have all been performed on arteries, and therefore, we will consider arterial stiffness and its measures (described in a subsequent paragraph) to determine blood vessel adaptations to exercise.Extensive literature has analysed the effects of RT on arterial stiffness, producing several systematic reviews and meta-analyses –91. DespI2 = 64%).However, common points of the analysed records are that high-intensity RT increases arterial stiffness, while low load may potentially decrease it, in both the elderly and young. The study of Beck et al. was the only one to also include pre-hypertensive patients  who undeThe effects of RT on arterial stiffness seem to be mainly influenced by exercise intensity, with low loads with the potential effect of decreasing stiffness and high loads with the potential effect of increasing stiffness. Encouraging results in hypertensive patients are observed after RT protocols. Different forms of AT were retrieved. These included walking, jogging, running, dancing and cycling on a cycle ergometer, using a non-weight-bearing air-braked ergometer.Despite the heterogeneity of exercise interventions, arterial stiffness in all cases decreased as a consequence of exercise. Findings were also confirmed by other systematic reviews and meta-analyses , 95.A walking and brisk running intervention was carried out for 16 weeks in healthy untrained participants . The int2 max for 45 min. As a consequence of the intervention, a decrease in brachial–ankle PWV was observed. The intervention was carried out on middle-aged adults who were classified as having a high or low fitness level according to their peak oxygen uptake. It is to be noted that no differences were observed between the two groups, suggesting the independent effect of the intervention on arterial stiffness.A cycle ergometer was adopted in two investigations , 99 and 2 Max and a HIIT  protocol also performed on a cycle ergometer consisting of 6–7 sets of 20 s cycling at an intensity of 170% of VO2 Max, both applied to untrained healthy young men. For both protocols, a decrease in carotid–femoral PWV was obtained . The second investigation [Two studies compared continuous aerobic exercise to intermittent exercise , 101. ThAnother form of AT was provided in the form of dancing . The actAn observational study evaluating the effects of lifelong exercise frequency (exercisers were defined as subjects who practised periods of aerobic exercise of at least 30 min per session) on arterial stiffness  highlighAlthough this review did not evaluate combined forms of exercise, it is of valuable interest to highlight that when combined forms of exercise (AT plus RT) are evaluated, and in particular, regarding the exercise order, a decrease in arterial stiffness is observed only when the aerobic activity follows the RT protocol . TherefoFour interventions took into account pathological cohorts  have been performed, both from the same research group, both concluding that a decrease is observed as a consequence of the stretching interventions. Both studies have applied static stretching to the gastrocnemius and the biceps femoris to target the sciatic and tibial nerves. Tissue stiffness was measured in both cases through SWE. The first study  providedIt is still premature to infer that exercise may be able to chronically modulate peripheral nerve stiffness; however, mechanical elongation seemingly has some effects. Future studies should consider these less investigated aspects since nerve stiffness increases either following incorrect postures  or in moThis review has highlighted that exercise may drive specific stiffness adaptations to tissues following a period of intervention. These findings have arisen mainly from healthy but also from clinical populations, despite evidence and intervention studies regarding these latter conditions being limited. However, we believe that further considerations are needed to understand possible tissue adaptations to specific conditions. For this reason, comparison studies between healthy and pathological individuals have also been evaluated and differences caused by specific pathologies arose. For example, in populations affected by musculoskeletal conditions such as low back pain –123, necOther examples can be also applied to vascular and nerve pathology. Patients presenting abdominal aortic aneurysm , heart fTissue stiffness adaptions have been also observed in other functional pathologies such as migraine  or urinaIn all above-listed pathological conditions, except for urinary incontinency, stiffness of the different evaluated tissues increased. It is possible that increased stiffness represents a homeostatic mechanism, mediated by inflammatory processes which increase the production by fibroblasts of extracellular matrix components , with thThis review aimed to identify the effects of exercise on tissue stiffness. We need to highlight that the term stiffness was adapted to fit the needs of each tissue since the assessment methods and the outcomes measured may have differed significantly. A common method adopted by studies pertaining to muscular or connective tissue to determine stiffness was indirectly through a calculation fitted within regression equations of the gradient of force and elongation of muscle fibres during a given task performed through isokinetic dynamometers . Through this, a differentiation between active and passive stiffness in some cases was also calculated, considering the difference between active and passive forces produced by a specific