The public health system is extremely dependent on the use of vaccines to immunize the population from a series of infectious and dangerous diseases, preventing the system from collapsing and millions of people dying every year. However, to develop these vaccines and effectively monitor these diseases, it is necessary to use accurate diagnostic methods capable of identifying highly immunogenic regions within a given pathogenic protein. Existing experimental methods are expensive, time-consuming, and require arduous laboratory work, as they require the screening of a large number of potential candidate epitopes, making the methods extremely laborious, especially for application to larger microorganisms. In the last decades, researchers have developed in silico prediction methods, based on machine learning, to identify these markers, to drastically reduce the list of potential candidate epitopes for experimental tests, and, consequently, to reduce the laborious task associated with their mapping. Despite these efforts, the tools and methods still have low accuracy, slow diagnosis, and offline training. Thus, we develop a method to predict B-cell linear epitopes which are based on a Fuzzy-ARTMAP neural network architecture, called BepFAMN . This was trained using a linear averaging scheme on 15 properties that include an amino acid ratio scale and a set of 14 physicochemical scales. The database used was obtained from the IEDB website, from which the amino acid sequences with the annotations of their positive and negative epitopes were taken. To train and validate the knowledge models, five-fold cross-validation and competition techniques were used. The BepiPred-2.0 database, an independent database, was used for the tests. In our experiment, the validation dataset reached sensitivity = 91.50%, specificity = 91.49%, accuracy = 91.49%, MCC = 0.83, and an area under the curve (AUC) ROC of approximately 0.9289. The result in the testing dataset achieves a significant improvement, with sensitivity = 81.87%, specificity = 74.75%, accuracy = 78.27%, MCC = 0.56, and AOC = 0.7831. These achieved values demonstrate that BepFAMN outperforms all other linear B-cell epitope prediction tools currently used. In addition, the architecture provides mechanisms for online training, which allow the user to find a new B-cell linear epitope, and to improve the model without need to re-train itself with the whole dataset. This fact contributes to a considerable reduction in the number of potential linear epitopes to be experimentally validated, reducing laboratory time and accelerating the development of diagnostic tests, vaccines, and immunotherapeutic approaches. To prevent the health system from collapsing and millions of people dying each year, it is important to develop accurate diagnostic tests that help with control and monitoring, as well as efficient vaccines that immunize people against dangerous and highly infectious diseases.Vaccination has been applied systematically for decades, culminating in the eradication of many infectious diseases, such as mumps, rubella, whooping cough, polio, measles, and tetanus ,2. In adTo develop these vaccines and offer greater safety, potency, and efficacy , it is fB-cell epitopes are classified as linear or conformational. Linear epitopes are formed by sequential (contiguous) amino acids of the protein, while conformational epitopes are composed of amino acids that are not contiguous in the primary sequence but are joined in the folding of the protein .Although most epitopes are conformational, their identification requires information based on their three-dimensional structure, which is still very limited compared to the number of protein sequences currently available in public databases . TherefoThe conventional and most reliable methods used for mapping these epitopes, such as crystallography  and nuclDue to the simplicity in generating embeddings, the study of prediction of linear epitopes of B-cells has been taking place for several years, and already has many tools available in the literature, in which they use machine learning techniques in their predictions, such as ABCPred , BCPred However, because epitopes do not have intrinsic characteristics in the protein, but only when they interact with the antigen paratope, the task of predicting epitopes in silico becomes challenging . This faAnother factor that influences performance is the low quantity and quality of data available for training. Thus, with the constant updating of one of the main public databases, the Immune Epitope Database (IEDB) , togethehttp://tools.iedb.org/main/bcell, accessed on 2 March 2022), demonstrating the difficulty that these tools have in their predictions.Two tools frequently used to predict linear epitopes of B-cells are BepiPred-2.0  and EpiDIn this context, we developed BepFAMN, a tool that uses a new approach to identify and locate linear epitopes of B-cells in amino acid sequences. The tool uses an ANN from the ART (adaptive resonance theory) family that allows online training and promotes great ease in learning new patterns, without losing the memory of already learned patterns (plasticity and stability).The ART family networks are very suitable for complex classification problems, where useful knowledge about the features of the objects to be classified is quite limited . With enIn the tests of the tool, the AUC of the ROC curve and the AUC of the precision-recall curve were used, because, according to , in situFor the target problem, the number of false positives and false negatives impact our model in different ways. In this way, we use the F1-score metric, in the test dataset, to create a result from these divergences.BepFAMN was able to identify linear B-cell epitopes with a ROC curve AUC of 0.7831 and a precision-recall curve AUC of 0.8343, which significantly exceeds previous methods . This helps to considerably reduce the number of potential linear epitopes to be experimentally validated, reducing costs and services, as well as accelerating the process of vaccine development and diagnostic tests.The ABCPred  used recThe BCPred  used SVMThe BayesB  is a proThe SVMTriP  is a metThe BEEPro  is a proThe SVM BEEProPipe  is a linThe BepiPred-2.0  is a webEpiDope  is a tooIn this section, the dataset and the methodology used for training the proposed tool are presented and briefly described, in addition to the validations performed.The first stage of this research consisted of searching for linear B-cell epitopes, available in public databases. Due to the quantity, diversity, experimental validation, and many fine filters of research available in the IEDB database, it was decided to provide both positive and negative epitopes to the training and testing experiments.Positive epitopes are those that have been experimentally validated and have positive assays (results) in at least one experiment. The negative ones were also experimentally validated and must not present a positive test in any experiment.Obtaining the positive and negative epitopes of the IEDB was carried out on 5 April 2021, using the following filters for the query: linear epitopes, infectious diseases of humans, B-cell assays, no MHC restriction, and bacteria, viruses, and protozoa.In the end, the database contained 11,509 positive epitopes and 28,080 negative epitopes, as described in DB_bac: composed only of positive and negative bacterial epitope residues;DB_vir: composed only of positive and negative virus epitope residues;DB_prot: composed only of positive and negative epitope residues from protozoa;DB_all: composed of positive and negative epitope residues from all three taxa.Four databases are created, consisting of three independents, each formed by the residues of a taxon, and one dependent, formed by the union of the residues of all three taxa. The amount of positive and negative epitopes of each base is shown in A very important piece of data that was missing in the IEDB database metadata is the complete protein sequence of each antigen. Because machine learning algorithms use the context of a protein’s amino acids to try to find correlations and predict possible epitopes, it was necessary to obtain this information. With the code of each antigen, obtained from the IEDB database, the NCBI database  was acceThe validation database is independent of the training/testing database and was taken from . The basAmong the 30,556 sequences, 11,834 are positive epitopes and 18,722 are negative epitopes. The subset of positive epitopes has an average length of 13.99 (number of consecutive amino acids), while the subset of negative epitopes has an average length of 13.20, as shown in The epitopes that had similarities greater than or equal to 80% , evaluatNext, it was necessary to perform some adjustments to eliminate irrelevant data, in addition to enriching and transforming essential data into “cleaner” and “friendly” data. Thus, in addition to epitope information, only the metadata about the initial and final position of each epitope and the antigen sequence was kept. To this dataset, only one new metadata was added, the complete protein sequence of the antigen.Initially, a great imbalance between the classes was found. To prevent the knowledge models generated from being biased and important epitopes from being discarded, prevalence correction techniques were carefully applied, such as Stratified Sampling, for example. Furthermore, it was identified that some records had missing important data and, therefore, were also eliminated. Other records removed were those with epitopes greater than 30 and smaller than 5 amino acids, as they occurred sporadically in the database. In the end, the total database was composed of 9,968 positive epitopes and 10,766 negative epitopes, as described, by taxon, in From the raw dataset of , a set iFirstly, the amino acid sequences of identical proteins were joined together, but keeping the information about their verified regions  A,B, resuIt is important to mention that this entire procedure is necessary to avoid overrepresentation in the data, which can directly influence the training of the machine learning algorithm.The prediction strategy of this work was inspired by the BEEPro tool , which pAs PSSM calculations for very large proteomes are time-consuming, with any possibility of providing a web-based epitope prediction tool, this work chose not to use PSSM, and only use the other properties.Several studies in the literature ,19 showeα_i amino acid in positive epitopes of the protein;α_i amino acid in negative epitopes of the protein.Therefore, the proportion rate was calculated for each of the 20 amino acids present in the database, through Equation (1). However, to determine how often each amino acid occurs in positive and negative epitopes, Equation (1)  was applTo attenuate the frequency dominance of some amino acids and to prevent the machine learning algorithms from becoming biased, Equation (2)  normalizAfter testing a combination of various physicochemical and biochemical properties in predicting linear B-cell epitopes, reference  found thj, for j = 0, 1, ..., 14, according to Equation (3) .It is important to point out that each instance represents the calculations of the 15 properties applied to an amino acid of interest, being either for the subset of positive epitopes or for the subset of negative epitopes.After all the pre-processing procedures were applied to the datasets, the cross-validation technique was used, with K-folds equal to 5, to divide each dataset into k mutually exclusive subsets of the same size and, from then, to use a subset k for testing and the other subsets (k−1) for training.The FAM ANN technique has been used quite frequently in prediction work, mainly in the field of electrical engineering, because of its plasticity, stability, and ability to converge a few times ,38,39.aρ, bρ, and abρ. An inadequate choice can result in a loss of accuracy of the results, as values close to zero allow little identical patterns to be grouped into the same category, and values close to one allow small variations in input patterns, leading the ANN to create new classes [Although FAM ANN is a technique that presents good generalization rates, the performance of its prediction depends on the choices of the values of the following vigilance parameters:  classes .