However, some scion/rootstock combinations are considered as incompatible due to poor graft union formation and subsequently high plant mortality. The early identification of graft incompatibility could allow the selection of non-viable plants before planting and would have a beneficial impact on research and development in the nursery sector. In general, visible phenotypes of grafted plants  are poorly correlated with grafting success, but some studies have suggested that some polyphenols could be used as markers of graft incompatibility several months or years after grafting. However, much of the previous studies into metabolite markers of grafting success have not included all the controls necessary to unequivocally validate the markers proposed. In this study, we quantified 73 primary and secondary metabolites in nine hetero-grafts and six homo-grafted controls 33 days after grafting at the graft interface and in both the scion and rootstock woody tissues. Certain biomarker metabolites typical of a high stress status  were particularly accumulated at the graft interface of the incompatible scion/rootstock combination. We then used correlation analysis and generalized linear models to identify potential metabolite markers of grafting success measured one year after grafting. Here we present the first attempt to quantitatively predict graft compatibility and identify marker metabolites (especially asparagine,  In horticulture, grafting is a common vegetative propagation technique, allowing us to combine two different genotypes together to generate one plant. The mechanisms involved in graft union formation are complex and include the formation of a callus and new vascular tissues between the two genotypes. However, despite many studies and use of grafting for millennia, the mechanisms underlying the cohabitation of the two genotypes of grafted plants are still poorly understood .The commercial use of grafting depends upon the degree of graft compatibility, i.e. the ability of the assembled scion/rootstock to form and sustain a successful graft union. Graft incompatibility is often characterized by anatomical irregularities at the graft interface, which induce mechanical weakness and subsequently death of plants. Graft incompatibility can occur soon after grafting, reducing grafting success in nurseries, or can appear several years after planting in the field . These incompatibilities can be due to the presence of a pathogen or to genetic distance, but poor grafting success can be also due to graft quality or climatic conditions during the growth . The sigIn viticulture, after grafting, the grafts produced are planted in the nursery for one year, and then up-rooted and marketable grafts are selected. Many plants will not be considered marketable due to too insufficient root and/or shoot growth and/or problems with the development of callus at the graft interface; the proportion of marketable versus non-marketable plants gives an indication of grafting success. Graft incompatibility is relatively rare once grapevines have been planted in the vineyard, but of the 233.5 million plants grafted in France in 2017, only 155–165 million plants were commercialized . TherefoThe accumulation of secondary compounds at the graft interface has long been described as an explanation of incompatibility mechanisms , 6. The Vitis vinifera L. cv. “Touriga Nacional”, which have different degrees of grafting success [V. vinifera cv. Syrah known to die back in the vineyard after grafting with certain rootstock genotypes and showed that the concentration of gallic acid was higher, and ferulic and sinapic acid was lower in the incompatible scion genotype [In grapevine, the concentration of phenolic compounds at the graft interface three years after grafting was compared between two clones of  success . Gallic  success . Similargenotype , 12. Howgenotype .V. vinifera cv. Merlot (MN)/Vitis berlandieri x V. riparia cv. Selection Oppenheim 4 (SO4) and V. vinifera cv. Ugni Blanc (UB)/V. berlandieri x V. riparia cv. Rességuier Sélection Birolleau 1 (RSB1) are very commonly produced by wine nurseries (6 and 5.3 million plants were produced in 2017 in France respectively) [V. vinifera cv. Négrette (NG)/V. berlandieri x V. rupestris cv. Ruggeri (140Ru) was studied because it often has developmental problems producing large calluses and bulging graft unions and/or bad connections between the scion and rootstock that can result in dieback in the vineyard a few years after plantation  [The aim of this study was to identify marker metabolites that could be used to predict grafting success in grapevine; the objective was to graft different scion/rootstock combinations together, to analyze metabolites (24 primary and 49 secondary metabolites) in the scion, graft interface and rootstock tissues 33 DAG and quantify grafting success after one year in the nursery. These compounds were selected based on the literature . The difctively) . A third Ruggeri 0Ru was s Ruggeri 0Ru was sThe graft combinations used in our study had very variable grafting success rates (ranging from 20.8% for UB/RSB1 to 77.1% for 140Ru/140Ru) . InteresTo characterize the metabolite profiles of the nine hetero- and six homo-grafts studied, metabolites were quantified in the scion wood, graft interface and rootstock wood 33 DAG. Amino acids, starch, total proteins and soluble sugars were quantified as well as 49 secondary metabolites that were identified by HPLC-QqQ. The main secondary metabolites quantified were stilbenes . Overall, the metabolic profile of the graft interface was very different compared to scion and rootstock  and 1C. Figure 1B and trans-ω-viniferin, and trans-ε-viniferin as well as two tetramers of stilbenes (r-viniferin et r2-viniferin). Conversely, MN was on the positive side of PC2 and PC1, and strongly correlated with high concentrations of flavanols and some flavonols, especially epicatechin, flavanol dimers, quercetin-3-glucoside, naringenin-glucoside, and kaempferol-3-glucoside  while the other genotypes had lower concentrations, especially RSB1/RSB1, which had the lowest concentration at the graft interface . Despite these low concentrations, the accumulation of kaempferol 3-glucoside in NG/NG and UB/UB, and quercetin 3-glucoside in NG/NG, UB/UB, RSB1/RSB1, and NG/140Ru, was measured at the graft interface. Interestingly, a specific and strong accumulation of naringenin and taxifolin at the graft interface of MN hetero-grafts was found, contrary to MN/MN and non-vinifera homo-grafts . By focusing on UB/RSB1, a specific accumulation of GABA, Pro and Asp at the interface could be identified compared to homo-grafted controls and other hetero-grafts .Concerning secondary metabolites, no specific accumulation could be observed at the graft interface of UB/RSB1 in comparison to its homo-grafted controls except for pallidol. In fact, this compound was found at a higher concentration at the graft interface of UB/RBS1 than any other scion/rootstock combinations studied . However2 = 0.48) between a metabolite concentration and grafting success was found for t-piceatannol.To assess whether any metabolite concentration correlated with grafting success rate, correlation matrices were made between the different tissues  and percentage of successful grafts produced; either the data from all scion/rootstock combinations were used or it was separated into either homo- or hetero-graft data . When co2 = 0.49 and 0.51 respectively). In rootstock tissue, the strongest correlation was between grafting success and t-piceatannol (r2 = 0.55). The correlations were highest in the scion tissue between grafting success and t-resveratrol, t-piceatannol (positively correlated) and α-viniferin (negatively correlated) concentrations  In addition, the percentage of water and total stilbene trimers were negatively correlated with the grafting success rate (r2 = 0.54 and 0.49 respectively) while the amount of total protein, and Asn, total stilbene tetramers and hopeaphenol concentrations were positively correlated . . .c-piceid (r2 = 0.5)  .t-resveratrol, t-piceatannol, α-viniferin as well as Asn  