The world is witnessing a global rise in numbers of emerging invasive alien species, but identifying which species pose a pandemic threat remains poorly understood. The disjointed international regulatory environment presents a significant challenge to biosecurity interventions at a global scale. A novel way forward is through One Biosecurity, an interdisciplinary approach to biosecurity policy and research that enhances the interconnections between human, animal, plant, and environmental health to prevent and mitigate the impacts of invasive alien species. One Biosecurity underpins three initiatives essential to deal with the pandemic risks from biological invasions: new risk assessment tools that look beyond national borders toward biosecurity risks of international concern, a stronger regulatory instrument to address biosecurity threats at a worldwide scale, and the establishment of a multilateral biosecurity convention responsible for biosecurity governance. Together, these initiatives will drive a new science and policy agenda to deliver evidence-based governance of global biosecurity.The global response to prevent and contain the spread of the SARS-CoV-2 coronavirus undoubtedly represents the most dramatic biosecurity response ever undertaken at an international scale. This response contrasts markedly with the more muted international and national biosecurity strategies to manage emerging pests, pathogens, and weeds that negatively affect livestock, crops, or native species. This relative underappreciation of the risks arising from biological invasions doubtless reflects the fact that the global economic costs of recent emerging infectious diseases of humans have been huge: $40 billion for SARS-CoV in 2002–2003, $55 billion for H1N1/09 (swine flu) in 2009, and $53 billion for the Ebola outbreak in 2014 . To qualify as such a threat, an event must meet at least two of the following four criteria: The impact must be serious, it should be unusual or unexpected, there should be a significant risk of international spread, and it could pose a significant risk to international trade or travel restrictions , the species is initially identified before any impacts are documented within the territory and these impacts may occur several years after the first arrival of the species . Data on potential impacts of an invasive alien species are often assembled from existing literature and risk assessment tools rely heavily on evidence of invasion elsewhere Hulme . The depThird, although the majority of risk assessment approaches addressing biosecurity threats evaluate the extent to which an invasive alien species might spread within a specific target region, they do not address PHEIC criterion 3 in relation to the likelihood that a species could subsequently spread internationally . Furthermore, apart from the SPS agreement, much of the emphasis of the management of pathways focuses on reducing the risk of importing invasive alien species rather than the risk of re-exporting these species to other countries. As a result, invasive alien species posing pandemic risks such as the Asian house gecko (Hemidactylus frenatus) that continues to spread to countries across five continents as a stowaway in shipping containers and luggage Hoskin  or the AThe value in declaring that an emerging invasive alien is a Biosecurity Risk of International Concern is strongly dependent on a regulatory environment that facilitates a suitable response to a pandemic risk. Once again public health initiatives may provide a useful model for such an international regulatory environment. The dramatic global response to the SARS-CoV-2 pandemic has in part reflected the effectiveness of the World Health Organization's (WHO) International Health Regulations (2005), a set of legally binding obligations, that commit the 196 contracting parties to build capacity in order to prevent, protect against, control, and provide a public health response to the international spread of disease Jenkins . In partIn contrast, legal instruments addressing biological invasions by invasive alien pathogens, plants, and animals are ineffective or insufficient at both the national and international levels, which has led to breaches in authority and poor enforcement capacity  to combat invasive alien species of global significance, generating forecasts of future global biosecurity risks under different climate or socioeconomic scenarios, unambiguously identifying an invasive species incursion as posing a pandemic risk requiring coordinated international action, verifying compliance with norms established under the convention, negotiating with industries associated with the dissemination of potentially invasive species  to limit such trade, instigating accredited training programs for biosecurity professionals, and mobilizing capability in times of crisis by assembling the International Biosecurity Expertise Network. This may appear a huge task, but there is already ongoing research to develop standards and protocols for global monitoring networks . This would then need to be followed up by projections of how these costs could increase in the future without the intervention of dedicated biosecurity convention to illustrate the considerable return on investment on a multilateral binding agreement. Statements regarding impacts should be put into a context that the public can relate to more personally and this means that global estimates may be less effective than national or regional cost estimates that affect the taxpayer. However, statements such as “biological invasions cost the global economy $2 trillion” while sounding impressive present the issue in abstract terms and most people find it hard to process such large sums and compare their relative value . One Biosecurity seeks the development of a suite of more holistic risk assessment tools for the comparative assessment of biosecurity threats across the human, animal, plant, and environmental health sectors. Invasion scientists need to work with veterinarians and epidemiologists to devise a standardized quantitative approach to rapidly assess actual and potential impacts of emerging invasive alien species across all sectors, rather than being sector specific  must recognize that the sectorial approach to managing these risks in ineffective and that a more holistic view is essential to address this problem figure . HoweverHow would the International Biosecurity Convention be implemented (step 6)? Today, biosecurity threats are managed through inspection of cargo, mail, commodities, and shipping vessels; surveillance of new species incursions; and, in some cases, black or white lists relating to trade restrictions of specific species Hulme , 2014. TFinally, the International Biosecurity Convention would need to be evaluated every 5 years (step 7) to ensure it remains fit for purpose. The criteria for evaluation could include progressive reduction in the vulnerabilities of countries to biosecurity threats  as their national capabilities improve, a reduced rate of successive incursions of high pandemic risk pests, pathogens, and weeds from country to country , greater international cooperation through the International Biosecurity Expertise Network to address invasive alien species and an expanded catalogue of invasive alien species for which biosecurity risk assessments have been undertaken. It is impossible to know precisely how long each of the steps toward implementation might take but the entire process would certainly take no less than a decade and likely longer. Therefore, scientists and policymakers working with biosecurity threats must be prepared to a long-term commitment to delivering One Biosecurity.There must be a better way to manage the increased threat of invasive alien species to human health, agriculture, and the environment worldwide. A novel way forward is through the application of One Biosecurity, an interdisciplinary approach to biosecurity policy, and research that builds on the interconnections between human, animal, plant, and environmental health to prevent and mitigate the impacts of invasive alien species more effectively. At an international level, One Biosecurity leads the way to a clearer focus on the pandemic risks of invasive alien species and provides an opportunity to learn from the successes and failures of similar initiatives targeting global public health. Such a comparison highlights that the global governance of biosecurity threats requires the implementation of three interrelated initiatives: an improved approach to risk assessment that looks beyond national borders toward the pandemic risk of an invasive alien species and identifies when it might become a Biosecurity Risk of International Concern; a specific international regulatory instrument, modeled on the International Health Regulations, to enforce proactive surveillance and response to biosecurity threats worldwide; and the establishment of a dedicated, multilateral biosecurity convention responsible for international biosecurity governance. None of these initiatives will be sufficient on its own. Developing effective tools to assess the likelihood that an emerging invasive alien species poses a Biosecurity Risk of International Concern underpins the deployment of international regulations committing nations to the monitoring and reporting of these risks. Once international regulations can be agreed and ratified by a sufficient number of nations, the International Biosecurity Convention could come into force and initiate the multiple activities expected of its mandate. However, the essential groundwork for these activities such as data management systems, gap analyses of biosecurity capability, improved regional coordination of biosecurity response should be in place beforehand. These activities would give a stronger global direction to research on biological invasions, bringing different sets of expertise together across different countries in a more coordinated and applied context. Building belief among scientists, industry, policymakers, and potential funders that there is a long-term strategy to manage biological invasions will be key.