ρabaseline) 0.8, and 0.99 were used for the parameters aρ, bρ, and abρ, respectively.In this research, values of 0.61 . Furthermore, the number of neurons must be less than or equal to the values of the parameters am, bm, and n.n: number of input patterns.The weight matrices am and bm parameters, and, therefore, the values of these parameters must be doubled.It is important to point out that the complements of the input and output patterns are also accounted for when defining the values of the ma and bm were defined in 30 and 2, respectively. The value of n was set to the values of 35,157, 97,001, 109,120, and 241,278 for the DB_bac, DB_prot, DB_vir, and DB_all datasets, respectively.In this work, as already mentioned, 15 properties were used to predict whether an amino acid of an antigen protein is an epitope (1) or if it is not an epitope (0). In this way, the values of the parameters bw weight matrix was optimized and defined according to Equation (6).As it is a binary classification problem, the The order of presentation of the ANN input and output patterns can be used sequentially or in a random/pseudorandom order. However, from a mental point of view, it is more sensible to adopt the random/pseudorandom order. Therefore, in this work, the second approach was adopted.wa weight matrix contains the first input pattern, line one contains its complement. This process is repeated for all other input and output patterns, and at the end, the even lines will contain the input or output patterns, and the odd lines their respective complements.To calculate the complement of the matrix pairs (input and output) the strategy of doubling the number of rows of the respective matrices instead of doubling the number of columns was used. Thus, if line zero of the β = 1 (fast training) each knowledge model was generated from a single epoch. To improve the prediction of epitopes, for each fold created by cross-validation, three knowledge models are generated, and for the training of each one, the input and output patterns are presented in a pseudo-random way and in a different order, allowing that each model learns in a way and generates different amounts of neurons.Using the training parameter aw and wab weight matrices, since the bw weight matrix has only two neurons .To generate each group of knowledge models for each partition of each dataset, the same quantities and the same input data were used, changing only the order in which the inputs were presented to the ANN.aρ. However, it is possible that input patterns appear with very different characteristics from the existing categories, so none of the categories are similar enough to meet the vigilance test.In the diagnosis, ANN activates the category that best represents the entry pattern, through the choice function, and checks whether the degree of similarity between the entry pattern and the active category meets the value defined in the parameter aρ by parameter ε was adopted, until the closest category became compatible enough and passes the vigilance test. Empirically, the value of ε was set at 0.1.For these cases, the strategy of automatic gradual decrement of parameter aρ parameter returns to its original value of 0.61 and the execution passes to the inter-ART module. At that moment, it is verified which column of the abw weight matrix is active, based on the index of the winning neuron of the aw weight matrix (in this same row in abw). With the abw column index, it is verified which category (row) belongs to this index in the bw weight matrix, this category being the final diagnosis of the FAM ANN.After passing the vigilance test, the value of the aρ. However, it is possible that input patterns appear with very different characteristics from the existing categories, so none of the categories are similar enough to meet the vigilance test.aρ by parameter ε was adopted, until the closest category became compatible enough and passes the vigilance test. Empirically, the value of ε was set at 0.1.For these cases, the strategy of automatic gradual decrement of parameter For the diagnosis process, cross-validation and the competitive strategy were used, in which each generated knowledge model (three) performs its own process, and in the end, there is competition with its results, such as the result that prevails, is the final diagnosis of ANN, on the current input pattern. It is noteworthy that this approach was applied to each dataset independently, and only the input patterns were presented to the ANN, meaning that the output patterns were used only to validate the prediction results. For the validation database, the same competition process was applied, but in 100% of the dataset, in addition to using the same knowledge models generated by the training dataset DB_all.TP: number of true positives. Residues predicted as epitopes and actually are epitopes;TN: number of true negatives. Residues predicted as negative epitopes and actually are negative epitopes;FP: number of false positives. Residues predicted as positive epitopes and are negative epitopes;FN: number of false negatives. Residues predicted as negative epitopes and are positive epitopes;sen: the rate of positive epitopes that are correctly predicted as positive epitopes;Resp: the rate of negative epitopes that are correctly predicted as negative epitopes;Rpres: among all epitope classifications, how many are correct. Ideal in situations where FPs are considered more harmful than NFs;Racc: indicates overall performance. How well did the model rank;Rmcc: is a measure considered balanced and was proposed by Matthews in 1975. It assumes values between −1 and 1, where:Value close to −1, corresponds to a bad prediction;Value close to 0, corresponds to a random prediction;Value close to +1 corresponds to an excellent prediction.RTo measure the performance of the knowledge models generated for each dataset, the following metrics commonly used to assess the quality of prediction tools are used: sensitivity, specificity, precision, accuracy, and MCC, which are defined, respectively, by the Equations (7)–(11) ,40.(7)RIn addition to the aforementioned metrics, a graphical method is used, capable of demonstrating the relationship with sensitivity and specificity, called ROC curves (receiver operating characteristics). However, to make it possible to compare the results of the classifiers, it is necessary to reduce the ROC curve to a simple scalar. The method usually used for this purpose is called AUC, the function of which is to calculate the area under the ROC curve.For the validation dataset, the performance of the proposed tool is compared with the EpiDope tool  and withAlthough all the metrics mentioned in After the process of diagnosis and possession of the results from the DB_bac, DB_vir, DB_prot, and DB_all databases, the metrics discussed in According to Seeking to assess the ability of the implemented method to identify positive epitopes, an average of the ROC curve is calculated for each subset of each database. This was necessary, as five-fold cross-validation is used and, therefore, for each partition, a ROC curve is calculated, and at the end, an average of the ROC curve is generated. All sets used presented a ROC curve with accuracy greater than 86%, and the protozoan database (DB_prot) presented the best accuracy index, at approximately 99.45%. The worst rate was revealed by the viral database, a figure of approximately 86.89%. The other sets were within this range, obtaining approximately the values of 94.54% and 92.89% for DB_bac and DB_all, respectively.The validation database was used solely and exclusively to validate the knowledge models generated by the DB_all dataset. This way, there is no need to combine several forecasts to generate an average of the ROC curve, since the cross-validation technique is not applied and the data is not used for training. At this stage, the 15 knowledge models were used, together with the competition technique, to predict the linear B-cells epitopes.From the ANN diagnosis, all the metrics briefly discussed in According to The F1-score represents a harmonic mean between precision and recall, combining both in order to bring a single value that indicates the overall quality of the model . To presThe main tools available in the literature do not allow for large-scale testing and limit access to many features. Thus, the results of reference  are usedIn this work, the AUC of the ROC curve and the precision-recall curve are used to compare the performance of EpiDope with the performance of other tools commonly used in predicting linear B-cell epitopes.From the results obtained with the validation database, a set of data that went through all the pre-processing procedures, as described in reference , was triThere are several computational tools available in the literature for in silico identification of linear epitopes of B-cells. As described in However, many of these tools have a low rate of accuracy in predicting epitopes, or when they improve the results, they involve high computational costs, making large-scale use unfeasible, especially via the Internet.One factor that directly influences the reduction of these results is the bias inherent to this type of dataset. To obtain an unbiased epitope prediction it is essential to have a wide variety of known epitopes and non-epitopes from different organisms in the training set. Therefore, analyzing the taxonomic variety of the proteins provided by the database , eliminating redundancy, and balancing the classes is fundamental.In the extraction of training data, a similarity reduction filter (BLAST) was used, with a threshold of 80%, along with prevalence correction techniques to attenuate the number of redundant epitopes and balance the amount of data in each class to avoid bias to ANN. The final training dataset contained 18.81% bacteria, 42.69% viruses, and 38.50% protozoa. Overall, the training data exhibited a sufficient degree of taxonomic diversity.The same process occurred for the test data. However, these data were taken from the works ,13. To rThe method implemented for epitope prediction, as mentioned in The Fuzzy-ARTMAP ANN was trained and tested on datasets formed by a single taxon and one formed by the union of the three taxa. For each set, 5 times cross-validation and 3 times competition technique were applied, totaling 15 knowledge models for each dataset. In this way, for every 3 models generated, the diagnosis was made and the metrics calculated, and at the end of the cross-validation process, an average for each metric was calculated.The competition technique added about 5 to 15% to the final value of each metric. This is justified because the network creates independent models that learn in different ways, for the same dataset, in each execution. The order in which the input patterns feed the network and the value of its parameters influence how models