were created to predict the percentage of grafting success using all the metabolomic data (primary and secondary metabolites). Two parameters were taken into these models, which were the correlation between the model created and real values with the grafting success, and the contribution of each compound to participate in model construction. Variables appearing the most times in the models were considered as reliable predictors. Models were created for all data combined , then separated into different datasets: scion, interface, or rootstock, using only homo-grafts or hetero-grafts combinations . Additiol as Asn . In compl as Asn . Predictl as Asn .Here we identified and quantified part of the metabolome of the graft interface and surrounding woody tissues at 33 DAG in different scion/rootstock combinations with the objective to identify markers of graft incompatibility and compounds correlated with grafting success. Grafting success is a complex trait to phenotype, in this study, grafts were considered successful if the shoot had developed and the stem had lignified, if roots (at least 3) were present and homogeneously distributed, and if the graft interface was resistant to a force being applied to it (“the thumb test”), which are the same criteria used in grapevine nurseries. As such, it is difficult to separate grafting success from rooting success (which is the ability to produce roots) in the first year in the nursery. Here we define graft incompatibility as a lower rate of plant survival of a hetero-graft in relation to its corresponding homo-graft controls: based on this criterion only UB/RSB1 is incompatible, but other scion/rootstock combinations studied here also showed large variation in grafting success rates. The scion/rootstock combination MN/SO4 had the highest grafting success in agreement with the known behavior of this scion/rootstock combination.The analysis of primary metabolites revealed the high concentration of sugars and several amino acids like Gln, GABA, Ser, Ala, Asp, Pro, and Ile at the graft interface 33 DAG, which presumably comes from the mobilization of wood starch reserves. Cell proliferation for callus formation at the graft interface is one of the key steps to generate a successful graft union formation and is probably very energetically expensive. High concentrations of fructose were observed at the graft interface of highly compatible hetero-grafts, potentially suggesting a higher metabolic activity than homo-grafts and scion/rootstock combinations with lower grafting success. Low concentrations of starch were found in the wood and the graft interface of RSB1/RSB1, which suggests that the wood used for grafting had fewer starch reserves, which could explain the low soluble sugar concentrations in wood and its poor grafting success. However, this did not seem to have an impact on grafting success of RSB1 hetero-grafts. Furthermore, high levels of soluble sugars were found in MN/SO4 as well in UB/RSB1, which showed very different levels of grafting success suggesting that the mobilization of carbon reserves at the graft interface is not what limits graft union formation.WOUND INDUCED DEDIFFERENTIATION1 (WIND1), a gene involved in wound induced cellular reprogramming, triggers the accumulation of both GABA and Pro [in vitro [Some potential incompatibility markers were identified in the UB/RSB1 hetero-graft, such as a high accumulation of Asp, Pro and GABA at the graft interface. γ-aminobutyric acid is well known for its role in plant development . In addi and Pro . Prolinein vitro –24. ThisV. vinifera cv. Cabernet sauvignon at 28 DAG. In our data, only MN/MN homo-graft had higher total stilbene concentration at the graft interface compared to scion and rootstock tissues. This could be due to the genotypes studied, or the different stratification methods used, in our study grafts were placed in boxes with 1 cm of water in the bottom, while Prodhomme et al. (2019) used humid sawdust filled boxes [Both grafting and wounding stimulate defense mechanisms and local secondary metabolite synthesis , 25, 26.ed boxes . Anothered boxes –12. In oPrevious studies have tried to identify markers of graft incompatibility in several species by targeting secondary metabolites. Several metabolites, in particular flavanols or phenolic acids, have been quantified in higher concentrations at the graft interface in incompatible combinations several months or years after grafting as reviewed by Loupit and Cookson, 2020 . StudiesFinally, an accumulation of naringenin and taxifolin was found at the graft interface of MN hetero-grafts in comparison to rootstock homo-grafts and MN/MN. In addition to the antioxidant capacity of naringenin and taxifolin , naringeStilbenes are well known in grapevine as participating in defense mechanisms thanks to their antioxidant and antifungal properties . Concerntrans-resveratrol and trans-piceatannol, which were positively related with grafting success) and one stilbene trimer α-viniferin, which was negatively related to grafting success) in the scion tissue just above the graft interface 33 DAG were the most important compounds used to predict grafting success. Presumably, finding markers of homo-grafting success is simpler than in hetero-grafts as the effect of the different genotypes on the grafting success is stronger as the phenotype is not affected by the interaction with another genotype of the graft.In grapevine, to date no markers have been quantitatively related to grafting success across different scion/rootstock combinations. Previous studies have been limited to comparing either two  or four trans-resveratrol and from 96 h after wounding for α-viniferin [These two stilbene monomers and trimer are well known for their accumulation in response to abiotic stresses, particularly in the leaves, i.e. from 6 h after wounding for iniferin , but alsiniferin , 36. As V. vinifera as well as in rootstock homo-grafts. However, the identification of markers of grafting success remains complex and may only be valid at a certain time point studied. This underlines the importance of doing a time course study to understand the kinetics of metabolome changes at the graft interface, such as changes in sugar and amino acid remobilization, and stilbene accumulation to select the best time point for grafting success prediction.Here we present an approach to identify metabolite markers of grafting success in grapevine; this is the first attempt to study so many metabolites across such many scion/rootstock combinations and to use correlation analysis and GLMs to interpret the data. We have tentatively identified some differences between the incompatible scion/rootstock combination UB/RSB1 and its homo-grafted controls and other scion/rootstock combinations, which highlighted the accumulation of amino acids known to be involved in stress responses at the graft interface. These results are summarized in Standard amino acids , arginine (Arg), aspartate (Asp), asparagine (Asn), γ-aminobutyric acid (GABA), glycine (Gly), glutamate (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tyrosine (Tyr) and valine ) were purchased from Sigma.trans-piceid, trans-resveratrol and trans-piceatannol) and other compounds  were purchased from Extrasynthesis (France). Stilbenes, for the majority, have been purified in the MIB laboratory .Standards flavonols ; phenolic acids ; flavanols and procyanidins ; stilbenes , Chauvin) and placed in plastic crates. During the first 7 DAG, plants were kept at room temperature and then placed in callusing room . Two cm of water (with 0.2% dichlorobenzoic acid and 40 mg L of copper sulfate) was added to the crates. Callusing lasted between 8 and 14 DAG depending on scion/rootstock combination. Just before plantation, grafted plants were again dipped in melted wax, containing indolebutyric acid (Rhizopon).Fifteen scion/rootstock combinations were omega bench grafted (n = 200); the list of different combinations is given in th April 2019, 33 DAG, 5 pools of 5 plants were harvested randomly. Pieces of about 1 cm in length were taken from the graft interface, and above (scion), and below (rootstock) the graft interface (without nodes) and directly snap frozen in liquid nitrogen. Samples were ground to powder in a ball mill (MM400 RETSCH) in liquid nitrogen at 30 Hz during 30 s and kept at −80°C.On 30th December 2019, grafting success was measured on all scion/rootstock combinations. After verifying that the plant has enough roots and a lignified stem, the “thumb test” was performed to test the strength of the callus. The success rate represents the percentage of plants that validate all these criteria.On 16−1 fresh weigh (FW), mg g−1 FW and μmol g−1 FW respectively.The analysis was preceded by an ethanolic extraction as used by Hendriks et al., 2003 . The pel−1 FW.The ethanolic extract used for soluble sugars quantification was also used for quantification of free amino acids. Measures was made using an UltiMate 3000 ultra-HPLC system coupled with an FLD-3000 Fluorescence Detector after derivation with 6-aminoquinolyl-N-hydroxy-succinimidyl-carbamate  with an excitation energy at 250 nm and a emission energy at 395 nm . All res−1 made it possible to build the calibration curve and quantify the different compounds. All compounds were quantified with their corresponding standards except for gallocatechin, procyanidin B3, B4, B1 gallate, cis-piceid, cis-ε-viniferin and cis-astringin, which were quantified as epigallocatechin, procyanidin B1, B2, B1, trans-piceid, trans-ε-viniferin and trans-astringin respectively. Also, one flavanol and four stilbene dimers have been identified with an attempted assignment, which are trimers1, and dimer diglycoside, dimer glycoside A, dimer glycoside B and dimer glycoside C, quantified as procyanidin B1 and trans-ε-viniferin respectively. All results are expressed as g kg−1 FW.About 250 mg of ground sample was extracted into 4 mL of methanol in an ultrasound bath for 15 min. Then, after centrifugation, the supernatants were collected and diluted with milli Q water . Analysis of phenolic compounds was carried out using a high performance liquid chromatography  thermostated at 35°C) coupled with a triple quadrupole mass spectrometer (Agilent Technologies 6430 triple quadrupole detector) as described and used by Loupit et al., 2020  with somPrinciple component analysis (PCA), HeatMap, boxplot and statistical tests were made on the software R (version 4.0.4) and RStudio (version 1.2.5019) using ggplot2, gplot, FactoMineR and agricolae packages . FurtherWeb_Material_uhab070Click here for additional data file.


Most of the studies related to human mobility are focused on intra-country mobility. However, there are many scenarios  in which timely data on international commuters are vital. Mobile phones represent a unique opportunity to monitor international mobility flows in a timely manner and with proper spatial aggregation. This work proposes using roaming data generated by mobile phones to model incoming and outgoing international mobility. We use the gravity and radiation models to capture mobility flows before and during the introduction of non-pharmaceutical interventions. However, traditional models have some limitations: for instance, mobility restrictions are not explicitly captured and may play a crucial role. To overtake such limitations, we propose the COVID Gravity Model (CGM), namely an extension of the traditional gravity model that is tailored for the pandemic scenario. This proposed approach overtakes, in terms of accuracy, the traditional models by 126.9% for incoming mobility and by 63.9% when modeling outgoing mobility flows. In modern societies, understanding international human mobility is crucial under multiple perspectives . For insThe rapid diffusion of technologies such as mobile phones, devices with GPS receivers, social media  generate an enormous amount of data that we can utilize to investigate human movements –11.FigurWhile human mobility has been widely investigated at country and city scales, there are fewer studies regarding mobility across national borders. In such cases, official statistics  have been widely used both to understand mobility trends and types of mobility of international travelers –14, and Hence, using mobile phone data to quantify international commuters may represent a potential solution to the challenges mentioned above. Mobile phone data have been rarely used to deal with international commuters –31. AlsoIn this study, we use pseudo-anonymized and aggregated mobile phone data collected from a large mobile operator in UK (with a 28% market share in 2020) to model incoming and outgoing human mobility before and during the pandemic. More precisely, incoming mobility corresponds to the number of new foreign mobile phone SIMs  while outgoing mobility is the number of mobile phone SIMs registered in the UK that travel abroad  in UK, with 27.2 million subscribers as of May 2021. In particular, we detail the dataset we have built and the metrics we use to capture the international activity of smartphone devices.In this study, we use a passive measurement approach to retrieve some anonymized information about the devices attached to the antennas of the mobile network operator that provided the data. Each measurement carries the (1) anonymized user ID, (2) the SIM mobile country code (MCC) and mobile network code (MNC), (3) the first eight digits of the device International Mobile Equipment Identity (IMEI), (4) the timestamp, and other information. We also collect a device’s unique ID assigned by the Global System for Mobile Communications Association that describes some properties of the device like manufacturer, brand and model name, operating system, radio bands supported, etc. In this way, we can distinguish between smartphones (likely used as primary devices by mobile users) and Internet of Things devices. In this study, we use only the measurements related to smartphones. Additional information on the measurement infrastructure can be found in .at least a device that uses Radio Base Stations (RBSs) to interact with other devices . Whenever people traveling with connected devices cross a border, their devices need to connect to the radio network of another  operator to continue working correctly. For example, a person with a mobile phone traveling from Italy to UK will have to connect to a UK telecommunication operator network. The telecommunication operator will collect information about that device, including the country where the connected SIM is registered. The latter can be extracted using the MCC, a three-digit code that allows us to identify the origin of the SIM ),We acknowledge there are other more sophisticated models based on deep learning techniques as explained in a recent survey . ExampleWhile human mobility is an active research area both at national and local scales, there are fewer studies regarding international mobility patterns and their challenges . Tackling such challenges requires timely data with a proper spatial aggregation that roaming activities can provide. In this paper, we have proposed to use roaming network data to capture international mobility. Then, we use the gravity and radiation models to model international mobility. The incoming mobility is modeled better by a gravity model with an exponential decay both before and during the non-pharmaceutical interventions introduced for contrasting the spread of the COVID-19 pandemic. Instead, the outgoing mobility is captured equally well by the various models before the mobility restrictions were introduced. On the other side, after the second week of March, the radiation model is the one that captures mobility better. However, by explicitly modeling the COVID-19 restrictions for the origin and destination countries, the COVID Gravity Model (CGM) outperforms all the other models both for the incoming (improvement up to 126.9%) and outgoing (improvement up to 63.9%) mobility scenarios. These findings may have significant impact on how we should model international mobility in times of crises and can help policy makers in taking more accurate decisions. As part of future works, we will evaluate CGM also at a national and sub-national scales.