The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has turned into a pandemic with about thirty million confirmed cases worldwide as of September 2020. Being an airborne infection, it can be catastrophic to populous countries like India. This study sets to identify potential cytotoxic T lymphocyte (CTL) epitopes in the SARS-CoV-2 Indian isolate which can act as an effective vaccine epitope candidate for the majority of the Indian population. The immunogenicity and the foreignness of the epitopes towards the human body have to be studied to further confirm their candidacy. The top-scoring epitopes were subjected to molecular docking studies to study their interactions with the corresponding human leukocyte antigen (HLA) system. The CTL epitopes were observed to bind at the peptide-binding groove of the corresponding HLA system, indicating their potency as an epitope candidate. The candidacy was further analyzed using sequence conservation studies and molecular dynamics simulation. The identified epitopes can be subjected to further studies for the development of the SARS-CoV-2 vaccine. Betacoronavirus and family Coronaviridae with symptoms ranging from a runny nose to severe acute respiratory syndrome and kidney failure1. It has infected over 150 countries worldwide and the World Health Organization has declared it a pandemic. An airborne infection like SARS-CoV-2 in populous countries such as India can escalate very rapidly. Thus, finding a vaccine candidate for this deadly disease is of priority to researchers worldwide.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus of the genus 2.It is known that cytotoxic T lymphocytes (CTL) with the help of specialized proteins known as T cell receptors (TCR) on their surface help fight virally-infected cells. Using this natural mechanism of the immune system to fight this dreadful virus can be of great importance in vaccine development. Each TCR that is present on the surface of the CTL can specifically identify antigenic peptides bound to the major histocompatibility complex (MHC). Whenever the TCR detects a viral antigen, the CTL releases cytotoxins to kill the virus-infected cell thus helping in immune responseVaccine development is a tedious and complex process lasting for several years. With the advancement of bioinformatics, sequence‐based techniques have rapidly improved providing information related to the genome and proteome of different viruses. Employing these bioinformatics tools, scientists and researchers are greatly reducing the time and cost required for the experiments. In vaccine development, by analyzing the genomic and proteomic data, the conserved and immunogenic epitopes from a pathogen can be predicted effectively by sequence similarity studies. This helps in reducing the time required for epitope identification before preclinical and clinical trials. The computational techniques can also be employed to study the effects of the epitopes towards various cells and entities involved in the immune response by simulating an in-vivo environment thus increasing the efficiency of vaccine development.This study aimed to identify cytotoxic T cell (CTL) epitopes of SARS-CoV-2 Indian isolate for designing potential vaccine candidates that are effective on the Indian population using an in-silico approach. We predicted the CTL epitopes for all human leukocyte antigen supertypes (HLA) which have a high allelic frequency in the Indian population. We further studied the immunogenicity, foreignness, and interactions between the epitopes and the HLA molecules by the means of molecular docking studies. The stability of the docking studies was confirmed by employing molecular dynamics simulation analysis.The amino acid sequence of the complete genome of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate of India (MT050493.1) with 9950 AA was retrieved from NCBI. All the coding regions of the genome were retrieved from the features section containing the coding sequences of orf1ab polyprotein, surface glycoprotein, orf3a protein, envelope protein, membrane glycoprotein, orf6 protein, orf7a protein, orf8 protein, nucleocapsid phosphoprotein, and orf10 protein.3 was used to predict the CTL epitopes across the proteins coded by the SARS-CoV-2 Indian isolate. NetCTLpan uses a neural network to predict TAP-transporter binding and C terminal cleavage predictions in addition to HLA binding prediction. Considering the HLA supertype variation across populations, we predicted the epitopes only for those HLA supertypes which constitute the majority of human leukocyte antigen (HLA) distribution in the Indian population keeping cutoffs and parameters of NetCTLpan as default. The study on the evolution of HLA-A and HLA-B polymorphisms reveals that HLA A3, B7, and B44 are the major HLAs present in the Indian population4 with HLA A3 constituting HLA-A type in 47 percent of the Indian population and HLA B7, B44 constituting HLA-B type in 30 percent and 28 percent of the Indian population respectively.NetCTLpan version 1.16 to predict their immunogenicity score. IEDB immunogenicity tool relies on physicochemical properties such as side chain composition, amino acid position to predict the immunogenicity of the peptide sequence.Although the binding affinities of the peptides towards HLA help in predicting the epitopes, the immunogenicity plays an important role in the immune response. All the predicted epitopes were subjected to the Immune Epitope Database (IEDB) immunogenicity tool7 against human reference proteome with Proteome ID UP000005640. The Peptide Match tool is a search engine based on Apache Lucene and is designed to quickly retrieve all occurrences of the given query peptides from a reference or specified proteome.As the healthy human body majorly shows immune response only towards foreign antigens except under certain conditions such as autoimmune disorders, it is of great importance to consider only those epitopes which are foreign to the human body as a potential vaccine epitope candidate. To identify the vaccine epitope candidates that are foreign to the human body, all the epitopes that show positive immunogenicity are subjected to the Multiple Peptide Match tool8. The docking was performed without specifying the binding site residues to investigate if the studied epitopes would bind at the peptide-binding groove without any lead. The interaction diagrams are generated using LigPlot+9.The top two percent of the foreign epitopes based on immunogenicity scores were selected. To further confirm the candidacy of the foreign epitopes for vaccine development, these epitopes were subjected to molecular docking studies to confirm their interactions with the specified HLA at the peptide-binding groove considering PDB IDs 6O9C, 6AT5, and 3KPS for the structures of HLA-A3, HLA-B7, HLA-B44 respectively. This molecular docking study was performed using HPEPDOCK Server10 and aligned using the MAFFT sequence alignment server11. All the 2084 genomic sequences as of 15 September 2020 were downloaded from the EpiCoV repository of the GISAID. Later, the alignment was translated using the standard genetic code and the locations containing the epitopes were extracted from the alignment. The extracted alignment was subjected to WebLogo12 to generate sequence logos for visualizing their conserved nature in the Indian isolates.To check the conserved nature of the selected epitopes from Indian isolates of the virus, The SARS-CoV-2 genomic sequences isolated in the Indian region were obtained from GISAID13 and GROMACS (Version 2018.2)14. The complex was solvated using TIP3P explicit water molecules and in-house ad hoc scripts were used to neutralize, minimize, and equilibrate using GROMACS. The neutralization was performed using Cl− and Na+ ions as needed while the minimization was performed using the steepest descent algorithm until the maximum force is less than 10.0 kJ/mol. The system was equilibrated using NPT conserved ensemble under constant pressure and temperature of 1 Bar and 300 K respectively. Further, the molecular dynamics simulations of 50 ns were performed using the leapfrog algorithm with an integration time step of 2 fs. The generated trajectories were analyzed using GROMACS analysis utilities to derive results. The graphs showing the root mean square deviation (RSMD) between the initial and the simulated structure, the change in coulombic interaction energies between HLA and epitope over the simulation time, and the change in the number of hydrogen bonds formed between HLA and epitope are plotted to determine the stability of the HLA-epitope complex.To study the stability of the HLA-epitope interactions, the structures from the docking studies were subjected to all-atom molecular dynamics simulations to explore their stability and conformational flexibility using CHARMM36 all-atom force field253 CTL epitopes are predicted by NetCTLpan across the proteins coded by SARS-CoV-2 Indian isolate towards HLA A3, B7, and B44. The epitopes as predicted by NetCTLpan are shown in Supplementary Table 16. The MHC class I are also known to be ubiquitous while MHC class II molecules are known to be present only on select antigen-presenting cells17. There are also instances of viruses inhibiting MHC class II antigen presentation18.While there are two classes of MHC molecules namely MHC I and MHC II, in this study only HLAs corresponding to MHC class I are considered due to their widely known functions of viral peptide presentation to CD8 + T cells during viral infectionsThe IEDB immunogenicity tool was used to calculate the immunogenicity of the epitopes as it plays an important role in examining the immune response. IEDB immunogenicity tool returned 139 epitopes with positive scores. The data showing the immunogenicity score of the epitopes are shown in Supplementary Table All the epitopes with positive immunogenicity scores are subjected to the Multiple Peptide Match tool to identify the epitopes that are foreign to the human body. It was observed that all the epitopes that showed positive scores for immunogenicity calculations were foreign to the human proteome. The peptide matching step was performed on the sequence dataset ‘UniProtKB release 2020_01 plus isoforms |SwissProt| Isoform’ with the target organism set as ‘Homo sapiens [9606]’. The output log had stated that 0 out of 139 unique peptides had matches in 0 protein(s) found in 0 organism(s) confirming their foreignness to the human body. Ultimately, to select the promising epitopes, the NetCTLpan score and immunogenicity score were employed. The NetCTLpan score was used to filter the highly likely CTL epitopes by eliminating the epitopes with a negative NetCTLpan score. The remaining epitopes were sorted based on their immunogenicity score to identify the top epitope candidates.Mycobacterium tuberculosis that showed promising results in human subjects as a control epitope in this study19. The control epitope and the top five vaccine epitope candidates amounting to the top two percent of the total 253 epitopes as predicted by the above steps along with their epitope prediction score by NetCTLpan and immunogenicity score are given in Table Although there were many peptide-based vaccines against various infections in different phases of development, none of these have either been concluded or had their sequence data directly available. So, we have considered an epitope from protein Superoxide Dismutase of The top three vaccine epitope candidates based on their immunogenicity score are subjected to molecular docking studies using HPEPDOCK Server. Although the top two percent of the predicted epitopes were selected for docking studies, coincidentally the top three selected epitopes were against different HLAs covering all the HLAs from the study. So, the docking studies were performed only for the best epitope candidate for each HLA. The interaction diagrams revealed that the peptide epitopes bind to the peptide-binding groove even though they were subjected to blind docking. Thus, confirming their vaccine candidacy. To visualize the equivalent interactions between the reference and the epitope towards the corresponding HLA, the interaction plots of the docked epitopes and the references are