learn. Thus, for each partition built by cross-validation, on each dataset, the algorithm is executed three times and, in the end, the result that occurs most frequently, for each input pattern, is the ANN prediction.The Fuzzy-ARTMAP ANN used rapid learning in the training stage, using only one epoch for each knowledge model generated. This characteristic is one of the great advantages of the ART family algorithms concerning other approaches available in the literature, which generally use several epochs to converge.Tests performed on the DB_prot dataset showed the highest performance ratings, all above 99%. However, the viral database (DB_vir), even with good rates, obtained the lowest values for all metrics, indicating that the Fuzzy-ARTMAP ANN had problems generalizing it. This indication can be seen in The same conditions used to create the training dataset were applied to the validation dataset, i.e., all proteins that tested positive in at least two assays were stored as epitopes, and all that tested non-positive in at least two assays were stored as non-epitopes.Compared to the training data, the validation set has a higher proportion of bacteria verified regions (18.81% vs. 42.69% against 25.10%). Furthermore, the proportion of epitope versus non-epitope regions changed from 48.08% of epitopes in the training dataset to 34.62% in the validation dataset.For the validation dataset, the knowledge models reached a sensitivity rate of 81.84%, even with the number of negative epitope residues much higher than the number of positive epitopes. Therefore, deterministically saying that all residues are negative epitopes may provide a high rate of specificity but not sensitivity. On the other hand, a specificity of 74.75% also ensures that the model did not predict all residues as positive epitopes, otherwise it would have very low specificity.We evaluated the performance of BepFAMN with competing methods in the validation dataset for the verified regions, using the AUC of the ROC curve and the precision-recall curve. In both, BepFAMN outperformed competing tools’ prediction approaches. EpiDope, which had the best values for both metrics, was outperformed by 18% and 17%, respectively. The second best method (Beta-turn) was surpassed by 22.10% and 27%, respectively .These results indicate that BepFAMN was able to generalize the prediction task and can even predict data relatively distinct from the training data, with greater accuracy than competing methods.This research has contributed by offering a new approach to solving the target problem based on ANN using adaptive resonance theory (ART). Although this architecture was proposed a long time ago (the 1980s), it is certainly current and quite competitive in relation to new techniques available in the specialized literature, such as “deep learning”. It should be noted that the ANNs of the ART family are also susceptible to the implementation of the “deep learning” concept and, in fact, it is an approach that is being developed by the research group.In this sense, it opens the perspective of applications in several areas of human knowledge, including, in particular, continuing research in the health context, for example in aiding the development of immunizing agents. It is considered an important contribution to the technological area of public health, seeking to establish a healthy “symbiosis”, providing a great opportunity to develop new ANN architectures.In this study, we developed a new method for predicting linear epitopes of B-cells, which, despite requiring only an amino acid sequence as input, has shown to have the best performance among the current tools used for this type of prediction.The BepFAMN technique is based on an ANN from the ART family, and was trained on a dataset of approximately 21,000 experimentally verified epitope and non-epitope regions. We used the training dataset to understand the behavior of ANN on each taxon, and on the set of three taxon. In these sets, five-fold cross-validation was used to ensure the reliability of the experiment. Furthermore, all proteins in a subset have a sequence identity of less than 80% of the other proteins in the remaining 4 subsets.The second dataset, the one used for validation, consisted of almost 25,000 new epitopes and non-verified epitopes, both of which were not present in the training dataset. We used this dataset to compare the performance of BepFAMN with other current tools available in the literature and frequently used ,13,19. IWhen analyzing the validation set, it is possible to notice that the specificity value was slightly below the sensitivity value, because the number of false negatives is greater than the number of false positives. This event demonstrates that ANN makes a more conservative forecast. In practice, a high true positive rate and a low false positive rate are much more important to reduce laboratory work.The performance of BepFAMN can be attributed to the stability and plasticity characteristics provided by the ART family networks, its good generalization capacity for this type of data, and for the contribution of the competition technique. Furthermore, as the PSSM property was not used in the generation of the embeddings, we plan to develop a web and/or standalone version for the community, without a loss of performance.Finally, epitope prediction tools should primarily serve as filters to rule out regions that are unlikely to be epitopes and, thus, eliminate unnecessary experimental analysis. In this way, new tools should emerge that increasingly improve sensitivity and specificity, in order to allow these experiments to be more precise and targeted.


The optimal revascularization strategy remains uncertain in multivessel coronary artery disease (MVCAD). The durability of the surgical grafts should be weighed against the decreased invasiveness of percutaneous coronary intervention (PCI). Hybrid coronary revascularization (HCR), a combination of PCI and surgery, could be a feasible alternative. This study aimed to investigate the occurrence of major adverse cardiac and cerebrovascular events (MACCE) and all-cause mortality after both endoscopic coronary artery bypass grafting (Endo-CABG) and the HCR procedure.In this single-center retrospective observational study, 347 consecutive patients have been subjected to an Endo-CABG procedure, of which 103 underwent HCR between January 2016 and January 2018. A propensity score matching analysis was performed to match 103 Endo-CABG alone patients to the 103 HCR patients. The Endo-CABG procedure was performed through 3 endoscopic ports (5 mm) in the 2nd, 3rd, and 4th intercostal space and a utility port of 3 cm.p = 0.155). Additionally, the occurrence of MACCE after a mean follow-up of 1188 ± 538 days was similar in both groups . Still, the long-term all-cause mortality over this period was significantly higher in the matched Endo-CABG alone group .In both the HCR and matched endo-CABG alone group, the 30-day mortality was acceptable (0% in the HCR group and 1.94% in the matched Endo-CABG alone group, HCR has some advantages over Endo-CABG alone regarding the all-cause mortality, cross-clamping time, intensive care unit, and hospital length of stay. Therefore, HCR may be a suitable alternative therapy for patients with MVCAD. Coronary artery disease (CAD) has typically been treated either medically, with percutaneous coronary intervention (PCI), or with coronary artery bypass grafting (CABG). As advances in stent technology and minimally invasive surgery have developed, hybrid coronary revascularization (HCR) has emerged as an alternative treatment for multivessel coronary artery disease (MVCD) , 2. ClasIn this single-center observational study, the clinical outcomes after HCR and Endo-CABG alone were retrospectively investigated. The protocol conforms to the ethical guidelines imposed by the 1975 Declaration of Helsinki. The study was approved by the ethics committee of the Jessa hospital and Hasselt university on February 8, 2021 (2020/188).All 347 consecutive patients with MVCD who underwent coronary surgery in Jessa Hospital from January 2016 to January 2018 were enrolled. Altogether, 244 patients have been subjected to Endo-CABG alone and 103 to HCR  and Clopidogrel (75 mg) once daily. When PCI was performed after the Endo-CABG, 600 mg Clopidogrel was administered at hospital admission.The primary endpoints are the occurrence of MACCE at 30 days and the long-term all-cause mortality. MACCE is a composite of cardiac death, myocardial infarction, stroke, and revascularization. Cardiac death is defined as death after myocardial infarction, sudden cardiac death, heart failure, stroke, cardiac hemorrhage, and death due to cardiac procedures . SecondaStatistical analyses were performed using R Core Team (2021) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Statistical significance is defined as a p-value of < 0.05. Data normality was evaluated using QQ-plots and Shapiro–Wilk tests. All data were represented as mean ± standard deviation (SD), median (interquartile range (IQR)) or numbers (n), and percentages (%) as appropriate. Differences between groups were compared using a chi-square test, Mann–Whitney U test, Wilcoxon Signed-Rank test, and t-tests also as appropriate. A Kaplan–Meier analysis was performed to estimate the probability of survival and occurrence of MACCE past given time points. A propensity score matching was implemented using nearest pairing to correct for baseline differences between the two groups.p = 0.04), and 85.01% of all enrolled patients were male. The mean European System for Cardiac Operative Risk Evaluation (Euroscore) II was 2.24 ± 2.60 in the HCR group and 2.78 ± 2.97 in the Endo-CABG alone group. This Euroscore II corresponds to a moderate operative risk in both groups. A significant difference was found in baseline characteristics between HCR and Endo-CABG alone patients for age, preoperative PCI, chronic kidney disease, and left main disease . After propensity score matching, there were no differences in baseline characteristics. All demographic variables are represented in Table In total, 347 consecutive patients were enrolled in the analysis, of which 244 (70.32%) were in the Endo-CABG alone, and 103 (29.68%) were in the HCR group. The mean follow-up time was 1188 ± 538 days. Of the patients undergoing HCR, 44 (42.27%) received PCI first (reverse HCR) and 59 (57.28%) CABG first (standard HCR). The mean age of the included patients was 67.62 ± 10.08 and 67.67 ± 10.15 years for respectively the Endo-CABG alone and the HCR group  versus 12 (11.65%) for HCR and matched Endo-CABG patients; p = 0.652). The freedom of MACCE at the end of follow-up is 83.0% after matched Endo-CABG alone and 89.5% after HCR . The estimated overall survival at the end of follow-up is 72.5% after Endo-CABG and 96.3% after HCR  versus 3 (2.91%) for HCR and matched Endo-CABG patients; In addition to the four patients that suffered cardiac death, the cause of death was not defined in two patients. Moreover, a stroke caused the death of two patients, and another one died due to postoperative bleeding after carotid endarterectomy. Additionally, one patient died due to coronavirus disease 2019 (COVID-19) and one due to lung disease. One patient of the HCR group passed away due to multiorgan failure and another from a myocardial infarction. The last patient of the HCR group died of an unknown cause. All outcomes are represented in Table The intensive care unit LOS, ventilation time, and hospital LOS were not significantly different between the matched Endo-CABG and HCR groups. Moreover, the study groups