Mycobacterium leprae and host components in the induction of autoimmune response in leprosy. Based on our previous findings, we predicted and synthesized a total of 15 mimicking linear B cell epitopes (BCE) and 9 mimicking linear T cell epitopes (TCE) of keratin and MBP. Humoral and cell-mediated immune responses against these epitopes were investigated in Non-reaction (NR), Type 1 reaction (T1R) leprosy patients, and healthy controls. We observed significantly higher levels of antibodies against 8 BCE in T1R in comparison to NR leprosy patients. Further, we also found 5 TCE significantly associated with lymphocyte proliferation in the T1R group. Our results indicated that these epitopes play a key role in the induction of autoimmune response in leprosy and are also strongly associated with the inflammatory episodes of T1R. Conclusively, these molecules may be employed as a biomarker to predict the inflammatory episodes of T1R.Several Mycobacterial infections including leprosy and tuberculosis are known to evoke autoimmune responses by modulating homeostatic mechanism of the host. Presence of autoantibodies like, rheumatoid factor, anti-nuclear factor and antibodies to host, collagen, keratin, myelin basic protein (MBP) and myosin, have been earlier reported in leprosy patients. In the present study, we detected the role of mimicking epitopes between  Mycobacteria have been found to be able to modulate host immune responses both in humans and experimental animalsMycobacterium leprae, affecting mainly peripheral nerves, skin, and mucous membrane. Reactions in leprosy are immunological complicated state that occurs either before, during, or after treatment and affect 30–50% of patients with leprosy3. Type 1 reaction (T1R) is characterized by acute inflammation of skin lesions and/or nerves and is a major cause of neuritis often leading to nerve damage resulting in deformities of mainly hands and feet. The immune response is characteristic of delayed type hypersensitivity (DTH) reaction with an influx of peripheral blood lymphocytes demonstrating an increased reactivity to M. leprae antigens in a lymphocyte transformation test (LTT)4. This may lead to a local decrease in bacillary load and augmentation of T cell reactivity in the affected nerve leading to nerve damage5.Leprosy is a chronic granulomatous disease caused by 6. Antigenic mimicry might be one of the reasons for escaping the immune surveillance of M. leprae and its long period of incubation before the onset of the disease. The resemblance of epitopes may facilitate the survival of M. leprae in the human hosts when the antigens are not recognized as “non-self”, a situation that seems to occur in lepromatous leprosy when the patient’s tissues are loaded with bacteria virtually without any protective immune response. On the other hand, M. leprae antigens which mimic host antigens may induce an autoimmune reaction against the host’s own antigens. The antigenic molecular mimicry between pathogen and host may play a role in the pathogenesis of leprosy as well as it may be one of the reasons for immunological complications. Earlier, highest level of anti-keratin, anti-MBP and anti-myosin antibodies has been reported in T1R group of leprosy9. However, mimicking epitopes responsible for immunogenicity has not been studied extensively in leprosy. Hence, the present study was carried out to understand the immunological response to mimicking B and T cell epitopes across two different clinical spectra of leprosy and to elucidate their role in the induction of autoimmune response in T1R group of leprosy.Antigenic/molecular mimicry between host and pathogen has been suggested as a way to escape detection and destruction of the pathogen by host immunityM. leprae protein/s. Hence, the major objectives of this study were, firstly to evaluate antibody level against mimicking B cell epitopes across two groups, i.e., T1R and borderline leprosy patients without reaction (NR) for their humoral immune response to these mimicking epitopes and secondly to evaluate lymphocyte proliferation activity against the mimicking T cell epitopes in these groups to probe into their state of cell-mediated immune response. The healthy group was taken as the control variable group.Our main research question was to evaluate whether autoantibodies in the T1R group of leprosy produced because of mimicking epitopes of host protein/s and This work was carried out after getting approval from the Institutional Ethics Committee (IEC) of The Leprosy Mission Trust India, New Delhi . Blood samples were obtained from leprosy patients and healthy controls after obtaining written informed consent at the outpatient department at The Leprosy Mission Community Hospital, Delhi. Sample collection protocols and all methods in the study were performed with strict adherence to the guidelines and regulations of IEC, The Leprosy Mission Trust India (TLMTI), New Delhi and the Indian Council of Medical Research (ICMR), New Delhi.A total of 100 clinically diagnosed cases  consisting of 50 untreated leprosy patients in T1R and 50 untreated NR were recruited for the study. The inclusion criteria for patients were leprosy patients (NR and T1R) between ages of 18 years and 60 years, the patients below the age of 18 years and above 60 years were not included. Leprosy patients co-infected with other infections like dermatological infections, TB, HIV, Immune disorders, autoimmune diseases, and pregnant women were excluded from the study. Twenty-two non-occupational healthy controls without any clinical history of leprosy, TB, and other dermatologic infections and other diseases were included in the study from non-endemic areas. Lymphocyte proliferation assay was performed with 10 healthy controls, 20 untreated NR, and 20 untreated T1R patients. The demographic characteristics and clinical data of patients are given as below  were aseptically collected from all the subjects. Sera were separated and stored at − 20 °C till these were further used for antibody estimation against mimicking epitopes and for the second objective blood samples (≈ 8 ml each) were aseptically collected from all the subjects and processed further for PBMC isolation and then the 3--2,5-diphenyl -tetrazolium bromide (MTT) assay was performed against mimicking T cell epitopes.M. leprae predicted by BCPREDS server 1.0 and host protein/s identified by ClustalW multiple sequence alignment server were identified and the linear peptide epitopes/ were synthesized commercially by Thermo-Scientific, USA. A total of eleven B cell mimicking epitopes of protein HSP65 (M. leprae) and protein keratin (host) as well as four B cell mimicking epitopes of protein 50S ribosomal protein, lysyl tRNA synthetase (M. leprae), and myelin basic protein (host) were synthesized. In brief, The B cell epitopes/linear peptides synthesized included seven mimicking B cell epitopes of HSP65 of M. leprae (similar with the keratin of host) namely HSP1, HSP2, HSP3, HSP4, HSP5, HSP6, and HSP7; four mimicking B cell epitopes of keratin (similar with HSP65 of M. leprae), i.e. KER1, KER2, KER3, and KER4; two mimicking B cell epitopes of myelin basic protein  MBP50SB1 and MBP50SB2; two mimicking B cell epitopes of myelin basic protein (similar to lysyl tRNA synthetase of M. leprae) MBPLMB1 and MBPLMB2 were synthesized. All the peptides/ epitopes synthesized were of > 98% HPLC grade