shown in Figs. As it is observed that the Interactions between the identified epitopes are similar to that of the reference peptides, the conserved nature of the selected epitopes was studied and the sequence logos as generated by WebLogo is shown in Fig. To check the HLA-epitope stability of the conserved epitopes, a molecular dynamics simulation of 50 ns was performed. The RMSD plot as shown in Fig. Further, the short-range coulombic interaction and hydrogen bonding between the epitope and the HLA were analyzed to reveal the interaction stability of the epitope towards the corresponding HLA. The interaction plots as shown in Fig. 20. Apart from immune camouflaging, cross-reactivity of the T cells is also a major concern where the TCR recognizes more than one peptide-MHC complex. Although cross-reactivity is an essential feature of the immune system for biological robustness, in this case where only completely identical peptide sequences are filtered out to identify the epitope candidates, the autologous peptides could also be identified for an immune response especially in the case of an engineered TCR22.While identifying the potential vaccine epitopes from SARS-CoV-2, this study considers all the epitopes that are not an identical subsequence to the human proteome as a foreign sequence. This could lead to the epitopes showing immune camouflaging that is generally seen in pathogens as an immune evasion strategy. This immune evasion is achieved by adopting amino acid configurations recognized by autologous regulatory T cells of the hosts which are known for their immune-modulating capabilities23.The identified epitopes could be further subjected to in-vitro and in-vivo analysis after considering the limitations. Although epitopes play an important role in immune response, epitopes alone are not sufficient to develop vaccines as they cannot stimulate the immune system sufficiently. So, adjuvants such as biopolymers and nanoparticles are used besides an epitope in developing a peptide-based vaccine prototype for high immune response. These peptide-based vaccine prototypes should be further tested on a cell line for their physiological, biological, and chemical effects leading to cytotoxicity using in-vitro cytotoxicity assays such as in-vitro titration of live organisms, enzyme-linked immunosorbent assay (ELISA), and in-vitro antigen-quantification tests. The promising prototypes could be further subjected to in-vivo studies using animal testing techniques to identify the immunogenic prototype through immunization of laboratory animals and titration of immune sera to measure their antibody response. In-vivo serology analysis methods can be used to measure antibodies in blood samples employing techniques such as enzyme-linked immunosorbent assay (ELISA) and a multiplex assay that could analyze multiple antigens simultaneously before proceeding to further clinical phase studies26, identifying epitopes from SARS-CoV which shows high sequence similarity along with sequence conservation to SARS-CoV-227 or considering viral sequences from diverse geographical regions28. Unlike the other studies, our analysis primarily targets the Indian population where an infectious disease could escalate very rapidly. We have also analyzed the conserved nature of the identified epitopes along with their interaction stability towards the HLAs at the peptide-binding groove considering a known bound peptide as a reference. This could significantly increase the predictability in an in-vivo environment.Over the last few months, there have been various studies on the identification of immunogenic epitopes from SARS-CoV-2 using computational techniques. But, these studies focus on a particular viral proteinDesigning a vaccine is of top priority in the time of a pandemic. In this study, we attempted to identify potential CTL epitopes from the SARS-CoV-2 Indian isolate for the Indian population using a bioinformatics approach.The list of CTL epitopes was predicted using the NetCTLPan server which considers HLA binding affinity, TAP transport efficiency, and C-terminal cleavage to identify the epitopes. Further, Immunogenicity scores were calculated using the IEDB immunogenicity tool to identify the potential vaccine epitope candidates. The epitopes with positive immunogenic scores are subjected to peptide matching against the human proteome to check their foreignness to the human body. These unique immunogenic epitopes were further docked with their respective HLA molecule to study their interactions with the HLA molecule. The docking studies revealed that all the studied epitopes bind at the peptide-binding site of the HLA confirming their epitope candidacy. The identified epitopes were checked for their sequence conservation in the viral isolates from India and the HLA-epitope complexes were subjected to molecular dynamics simulation studies to analyze their interaction stability. The epitopes were observed to be highly conserved and the interactions between the HLA and the epitopes were seen to be very stable, further confirming their potency in vaccine development. The epitopes identified in this study can be further subjected to in-vitro and in-vivo studies to design a vaccine against the dreadful SARS-CoV-2.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


The infection mechanism and pathogenicity of Human T-lymphotropic virus 1 (HTLV-1) are ambiguously known for hundreds of years. Our knowledge about this virus is recently emerging. The purpose of the study is to design a vaccine targeting the envelope glycoprotein, GP62, an outer membrane protein of HTLV-1 that has an increased number of epitope binding sites. Data collection, clustering and multiple sequence alignment of HTLV-1 glycoprotein B, variability analysis of envelope Glycoprotein GP62 of HTLV-1, population protection coverage, HLA-epitope binding prediction, and B-cell epitope prediction were performed to predict an effective vaccine. Among all the predicted peptides, ALQTGITLV and VPSSSTPL epitopes interact with three MHC alleles. The summative population protection coverage worldwide by these epitopes as vaccine candidates was found nearly 70%. The docking analysis revealed that ALQTGITLV and VPSSSTPL epitopes interact strongly with the epitope-binding groove of HLA-A*02:03, and HLA-B*35:01, respectively, as this HLA molecule was found common with which every predicted epitope interacts. Molecular dynamics simulations of the docked complexes show they form stable complexes. So, these potential epitopes might pave the way for vaccine development against HTLV-1. The total genome size of HTLV-1 is 8507 nucleotides in length encoding 6 different proteins. In one stage of the HTLV-1 lifecycle, the RNA is utilized to synthesize double-stranded DNA which inserted into the genomic DNA of a host cell . mRNA of+ and CD8+ T cells, therefore, the immune system elicits a T-cell mediated immunity, which binds to MHC class I or class II complexes. Activated, CD4+ and CD8+ T cells secrete cytokines asserting an immune response against HTLV-1. Cytotoxicity and the lysis of the infected cells are two concurrent events mediated by CD8+ T cells. However, an effective vaccine against HTLV-1 is yet to be examined. In this study, all the possible strains of HTLV-1 were considered and explored to predict an efficient vaccine against a unique envelope. Glycoprotein GP62 of HTLV-1 by using different data-driven tools, including immunoinformatics – that is one of the branches of data-driven tools that uses various sequence- and structure-based methods to solve immunological data and problems , t, t31], t) method . MD simu2.7http://tools.iedb.org/bcell/) as an antigenicity prediction method. Here, Kolaskar and Tongaonkar antigenicity prediction method has been selected because this method is based on a table that corresponds to the occurrence of amino acid residues is experimentally have known as segmental epitopes. This method can ensure approximately 75% accuracy in antigenic peptides prediction [B-cell epitopes (Linear) of the given protein sequence were predicted from the B-cell epitope prediction tool available at  and (http://tools.immuneepitope.org/mhcii/) to analyze the binding prediction of MHC class I and MHC class II, respectively. Throughout the analysis, it has been found that only 10 epitopes having an ANN IC50 score of less than 50 among previously primary selected 52 epitopes for MHC I and 5 epitopes for MHC II out of 17 epitopes  was followed by the analysis of the variability of Envelope Glycoprotein GP62 of HTLV-1 using a Protein variability server (PVS) where the Shannon variability method was selected for sequence variability options and a conserved fragment of length was selected, 9 with variability threshold of 0.5. Envelope Glycoprotein GP62 of HTLV-1 was consensus under a variability threshold of 0.5 .st and 25th position and some of the amino acid positioned near 70, 140, 330, 345, and 475 are slightly variable. Otherwise, the overall position of amino acid positions is highly conserved. Finally, we have selected only those epitopes that specifically matched (consensus) with their full length of the generated sequences and have found a total of 9 epitopes for MHC I and 5 epitopes for MHC II that meet up these criteria.This plot is drawn putting the amino acid's position in the x-axis and the Shannon variability in the y-axis. A higher value in the y-axis means the higher variability in amino acid for a specific position. From those variability plots, it is easily noticeable that for Envelope Glycoprotein GP62 of HTLV-1, amino acids between 13.4http:/tools.immuneepitope.org/tools/population) from the IEDB analysis resource. It has been a set of 14 epitopes for the calculation and interacting alleles were put against each epitope as MHC Restricted Allele(s)  . The comA total of 11 regions of the world and overall world coverage were calculated. Among 11 regions, 7 regions were found to have population coverage of more than 70% and overall world population coverage found to be 81.27% and the highest population coverage was found to be 87.54% and 85.87% in West Africa and Europe, respectively. From the population coverage graph, it is observable that the highest population coverage in North America is 81.28%. Central America, Europe, and West Africa also show a high population coverage which is more than 70% .3.5PEP-FOLD was used to generate the 3D structure of the predicted epitopes. Both linear and disulfide bonded cyclic peptides containing 9–36 amino acids were allowed during the model prediction . Nine po3.6th positioned residues (2.5 Å–3.4 Å) of the ALQTGITLV-4PRN complex and also in the Arg17 (4.1 Å) residue of the VPSSSTPL-3OX8 complex. Both the 41–44th residues and the Arg17 residue are located in the beta-beta joining loops of the MHC peptide-binding site. One possible reason for the fluctuation observed in these regions could be due to the adjustment of the beta-sheets in the binding site to better accommodate the peptides.To understand the dynamics of the bound peptides in binding grooves of the HLA proteins within an aqueous environment, we performed 50 ns MD simulation of the docked complexes. The average RMSD values of the Cα atoms of ALQTGITLV-4PRN and VPSSSTPL-3OX8 complexes are 1.81 Å and 2.02 Å, respectively. The RMSD values for both the complexes remain relatively constant after 35 ns at around 2 Å values A which s3.7To show whether the Envelope Glycoprotein GP62 of HTLV-1 elicits B cell immunity we have employed Kolaskar and Tongaonkar prediction method based on the physicochemical properties of amino acids in the proteins and the abundances in the experimentally known epitopes . The amiNineteen peptides  are foun4Our study has predicted 14 epitopes that show good MHC binding profile, as well as, the overall population coverage was found good enough to make them suitable candidates. Using the predicted peptides and addition of potential molecule(s), if required, to enhance the immunogenicity might pave the way to designing a universal epitope-based vaccine against HTLV-1. Furthermore, collaboration with a wet laboratory is important to identify and validate the best peptide among the candidates as a peptide-based vaccine.