did not differ between postoperative parameters among new-onset atrial fibrillation, neurological dysfunction, and perioperative and 24 h bleeding. Blood chemistry and hematology data are shown in Table p < 0.001). The CPB time was significantly shorter in the reverse HCR group (p = 0.034). Additionally, there was a significant difference in graft failure. It occurred in 10.17% after standard HCR while no graft failure was seen in the reverse HCR group (p = 0.029). However, there was no significant difference in peri- and postoperative bleeding between standard and reverse HCR . All outcomes of standard and reverse HCR are represented in Table For patients of the HCR group who underwent reverse HCR N = 44), the mean number of days between the two interventions was 46.23 ± 39.48 days. For those who received standard HCR (n = 59), the mean number of days in-between was 59.07 ± 57.42 days  showed that in patients with multivessel coronary disease, the risk of MACCE after HCR and CABG was similar between the two groups  [CR group .In the previously conducted trials, HCR patients had a shorter hospital LOS , 8 which., respectively) [Our baseline characteristics resemble the ones found in previous studies , 8. Othectively) . This reThe ideal sequence of PCI and CABG is an important debate. The graft patency can be checked during the PCI when standard HCR is applied, but there is a risk of lesion instability in the waiting period between surgery and PCI. In reverse HCR, the dual antiplatelet therapy is continued to avoid stent thrombosis, increasing the risk of surgical bleeding . HoweverWhen further comparing the clinical outcomes, we found a significantly lower CPB time in the reverse HCR group. The other postoperative results, i.e., clamping and ventilation times, are not significantly different. Interestingly, the peri- and postoperative bleeding was not higher in the reverse HCR group despite the use of Clopidogrel. Prolonged CPB time has been associated with postoperative complications, such as heart failure, impaired coagulation, and renal, pulmonary, and neurological dysfunction . NeverthOverall, the procedure sequence did not dramatically influence the clinical outcomes of the patients in this observational study. Though, it is important to mention that the preferred applied sequence of HCR depends not only on the best postoperative outcomes it would provide but also on the patient's clinical presentation, such as the location and severity of the lesions .Due to the retrospective nature of this study, it is questionable whether the decision for the HCR treatment causes the observed difference in mortality. Therefore, interpretation of these survival curves needs to be done carefully. Additionally, this study is prone to a high amount of selection bias since the order and timing of the Endo-CABG and PCI were determined by the patient's coronary anatomy and are joint decisions between the surgeon and interventional cardiologist. Moreover, twice as many patients were enrolled into the Endo-CABG group compared to the HCR group. The assessment of the lesions was mainly performed visually. However, a hemodynamic evaluation would improve the decision-making. Another limitation of this study is that some patients were followed up at another hospital, so follow-up data was unavailable.A prospective randomized controlled trial is needed to investigate the aforementioned findings further.This observational trial is the first to examine the value of on-pump totally endoscopic pan-arterial surgical revascularization without robotic assistance in a setting of HCR or coronary surgery alone. HCR with Endo-CABG seems to be a suitable alternative therapy for elective patients with CAD since it provides an excellent short-term outcome. Additionally, HCR has some advantages over Endo-CABG alone regarding the cross-clamping time, intensive care unit, and hospital LOS. However, a prospective trial should be performed to confirm these results.


High positive correlations were observed between the short and standard protocols in the stiffness of the VL , the RF  and the PT  and no differences were found between both protocols in the stiffness assessment of the VL, RF and PT (p > 0.05). Therefore, the five-compressions protocol is a valid protocol for the assessment of lower limb mechanical properties.The objective of this study was to evaluate the validity of a short assessment MyotonPRO protocol to measure the stiffness of the superficial muscles and tendons of the lower limbs. The stiffness of the dominant lower limb vastus lateralis (VL), rectus femoris (RF) and patellar tendon (PT) was evaluated in 52 healthy participants (26.9 ± 3.4 years) with two MyotonPRO protocols: the standard protocol  and the short protocol . The myotonometry was performed at the midpoint of the length from the upper pole of the patella to the greater trochanter for the VL, and to the anterior superior iliac spine for the RF. The PT was evaluated 1 cm caudal from the inferior pole of the patella. Pearson’s correlation coefficients were calculated to determine the relationships between protocols. The validity of the short protocol was evaluated with Student’s  Stiffness is one of the most studied mechanical properties of muscles and tendons because of its positive relation with the rate of force development and performance in fast stretch shortening cycle activity . ElastogSeveral studies have shown the feasibility of using the MyotonPRO device to monitor the mechanical properties of skeletal muscles in athletes, healthy young and older adults, and individuals with some pathology ,7,8. TheReducing the time duration of the standard measurement protocol would facilitate the application of the MyotonPRO within more complex assessment processes and help to increase its efficiency, as overall test time would be lower. Some authors have employed shorter protocols for measuring skin mechanical properties, and have obtained good reliability results , althougA total of 52 physically active people volunteered to participate in this investigation ((mean ± standard deviation) age: 26.9 ± 3.4 years, height: 173.8 ± 12.7 cm, body weight: 72.9 ± 11.6 kg). The sample size was previously calculated based on Schneebeli et al. , who use® Pro . All the measurements were taken by the same expert operator (ABS). During the assessment with the myotonometer, the testing probe was placed perpendicular to the skin at the reference point. The MyotonPRO was set up with a multiscan sequence (1 s apart) of a short duration (15 ms) impulse involving minimal mechanical force (0.4 N) and a light precompression force (0.18 N). The difference between protocols consisted of the number of taps or impulses that composed the multiscan sequence. The first protocol (standard protocol) consisted of 10 mechanical taps whereas the second protocol consisted of 5 mechanical taps (short protocol). The mean value of the data from the 10 and 5 compressions protocols was used to describe the oscillation frequency, logarithmic decrement and stiffness of the examined muscles and tendon. Two protocols were employed to assess the mechanical properties of three dominant lower limb regions: the Vastus Lateralis (VL), the Rectus Femoris (RF) and the Patellar Tendon (PT), using a hand-held myotonometer MyotonThe anatomical locations of the measurement points and participants’ positions were standardised for all subjects. MyotonPRO exams were performed at: the midpoint from the upper pole of the patella to the greater trochanter for the VL; at the midpoint from the upper pole of the patella to the anterior superior iliac spine for RF and at 1 cm caudal from the inferior pole of the patella for PT.The participants were evaluated twice in random order, using different protocols each time (standard and short protocol), to be able to compare the validity of the short protocol. All the measurements were performed in a relaxed condition, and the participant was requested to not contract their muscles. For the assessment of the VL and RF the participants were laid in a supine position with the knee full extended and, for the PT assessment the participants were laid in a supine position with 30° of knee flexion, using a foam pad to support the position .p > 0.05), the validity of the short protocol was evaluated with Student’s t-test and the Bland-Altman plot. Pearson correlation coefficients were used to examine the relations between the standard and short protocols. A paired sample Student’s t-test was performed to analyse the inter-region differences in stiffness values measured with both protocols. The inter-region differences in stiffness  were calculated for each protocol result as follows:Data analysis was performed using SPSS v 28.0 . All data were expressed as a mean with standard deviation. The data were tested for normality with the Shapiro-Wilk’s test. Given the assumption of normality . The effect size was interpreted using Cohen’s d [p < 0.05 was accepted as the level of significance.A paired sample Student’s ohen’s d : an effet-test did not show significant differences between protocols (standard protocol vs. short protocol) for any mechanical properties (p > 0.05) (Student’s  > 0.05) . The Bla > 0.05) .p < 0.001), VL stiffness , RF oscillation frequency , RF stiffness , PT oscillation frequency , PT stiffness .Significant positive correlations were revealed between the standard and short protocol for VL oscillation frequency  and the RF  in the short protocol, and the PT stiffness was also significantly higher than the VL stiffness  and the RF stiffness  in the standard protocol (p < 0.001) and standard protocol   .p > 0.05).The current study aimed at validating a new measurement protocol of the MyotonPRO application for assessing myotendinous mechanical properties. Reducing the measurement time of the MyotonPRO application would improve efficiency in research studies and increase the possibilities of the MyotonPRO application for the assessment of myotendinous mechanical properties in different research environments. The short measurement protocol was applied previously in the study of the Achilles tendon mechanical properties with different levels of Triceps Surae muscle contraction  and duriSeveral studies showed potential differences in MyotonPRO parameters between the evaluated regions (inter-muscle differences) and sex-related differences ,18, and Finally, we evaluated the relationship between the two measurement protocols using Pearson’s r statistic, which allowed us to establish a linear relationship between both protocols. The results obtained in our research have shown a significant high positive relationship between both measurement protocols for all the analysed variables. According with Schober et al. , r valueThis work has some limitations. MyotonPRO measurements could be conditioned by the thickness of the adipose tissue under the probe, so our results should be corroborated for other body regions with thicker adipose tissue. In addition, only a specific sample of healthy and young people was included in this study, so it would be necessary to apply the study in a population sample with different characteristics. Finally, all the exams were done in a relaxed condition, without muscle contraction, so new research is required in dynamic conditions, during muscle contraction.In conclusion, our study demonstrated that the five-compressions protocol (short protocol) is valid for the assessment of lower limb myotendinous mechanical properties. Therefore, the time for myotonometer assessment could be reduced in future research works and daily clinical practice.