joint from a neutral to a different position and the related tendon or muscle length, and joint angle changes. Isokinetic dynamometry is considered the gold standard for joint stiffness assessment . Limitatβ-stiffness index, which can be measured through carotid ultrasound, which is used to detect the internal luminal diameter during end diastole and end systole of the carotid artery. A ratio is then performed between the ratio of systolic and diastolic pressure, multiplied by its natural logarithm and also by the difference between end systole and end diastole. The obtained number is finally multiplied by the end-diastole diameter [Other methods are employed to assess arterial stiffness. The most commonly employed is tonometry, which evaluates the time it takes for a pulse wave to travel between two points. These are usually the carotid and the femoral arteries, or the brachial and ankle arteries, to determine central and systemic stiffness, respectively. Knowing the distance between the two points, velocity is calculated, and based on tissue stiffness, the pulse wave velocity may increase (stiffer arteries) or decrease (softer arteries). Derivate measures, as those above described for the muscular tissue, can be calculated for arterial stiffness evaluation. An example is the diameter . Anotherdiameter .The differences between the assessment methods, as well as the heterogeneity for each exercise protocol within each form of exercise (i.e. (1) resistance training performed through concentric, eccentric or isometric contractions with high or low loads, (2) static, PNF or ballistic stretching and the velocity the stretches are applied, (3) continuous or intermittent aerobic activity) or differences between the analysed populations, prevent defining a dose–response adaptation or in some cases (i.e. stretching for connective tissues) the effect of the exercise interventions.Longitudinal studies taking into account measures of stiffness as the result of an exercise regime are also very limited.Specific tissue adaptations related to each exercise modality seem to be present. Resistance training increases tissue stiffness of tendons and depending on exercise intensity (high or low loads) may also increase or decrease arterial stiffness. However, the effects of resistance training on muscles and muscle–tendon units provide conflicting results related to different exercise modalities.Plyometric training was seen to increase the stiffness of the muscular tissue; however, no conclusive evidence could be drawn for tendons. No evidence regarding other tissues is to date present.Aerobic activity was investigated pertaining only to arterial stiffness, which decreases following the exercise protocols.Passive stretching decreases muscle tendon unit, vessel and nerve stiffness, while unclear effects are seen for muscles and tendons. The effects which were discussed are specific for healthy people.When analysing pathological populations, it seems that all forms of exercise drive adaptations towards a physiological stiffness state of the targeted tissues. Of interest, resistance training and aerobic training were able to decrease arterial stiffness in hypertensive patients. However, very limited evidence is currently available. Studies evaluating tissue stiffness adaptations using standardized assessment procedures, following specific exercise modalities, should be carried out. Further attention should be paid to peripheral nerves and pathological conditions.


Mycobacterium leprae and Mycobacterium lepromatosis. Most of the affected population lives in low-income countries and may take up to 10 years to show any clinical signs, which is how physicians diagnose it. However, due to progressive cell damage, early diagnosis is very important. The best way to confirm leprosy is through bacilloscopic, which only confirms the diagnosis and has low accuracy or PCR, that requires specialized operators and is expensive. Since the bacteria are fastidious and do not grow in any culture media, therefore, diagnosing leprosy in the lab is still a challenge. In this concern, a recombinant multi-epitope protein can be a beneficial strategy in the management of the diagnosis, as diverse immunogenic epitopes are precisely selected to detect specific antibodies. Therefore, the purposes of the present study were to select immunogenic epitopes from different relevant proteins, with immunogenic properties, and then to construct a recombinant multi-epitope protein that accuses the presence of the antibodies in the early stages of the disease, making it more than appropriate to be applied as a diagnostic tool.Leprosy is caused by We selected 22 common proteins from both species and, using bioinformatics tools, predicted B and T cell epitopes. After multiple filtering and analyzing, we ended up with 29 epitopes {MHC-I  and MHC-II } from 10 proteins, which were then merged into one construct. Its secondary and tertiary structures were also predicted and refined to comprise the amino acid residues in the best conformation possible. The multi-epitope protein construct was stable, non-host homologous, non-allergic, non-toxic, and elicit humoral and cellular responses. It has conformational B cell epitopes and potential to elicit IFN-γ, IL-4, and IL-10 secretion.M. leprae and M. lepromatosis has a huge immunological potential, is stable, and can be lyophilized to be used in ELISA plates or even in biosensors, which are user-friendly diagnosis tools, facilitating translation into human sample