purity.Mimicking B cell epitopes. of M. leprae and host protein/s were identified by ClustalW multiple sequence alignment server. A total of nine T cell mimicking epitopes/linear peptides of protein 50S ribosomal protein and Lysyl tRNA synthetase of M. leprae similar to myelin basic protein (host) were predicted. Similar to B cell epitopes, the T cell epitopes/linear peptides were also synthesized commercially by Thermo-Scientific, USA. In brief, The T cell epitopes/linear peptides synthesized included two mimicking T cell epitopes of 50S ribosomal protein of M. leprae (similar with MBP), i.e., 50ST1, 50ST2; two mimicking T cell epitopes of Lysyl tRNA synthetase of M. leprae (similar to MBP) i.e. LMT1, LMT2; three mimicking T cell epitopes of MBP , i.e., MBP50ST1, MBP50ST2, MBP50ST3 and two mimicking T cell epitopes of MBP (similar to lysyl tRNA synthetase of M. leprae), i.e., MBPLMT1 and MBPLMT2.T cell epitopes were predicted by Syfpeithi epitope prediction server and among them mimicking T cell epitopes of Standardization of ELISA with B cell epitopes and MTT assay with T cell epitopes was performed by using checkerboard analysis and we found that 2.5 µg/ml concentration of predicted B cell epitopes/peptides and 1:1600 ratio dilution of serum sample were most suitable parameters to perform the test. Similarly, it was observed that 10 µg/ml concentration of T cell epitopes was the appropriate dose for MTT assay.2O2 and incubated for 20 min in dark at RT. Thereafter, the reactions were stopped with a solution of 2 N H2SO4, and OD was determined at 492 nm by using MULTISKAN FC ELISA Reader .Thereafter, ELISA was performed to evaluate humoral immune response against mimicking B cell epitopes across groups i.e., T1R, NR and healthy control. In brief, 96 well Microtiter plates were coated with mimicking B cell epitopes/peptides (Conc. 2.5 µg/ml) dissolved in 0.05 M Carbonate—bicarbonate buffer (pH 9.6) and incubated overnight at 37 °C. Thereafter, the plates were washed with PBS (pH 7.4), then blocked with 2% BSA in PBS and incubated at 37 °C for 1 h. Followed by washing with PBS the diluted serum samples were added [Dilution factor 1:1600 in 1% BSA in PBS-Tween 20 (0.05%)] and plates were incubated at 37 °C for 2 h. Later on, the washing was done with PBS -Tween 20 (0.05%) [PBST], then Anti-human IgG peroxidase conjugate  diluted in 1% BSA in PBS-Tween 20 (0.05%) was added and plates were incubated at 37 °C for 1 h. Afterward, plates were washed with PBST, and substrate solution of O-Phenyldiamine (Conc. 0.5 mg/ml) was added with 0.5% H10. In brief, PBMCs isolated from leprosy patients and healthy controls were suspended in culture media RPMI—1640  containing 5% of heat-inactivated fetal bovine serum (FCS) , 2 mM of l-glutamine , 2 mM of antibiotic antimycotic solution , and a suspension containing 2 × 106 viable cells/ml was made. The cells were dispensed 2 × 105 cells per well in 96 well flat-bottomed plates and stimulation was given by adding 10 µg/ml concentration of T cell epitopes/peptides. The plates were incubated in humidified environment at 37 °C in a CO2 incubator  at a 5% CO2 level, for 72 h. Thereafter, lymphocyte proliferation assay was performed by adding MTT  solution (5 mg/ml) to each well and incubated for 4 h at 37 °C. The formazan crystals thus formed were dissolved with 100 µl of lysis buffer . After a few minutes of incubation at RT, the OD was determined at 540 nm by using MULTISKAN FC ELISA Reader . The stimulation index was calculated by using the formula: Stimulation index = A 540 nm with stimulant/A 540 nm without stimulant, where A 540 = Absorbance at 540 nm.Similarly, lymphocyte proliferation activity against the mimicking T cell epitopes was performed to investigate cell-mediated immune response across the same groups. PBMCs were isolated by the Ficoll-Hypaque density gradient centrifugation methodThe study was approved by the Institutional Ethics Committee (IEC) of The Leprosy Mission Trust India, New Delhi . The IEC of The Leprosy Mission Trust India is associated with the The Leprosy Mission Community Hospital, Delhi. All participants were recruited after obtaining written informed consents at the outpatient department.The humoral immune response against each mimicking B cell epitope of keratin and myelin basic protein was estimated by the level of auto antibodies. Antibodies against HSP1, HSP2, HSP3 were found to be higher in NR when compared to T1R ; however, the mean level of antibodies against HSP1 was found to be significantly higher in both T1R and NR when compared to HC (p < 0.0001). Among all mimicking HSP65 peptides screened, it was observed that the mean level of antibodies against HSP4 and HSP5 were significantly higher in T1R when compared to NR  and HC . The mean level of antibodies against HSP6 was higher in T1R when compared to NR (p < 0.0001) and HC (p = ns). The antibody levels against HSP7 were found to be higher in HC followed by NR (p < 0.01) and T1R (p < 0.0001).  and HC (p < 0.0001). Only one peptide i.e. KER3 showed no significant difference between the groups (p > 0.05). (Table M. leprae) MBP50SB1 and MBP50SB2, we observed significantly higher antibody levels in leprosy patients when compared to HC . We also found a higher level of antibodies against MBP50SB1 in T1R and a lower level against MBP50SB2 when compared to NR . Similarly, we observed a higher level of antibodies in leprosy patients when compared to HC for two mimicking B cell epitopes of MBP (similar to lysyl tRNA synthetase) MBPLMB1 and MBPLMB2. We also found a significantly lower level of antibodies in T1R for MBPLMB1 (p < 0.05) and a significantly higher level for MBPLMB2 (p < 0.01) when compared to NR,  in the T1R group was found with 50ST2 and LMT1 when compared to NR  and HC .We observed that among four mimicking T cell epitopes of 50S ribosomal protein and Lysyl tRNA synthetase of M. leprae), we found higher SI in the T1R group with MBP50ST2 and MBP50ST3, when compared to NR  and HC . Among two mimicking T cell epitopes of MBP (similar to lysyl tRNA synthetase of M. leprae), we found significantly higher SI in the T1R group with MBPLMT2, when compared to NR (p < 0.01) and HC (p < 0.001). We also found higher SI in the T1R group with keratin  and MBP , when compared to NR  and HC (p < 0.01 and p < 0.001)  Table , 7 and 813.The association of infectious agents with autoimmune disease has been confirmed by epidemiological, clinical, and experimental findings suggesting existence of cross-reactivity with host ‘self’ antigens and microbial determinantsM. leprae monoclonal antibodies cross-react with human nerve and skin components and it has been suggested that this antigenic similarity could also be liable for the event of autoimmune clinical manifestations of leprosy15. This antigenic similarity is known as molecular mimicry, it could be linear or conformational.It has been reported earlier that anti-16. Therefore, HSP may evoke an autoimmune phenomenon in vivo during chronic mycobacterial infections. Earlier, Mycobacterial 65 kDa heat shock protein has been reported to share a carboxy-terminal epitope with human epidermal cytokeratin 1/217. Further, molecular mimicry between cytokeratin-10 of keratin (host) protein and 65 kDa HSP (groEL2) of M. leprae was reported and seven B cell epitopes of cytokeratin-10 and HSP 65 were found to be similar7. In the present study, we predicted a total of eleven