This study was designed to connect aortic stiffness to vascular contraction in young male and female Wistar rats. We hypothesized that female animals display reduced intrinsic media-layer stiffness, which associates with improved vascular function. Atomic force microscopy (AFM)-based nanoindentation analysis was used to derive stiffness (Young’s modulus) in biaxially  unloaded aortic rings. Reactivity studies compatible with uniaxial loading  were used to assess vascular responses to a selective  The leading cause of death globally, cardiovascular diseases (CVDs), dimorphically affects men and women ,2. Sex dThere are multiple approaches to quantifying arterial stiffness. The most widely used technique, PWV, non-invasively derives stiffness by calculating the transit time of the CV pulse . HoweverAccordingly, we designed this study to connect aortic stiffness to vascular contraction in young male and female Wistar rats. We hypothesized that female animals display reduced media-layer stiffness, which associates with improved vascular function. AFM-based nanoindentation analysis was used to measure stiffness (Young’s modulus) in biaxially unloaded  aortic rings. Functional studies compatible with uniaxial loading  were performed to assess vascular responses to phenylephrine, a commonly used Compared with male Wistar rats, female animals present lower body weight at 16 weeks . The pre2, determined in pilot experiments, to map stiffness in biaxially  unloaded aortic rings , we deric rings B, which p < 0.05, p < 0.05, 2; p < 0.05, p < 0.05, Histological studies are key to determining the geometry of aortic rings ,26 whilep < 0.05, p > 0.05, p < 0.05, p < 0.05, We previously showed that the aortas of male and female Sprague Dawley rats respond differently to phenylephrine stimulation , a featup < 0.05, p < 0.05, p < 0.05). Additionally, we further explored the dynamics of reactivity by fitting the relaxation component of the transient part of the calcium protocol to Equation . On the other hand, we observed that male animals had enhanced collagen levels  to evaluate the function of isolated aortas. This set of experiments allowed us to analyze vessel response, accounting for the cellular (ECs and VSMCs) and non-cellular components (ECM). Additionally, aortic rings loaded circumferentially are stronger than counterparts loaded perpendicular to ECM fiber direction, particularly because of the anisotropic behavior of blood vessels . As expeκ D coupledκ . Before euthanizing the animals, we determined non-fasted glucose levels in male and female rats using a commercially available blood glucose monitoring system. We also confirmed that animals displayed similar mean arterial pressure (MAP), as follows. A sterile catheter connected to a pressure transducer  was inserted into the femoral artery , and blood pressure values were recorded for approximately 30 min.Male and female Wistar rats (14 weeks) were acquired from Envigo, and housed under alternating light/dark cycles (12-h). Animals were subjected to a 2-week acclimation period in which they were fed standard chow diet and water 2. The Young’s modulus (stiffness) was obtained by fitting the force-displacement curves to the Hertz Model [E is the Young’s modulus, v is the Poisson’s ratio, R is the tip radius, and Thoracic aorta sections .Aortic sections , which was gassed with carbogen  and kept at 37 + solution (120 mmol/L for 10 min) modified from PSS. Viable rings (force development > 50% of preload) were washed and allowed to return to baseline. Note that we did not observe differences between sexes for the maximum contraction tone elicited by KCl . Then, rings were stimulated with a single concentration of phenylephrine  for 12 min. Samples were washed and allowed to return to baseline. Afterwards, we obtained a concentration-response curve to phenylephrine (10−9–10−4 mol/L). In another set of experiments, following the viability test, we assessed vascular responses to calcium [−5 mol/L) was used to stimulate calcium release from internal stores. Contraction was recorded for 10 min. Then, we restored the calcium concentration to the PSS (1.56 CaCl2 * H2O mol/L) and observed force displacement in response to calcium influx for 15 min.Functional studies were performed in a DMT620M multi-wire myograph system  ,40,41,42 calcium ,42,43. B−5 mol/L. In the time-force and calcium protocol, data points were sampled every second and are reported as absolute force development from basal levels. The amplitude of the phasic  is equal to 0, and The ch group . The traAortic rings (10 ure time . Autofluure time . RepreseCollagen levels were assessed in aortic rings (10 t-test (unpaired), and statistical significance was considered for values of The software GraphPad Prism (version 9.0) was used to create the graphs, which are depicted as means ± SEM, unless stated otherwise, and to compute significance of differences between male and female samples. The statistical test applied was the two-tailed Student’s It seems that, under the conditions evaluated in this study, the observed sex-related differences in aortic function are due to alterations in the dynamics of vascular reactivity and ECM composition rather than a direct response to a shift in intrinsic media-layer stiffness. Broadly speaking, our work provides a critical piece of information  for researchers modeling arterial stiffness in non-pathological  and pathological  conditions.


Introduction: Living donor kidney transplantation (LDKT) is accepted as first-line treatment for patients with end-stage kidney disease with advantages over deceased donor kidney transplantation (DDKT). Still, how the known detrimental effect of HLA mismatch (MM) may hamper these advantages remains unsettled. We sought to determine the effect of the degree of HLA MM, separately in deceased and living donor renal allograft outcomes.Methods: We evaluated all adults submitted to LDKT and DDKT at our center between 2006 and 2018. Their HLA MM was classified according to the British Society of Transplantation system in low mismatch (LM) (level 1-2) and high mismatch (HM) (level 3-4). Acute rejection (AR) and global or censored graft survival were the outcomes of interest. Recipients were followed up from transplant until death, graft failure or the end of 2020. Results: One thousand sixty-eight kidney transplant recipients were analyzed, 815 (76%) received a DDKT whereas 253 (24%) received an LDKT. From those submitted to DDKT, 95 (12%) had an LM and 720 (88%) had an HM, whereas in LDKT 32 (13%) had an LM and 221 (87%) had an HM. The AR at one year was 9% in the full cohort. Significant risk factors for AR were HM DDKT  or HM LDKT  (LM DDKT as reference), calculated panel-reactive antibody (cPRA) ≥5%  and delayed graft function (DGF), . Censored graft survival (CGS) at five years was 96% in the full cohort. Independent predictors for censored graft failure (CGF) were HM LDKT  (LM DDKT as reference), AR  and DGF . Global graft survival (GGS) at five years was 91% in the full cohort. Independent predictors for global graft failure (GGF) were HM LDKT  (LM DDKT as reference), recipient age  and DGF . No AR, CGF or GGF episodes were observed in the LM LDKT group.Conclusions: In our cohort, the level of HLA MM increased the risk of AR independently of donor type. Considering short graft survival, our results support the advantage of living donor vs deceased donor even with an increased HLA MM. However, its effect on long-term graft survival remains to be settled, emphasizing the need for further studies on this matter. Living donor kidney transplant (LDKT) is the best treatment for suitable patients with end-stage renal disease. It offers several advantages as it allows a shorter waiting time with the possibility of transplant before dialysis (preemptive), a lower rate of delayed graft function and improved long-term graft survival, even from genetically unrelated donors, over those who receive deceased donor grafts ,2. The sPopulation and study cohortWe retrospectively evaluated all adults submitted to LDKT and deceased-donor kidney transplant (DDKT) at our institution between 2006 and 2018 (n=1193). After the exclusion of 63 patients whose degree of mismatching was unknown, 43 patients who had a primary loss of KT and 19 HLA incompatible transplants, the remaining 1068 recipients defined our study cohort.HLA matching classificationThe HLA mismatch was classified according to the British Society of Transplantation system as low mismatch (LM) level 1-2) and high mismatch (HM) (level 3-4) . The fou-2 and hiBaseline data and graft outcomesBaseline demographic and clinical data were collected from both recipients and donors. Transplant data were also collected and analyzed. Delayed graft function (DGF) was assumed when there was need for dialytic support during the first post transplantation week. Graft biopsies were performed by indication. All acute rejection (AR) episodes were supported with biopsy, classified according to Banff’17 criteria and recorded during the first year after procedure. Recipients were followed up from transplant until death, graft failure or the end of 2020. The study protocol was reviewed and approved by the institutional ethical review and hospital administration boards of Centro Hospitalar Universitário do Porto and Instituto de Ciências Biomédicas Abel Salazar  as per the recommendations of the Declaration of Helsinki and European Data Protection Regulations.Immunosuppression (IS) and desensitization protocolsInduction therapy with an anti-interleukin (IL)-2 receptor monoclonal antibody (basiliximab 20 mg twice on days 0 and four) or with polyclonal anti-thymocyte globulin (ATG) (3 mg/kg for five to seven days) was used in most patients. Anti-thymocyte globulin was used primarily in patients with cPRA >60% and in patients submitted to retransplantation. The great majority of recipients (>90%) received