However, its roles in regulating starch production in storage roots have not been investigated. In this study, we found that IbVP1 overexpression in sweet potato improved the photosynthesis ability of and sucrose content in source leaves and increased both the starch content in and total yield of sink tissues. Using 13C-labeled sucrose feeding, we determined that IbVP1 overexpression promotes phloem loading and sucrose long-distance transport and enhances Pi-use efficiency. In sweet potato plants overexpressing IbVP1, the expression levels of starch biosynthesis pathway genes, especially AGPase and GBSSI, were upregulated, leading to changes in the structure, composition, and physicochemical properties of stored starch. Our study shows that the IbVP1 gene plays an important role in regulating starch metabolism in sweet potato. Application of the VP1 gene in genetic engineering of sweet potato cultivars may allow the improvement of starch production and yield under stress or nutrient-limited conditions.Storage roots of sweet potato are important sink organs for photoassimilates and energy, and carbohydrate metabolism in storage roots affects yield and starch production. Our previous study showed that sweet potato H Ipomoea batatas (L.) Lam.) is a multifunctional starchy root crop species that is a primary resource for food, feed, and bioindustrial applications. The annual global production of sweet potato is approximately 92 million tons of fresh storage roots (FAOSTAT 2018), which is crucial for food security, malnutrition and poverty alleviation, and bioindustrial development2. With increasing demand for sweet potato production, storage roots need to be improved, especially in terms of yield and quality, both of which are positively correlated with the acquisition and partitioning of organic carbon4. In addition to a strong photosynthesis ability of the leaves, an efficient system of sucrose transport from source to sink via stem phloem can ensure sufficient access to carbohydrates7. Sucrose transporters (SUTs) are responsible for sucrose loading into the complex comprising phloem sieve elements and companion cells. Overexpression of SUT genes has been demonstrated to improve plant productivity by effective partitioning of carbohydrates in sinks in rice, pea, and wheat10. In sweet potato, overexpression of the H+-translocating inorganic pyrophosphatase (H+-PPase) gene IbVP1 increases plasma membrane H+-ATPase hydrolytic activities, SUT1 expression, and root growth11. Nevertheless, how IbVP1 regulates starch metabolism in sweet potato storage roots has not yet been determined.Sweet potato )25. Overexpression of H+-PPase in Arabidopsis results in the upregulated expression of the sucrose proton symporter SUC1, suggesting the involvement of H+-PPase in the transport of sucrose into the phloem19.HIbVP1 in sweet potato improves plant growth and development through altered carbohydrate metabolism in the leaves, transport of polar auxin from source to sink, and rhizosphere acidification to facilitate nutrient acquisition11. However, the underlying molecular mechanism for the function of IbVP1 in storage root starch metabolism remains unclear. In this study, we showed that overexpression of IbVP1 resulted in an increase in starch accumulation in, storage root numbers of and yield of sweet potato. Transgenic sweet potato plants exhibited increased phloem loading and long-distance transportation of sucrose through the acquisition and utilization of activated Pi, thereby increasing the starch content in and yield of the storage roots. Moreover, the starch physicochemical properties, including their composition and structure, were altered due to the increased transcript level of granule-bound starch synthase I (GBSSI). These findings advance our mechanistic understanding of the functions of IbVP1 in the starch biosynthesis process and provide a new target for adding value to root crops by improving starch content and quality.In a previous study, we showed that overexpressing IbVP1-overexpressing transgenic lines (IA lines) were produced as described by Fan et al.11. In early May 2014–2017, wild-type (WT) plants and transgenic lines were planted in a field at the Wushe Plantation for Transgenic Crops, Shanghai, China .Sweet potato cv. Taizhong 6, one of the edible yellow-fleshed cultivars grown in China, was used as a donor parent for generating transgenic plants. 26.The storage roots of IA lines and WT plants were harvested after the plants had grown in the field for 5 months. Freshly harvested storage roots of the sweet potato plants were washed, peeled, cut into small slices, suspended in distilled water, and blended with a commercial scale blender. The starchy liquid was passed through a 100 μm sieve, and the starch granules were allowed to sediment. The supernatants were decanted and replaced four times with distilled water. The final supernatants were discarded, and the samples were dried at 40 °C for two days27. The starch content was measured by the use of starch kit . After sugar extraction, the supernatant was centrifuged and transferred into a new glass tube for high-performance liquid chromatography (HPLC) analysis for the identification of the sugar fractions. The different fractions of sugar were identified on the basis of their retention time via known standards. The sugar concentrations were subsequently determined with a standard curve.Storage roots of WT and IA lines growing in the field for 5 months were harvested, ground into powder, and then heated at 80 °C for approximately 48 h to obtain a constant dry weight (DW). The dried samples (30 mg) were used to determine the sugar and starch contents, as described by Knutson et al.28. Pure amylose  and amylopectin (Sigma 10118) of potato starch were purchased and used to make known dilutions to establish standard curves.The amylose contents in the starch samples from 5-month-old field-grown WT and IA plants were measured by the colorimetric method by following a previously described protocolThe thermal properties of starch from 5-month-old field-grown WT and IA plants were assessed by the differential scanning calorimeter method . All the samples were prepared and kept in aluminum sealed pans for 2 h, after which scanning was performed in the temperature range from 30 to 95 °C. An empty, sealed aluminum pan was used as a reference. The measurements were recorded in triplicate for each individual sample.26.The pasting properties of starch from 5-month-old field-grown WT and transgenic plants were measured using a Rapid Visco-Analyzer (RVA) . A starch suspension (5% wt/vol) was produced by mixing in distilled water, and starch viscosity parameters were recorded at various temperatures as described by Zhou et al.26 and Blazek et al.29.The distribution of granule size of starch from 5-month-old field-grown WT and IA lines were determined by using a Master-sizer  in wet-cell mode as described by Zhou et al.3) were glazed with 1,3-diformal propane. Afterward, ultrathin sectioning was performed, and the samples were examined via TEM .The granular morphology of the starch from 5-month-old field-grown plants was observed via scanning electron microscopy (SEM). Starch samples were dispersed in distilled water and then spread onto double-sided adhesive tape, air dried, and coated with gold powder. The samples were observed via SEM  and imaged. For transmission electron microscopy (TEM), slices of freshly prepared storage roots . The debranched starch samples were then used to quantify the amylopectin content using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).Amylopectin CLD was measured by using the method of Nishi et al.28. Briefly, 5% starch paste (wt/vol) was liquified by boiling for 20 min. The samples were cooled, after which 0.5 mL of each sample was transformed into new 1.5 mL Eppendorf tubes. The weight of the starch in each sample tube was measured. All the samples were stored at –70 °C overnight and then put into a water bath to thaw for 60 min at 22 °C. Six replications from each starch tube were taken and centrifuged at 8000 х g for 10 min at 18 °C. The supernatant was separated, and the remaining paste was weighed. The obtained value  was considered a measure of syneresis. The tubes were subsequently stored at −70 °C for further freeze–thaw cycles and measurements.The stability of starch subjected to freeze–thaw cycles from 5-month-old field-grown plants was evaluated by a previously described method31, with some modifications. Stems with three unfolded leaves were cut and soaked immediately in a 15 mM EDTA solution. The stems were then recut to pieces 1.5 to 2 cm in length in 15 mM EDTA solution buffer to collect the phloem exudate. After the samples incubated for 12 h in the dark at 25 °C, phloem exudates were concentrated for sucrose measurements via HPLC.The phloem exudate assay was performed on plants grown for 3 months in a greenhouse as described previously13CO2 isotopes to investigate the diffusion rate of photoassimilates into the phloem, as described by Gui et al.32 and Koubaa et al.33. The first unfolded leaf was positioned in a gas-sealed transparent glass chamber filled with 13CO2. After 12 h of photosynthesis, treated plants were kept in the dark, and leaf samples were collected for sucrose isolation and measurements. Following derivatization, analysis was performed by gas chromatography–mass spectrometry (GC–MS). The proportions of 13C isotope mass spectral fragments were determined to measure the amounts of 13C-labeled sugars.The photoassimilates were labeled with stable 2) was measured using WSeen software. At least six replicates for each leaf were recorded for the transgenic lines and WT.Leaves from sweet potato plants that had been growing for 3 months (six different pots) were sampled to measure the leaf area. The leaves were imaged, and the leaf area . All measurements were carried out at 9:00 to 10:00 a.m. following the manufacturer’s instructions.For SPS analysis, the first unfolded leaves of 3-month-old plants were frozen in liquid nitrogen, after which total proteins were extracted, desalted using a spin column, and immediately assayed. SPS activity was determined using a sucrose phosphate synthase assay kit (Solarbio BC0600) according to the manufacturer’s specifications.