B cell mimicking epitopes of protein HSP65 (M. leprae) and protein keratin (host) and evaluated host humoral immune response to determine the antigenic determinant responsible for the autoimmune phenomenon in the T1R group of leprosy. In our observation, the mean level of antibodies against HSP4 and HSP5 were significantly higher in T1R when compared to NR  and HC . Among all mimicking HSP65 peptides screened, the mean level of antibodies against HSP1 was found to be significantly higher in both T1R and NR when compared to HC (p < 0.0001). The antibody levels against HSP7 was found higher in HC followed by NR (p < 0.01) and T1R (p < 0.0001). The HSPs are well known to express during inflammatory and pathological stress conditions and the occurrence of molecular mimicry at antigen level may induce altered humoral and cell-mediated immune response evoking an autoimmune reaction. The differential level of antibodies against mimicking peptides HSP4, HSP5, and HSP7 among the different groups could be utilized as a predictive biomarker for the development of T1R in leprosy. On the other hand, the epitope HSP1 could be utilized to predict leprosy disease.Heat shock proteins (HSP)s are a phylogenetically conserved family of proteins, which are expressed during chronic inflammation and other forms of physiological stress conditions that lead to both humoral and cellular immune responses against microorganisms19.High level of antibodies against self-protein, keratin could be an expression of the autoimmune phenomena in leprosy patients, as the majority of leprosy lesions are manifested within the skin, and the occurrence of keratosis is a common phenomenon especially during T1R of tuberculoid leprosyM. leprae antigens which mimic host antigens may induce an autoimmune reaction against the host’s own antigens, which could explain the immune reaction in tuberculoid leprosy and during a “reversal reaction”, when host tissue is lacking M. leprae, whereas, extensive granuloma formation can be seen14. Singh et al., have reported that a high level of anti-keratin antibodies (AkAbs) are present in leprosy patients in comparison to healthy controls and the highest level AkAbs was present in T1R followed by LL, BL, ENL, and TT/BT leprosy. It was very interesting to find out that level of AkAbs also clinically correlated with the number of lesions present in leprosy patients7. Similarly, in the present study the level of antibodies against three mimicking B cell epitopes of keratin i.e., KER1, KER2, KER4 were found higher when compared to NR  and HC (p < 0.0001). The antibody responses to these three epitopes were found significantly associated with T1R. Thus, our finding suggests future utilization of these mimicking epitopes as a predictive biomarker for reactions (T1R) in leprosy.7. In the present study we predicted mimicking B cell epitopes and showed a high level of antibodies against a few of these mimicking B cell epitopes. We propose that molecular mimicry between these epitopes of cytokeratin 10 and HSP 65 may potentially lead to acute inflammation in skin lesions. Skin lesions develop scaling in T1R which might also be caused by an autoimmune reaction against these mimicking peptides.It has been noted that the T1R in BT leprosy patients often occurs suddenly during treatment or after completion of therapy. We still lack data on the exact mechanism of pathogenesis of T1R. Doubts have been raised on the role of mimicking proteins/peptides in the initiation of the reaction. It has been shown earlier that a high level of antibodies against keratin is associated with T1R patients20. It has been shown by Vardhini et al. that there is a molecular mimicry between mycobacterial proteins  with myelin P0, a protein that aids in compacting myelin through homotypical interactions21. In the above study, it was noted only by using bioinformatics tools however, in the present study we performed wet-lab validation along with using bioinformatics tools. Earlier, it was reported that in LL patients circulating immune complexes (CIC) contain MBP as an antigen. Liberation of MBP after M. leprae nerve damage may elicit anti-MBP antibodies, which react with peripheral nerve MBP. This mechanism may be responsible for demyelination and destruction of nerves in leprosy22. Earlier, it was also reported that MBP antibodies are associated directly or indirectly with neuro-degeneration in leprosy patients23.It has also been reported that this immunological response also includes an autoimmune response to nerve antigens which leads to demyelinationM. leprae), and myelin basic protein (host), we found a higher level of antibodies against MBP50SB1 in T1R and a lower level against MBP50SB2 when compared to NR . Similarly, we observed a higher level of antibodies in leprosy patients when compared to HC for MBPLMB1 and MBPLMB2. We also found a lower level of antibodies in T1R for MBPLMB1 (p < 0.05) and a higher level for MBPLMB2 (p < 0.01) when compared to NR. MBPLMB2 was found to be significantly associated with T1R. Singh et al., reported a high level of anti-MBP antibodies in leprosy patients across the spectrum and also found that four B cell epitopes of myelin A1 and M. leprae proteins, 50S ribosomal L2, and lysyl tRNA synthetase were cross reactive8. Earlier reports have shown that M. leprae binds to a nerve protein, the myelin protein P zero (P0) which is specific to the peripheral nerve and may be important in the initial step of M. leprae binding and invasion of Schwann cells. For the first time, our data indicate that these mimicking B cell epitopes might be responsible for pathological destruction of myelin sheath which may lead to nerve damage and cause deformities and permanent loss of nerve function in leprosy patients.In the present study among four B cell mimicking epitopes of protein 50S ribosomal protein, lysyl tRNA synthetase  and HC .On the evaluation of lymphocyte proliferation activity against mimicking T cell epitopes we observed that two mimicking epitopes i.e., 50ST2 and LMT1 of M. leprae protein 50S ribosomal protein, significantly stimulated cells in the T1R group when compared to NR  and HC . Further, one mimicking epitope of MBP i.e., MBPLMT2, and M. leprae protein Lysyl tRNA synthetase, significantly stimulated cells in the T1R group when compared to NR (p < 0.01) and HC (p < 0.001) group. The purified host proteins Keratin and MBP were also evaluated for reference purposes and we observed that higher SI is associated with the T1R group when compared to NR  and HC (p < 0.01 and p < 0.001) group.Similarly, two mimicking epitopes i.e., MBP50ST2 and MBP50ST3 of host protein MBP and M. leprae sonicated antigens and this induced auto-reaction is transferrable to naïve mice by immune cells8. The higher SI by mimicking T cell epitopes suggests high cell mediated immune response against mimicking T cell epitopes and for the first time showed the involvement of mimicking epitopes associated autoimmune reaction in the pathogenesis of reaction as DTH induced by self-antigens.The T cell epitopes were found to be associated with high CMI for keratin and MBP in the T1R group of leprosy patients. We observed earlier that auto-reaction can be induced in experimental animals after hyper immunization with .7 and Singh et al.8 are mimicking B cell epitopes between host protein/s and M. leprae protein/s. Further, high levels of antibody