triple maintenance immunosuppression with oral tacrolimus, mycophenolate mofetil (MMF) and corticosteroids. Tacrolimus was started at a dose of 0.1-0.15 mg/kg/day, which was then adjusted to maintain a trough level between 8 and 12 ng/ml during the first post transplantation month, between 7 and 10 ng/ml after two to three months and between 5 and 8 ng/ml long-term. MMF was initiated at a dose of 2000 mg/day, with decreasing dose to 1000-1500 mg/day during the first month postoperatively, adjusted to white blood cells count. Both tacrolimus and MMF were started one week before transplant in LDKT recipients. Methylprednisolone was administered at doses of 500 mg on the transplantation day, 250 mg the two days after and 125 mg on day three and four after the operation. Oral prednisolone was then at the dose of 20 mg and tapered to 5-10 mg/day within two to three months.Statistical analysisContinuous data are described as mean ± standard deviation (SD) or median (interquartile range [IQR]). Categorical data are expressed in numbers and percentages. Categorical data were compared using Pearson’s chi-square test or Fisher’s exact test, and continuous variables were compared using Student’s t-test or Mann-Whitney U-test.AR and graft survival curves were visualized using the Kaplan-Meier method, with a comparison between patients' groups done by log-rank test. In the case of death with a functioning graft, time was censored at the time of death. Potential predictors of AR and censored and global graft failure were explored by univariate and multivariable Cox proportional hazards models. In all multivariable models, independent predictors were identified using a backward elimination method, with a P-value < 0.05 necessary for retention in the model. A 2-sided P-value < 0.05 was considered as statistically significant. Statistical calculations were performed using STATA/MP, version 15.1 .Characteristics of patientsOur study cohort comprised 1068 recipients; 815 (76%) received a DDKT whereas 253 (24%) received an LDKT. From those submitted to DDKT, 95 (12%) had an LM and 720 (88%) had an HM, whereas in LDKT 32 (13%) had an LM and 221 (87%) had an HM. Table The mean recipient age at the time of kidney transplantation was lower in LDKT groups compared to DDKT groups (P<0.001). Fifteen percent of all patients had a previous transplant and the percentage of preemptive kidney transplants was superior in LDKT groups (19% with LM and 23% with HM) compared to DDKT groups .ATG was used as an induction in 20% of the cohort, particularly in HM DDKT (25%). DGF occurred in 20% of all cohort and was more frequent on DDKT, regardless of HLA mismatch level.Acute rejection analysis by HLA match groupAcute rejection (AR) at one year was 9% in the full cohort and was superior in HM DDKT and HM LDKT groups, 10% and 12%, respectively P=0.039), as shown in Table 9, as shoAllograft and patient survival analysis by HLA match groupFive-year censored and global renal allograft survivals by donor type and HLA match group are shown in Figure As documented in the available literature, renal allograft survival is improved for recipients of living donors compared with deceased donors ,13. AddiThere are reports suggesting that HLA matching may not be as important in LDKT as it is in DDKT. A retrospective analysis of Casey et al. matched 83 0-HLA MM to 407 controls with ≥1 HLA MM in living donors. The authors reported that 0-HLA MM did not confer improved death-censored graft and patient survival . In anotIn contrast, an analysis based on the data of the International Collaborative Transplant Study showed a strong and statistically significant impact of HLA-A+B+DR MMs on the survival of pediatric transplants from living donors . FurtherOur study design was similar to the one conducted by Marlais et al. that addressed the effect of HLA matching on deceased and living donor renal allograft outcomes in pediatric recipients. They tried to understand whether it is better to wait for a closely matched organ from a deceased donor or to decide to provide an organ from a living donor with HM . The resOur results also showed that the censored and global allograft survivals were significantly better in the HM LDKT group compared to the LM DDKT group. Furthermore, the patients who received a poorly matched LD kidney transplant have an 82% lower risk of censored graft failure and an 80% lower risk of global graft failure, supporting the benefits of LDKT above DDKT. However, it is still early to try to answer if it is worth the wait for LM DDKT or if it is better to proceed to HM LDKT, also due to the lack of data on the impact of time of the waiting list in patient survival .In our study, HLA MM increased the risk of AR, independently of donor type with greater impact on the HM LDKT group. This finding raises concern about the potential immunosuppression risks in the short and long term, particularly infections and neoplasms, and stresses the problem of the increasing number of hypersensitive patients on the waiting list. This could be possibly overcome through the inclusion of compatible pairs in kidney-paired exchange programs, with the goal of obtaining a better HLA or eplets match. The pairing of donor and recipients through these programs increases the transplant opportunities for all patients on the waitlist and would favor highly sensitized and blood O type recipients ,23,24. DThe present study has several limitations. Firstly, it is unicentric and restricted to a five-year outcome timeframe on renal allografts, leading to the lack of assessment of the long-term effect of poorly HLA-matched transplantation. Secondly, it involved a prolonged recruitment time, which implicates both different immunosuppression and histocompatibility-testing protocols. Lastly, the population analyzed was exclusively Caucasian.In our cohort, the level of HLA mismatching increased the risk of AR, independently of donor type. Considering short graft survival, our results support the advantage of living donor vs deceased donor even with an increased HLA mismatch. However, its effect on long-term graft survival remains to be settled, emphasizing the need for further studies on this matter. Moreover, the absence of any outcomes of interest in the LM LDKT highlights the optimal choice represented by a low mismatch transplant, which in the case of LDKT could be reached by the introduction of compatible pairs in the kidney paired donation program.


In recent years, the demand for environmental sustainability has caused a great interest in finding novel polymer materials from natural resources that are both biodegradable and eco-friendly. Natural biodegradable polymers can displace the usage of petroleum-based synthetic polymers due to their renewability, low toxicity, low costs, biocompatibility, and biodegradability. The development of novel starch-based bionanocomposites with improved properties has drawn specific attention recently in many applications, including food, agriculture, packaging, environmental remediation, textile, cosmetic, pharmaceutical, and biomedical fields. This paper discusses starch-based nanocomposites, mainly with nanocellulose, chitin nanoparticles, nanoclay, and carbon-based materials, and their applications in the agriculture, packaging, biomedical, and environment fields. This paper also focused on the lifecycle analysis and degradation of various starch-based nanocomposites. In recent days, nanocomposites have gained much attention over traditional composite materials and are widely used in food, packaging, biomedical applications, electronics, energy storage, optics, the automotive industry, bio-sorbants for environmental remediation, textiles, and many other applications ,2. PolymThus, biopolymer-based nanocomposites can be a sustainable alternative for petroleum-based nanocomposites in many applications due to their biodegradability, eco-friendliness, renewability, relatively inexpensive, low toxicity, abundancy, and improved thermal, mechanical, physical, barrier, and functional properties ,4. VarioStarch is one of the most abundant natural polymers globally. Starch and its nanocomposites have been extensively studied for their abundance, low cost, ease of processibility, and chemical and physical properties ,4. FurthStarch can be modified into nanoparticles and can also undergo various physical  and chemical  modifications to produce materials with novel properties ,10,11,122 NPs, ZnO NPs, etc.) to achieve desirable properties and produce potential green sustainable nanocomposite materials  ,54 53,54Nanocellulose is widely used in various applications, such as biomedical engineering, the automotive industry, electronics, food packaging, cosmetics, construction, textiles, wood adhesives, and wastewater treatment applications ,57.Othman et al.  preparedChitin is the second most abundant natural polysaccharide next to cellulose and is found in the shell of crustaceans , the exoskeleton of arthropods, molluscan shells of squid, mushrooms, the cell wall of algae and fungi (yeast and mold). Chitin is composed of N-acetyl-2-amido-2-deoxy-β-D-glucose (N-acetylglucosamine) units linked with a β--glycosidic bond, in which acetamide groups (−NHCOCH3) consists at the C2 of cellulose monomer. Chitosan is derived from the alkaline deacetylation of chitin . Chitin Chitin nanomaterial can be prepared through top-down and bottom-up approaches. Chitin fibrils consist of amorphous and crystalline regions and thus can be converted into three types of nano-chitins in a top-down approach: nanocrystals (via acid hydrolysis), nanofibers , and nanowhiskers  ,62.Nano-chitin has been widely studied for its high aspect ratio, high surface area, good mechanical properties, lightweight/low density, good chemical stability, renewability, non-toxicity, and antibacterial properties, and it is used in biomedicine, packaging, water treatment, green electronics, cosmetics, and many other applications ,63.A combination of chitin nanofibers and starch nanoparticles showed higher emulsion stability over a range of pHs and temperatures and can be used as an emulsion stabilizer in various products, such as food, paint, coating, cosmetics, and pharmaceuticals .Chang et al.  reportedMucor indicus, Young’s modulus and the tensile strength of TPS were enhanced by 239% and 216%, respectively, and moisture absorption was reduced from 51% to 38%. However, the addition of CNF at a higher level increased moisture absorption and reduced the mechanical properties of TPS [Ac−]) significantly increased the tensile strength by 327%, Young’s modulus by 2484%, and elongation at break 82% (from 30 to 69%). Moreover, electrical conductivity was increased with MWCNT content (wt.