+-PPase activity was determined using the method of Fan et al.11.Vacuole H34. One unit of enzyme  activity was defined as the production of 1 nmol of ADP per min at 30 °C, while 1 unit of SBE enzyme activity was described as the amount of enzyme needed to increase the spectrophotometric absorbance by one unit at 540 nm in 1 min. The activities of α-amylase and β-amylase in the storage roots of sweet potato plants were also measured. A Ceralpha kit  was used following the manufacturer’s protocol, with the appropriate dilutions.The enzyme activity of starch biosynthesis-related enzymes  was measured in tuberous roots of the AI and WT plants by following the methods described by Nakamura et al.Actin gene as an internal control.The mRNA levels of starch biosynthesis-related genes in the storage roots of IA and WT plants were measured by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Gene-specific primers were designed Table  to analyg for 5 min. The precipitate was subsequently resuspended in chilled 80% ethanol and centrifuged. The supernatants were collected and dried with a centrifugal evaporator. The dried extract was resuspended in water, and water-soluble compounds were extracted. PPi was measured using a pyrophosphate assay kit (Sigma MAK168-1KT) according to the manufacturer’s specifications. All glassware was pretreated overnight with 0.1 M HCl to remove residual Pi.For the quantification of pyrophosphate (PPi), roots from 5-month-old field-grown plants were collected and immediately frozen in liquid nitrogen to avoid PPi hydrolysis. The frozen roots were homogenized in chilled 80% ethanol, vortexed for 10 min, heated at 80 °C for 30 min, and then centrifuged at 20,000 x 2SO4–H2O2. After 2 mL of H2SO4 was added to the samples followed by thorough shaking (including the blowing of air through a pipette), 1 mL of H2O2 was added two times via multiple small drops. After the reaction was completed, the sample was placed in an electric heater (nitrogen blowing at 120 °C for 15 min) until the solid disappeared into an essentially transparent solution. The digested solutions were subsequently diluted with ddH2O. Aliquots of diluted solutions (0.5 mL) were added to 4 mL of a solution consisting of 0.5% (w/v) ammonium molybdate, 0.6 M H2SO4, and 2% (w/v) ascorbic acid. The absorbance was read at 660 nm, and the Pi content was measured as milligrams of Pi per gram of fresh weight.The Pi content in roots and shoots of 5-month-old field-grown plants was determined by digesting the tissues in Ht test via SigmaPlot 10.0 . The differences among the treatments were statistically significant when P was <0.05 or 0.01.At least three biological replicates were used for each experiment. The data are reported as the means ± SDs, with three replicates for each individual experiment. Significant differences were identified for each individual treatment using Student’s IbVP1 transgenic sweet potato lines and characterized the impacts of IbVP1 overexpression on shoot and fibrous root growth11. However, the storage roots of the IbVP1 transgenic lines have not been thoroughly studied. To understand the impact of IbVP1 overexpression on storage root yield, mature storage roots from 5-month-old IbVP1-overexpressing sweet potato plants (IA lines) were harvested from the field. Compared with the WT plants, all IA lines exhibited higher yields and increased numbers of storage roots  in the storage roots of IA and WT plants were measured. The sucrose contents in the IA lines were 28–67% higher than those in the WT plants  within the starch were observed for both IA lines and WT Fig. . Howeverpes Fig. .The pasting profiles of IA and WT starch were measured with RVA. As the temperature increased, granules of the WT starch swelled, with a rapid increase in viscosity; the granule structures of starch also became distorted, resulting in polymer dispersion, and finally, starch gel formed upon cooling. As shown in Fig. To), top melting temperature (Tp), end melting temperature (Te), and enthalpy (ΔH) were observed in the IA starch compared with the WT starch. During the melting process, the To decreased slightly for all IA starch, ranging from 63.5 °C (in IA4) to 60.32 °C (in IA8), compared to the WT To of 64.77 °C, with a ΔH of 11.32 J g−1. Notable decreases in Tp and Te were also detected in the IA starch compared to the WT starch  was determined and normalized based upon the peak area  consistently have more hexose and starch in rosettes due to improved phloem transport accompanied by upregulated Suc gene expression17. Overexpression of the AVP1 gene in Arabidopsis and barley results in an increase in phloem loading, transport, and unloading of sucrose into sink organs, which leads to biomass enhancement42. Improved transport of sugars from source to sink in H+-PPase-overexpressing plants may explain the increased biomass production as well as increased fixed carbon in the root system25. In our study, compared with the field-grown WT plants, the field-grown IA transgenic lines showed enhanced growth and vigor as well as increased yields of and total starch contents in the storage roots  for sink biomass production through increased photosynthesis, enlarged leaf areas, improved SPS activity, and decreased starch levels in the leaves of ent Fig. . The subsupplemental file


Uterine transplantation restores fertility in women with absolute uterine factor infertility and allows the opportunity to conceive, experience gestation, and acquire motherhood. The number of cases being performed is increasing dramatically, with detailed outcomes from 65 cases now available. Pregnancies achieved through uterus transplantation and following in vitro fertilization (IVF) are associated with an increased risk for further mother and newborn babies. This traditional review is focused on the safety and efficacy features of the treatment. However, it is associated with significant risk, with approximately one-quarter of grafts are removed because of complications. Uterine transplantation is realizable in women with uterine factor infertility but is associated with a significant complication risk. The risk of the procedure and gestational and delivery complications deserve important consideration before receiving such treatments. Nevertheless, these observations are preliminary and should be revised after a larger series of data are published.  Historically, society believes that for girls to become "real mothers," they must carry a pregnancy themselves. The desire for pregnancy is so strong that some women are willing to risk their health to have a baby . Absolute uterine factor infertility (AUFI), which refers to infertility that is completely attributable to the uterine absence  or an abnormality , affects approximately one in 500 women of childbearing age, or 1.5 million women worldwide . UterineMotherhood choices traditionally offered to women with AUFI spin around two options: adoption or surrogacy. Adoption provides legal motherhood, while a surrogacy agreement confers genetic motherhood and, after adoption, legal motherhood to infertile mothers. During this process, multiple medical and legal issues may arise. Maternal surrogacy is forbidden in many countries, including France, Germany, Bulgaria, Croatia, Estonia, Finland, Hungary, Italy, Lithuania, Portugal, Slovakia, Australia, Holland, Spain, Sweden, and Norway. However, there is no legislation or regulation in other countries, leading to conflict between the involved parties . The evolution of uterine transplantation (UTx) was primarily motivated by the potential to help women suffering from the discrepancy between procreative ability and reproductive aspirations because of AUFI. Although this procedure is associated with greater risk from multiple major surgeries and exposure to immunosuppressant drugs, UTx is the only option that allows women with AUFI the opportunity to become pregnant and give birth to genetically correlated offspring themselves . Women who are inspired to pursue uterus transplantation take risks from immunosuppression, a high‐risk pregnancy, and at least three surgical procedures. The first procedure includes allotransplantation, then the caesarian section to deliver the child (and a second section if a second child is desired), and a graft hysterectomy after the delivery . Steps required pre/post the uterus transplantation procedureThe transplant process to successful birth fluctuates from person to person but can take approximately two to five years for many participants -7. It in. Medical evaluation of donor and recipient [1ecipient -7. 2.Embryo generation: Before the surgery, a woman generates embryos through in vitro fertilization (IVF). During this process, she should take fertility drugs to produce eggs removed from her ovaries and fertilized outside of her body and frozen for later use -7. 3.Transplantation: The uterus is removed from a donor during a three-hour operation and surgically placed into the recipient in a six-hour operation. The recipient starts taking immunosuppressive drugs to prevent rejection of the transplant. These medications should be taken while the transplant is in place and during pregnancy -7. 4.Pregnancy: Several months or a year after the transplant surgery, one of the recipient's embryos will be placed into the uterus. If implantation of the embryo is successful, the recipient will become pregnant. Both baby and mother's health are monitored closely at frequent prenatal care visits with a high-risk obstetrician, known as a maternal-fetal medicine specialist -7. 5. Delivery: The baby is born as close to term as is possible via a planned cesarean section. If the pregnancy has gone without any complications, and the patient wants one more child, the uterus is left, and immunosuppression medications are continued. Another embryo transfer can be attempted after approximately six months after delivery . 6.Uterus removal: When childbearing is complete, the transplanted uterus must be removed, and immunosuppressive medications should be stopped -7.  Figure Possible complications following uterus transplantation All surgeries have risks and benefits; therefore, it's important to fully consider them before deciding whether the procedure is appropriate for the patient or not. As with any operation, uterus transplantation also puts the donor and the recipient at risk. The Czech Republic team reported vaginal stenosis, vesicovaginal fistula, cytomegalovirus, and herpes infection in recipients . The donThe likelihood of rejection for a transplanted uterus is still unclear; however, rejection has been detected in cervical biopsy for many transplanted uteruses after UTx. Some patients develop rejection before and during pregnancy, which is managed with immunosuppressant medication . Figure This review article aimed to analyze the first uterus transplantations done in the past 20 years. Additionally, we tried to investigate the complications faced by specialists following this sophisticated procedure. Methods An extensive literature search on Google, Google Scholar, PubMed, and non-PubMed indexed journals was performed. Literature databases were searched to identify the information available about uterus transplantation, using the following terms: "uterus transplantation," "uterus transplantation and complications," "first uterus transplantation," "babies born after uterus transplantation." The full text of the articles eligible for our study was reviewed and included in the review. We only included articles written in the English language and were done on human models, while studies on animals were excluded. Only the most relevant research articles are included. The first uterus transplantation, Saudi Arabia The first human uterine transplantation was performed on April 6, 2000. The recipient was a 26-year-old female who lost her uterus six years earlier due to delivery complicated with post-partum hemorrhage; the donor was a 46-year-old patient with multiloculated ovarian cysts who underwent a hysterectomy to preserve tissue and vascular integrity. The donor uterus was connected to the recipient's vaginal vault with additional fixation using a shortened uterosacral ligament. The uterine arteries and veins were connected to the external iliac arteries and veins using segments of the great saphenous vein . Immunosuppression was maintained by oral cyclosporine A, azathioprine, and prednisolone. An acute rejection episode was treated and controlled on the ninth day with anti-thymocyte globulin (ATG). The transplanted uterus responded well to combined estrogen-progesterone therapy, with endometrial proliferation up to 18 mm, which resulted in withdrawal bleeding upon cessation of the hormonal therapy. Unfortunately, 99 days after transplantation, vascular thrombosis developed, and hysterectomy was necessary. Macro- and microscopic histopathological examination showed acute thrombosis in the uterine body vessels, which resulted in infarction. Both fallopian tubes were unchanged, with no evidence of rejection. Inadequate uterine support caused acute vascular occlusion, which led to probable tension, torsion, or kinking of the connected vascular uterine grafts [This first human case, although a failure, inspired researchers around the world to initiate new studies on uterus transplantation, immunosuppression, rejection, and pregnancy outcome. Uterus transplantation, Turkey The second uterus transplantation, and the first from a deceased donor, was performed in Turkey in 2011. The recipient was a 22-year-old woman diagnosed with MRKH syndrome, and the donor was a 22-year-old nulliparous woman with brain death after a car accident. From past medical history, she also underwent vaginal reconstruction surgery with jejunum segment