response by the host during T1R to the mimicking epitopes of M. leprae might strongly indicate that antibody-dependent cell-mediated cytotoxicity (ADCC) might be also involved in tissue damage in type 1 reactions in leprosy.This study indicated that the possible key factors responsible for the high autoantibodies against keratin and MBP observed earlier by Singh et alHence, we conclude from our study that these mimicking B and T cell epitopes are responsible for auto-reaction observed in the T1R group. These mimicking B and T cell epitopes might be responsible for acute inflammation of skin lesions or nerves or both.M. leprae specific B cell and T cell epitopes could be utilized for the development of a serological test for diagnosis of Type 1 reaction. Early identification of a case of leprosy with type 1 reaction may facilitate early diagnosis and treatment before the onset of deformity consequent to nerve damage in T1R leprosy. Further, this study also indicated that T cells might also be involved in ADCC to explain the mechanism of tissue damage. However, to establish such a mechanism of T cell cytotoxicity further studies have to be carried out to explain the tissue damage in T1R.This study indicated that mimicking host and


The effect of chitosan ratios on the physical, mechanical, water-vapor barrier, and thermal properties of the film was studied. Chitosan-protonated amino groups promoted the formation of intermolecular bonds, improving tensile strength, thermal stability, hydrophobicity, water adsorption capacity, and the gas barrier of starch films. The results show that the film composed of TPS25-CH75 proved to be the best barrier to water vapor; thus, these composite films are excellent choices for developing biodegradable packaging for the food industry.Starch is a biopolymer with enormous potential for generating new biodegradable packages due to its easy availability and low cost. However, due to its weak functional properties, limitation of its interaction with some hydroxyl groups and evaluation of blends with other polymers are necessary in order to improve its performance. Glycerol-plasticized acetylated corn starch films were developed using the casting method, and the impact of incorporating chitosan (TPS:CH) in various proportions (75:25, 50:50, and 25:75  The world is well aware that humanity is facing a critical pollution problem. Plastic, particularly as used in the food industry for packaging, conservation, transport, and consumption, is one of the most commonly discarded materials. Thus, several research propositions have been made to develop more environmentally friendly materials to replace conventional synthetic plastics. These new materials can be obtained from various mixtures of biodegradable polymers with additives such as plasticizers, antioxidants, and antimicrobials.Starch, a natural polymer, is an excellent choice for making low-cost biodegradable packaging materials due to its affordability and wide availability. Starch is composed of two molecules: amylose and amylopectin. Amylose is a linear polysaccharide comprising α-1,4-linked D-glucopyranose, while amylopectin is a highly branched molecule consisting of chains of α-D-glucopyranosyl residues connected by 1,4 and 1,6 bonds. However, starch-based materials are brittle and susceptible to moisture; thus, several methods have been developed to improve their functional performances. Starch can be modified in various ways to change its processing properties, as well as the materials generated. One method of modification is through chemical processes such as esterification and acetylation. Acetyl groups break the inter- and intramolecular bonds of the starch, weakening its structure and allowing a higher mobility of the polymeric chains of the amorphous region . AcetylaChitosan is a natural linear polysaccharide derived from chitin extracted from the exoskeleton of crustaceans, a byproduct of waste from the fishing industry . It is bv/v).The objective of this study was to evaluate how acetylated corn starch-chitosan (TPS:CH) affected the mechanical, gas barrier, thermal, and water-vapor-sorption properties of a biodegradable film based on modified (acetylated) starch films plasticized with glycerol in three different ratios , magnesium chloride (MgCl2), potassium carbonate (K2CO3), sodium bromide (NaBr), sodium chloride (NaCl), potassium chloride (KCl), and barium chloride (BaCl2)  were used to prepare supersaturated saline solutions to obtain environments with an equilibrium relative humidity (RH) of 11%, 22%, 32%, 43%, 57%, 75%, 84%, and 90%, respectively.Modified food starch  obtained from Grain Processing Corporation  and chitosan (90% deacetylation degree) from Alfadelta Materias Primas  were used as biodegradable polymers. Anhydrous glycerol from J.T. Baker  was used as a plasticizer. Productos Quimicos Monterrey  provided glacial acetic acid. Lithium chloride (LiCl), potassium acetate (CHw/v) was dispersed in distilled water, and the plasticizer was added at a concentration of 30% . When the starch dispersion reached 50 °C, a corresponding volume (mL) of a 1% (w/v) solution of chitosan (dissolved in 1% v/v acetic-acid solution) was added. The filmogenic solutions were constantly stirred, and the temperature was increased to 90–92 °C for 7 min to allow the starch to gelatinize. Then, 100 mL of the filmogenic solution was poured into 150 × 150 mm acrylic molds and dried at 65 °C for 5 h in an oven . Before evaluating the functional properties, the dried films were peeled and stored in desiccators containing a supersaturated solution of NaBr for 48 h at room temperature (~25 °C) and 54% RH.To prepare the film-forming solutions, five starch:chitosan (TPS:CH) ratios were developed: 0:100, 75:25, 50:50, 25:75, and 0:00. They were denoted as TPS100-CH0, TPS75-CH25, TPS50-CH50, TPS25-CH75, and TPS0-CH100, respectively. The corresponding amount of starch . The average and standard deviation of 10 film samples were obtained.The capacity and resistance to being in contact with the liquid water of the materials were determined with the solubilization capacity in water at room temperature (25 °C), following the Fonseca-García and Jiménez-Regalado  methodolTo determine the hydrophilicity of the films, the contact angle formed by a 3 µL drop of distilled water on the opaque side of the films (mold contact) was measured. To perform this analysis, we used an instrument from Rame-Hart Co.  coupled with a camera; a video of the raindrop formed on the films was recorded. The angle formed in the water drop was analyzed 20 s after it had been in contact with the films. The contact angle was measured in triplicate for each film.The chemical structure of films was analyzed by Fourier transform infrared (FT-IR) using a NICOLET iS10 spectrometer of Thermo Fisher SCIENTIFIC . The measurements were performed with attenuated total reflection (ATR).The arrangement of the structural matrix of the films was determined using a Siemens D500 powder diffractometer, following the methodology proposed by Fonseca-García and Jiménez-Regalado .The thermal behavior of the materials was determined using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) techniques with DSC 2500 Discovery  and TGA Q500  analyzer equipment, as well as the methodology proposed by Fonseca-García and Jiménez-Regalado .2 (90% RH), generating a water-vapor differential pressure