%) and reached a maximum (56.3 S/m) at 5 wt.% MWCNT. MWCNT/starch plasticized with [emim+][Ac−] showed electroconductive properties because of its ionic nature of ionic liquids and the excellent electrical conductivity of MWCNT . A starcGraphene is a two-dimensional material arranged in a hexagonal lattice. Plasticized starch incorporated with reduced graphene oxide (rGO), a derivative of graphene, exhibited increased conductivity and dielectric properties, which could make it a potential candidate for producing sustainable bio-friendly electronic devices .Investigation of poly(lactic acid) (PLA)/thermoplastic starch (TPS)/graphene nanoplatelets (GNP) blends revealed that the addition of GNP increased the crystallinity of the PLA/TPS blend, and the maximum crystallinity (68.39%) was observed with PLA (70%)/TPS (30%)/GNP (1%). Further increases in GNP resulted in the reduction of compatibility .Biodegradable starch-based nanocomposites have been used in agriculture, packaging, biomedical, environment, and many other fields .In recent years, biodegradable films have been developed for agricultural purposes, particularly for mulching applications, the coverings of a greenhouse, and the controlled/slow release of agrochemicals such as fertilizers and pesticides ,83,84.Agricultural mulches are used to prevent the hindrance caused by the weeds’ growth, maintain soil wetness, and regulate soil temperature . InteracPesticides protect the crop from pests, pathogens, weeds, and insects by destroying, attacking, mitigating, or repelling activity, whereas fertilizers are essential in agriculture to increase crop yield. However, in conventional applications, the efficiency of reaching their target sites is relatively low as they are hindered by immobilization, erosion, volatilization, leaching, surface runoff, or scavenging by soil. In addition, water is also an essential factor in crop growth and driving off fertilizers. Therefore, management of nutrient/pesticide active compounds and water loss is essential for crop production. To reduce the loss and improve their utilization efficiency, slow-release fertilizers or controlled-release pesticides with improved water retention and water holding capacity can be formulated by incorporating nanomaterial into biopolymers ,84,87,88Merino et al.  investigPhilodendron spp. compared to the direct application of urea, suggesting the efficacy of nanocomposites in slow-release fertilizer formulation [Superabsorbent hydrogels are widely used in bi-functional (retain and supply water and nutrient over a long period) slow-release fertilizers due to their water retention properties. The addition of natural char nanoparticles (NCNPs) into corn starch-g-poly(AA-co-AAm) encapsulated urea provided high biodegradability and improved the soil water-retention capacity along with the slow release of urea . Chitosamulation . Urea enmulation . Modifiemulation .Food packaging protects food from humidity, high/low temperatures, and other physiological factors and aids in food quality monitoring and control in the food supply chain and during storage  . Starch 2, Ag2O, MgO, CuO, SnO2) [Organic or inorganic nanofillers have been widely studied for food packaging applications, whereas organic nanofillers include nanoclay , natural biopolymers , and natural antimicrobial agents (nisin), and inorganic nanofillers includes metals , and metal oxides  ,52,91.2 NPs reinforcement in potato starch-based composite films led to a reduction in water solubility, moisture uptake, and water vapor permeability, and an increment of UV barrier properties and tensile strength of the film, showing its potential for food packaging [2 NPs reduce the hydrophilicity and improve mechanical, water vapor, and UV barrier properties in cross-linked wheat starch, which makes them a suitable material for food packaging [Staphylococcus aureus, Salmonella typhi, and Escherichia coli and can be used as an antibacterial food coating material [Proteus mirabilis, Candida albicans, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, among others, which shows the potential applications in antimicrobial packaging [Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis [The suitability of a film for packaging materials is mainly assessed by water vapor and oxygen barrier properties and good heat salability . Furtherackaging . Na-MMT ackaging . UV barrackaging . Starch material . Linseedackaging . Poly for more than three months without affecting the oil quality in terms of rancidity, viscosity, and color .Starch from potato, wheat, and corn blended with carboxyl methylcellulose (CMC)/Na-MMT has potential applications in food packaging . CelluloEscherichia coli due to the synergistic antibacterial effect of carvacrol essential oil and MMT suggesting the applications in antimicrobial packaging [Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, and significantly inhibited the microbial growth in pork meatballs stored under ambient and refrigeration conditions [TPS/MMT/carvacrol essential oil showed biocidal effects against ackaging . Packagiackaging . They renditions .S. putrefaciens) with reduced flexibility. The oxygen and water vapor permeability reduced at 1.0 and 2.5% MFC but increased at higher concentrations. In addition, MFC, at 1 and 7.5%, controlled the release of cinnamaldehyde.The addition of potassium sorbate, a commonly used preservative, into starch/nanoclay films controlled the migration of sorbate, resulting in the retention of antimicrobial activity for a long period . Chen et2, SnO2, Ag2O, MgO, ZnO, CuO) can be used in gas sensors to monitor food quality [Smart packaging materials for monitoring the spoilage of milk packed in a bottle were developed by incorporating pH indicators, including bromocresol green (BG) and methyl orange (MO), into a starch/nanoclay nanocomposite . Further quality .Biodegradable polymers, including starch-based bionanocomposites, are widely used as scaffolds for tissue engineering, drug delivery systems/drug carriers, wound dressing, surgical sutures, and implants due to their mechanical properties, biocompatibility, biodegradability, and also the generation of non-toxic, biodegradable products ,104,105.Biopolymers in the repair of healing tissues accelerate treatment processes and eliminate implant removal surgery. Furthermore, implant materials and their biodegradable products must be non-cytotoxic and biocompatible . IncorpoHydroxyapatite has been used widely in biomedical applications due to its biocompatibility and osteoconductive (cell regeneration process) properties. However, brittleness and lack of flexibility limit the applications. The combination of hydroxyapatite with starch materials can reduce brittleness, and the polar nature of starch encourages a good adhesion between starch and hydroxyapatite. Sadjadi et al.  synthesiAspergillus flavus and Candida albicans) and bacteria (Staphylococcus aureus and Escherichia coli). PVA/starch incorporated with Ag NPs synthesized from green methods (Diospyros lotus fruit extract) has the potential to be used in wound dressing as it shows increased swelling and moisture retention capacity and reduced water vapor transmission that prevents the wound from dehydration and better antimicrobial activity against Escherichia coli and Staphylococcus aureus [Abdel-Halim and Al-Deyab  reporteds aureus .Escherichia coli and Staphylococcus aureus), and drug release of PLA/S/PCL film. An increase in nHA content (1–7%) improved the biodegradation (13–10 months), and the antibacterial triclosan release rate of PLA/S/PCL/nHA film at 37 °C in buffer solution was increased (0.12–0.18 μg/mL every day), which is in the range of MIC of triclosan (0.025–1 μg/mL). Furthermore, the degradation and release time of PLA/S/PCL/nHA (3 wt.%) nanocomposite showed similar profiles that ensure continuous drug release during the application [The ternary blend was developed by mixing polylactic acid (PLA)/starch (S)/poly-ε-caprolactone (PCL) with nano-hydroxyapatite (nHAp) for controlled release of antibacterial triclosan. The incorporation of nHA (3%) improved the hydrolytic hydrophilicity, hydrolytic degradation, antibacterial activity  for the controlled-release of guaifenesin (GFN) with low cytotoxicity. A nanocomposite with magnetic Fe3O4 NPs at 0.83% significantly enhanced the drug release from 54.1 to 90.4% within 24 h in pH 7.4 [Nezami et al.  fabricatn pH 7.4 .Starch-based-fluorescent organic nanoparticles (FONs) reported high water dispersibility and excellent biocompatibility (cell viability was 99.69% at the concentration of FONs 100 µg/mL after 24 h). Thus, FONs are a promising candidate for biomedical applications that can be potentially used as fluorescence probes and carriers for delivering biologically active components .6+, Hg2+, Zn2+, Pb2+, Co2+, Cd2+, Cu2+, etc.), dyes, organic substances , pathogenic microbes , and toxic gases  in water, soil, and air [Extensive agricultural and industrial practices lead to the accumulation of various contaminants, including heavy metals and metalloids , nanoscale zero-valent iron (nZVI) (Fe⁰), carbonaceous materials , nanoclays , and polymers  are used in materials as recyclable and reusable filters, absorbents, reductants, photocatalysts, coagulants and flocculants, disinfectants, and gas sensors to detect or remediate contaminants, such as dyes, heavy metals ions , various aromatic derivatives, fertilizers (urea), and other organic pollutants [Starch-based nanocomposites with various nanofillers, including metal , bimetal (Ag/Au), metal oxides  reported catalytic activities that involve the reduction of 4-nitrophenol to 4-aminophenol and degradation of azo dyes  by cleavage of −N = N-bond thus can be used in water treatment . Gomes eStarch-graft-poly(acrylamide) (PAM)/graphene oxide (GO)/hydroxyapatite NPs (nHAp) nanocomposite was developed as a recyclable adsorbent for efficient removal of malachite green (MG) and other cationic dye from aqueous solution. The introduction of nHAp improved the biocompatibility of the PAM/GO composite, whereas the biodegradability, porosity, water content, and water uptake decreased with increasing nHAp content. Adsorption capacity increased with agitation time, pH, nHAp content, and initial dye concentration, and the optimum conditions were 60 min, pH 10, 5% nHAp, and 100 mg/L. PAM/GO and nHAp at 1–5 wt.