two years before transplantation ,11. The retrieval procedure resembled radical hysterectomy and dissection of vascular pedicles, including hypogastric, uterine artery, and ovarian vessels, from surrounding tissues. Additionally, the bladder peritoneum's anterior reflection was dissected and included within the retrieved uterus. The anastomosis was performed between the recipient's external iliac vessels and the hypogastric artery pedicle of the donor's uterus. By the 20th month of the postoperative period, the recipient did well with no signs of rejection, continuing to menstruate. Six IVF cycles were attempted, with one chemical and three missed abortion pregnancies  . They elected to use a deceased donor as its main advantage is the possibility of achieving longer and enhanced vascular grafts within a significantly short time . This was the first heart beating pregnancy following deceased donor transplantation, an important step in uterus trial attempts. Although these pregnancies ended with missed abortion, they were still direct evidence of the viability of a uterus retrieved from a deceased donor. On the other hand, the donor's nulliparous state with unproven fertility may have adversely affected pregnancy chances. Moreover, previous vaginal reconstruction with an intestinal flap might have impeded the implantation process, similar to the adverse effect of hydrosalpinx on in vitro fertilization (IVF) cycles. Although the intestinal neovagina no longer performed a fecal storage function, normal bacterial flora may have diminished endometrial receptivity and abnormal expression of factors related to implantation . Based on Daily Sabah News, in her 28th week of pregnancy, Derya Sert gave birth on June 4, 2020, to a healthy baby boy, with the help of plastic surgeon Dr. Ömer Özkan of Akdeniz University in Antalya province. After successful birth, the uterus was removed . Uterus transplantation, Sweden The first clinical trial of uterus transplantation was started in Sweden in 2013 and involved nine live donor procedures. This trial included nine recipients (27-38 years of age), with eight having MRKH syndrome. In contrast, the ninth patient had a radical hysterectomy because of cervical cancer seven years before UTx. The donors were in seven cases genetically related, and two were non-genetically related. The donors' age varied from 37 to 62 years, with five being postmenopausal. Surgical success was established in eight of nine, but one graft was removed three days postoperatively due to bilateral arterial and venous thrombosis. Another recipient developed an intrauterine infection two months after UTx. Despite treatments with antibiotics and surgical drainage, a hysterectomy had to be performed three and a half months later when septicemia developed. Seven of nine patients had regular menstruations during the initial year and were scheduled for embryo transfer 12 months post-transplantation [The first uterus transplantation was done for a 35-year-old woman with a congenital absence of the uterus (Rokitansky-Mayer syndrome). The uterus donor was a living, 61-year-old, two-parous woman. The first menstruation occurred 43 days after uterus transplantation, and she continued to menstruate at regular intervals afterward at the median of 32 days. One year after transplantation, the patient underwent her first single embryo transfer, which was successful and resulted in a viable pregnancy. Triple immunosuppression  was given and continued during pregnancy. She had three mild rejection incidents, one of which occurred during pregnancy which was managed by corticosteroid treatment. At 31 weeks of pregnancy and five days, the patient was admitted with preeclampsia, and a cesarean section was done because of abnormal cardiotocography. A healthy baby boy with normal birth weight for gestational age  and with appearance, pulse, grimace, activity, respiration (APGAR) scores of 9, 9, 10 was born . AdditioThe first uterus transplant involving surgeon-operated robots was performed in Sweden in October 2017 without any complications. The robot-assisted procedure involved making a one-centimeter incision in the abdomen of the donor, who was the recipient's mother. Two robot arms performed the procedure, managed by a human surgeon. The uterus was removed through that incision and directly transferred to the recipient via open surgery carried out by humans. Ten months after transplantation, the recipient became pregnant via IVF. The pregnancy went without any complications, and a baby boy was delivered by cesarean section . Additionally, another uterus transplant was done in December 2019 in Sweden, for the first time from a deceased donor. The recipient was a 30-year-old woman, and the donor was a woman who had previously given birth and was of fertile age at the time of her sudden death. Ten months after surgery, attempts will be started to make the woman pregnant . Transplantation in June 2020 that >10 babies were born in Sweden from uterus transplant recipients [Since 2014, when the first baby was born, another eight babies, including three sibling pairs, were born in Sweden; the latest birth took place in January 2020 . Howevercipients . Uterus transplantation, ChinaChina's first human uterus transplant was done in 2015; the recipient was a 22-year-old woman, and the donor was her mother of 43 years . The proUterus transplantation, USA Candida infection from the transplanted uterus [The first uterus transplantation in the USA was performed in February 2016 in Cleveland Clinic. The recipient was a 29-year-old mother of five adopted children, and the donor was a deceased woman in her 30s. Unfortunately, on March 7, the patient began bleeding, and doctors discovered that an artery connected to supply the uterus was damaged and had no choice but to remove the organ. The cause was revealed to be d uterus . Doctors at Baylor University Medical Center, USA, performed four uterus transplantations in September 2016, but only one has proven successful. This was the first living-donor uterine transplant performed in the USA . In NoveTwo important modifications from the previously reported live births characterized how this uterus transplant was done. First, the transplanted uterus had only the utero-ovarian vein as venous outflow; second, the time from transplantation to embryo transfer was shortened from prior protocols, allowing for shorter exposure to immunosuppressant drugs by the patient, which minimized the risk for potential adverse effects from these medications . From 2016 to 2020, Baylor University Medical center reports that 20 uterus transplants were performed. Twenty women were enrolled in the clinical trial, with a median age of 29.7 years, with 10 in the first and second phases. All but two recipients had a congenital absence of the uterus. Eighteen recipients received a uterus from living donors and two from deceased donors. In phase one, 50% of recipients had a technically successful uterus transplant, compared to 90% in phase two . The insOne of the uterus transplantation recipients from the Baylor University Medical Center clinical trial gave birth to the second baby girl in December 2020. She was the fifth woman to undergo uterus transplantation from a living donor in the clinical trial. The procedure went without any complication, and the first baby girl was born in 2018 . To date, 20 women have undergone uterus transplantation at Baylor University, and 13 babies were born as part of Baylor's uterine transplant program . In October 2015, Cleveland Clinic began an interventional clinical trial to perform 10 uterine transplantations from October 2015 to October 2023. This study has seven phases: primary and secondary screening, medical evaluation, IVF, transplantation, embryo transfer, pregnancy/delivery, and follow-up after delivery. Women will undergo deceased donor uterine transplantation after IVF . AlthougSince Cleveland Clinic started the clinical trial, the team has completed eight uterus transplants; six were successful, and two resulted in hysterectomies soon after transplantation . The firFigure Uterus transplantation, Czech Republic The Czech UTx trial was established in July 2015 after approval by the Ministry of Health, Czech Republic. The first UTx of that trial was performed in April 2016 with a living donation from mother to daughter, and the first deceased donor (DD) UTx was performed in June 2016. As of November 2017, another seven uterine transplantations had been performed, in total, nine transplantations, four from deceased and five from living donors (LDs) . All the recipients were healthy, and all of them had MRKH syndrome. Donors aged 18 to 60 years old were used, LDs underwent a thorough examination, having a normal uterus, without a history of cancer, diabetes, hypertension, or any other serious disease, including infectious diseases. DDs had a normal uterus and no history of cancer, diabetes, or other chronic or infectious diseases. A maximum of four deliveries, including one cesarean section, were accepted for uterine donation. Both LDs and DDs had transvaginal ultrasound examination, hysteroscopy, and Pap smear before UTx. In two of the recipients, the uterus was transplanted from nulliparous DDs. Mild rejection episodes occurred seven times in three recipients, while moderate rejection occurred only once. Two uterine grafts were removed on day seven (DD) and day 15 (LD) after the UTx because of vascular thrombosis discovered by regular clinical examination and transabdominal Doppler ultrasound . One graft from a surgically successful DD UTx was removed on post-transplant month seven due to Herpes simplex virus‐2 (HSV‐2), which destroyed the uterine cavity and cervical canal. Histopathologic examination of the graft showed fibrotic obliteration of the cervical canal with signs of rejection in the myometrium . Partial postoperative stenosis of the uterine‐vaginal  anastomosis developed in three recipients, which were found between the first and second months after UTx. The stenosis was most likely caused by the discrepancy between the large uterine cervix and the narrow and tough vaginal vault. Spontaneous menstruation started within five to 14 weeks post-transplant in all six recipients with a functional uterus . In August 2016, a 26‐year‐old woman with MRKH syndrome was the fourth person worldwide who received a uterine transplant from a deceased donor. The donor was a 24‐year‐old brain‐dead nulliparous woman. The fifth frozen embryo transfer was successful, which resulted in a clinical pregnancy, and three weeks later, an intrauterine gestational sac containing an embryo with a heartbeat was detected. However, the pregnancy resulted in missed abortion . Uterus transplantation, Brazil The first worldwide case of a live birth following uterine transplantation from a deceased donor happened in Brazil. A 32-year-old woman with MRKH syndrome underwent uterine transplantation in São Paulo, Brazil, in September 2016, from a 45-year-old woman who died of subarachnoid hemorrhage. The donor had three previous vaginal deliveries . The recipient had satisfactory postoperative recovery and was discharged after eight days of observation in the hospital. Immunosuppressive drugs were begun with prednisolone, thymoglobulin, and continued via tacrolimus and mycophenolate mofetil (MMF). Five months post-transplantation, azathioprine replaced MMF .First menstruation started 37 days post-transplantation, which was regular (every 26-32 days). Pregnancy occurred after the first embryo transfer seven months post-transplantation. No abnormalities on blood flow velocity waveform were detected by Doppler ultrasound of uterine arteries, fetal umbilical, middle cerebral arteries, or any fetal growth impairments during pregnancy. No rejection episodes were noted after transplantation or during gestation. Cesarean delivery occurred on December 15, 2017, near week 36 of pregnancy. The baby girl weighed 2,550 g at birth, with APGAR scores of 9, 10, 10 and developing normally