tests.This novel recombinant multi-epitope protein constructed using the common epitopes from The online version contains supplementary material available at 10.1186/s43141-022-00411-7. Mycobacterium leprae and Mycobacterium lepromatosis, which can invade Schwann cells [Only in 2019 the WHO  reported 202,226 new cases of Leprosy worldwide, with almost 80% of the cases in just 3 countries: India, Brazil, and Indonesia  [The immunological response in leprosy is highly dependent on the host’s genetic background, and it drives its clinical manifestations , 7. The M. leprae and are not sensitive enough, detecting only LL and symptomatic cases, but not PB [The leprosy diagnosis is mainly based on clinical and laboratorial evaluations. Due to the progressive cell damage, the early diagnosis is very important; however, most diagnoses are performed when there is already a significant nerve damage . The best not PB –15. The t not PB . Given tt not PB , 17.To try and stop the transmission of new leprosy cases, the WHO set a few objectives to be fulfilled from 2016 to 2020. One of the objectives was the development of a new diagnosis tool . Since mM. leprae and M. lepromatosis are not yet cultivable in any culture media [Bioinformatics tools are of the utmost utility to assess the immunogenic peptides within a protein, saving time, money, and even diminishing the use of animals, since it provides multiple filters before in vitro and in vivo tests are performed –25. Givere media , bioinfore media . Multi-ere media . Diseasere media , Chagas re media , cryptocre media , 30, tubre media , and toxre media  already The largest challenge of the multi-epitope diagnosis is to construct a chimeric protein with all the epitopes exposed to interact with the antibodies, as the coiling event predicted can be different in practice . FurtherM. leprae and the recently described M. lepromatosis were selected.There is an effective therapy against leprosy, the multidrug therapy (MDT) program ; howeverThe purposes of the present study were to select deeply immunogenic epitope proteins, with immunogenic properties or certified to be detected in diagnosis tests, and then to construct a recombinant multi-epitope protein that could be applied as a diagnostic tool. Advanced techniques in protein structure design and evaluation were performed to build a stable and safe multi-epitope protein. Also, in silico cloning was applied to arrange codon bias to get an idea about the capacity of the protein to be expressed.The complete workflow of the methodology used in this study is described in Fig. M. lepromatosis and M. leprae proteins ML0091, ML0405, ML1636, ML2055, ML2331, ML2346, and ML1556 were previously proved to detect leprosy at some level [The me level . ML2028,me level . The seqme level . The antme level . In totaThe epitopes able to bind to MHC I alleles and activate cytotoxic T lymphocytes (CTL) were predicted by two different platforms to improve the confidence of the prediction. The Immune Epitope Database and Analysis Resource (IEDB) contain thousands of high- and low-affinity epitopes used in training to enhance the accuracy of the predictor , 41. AimFor epitopes that activate helper T lymphocyte (HTL) (MHC II-binding epitopes), we also used two different predictors, IEDB tool  and NetMTo predict linear B cell epitopes, we used ABCpred , 48 whicin-house python script which compare the results from both programs for each epitope , and the instability index were analyzed through the final construct sequence using ProtParam [E. coli expression data.Molecular mass, theoretical pI, extinction coefficient, aliphatic index, grand average of hydropathicity (GRAVY), estimated half-life for three model organisms , three different programs were used, and the best 3D structure was chosen based on its structural quality. For the evaluation, PROCHECK was used through SAVES v6.0 , 59 to gTo enhance he local and global quality of the modeled 3D structure, we used GalaxyWeb Server which applies the methods for the refinement of amino acid side chains using light and aggressive relaxation approaches .The final construct sequence was analyzed for crucial aspects related to the induction of immune responses, toxicity, and allergenicity. We used VaxiJen to assess the antigenic capacity through the automatic cross-covariance method, thus analyzing the physical–chemical properties and predicting the ability to induce immune responses without the need to do alignments .The search for epitopes able to induce IFN-γ production was performed with the IFNepitope predictor, using MHC II epitopes. This predictor uses a SVM hybrid method based on motifs to perform the prediction . IL-4 anThe ElliPro web-based tool was used to predict conformational B cell epitopes from the refined predicted structure of our multi-epitope protein . These eE. coli K12 expression system’s codon usage through reverse translation. With the cDNA-optimized sequence, the codon optimization for E. coli k12 was performed, and it returned the Codon Adaptation Index (CAI), which must have a score higher than 0.8, and the GC content rate should be between 30 and 70%. Furthermore, to clone the final optimized gene sequence, we used the pET28a( +) vector obtained from the Addgene website (https://www.addgene.org/), with Blpi and BamHI restriction sites. Finally, the optimized sequence was inserted into the pET28a( +) vector using the SnapGene tool [To verify the capacity of cloning and expression of the multi-epitope protein in an appropriate