% reported excellent porosity (31–11%), degradability (41–11% after 15 days), the maximum adsorption capacity of 297 mg/g, excellent regeneration capacity after five consecutive adsorption-desorption cycles of dye with high removal efficiency (77–86%) .obs) and the influencing factors  are important to explore the suitability of materials in adsorption-based applications. Monodispersed starch stabilized magnetite nanoparticle (MSM) showed 70% absorption of antibiotic tetracycline within the first 5 min and reached 90% after 1 h. The degree of the time dependency of the kinetic coefficient (kobs) had a negative correlation with the initial tetracycline concentration [Adsorption is a basic principle of mechanism in targeted drug delivery, controlled release of pharmaceutically active compounds, and treatment of chemical water pollution . The degntration .2+  and Co2+  in comparison to the starch matrix  [6+.Chitin nanowhiskers (CNW) are better nano-adsorbents due to their high surface/volume ratio and abundant hydroxyl and acetamide functional groups on the surface . MMT is ctively) . Yang etctively)  studied 3O4 magnetic NPs has been successfully used to remediate wastewater containing different dyes in the textile industry [Enzyme immobilization is an emerging technology for environmental remediation which gives many advantages over free enzymes, which include the efficiency and stability of catalytic enzymes and their enhanced recovery and reusability . Furtherindustry . Immobilindustry . Mehde [industry  reportedPlasticized starch/reduced graphene oxide (rGO) nanocomposites with improved conductivity and dielectric properties can be used in bio-friendly flexible electronic devices . The maiWith increasing fossil depletion and environmental concerns, sustainable biobased materials have gained increasing interest. For biobased materials to be sustainable, preparation and processing should have limited environmental impacts .The environmental credentials of bionanocomposites are evaluated by assessing their material production, product manufacturing, and product end-of-life. Many tools, including environmental impact analysis (EIA), life cycle analysis (LCA), material flow analysis (MFA), and ecological footprint (EF), are used for analyzing the environmental impacts of materials and manufacturing processes . Life cycradle to grave” and “cradle to gate” [cradle to gate” system covers all the steps from raw material extraction and energy to product conversion and delivery at the factory gate, whereas “cradle to grave” covers all phases of the lifecycle of a product, i.e., includes all steps of “cradle to gate” and usage and disposal phase [The international organization for standardization (ISO) standardized the LCA via ISO 14040 series . The twoto gate” ,135. Theal phase . LCA canal phase ,137,138.al phase ,140. Fig2 eq./kg) and agricultural land use (0.3–1.3 m2yr/kg)  , eutrophication (1.2–1.9 g P eq./kg), greenhouse gas (GHG) emissions (1.8–3.7 kg COm2yr/kg) . CompareThe microwave-assisted technique can be an environmentally friendly alternative for glucose-reduced and starch-stabilized Ag NPs production .LeCorre et al.  comparedThe choice of starch sources and plasticizers influences the environmental impacts displayed by the production of composites. Corn starch/glycerol exhibited the lowest impact on the ecosystem, human health, and resources .Based on ASTM, biodegradable is defined as ‘capable of undergoing decomposition into carbon dioxide, methane, water, inorganic compounds, or biomass in which the predominant mechanism is the enzymatic action of microorganisms that can be measured by standard tests, in a specified period, reflecting available disposal condition’ . BiodegrThe type, nature, concentration, chemical modification, and antimicrobial properties of nanofiller, biodegradation test methods, and parameters, including temperature, moisture, humidity, pH, quantity and type of microorganisms, etc., can influence the biodegradability of nanocomposites ,145,147.2, graphene oxide, etc., reduce the biodegradability of starch-based nanocomposite because of their antioxidant potential [Starch modification and incorporation of nanomaterials as nanofiller have been shown to alter biodegradability. For example, the biodegradability of starch increased with the addition of MMT at lower concentrations because of increased hydrophilicity that permits the microorganisms to enter into the polymer. In contrast, chemical modification of starch, nanofillers such as TiOotential ,15,148.Crosslinked nanocomposite film produced from thermoplastic corn starch crosslinked with oxidized sucrose and reinforced with cellulose nanofibrils from a pineapple leaf was reported to have a 30% weight loss rate after 30 days of burial, much lower than that of thermoplastic starch (80%) . CrossliThe addition of MMT into sweet potato starch (SPS) hindered biodegradability in soil burial tests due to the strong hydrogen bond between the hydroxyl groups of SPS and MMT and decreased water solubility that prevents water diffusion into the film . HoweverThe cationic starch-based film incorporated with MMT and nanocrystalline cellulose degrade faster than the pure cationic starch film in composting at 58 °C, which may be attributed to hydrophilic nanocrystalline cellulose . Thyme e2 (0.01 and 0.05 wt.%) in maize starch/PVA composite films improved the tensile strength, water vapor, and UV barrier properties with little effect on biodegradability in soil [Micrococcus luteus culture at room temperature for 30 days [Incorporating fibrous TiO in soil . The add 30 days . Incorpo 30 days .3 in starch/polyethylhexylacrylate (PEHA)/PVA composite film improved the tensile strength, thermal stability, chemical resistance, and antimicrobial properties, which can be suitable for packaging. Starch/PEHA/PVA/CaCO3 degraded by 65% after 15 days in activated sludge water [The addition of CaCOge water . Food page water .−1) and compost media (0.09 to 0.08 d−1) [Poly(lactic acid) (PLA)/thermoplastic cassava starch (TPCS)/graphene nanoplatelets (GRH) nanocomposite film showed a lower biodegradation rate than PLA film in vermiculite (0.11 to 0.06 d.08 d−1) .In slow-release fertilizer formulation, the incorporation of natural char nanoparticles (NCNPs) into corn starch-g-poly (acrylic acid-co-acrylamide)/urea composite increased the degradation rate (23.9% after 30 days in soil), which may be attributed to the increment in water absorbance that promotes the soil microorganisms to enter into the polymer matrix .The biodegradability of nanocomposite film polylactic acid/starch/poly-ε-caprolactone/nano hydroxyapatite (nHAp) was increased with the nHAp content . HosseinIn vitro degradation tests performed in a simulated body fluid (SBF) showed that thermoplastic starch (TPS)/beta-tricalcium phosphate (β-TCP) NPs degraded 51% after 28 days, higher than that of TPS (47%) . Table 42, ZnO, CaCO3, etc.), have been widely used for the creation of new starch-based bionanocomposites and are promising candidates for various industrial applications.In summary, starch is a natural polymer with outstanding biocompatible characteristics and can be used as both a matrix and reinforcement material for the development of new bionanocomposites. Starch nanoparticles and other nanofillers, including nanocellulose, chitin NPs, nanoclay , carbon nanoparticles , metal and metal oxides , wound healing, scaffolds in tissue engineering, absorbents, filters, catalysts, or disinfectants for environmental remediation, electronic devices, lightweight architectural constructions, stabilizers in food and paints such as non-food applications, and many others.Modification of starch or reinforcement with other materials to form a nanocomposite may alter biodegradability. Therefore, regarding the biodegradability of starch-based nanocomposites is important for them to be claimed as being biodegradable materials. Life cycle assessment of starch-based biocomposite materials for their respective applications provides critical information regarding the environmental and ecological benefits of the materials over fossil-based synthetic polymers for developing sustainable nanocomposites. However, only few studies have focused on life cycle assessment. Therefore, further studies on life cycle assessment of starch-based nanocomposites needs to be investigated. Nanomaterials can also enter the human body through inhalation, contact, and ingestion, which can lead to their accumulation in the human body, Therefore, further investigations on toxicity and risk factor analysis are necessary to find the most suitable starch-based nanocomposite materials.