seven months postpartum. The uterus was removed, and immunosuppressive therapy was suspended . Uterus transplantation, Germany In October 2016, the first UTx was performed in Germany. In total, four pairs of uterus recipients and living donors underwent complete UTx procedures between October 2016 and January 2019. Recipients were from 23 to 35 years, and all had type 1 MRKHS. Donor ages were between 32 to 56 years, with only one postmenopausal donor. All donors gave birth to at least two children, to a maximum of four deliveries [The donor surgery involved harvesting the uterus with its blood vessels without the ovaries; the Fallopian tubes were removed to avoid extrauterine pregnancies. The arterial vasculature involved the deep uterine artery bilaterally with a segment of the internal iliac arteries, and the venous vasculature connected to the uterine graft to be harvested one or two deep uterine veins on both sides, connected to a segment of the internal iliac vein, and/or the proximal parts of the utero-ovarian branch, divided proximally to the inlet of the ovarian veins, to preserve the ovaries in situ . Recipient surgery comprised preparation by dissection of the vaginal vault and the external iliac vessels. Anastomoses were performed end to the side from the segments of the graft’s deep uterine arteries to the recipient’s external iliac arteries and the segments of the graft’s internal iliac veins to the recipient’s external iliac veins. In recipients with a thin deep uterine vein on one side, the proximal part of the utero-ovarian vein was used for an additional venous outflow, either by anastomosis onto the graft segment’s internal iliac veins or directly onto the external iliac veins. After proper reperfusion, the recipient’s vagina was opened, and vaginal-vaginal anastomosis was performed. The uterus was affixed to the sacro-uterine and round ligaments, while the bladder's peritoneum of the graft was sutured with the recipient’s bladder . There were no complications encountered intra- or postoperatively, except in one recipient, who showed impaired arterial flow on the right side intraoperatively, necessitating re-anastomosis. Suturing the right uterine artery end to side onto the external iliac artery in addition to a segment of the internal iliac artery with adjacent proximal vesical artery were performed. Immunosuppressive (IS) regimens for UTx were adopted from kidney transplantation . Pneumocystis jirovecii prophylaxis was prescribed. Two mild rejection episodes were observed in two recipients, and one recipient developed a CMV infection seven months post-surgery, which was successfully treated with valganciclovir [Induction therapy was begun with anti-thymocyte globulin (ATG) for three days, and the parallel start of a triple-drug IS regimen with tacrolimus, MMF, and prednisolone. MMF was replaced after approximately six months with azathioprine (AZA) for at least three to six months given a planned pregnancy. To improve individual tolerability, tacrolimus was replaced by cyclosporin. According to the donor’s and recipient’s cytomegalovirus (CMV) status, prophylaxis with valganciclovir for three to six months was done. Additionally, cotrimoxazole for six months for ciclovir . After the third single-embryo transfer, one recipient got pregnant, which was attempted at month 17 but ended in an early miscarriage at eight weeks and four days of pregnancy. However, following the next ovarian restimulation and oocyte fertilization procedures, she and another recipient underwent a fresh single-blastocyst transfer, resulting in successful intrauterine pregnancies in both .As a result, the two mothers (25 and 26 years old) gave birth to the 15th and 17th children globally, the first baby in March and a second in May 2019 . The firUterus transplantation, Serbia In March 2017, in Serbia at University Children's Hospital in Belgrade, a 38-year-old woman received a uterus transplant from her twin sister. The transplant was conducted in Belgrade by the Swedish medical team of Dr. M. Brännström, the pioneer of the uterus transplant technique, and assisted doctors from the Serbian hospital and physicians from Brigham Women's Hospital and Harvard Medical School, both in Boston, Massachusetts . According to Bologna Today, the uterus extraction lasted 10 hours, and the transplant lasted five hours without any complications. The sister who donated her uterus already has three children. After the transplant, the woman traveled to Stockholm to perform an IVF procedure (the post-transplantation management is not mentioned). Following an IVF procedure, she gave birth to a healthy baby boy at Sant' Orsola Hospital in Bologna, Italy . Uterus transplantation, India On May 18, 2017, a 21-year-old woman born with a congenital absence of the uterus became the first person to undergo a womb transplant in Pune, Inda. A team of doctors transferred her mother's uterus to her using a minimally invasive procedure. It took four-and-a-half hours to retrieve the uterus from the 41-year-old woman through minimally invasive keyhole surgery, medically known as laparoscopic surgery. In contrast, womb transplant surgery took nine hours . On May 19, 2017, a second uterine transplant was performed by the same team. The second recipient of a uterus was a 24-year-old woman, and the donor was her 45-year-old mother. The same procedure was successfully followed . The third successful uterine transplant was conducted in January 2018 by the same Galaxy Care Hospital team in Pune. While the first two womb transplants were completed in approximately nine hours, the third was completed in six hours. All three womb transplants at Galaxy Care Hospital were performed through laparoscopic surgery . In April 2018, another two successful uterus transplantation procedures were conducted in India at an urban, private, tertiary care hospital. Two patients, ages 30 and 24 years, diagnosed with MRKH syndrome, underwent LD uterine transplants; donors were their mothers of 48 and 47 years old, respectively. The IVF protocols and the results are not mentioned in the paper .Retrieval of organs was done through mini-laparotomy and laparoscopic intervention. The ovarian veins were used as outflow channels, which allowed to avoid the challenges involved in dissection along the internal iliac vein while harvesting the donor internal iliac artery reduced the tension on the vascular anastomosis . Oral immunosuppression with tacrolimus and mycophenolate mofetil was started 24 hours before surgery and continued postoperatively. Everolimus was commenced a month later to prevent rejection and allow for the decrease of the tacrolimus dose. Intravenous dexamethasone was administered postoperatively and gradually tapered to be replaced by oral prednisolone. Oral valganciclovir and cotrimoxazole were added on postoperative day one to prevent opportunistic infections. Subcutaneous heparin for three weeks, replaced by aspirin, was used for antithrombotic prophylaxis. Both recipients started menstruating within two months of surgery . In October 2018, the first baby girl was born via cesarean section at Galaxy Care Hospital in Pune, India. The baby was born 17 months after the uterine transplant, at 34 weeks of gestation. The uterus transplant was done on May 19, 2017. The baby was delivered prematurely as the mother's blood pressure began to rise suddenly, and the fluid around the baby had started to reduce . Uterus transplantation, Lebanon The first uterus transplantation in Lebanon was conducted by a team of doctors from Lebanon and Sweden on June 11, 2018 . The recPneumocystis jirovecci pneumonia was given for three months [The immunosuppression protocol consisted of induction by tacrolimus, azathioprine, thyroglobulin, and methylprednisolone. Thyroglobulin was given daily for five days, methylprednisolone was given perioperatively, and the prednisolone (per os) p.o. was given for six days. Maintenance immunosuppression was by daily administration of tacrolimus and azathioprine to maintain adequate immunosuppression. Mycamine was given as antifungal prophylaxis, daily valganciclovir for six months was used as CMV prophylaxis, and trimethoprim/sulfamethoxazole for prevention of e months . The first menstruation occurred three weeks after UTx, which was regular afterward, from 28 to 30 days. The patient developed vaginal stenosis, which required manual dilations three times. Due to persistent anemia, the patient received erythropoietin two times a week and oral ferrous sulfate with vitamin B12 three months before embryo transfer due to low hemoglobin levels . Embryo transfer was performed 10 months after uterus transplantation, which was successful and resulted in a positive pregnancy test after 14 days. Cesarean section was performed at 35 weeks gestation due to short uterus cervix and premature contractions registered on cardiotocography (CTG). A healthy female fetus was delivered 15 min after the skin incision . Table Limitations of this review This review article is a traditional review and, therefore, does not follow the standard Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews. Further researchUterus transplantation is rapidly spreading worldwide and is starting to be recognized as a new assisted reproductive technology and transplantation technique. However, it is still at the experimental stage and has many medical, technical, and ethical issues. The uterus transplantation procedure is a constant risk for further mothers, starting with transplantation, an immunosuppressive status, and ending with the cesarean section and hysterectomy.As observed, the babies born as a result of uterus transplantation should be born prematurely at less than 37 weeks of gestation, as specialists are concerned that as the uterus grows, this can affect the uterine vascular supply. Premature birth puts newborn babies at risk for apnea of prematurity, respiratory distress syndrome, infections or neonatal sepsis, intraventricular hemorrhage, necrotizing enterocolitis, retinopathy of prematurity, jaundice, and anemia. Some premature babies have to spend more time in a hospital’s newborn intensive care unit to get special medical care. Premature birth can also lead to long-term challenges for some babies, including intellectual and developmental disabilities. Long-term observation and control of babies born as a result of uterus transplantation from a living donor and a deceased donor are preferable, and case-control studies between babies born naturally and babies born after uterus transplantation.Further studies should concentrate on the safest operation method to minimize the complications on the donor and recipient. Additionally, further research is needed on the immunosuppressive protocol, which is used before and during the pregnancy period. Long-term observation of the patients (recipients) after hysterectomy is demanded. As they have to have a big intervention which changes the anatomy of pelvic floor muscles, the direction of ureters, and place of the ovaries, these may predispose to pelvic organ prolapse and urinary tract infections with further kidney damage. Additionally, these procedures can affect the vascular supply of pelvic organs and lower limbs.Until now, there is no clear information regarding how many weeks gestation a transplanted uterus can tolerate, newly formed vaginal vault, and deranged pelvic floor; further research is still needed. And is there any chance for a twin pregnancy? Uterus transplantation is a new treatment approach of absolute uterine factor infertility, which has proceeded rapidly from an experimental procedure in animals to a successful clinical application. However, at this time, uterus transplantation should be considered a clinical experimental procedure until sufficient experiences have been collected from clinical trials expected to enroll during the next years. There is a significant clinical demand for uterus transplantation; therefore, it is expected that the number of procedures will increase.