expression vector, we performed in silico cloning. Using JCat, we adapted the codon of our peptide according to the ene tool  to ensurin-house python script, we searched for overlapping epitopes that were predicted by both programs, in order to find common epitopes, and it returned 227 B cell shared epitopes  and B (227) epitopes, with similarity of at least nine sequential residues. At this screening step, we reduced the total number of MHC II and B epitopes to 40 overlapping epitopes  and MHC-II } epitopes, from 10 proteins, were selected for the final recombinant multi-epitope construction , and no significant similarity was found.Host homology was performed through NCBI BlastP with The protein’s molecular mass is 46,779.35 (46.7 kDa), with a theoretical pI of 9.38, which means its behavior is at basic pH. The protein is considered stable since the instability index is 29.99. The aliphatic index is 91.79, also showing stability in changes in temperature. Positive great average of the hydropathy value (GRAVY) scores mean hydrophobicity, as our result is 0.489.E. coli solubility, and scores higher than 0.45 has a higher solubility average.The solubility analysis performed through Protein-Sol described a score of 0.382, which is lower than the tool threshold of 0.45. The threshold corresponds to The application of multi-epitope protein in cell-based in vitro platforms depends on its ability to be antigenic and to induce cytokine production. The designed protein had a predicted antigenicity score of 0.5596 through VaxiJen which means it is probably antigenic. The tool IFNepitope predicted two epitopes as probable IFN-γ inducer, and IL-4Pred and ProInflam predicted five and IL-10Pred four epitopes Table . ElliProBipI and BamHI were inserted in the expression vector pET28a( +) (Addgene) totalizing 6310 base pairs in the complete clone length  index is 1.0. Both are within the parameter range, which is important to measure the cloning and expression potential. In order to construct the cloning vector, through the SnapGene tool, restriction site sequences of the enzymes M. leprae as the focus of their studies. Here, we chose to develop a universal chimeric protein that can identify not only M. leprae, but also M. lepromatosis infection, increasing the chance of diagnosis.In fact, there is a successful treatment for leprosy. However, the damage caused by the disease, due to the lack of accurate and early diagnosis, causes irreversible damage which highlights the need for highly sensitive detection tools for rapid diagnosis and epidemiological surveillance of the disease . Also, iM. leprae and M. lepromatosis were never cultivated in axenic media [For being fastidious bacteria, ic media , which hM. tuberculosis Rv3810 and Rv0455c proteins, but, as it was described, no homology was found in our construct. On the other hand, ML2346 has no known homology [ML0091, ML2380, and ML2346 were first described by Cole et al. in 2001 . The firhomology , 59. As homology .M. leprae) by Duthie et al. [Duthie et al. tested ML0091 and ML2346 against the patients’ sera from Goiânia, Brazil, having a positive response in 71% and 29% of the cases, respectively . The proM. leprae and two strains of M. lepromatosis. Since the definition of the core genome is to be present in all strains analyzed, it makes our recombinant multi-epitope protein a good candidate to diagnose the disease caused by any of them.The other proteins used were predicted by our group  in a revGiven that diagnosis of leprosy is essential for treatment initiation and the earlier it begins, the better the response , many arM. leprae in guinea pigs and armadillos [T. cruzi TR proteins [Immunoinformatics is an in silico approach that helps in predicting epitopes that have a greater chance to be immunogenic; nevertheless, it may not be accurate if we take into consideration that proteins undergo unique biological complex processes driven by genetics to be presented as an epitope by a cell , 22, 71.madillos . As to oproteins ; Yin et proteins ; Ebrahimproteins .Most of these tests are directed to humoral immunity, which is one of our goals, since it is less expensive and has a good accuracy in LL and MB leprosy, where antibodies are more present –10; howeT cell interferon-gamma release assays (IGRA) were developed as an alternative for delayed-typed hypersensitivity tests for latent tuberculosis diagnosis, reducing false-positive results , 75. SinWith results in both the humoral and cellular response, this protein will be able to diagnose leprosy without much difficulty due to the detection of both ends of its spectrum. Other studies using immunoinformatics to construct multi-epitope proteins for diagnosis purposes had good results in silico, but they lacked sensitivity and specificity in ex vivo  or had sThis novel recombinant multi-epitope protein has a huge immunological potential, is stable, and can be lyophilized to be used in ELISA plates or even in biosensors, which are user-friendly diagnosis tools, facilitating translation into human sample tests.Additional file 1: Table S1. List of Mycobacterium leprae and M. lepromatosis proteins selected for the analysis with their antigenicity value predicted through VaxiJen. Table S2. Number of B cell epitopes. Table S3. The total numbers of MHC I epitopes. Table S4. The total numbers of MHC II epitopes. Table S5. Number of epitopes in MHC II and I filtering. Table S6. The Immunogenicity Scores of MHC I predicted epitopes. Fig. S1. PSIPRED secondary structure prediction.