Phaseolus vulgaris) and to test their anti-inflammatory properties in a macrophage cell model. In vitro digestion of cooked beans released twenty times more polyphenols /g) than domestic processing (2.22 ± 0.1 mg GAE/g), with starch digestion contributing to the highest release (30.9 ± 0.75 mg GAE/g). Fluorescence microscopy visualization of isolated bean starch suggests that polyphenols are embedded within the granule structure. LC-MS analysis showed that cooked Borlotti bean contain flavonoids, flavones and hydroxycinnamic acids, and cooked bean extracts exerted moderate anti-inflammatory effects by decreasing mRNA levels of IL1β and iNOS by 25% and 40%, respectively. In conclusion, the bioaccessibility of bean polyphenols is strongly enhanced by starch digestion. These polyphenols may contribute to the health benefits associated with bean consumption.The consumption of beans has been associated with chronic disease prevention which may be attributed to the polyphenols present in the seed coat and endosperm. However, their bioaccessibility is likely to be limited by interactions with bean matrix components, including starch, protein and fibre. The aim of this project was to evaluate the effect of domestic processing and enzymatic digestion on the bioaccessibility of polyphenols from Borlotti beans ( Phaseolus vulgaris) are traditional staple foods in many countries around the world [Common beans  polyphenols and to determine the contribution of protein and starch enzymatic digestion to their release from the bean matrix. In this paper, we define bioaccessibility as ‘the quantity of a specific compound that is released from the food matrix during digestion and is available to the intestinal cells for potential absorption’ [The aim of this study was to evaluate the in vitro bioaccessibility of Borlotti bean (orption’ . FurtherP. vulgaris) were purchased from a supermarket in Leeds, UK. Beans were soaked overnight (16 h) in ultrapure water , drained and boiled for 10 min in water  followed by simmering for 1 h, as advised by the manufacturer. Aliquots of soaking and cooking water were kept at −20 °C for polyphenol analysis. Cooked beans were either subjected to digestion or freeze-dried for polyphenol extraction. Raw or freeze-dried cooked bean samples were milled to a fine powder and extracted with acidified methanol (3% trifluoroacetic acid) in a 1:20 ratio (w/v) by vortexing for 1 h. The supernatants of extractions were collected by centrifugation (9000× g) and kept at −20 °C for polyphenol analysis.Borlotti beans (2 (pH 6.0), according to McCleary [Aspergillus niger, Megazyme International, Bray, Ireland) for 60 min at 60 °C, pH 4.5. Samples were boiled for 5 min to stop further enzymatic activity and aliquots of the supernatants were kept at −20 °C for further analysis. For protein digestion, homogenized beans were digested with 30 U/mL protease  for 30 min at 60 °C, pH 6.0. Samples were boiled for 5 min to stop further enzymatic activity. The supernatants of digestion were collected by centrifugation and kept at −20 °C for polyphenol analysis.The in vitro digestion method used in this study was optimized to achieve the highest starch hydrolysis possible. Optimization was performed by treating the cooked bean with increasing concentrations (100–1000 units) of porcine pancreatic α-amylase at different incubation times  in combination with a fixed amount of amyloglucosidase (80 units) and a further protease (30 U) digestion . The optMcCleary , with soMcCleary . InitialThe residues of starch and protein digestion were collected by filtration through sintered glass, washed with series of acetone, dried in an oven at 80 °C and weighed to obtain a measurement of fibre content. The fibre was hydrolysed with 1 M HCl (10 mL) at 100 °C for 1 h. The supernatant of hydrolysis was collected by centrifugation and kept at −20 °C for polyphenol analysis.2CO3  and 750 µL of water. The mixture was incubated for 30 min in the darkness and the absorbance measured at 765 nm with a double beam spectrophotometer. Results were expressed as mg gallic acid equivalents (GAE) per g of dry weight.TPC content was determined by the Folin–Ciocalteu assay  with a fThe LC–MS analysis was carried out on Agilent 1200 LC with a Bruker HCT Ultra Ion Trap for MS detection and a photodiode array detector (DAD) for UV/V is measurements. The mobile phase consisted of acetonitrile/water, both with 0.1% formic acid. The gradient increased from 2–95% of acetonitrile over 1 min. The column used was Phenomenex Kinetex (50 × 2.1 mm and 2.6 µm particle size). The flow rate applied for the analysis was 1.3 mL/min. Putative identification of the polyphenols was done using information available on the phenol explorer database  and the g and 4 °C for 15 min. The pellet was washed three times using extraction medium, then twice in acetone, and centrifuged as mentioned previously. The final pellet was dried overnight at room temperature. Isolated bean starch was fixed in water and analysed with an Olympus BH2 fluorescence microscope . Pictures of native and gelatinized (from cooked bean) starch were taken under white and UV light.Starch isolation of beans was performed as described by Bustos et al. (2004) with a few modifications . Brieflyv/v) foetal bovine serum, and 1% antibiotics mix. Cells were grown in a humidified incubator at 37 °C and 5% CO2 and medium changed every second day. Methanolic extracts of cooked beans were evaporated with a Genevac EZ-2 ENVI and the resulting pellet was resuspended in 99% dimethyl sulfoxide (DMSO) to prepare a stock solution (200 mg/mL). Working solutions  were prepared by dissolving the stock solution in DMEM. The final DMSO concentration of the working solutions was < 0.1%.Murine RAW 264.7 macrophages were obtained from the European Collection of Authenticated Cell Cultures (ECACC). Macrophages were cultured in Dulbecco’s modified Eagle’s medium (DMEM), high glucose (4.5 g/L), supplemented with 10%  . BrieflyCells were incubated with increasing concentrations of bean extract  and sulforaphane as a positive inhibition control (5 µM) for 1 h, and then stimulated with LPS for 6 h (100 ng/mL). Cells were washed with PBS, lysed with Trisure  and frozen for later RNA isolation. Experiments were performed in three independent passages in duplicate.interleukin 6 (IL-6), interleukin 1 beta (IL1β) and nitric oxide synthase (iNOS). The housekeeper gene used was beta actin (ACBT). Forward (5′-3′) and reverse (3′-5′) sequences of the target genes used in this study are listed in TC method and expressed as the ratio of target gene to housekeeper gene.RNA was isolated following the manufacturers’ instructions and quantified using Nanoquant plate . iScript kit  was used for cDNA synthesis. Real time PCR was performed using the SensiMix SYBR High-ROX Kit and the StepOne real time cycler from ThermoFisher scientific. The primers used were for the following target genes: p < 0.05). Cell culture data were analyzed with one-way ANOVA followed by Dunnett’s t-test to compare treatments with control group. Significance was accepted at p < 0.05.Statistical analysis was performed using SPSS 24.0 statistical package. For digestion experiments and polyphenol determination, statistical differences between the means of three independent experiments were analyzed by one-way ANOVA, followed by multiple comparisons using the Tukey test , flavonoids (procyanidin B and petunidin derivatives) and flavones (apigenin derivatives) . A numbeWhen incubated with concentrations up to 200 μg/mL of cooked bean extract, macrophage viability was not affected a. The exP. vulgaris polyphenol studies [P. vulgaris beans [Vigna aconitifolia) [P. vulgaris) [Relatively small amounts of polyphenols were solubilised during soaking and cooking; these polyphenols are likely to originate from the coloured seed coat and may have functions in protecting seeds from environmental stress and pathogen attack. Our TPC values of whole Borlotti beans are comparable to other  studies . Around is beans . Soakingis beans . Polypheis beans . Satwadhtifolia) . In the ulgaris) . After bulgaris) ,47.P. vulgaris) increased the amount of free phenolic acids and flavonols measured [In the present study, cooking water presented higher TPC values than raw bean methanolic extract . A possimeasured . Cookingmeasured . Our resThe most significant finding of this work was that α-amylase hydrolysis was responsible for the release of around 76% of the polyphenols liberated upon in vitro digestion. This provides strong evidence that polyphenols interact with starch structures in nature, and therefore starch can be considered as the main limiting factor for polyphenol bioaccessibility in Borlotti beans, which has not been reported before. Fluorescence microscopy of isolated starch granules revealed strong fluorescence within the granules, providing further evidence that polyphenols are embedded within the starch granule structure and are liberated from the starch matrix during digestion. The inclusion of polyphenols into the starch granule may occur during granule formation. Legume starch has been shown to have a C-type polymorphic arrangement within starch granules, which in effect is characterized by the presence of both A- and B-type polymorphs within the same granule . We postThe mechanisms by which polyphenols interact with starch are not yet clear. Examples of non-covalent interactions between flavonoids and starch have been proposed ,53. Wu eThe digestion protocol used in this study was optimized to achieve the maximum possible digestion of starch . The panP. vulgaris) protein [Around 16% of the TPC released upon in vitro digestion were liberated after the protease hydrolysis of Borlotti beans. Polyphenols may bind with proteins mainly by non-covalent interactions  . In this protein . TherefoO-glycosides) or through carbon–carbon bonds (C-glycosides) [In this study, a further polyphenol extraction was achieved after acid hydrolysis of Borlotti bean fibre. It is known that polyphenols bind with fibre components such as cellulose, hemicellulose , lignin and pectin, and are normally present as glycosides, linked through a hydroxyl group with single or multiple sugars (cosides) . Under pcosides) .As shown by LC–MS results, the cooked Borlotti bean extract contained a mixture of polyphenols including catechin, apigenin and hydroxycinnamic acid derivatives, which are likely responsible for the anti-inflammatory effects observed in LPS-stimulated RAW264.7 macrophages. In a previous study, chlorogenic acid inhibited LPS-induced RAW264.7 macrophages . Kim et P. vulgaris) cultivars have reported anti-inflammatory effects in vitro and in vivo. Garcia-Lafuente et al. (2014) found that round purple bean extracts reduced NO production and reduced IL-1b and IL-6 mRNA expression of LPS-stimulated macrophages [Studies using extracts from common bean  the maximum concentrations of polyphenols in the blood stream after the consumption of polyphenol-rich foods are about 0.1–1 mM . The TPCThe present study presents interesting data regarding possible associations between polyphenols and bean starch, which have not been reported previously. Also, cooked Borlotti bean extract exerted a moderate anti-inflammatory effect indicating the possible health benefits of this legume. However, we consider that this study has a few limitations that should be addressed in future work, including: applying physiologically relevant digestion conditions, assessing bioavailability in vivo, testing the effect of lower concentrations of extract and single/combinations of compounds on NF–κB and downstream pathways.Our data provide new evidence that digestion, in particular starch enzymatic hydrolysis, may increase polyphenol bioaccessibility. It is currently unknown whether polyphenol/starch interactions are also naturally present in other beans and other plant foods. Further studies are required to understand the mechanism of starch polyphenol interactions, how these are formed in planta and how they are disrupted during food processing. Furthermore, the physiological relevance of these interactions is not yet clear. Further research, in vitro and in vivo, is needed to understand these interactions and their potential impact on human metabolism and health, and the implications of the interactions on polyphenol bioactivity. Furthermore, studies are needed to better understand the effect of individual polyphenols or combinations, as well as polyphenol-rich diets, on anti-inflammatory pathways and disease prevention.