of 2854.23 Pa. The cell was weighed seven times at 60 min intervals. The determinations were made in triplicate.Water-vapor permeability was determined according to the methodology proposed by Aguirre-Loredo and Rodríguez-Hernández  followin2) were placed in airtight plastic containers with supersaturated saline solutions, generating 11–90% RH. The containers were kept at room temperature (25 °C) for 14 days. The Guggenheim–Anderson–de Boer (GAB) mathematical model (Equation (1)) was used to describe the sorption isotherms of the films with different starch-chitosan ratios.X is the total moisture content in the material; wa is water activity; the GAB model parameters, Xm, are the moisture content in the monolayer; C is the constant related to the sorption in the first layer; and K is a constant related to the sorption of water molecules in the multilayer.The water-vapor adsorption isotherm of each material was determined using the static microclimate method, following the methodology developed by the COST90bis Project  and by A−1, with an initial separation of 25 mm, according to the D882-12 standard [The mechanical properties of 10 film samples with dimensions of 10 × 50 mm were measured using a TA.XT Express Enhanced texture analyzer  equipped with tension grips (A/TG) at room temperature (25 °C), operating at a cross-head speed of 1 mm·sstandard . Tensilep < 0.05 significance level, using the OriginPro 8.5.0 SR1 software .The results were analyzed for statistical significance by analysis of variance and Tukey’s test, with a A significant difference in the thickness of the polymeric materials  as a funOne of the most important factors considered when designing and using a completely biodegradable material is the susceptibility or damage of the material due to contact with water, as most biopolymers are hydrophilic. The biodegradable films developed in this study showed increased solubilization capacity in liquid water, as observed by the values shown in The hydrophobic behavior shown by acetylated starch films is congruent with the fact that this biopolymer was modified by substituting hydroxyl groups with acetyl groups that promote a relative hydrophobicity such as is shown in the pristine acetylated starch film with a contact angle of 90.58°. This angle corresponds to a hydrophobic material .Nevertheless, this behavior is apparently reduced in the composite films due to the addition of chitosan on the starch films. Even though in film TPS25-CH75, hydrophilicity was lower than in pristine chitosan film, this effect can be associated with polymeric chains that were ordered along the film, allowing hydroxyl groups of chitosan and starch to be on the surface of the film.−1, the stretching of O–H, which decreases its absorption chitosan concentration, increases. This absorption band is strong and broad, and it overlaps with –NH stretching vibration and intermolecular hydrogen bonds in the mixtures . At wavenumbers of 2924 and 2887 cm−1, symmetric and asymmetric stretching vibrations of methylene groups (CH2) are observed, respectively, in the spectrum of thermoplastic starch [−1, the bending vibration of OH (water) and carbonyl appears in chitosan at the same wavenumber. CH and CH2 deformations are evident at 1412 cm−1. In the region of 1242 and 1240 cm−1, O–H vibrations (bending) and C–O stretching of acetate are present. Some bands are also observed that could be attributed to the formation of hydrophilic colloids in the composite films at 1077 cm−1, 1018 cm−1, and 927 cm−1 (C–O stretching and O–H bending), as well as at 851 cm−1 (H-atoms), 651 cm−1, and 600 cm−1 (plane bending) [−1 it is related to the vibratory modes of the d-glucopyranosyl ring and the skeletal modes of the pyranose ring [−1, corresponding to the stretching vibrations of the C–O group, and at 1153 cm−1, corresponding to the asymmetric stretching of C–O–C bridge, also related to the saccharide structure [−1 for C=O stretch, 1554 cm−1 for flexion –NH2, and 1378 cm−1 flexion –CH2. The absorption band at 3409 cm−1 (strong and broad) was attributed to the stretching vibration of –OH, the stretching vibration of –NH, and the intermolecular hydrogen bonds of other polysaccharides. c starch . At 1646tructure . Typical−1, it could be inferred that this analysis is sensitive to changes in the structure at the molecular level of starch. In general, these bands exhibited broadening due to the multiple and stronger intermolecular interactions that influence the vibration of the C–O group. Differences between the 1047 cm−1 bands of the crystalline regions and the 1022 cm−1 bands of the amorphous region were observed between the complexes [−1 related to the double helices of the amorphous region allowed for estimation of the degrees of order  and double helices  in the mixtures  occurs, the glycerol evaporates (at approximately 290 °C), and the cleavage of the polymeric starch chains begins. The last event occurs beyond 300 °C, where the decomposition product residues are evident (CHOH group and starch cyclic structures arise). The best thermal performance was presented by the TPS75-CH25 blend, which manages to increase the value of the maximum degradation temperature by 11.4% for TPS100-CH0.g2 of the mixtures decreases due to the plasticizer content and in the presence of low starch contents, mobilizing chitosan chains through the protonation of the amino group (H-bond). The TPS75-CH25 sample exhibits a more significant restriction in molecular mobility, with a higher and more defined Tm for TPS100-CH00. This effect is consistent with the strong interactions reported in the FT-IR analysis, as well as with the DO. Modification by the acetylation of starch causes partial molecular hydrolysis and reduces the length of glucose chains. Hence, modified starch films have a lower decomposition temperature than that of those based on native starch [The thermal transitions  for diffe starch .−11 g·m−1s−1·Pa−1. The amount of water vapor permeating through the material decreased with an increase in the ratio of chitosan, the TPS25-CH75 ratio being the one that reduces it the most, with a value of 0.55 × 10−11 g·m−1s−1·Pa−1. The humidity of the material significantly modifies the barrier capacity of biodegradable materials. With higher the moisture content of the material, the barrier capacity allows for increased flow of gases. However, among the materials developed in this study, the film that absorbed the most moisture presented the best barrier to water vapor. The presence of water molecules and their interaction with the polar groups of the biopolymers through interchain hydrogen bonds progressively reduces the cohesive energy of the polymer, resulting in the plasticization of the matrix and an increase in the diffusion coefficient. Higher hydrophilicity in chitosan can explain this behavior due to the presence of –NH3+ groups, amino, and hydroxyl groups, which represent binding sites for water molecules in the chitosan chain [−11 g·m−1s−1·Pa−1) [Acromonia aculeata) flour films (0.17–0.20 × 10−11 g·m−1s−1·Pa−1) [−11 g·m−1s−1·Pa−1) [an chain . The mat−1·Pa−1)  and boca−1·Pa−1) . These d−1·Pa−1) ,29,30.w < 0.8 .The water-vapor adsorption behavior of the materials is an important parameter that needs to be evaluated when considering use as packaging film. The moisture that these packages can adsorb when exposed to certain environmental conditions is a factor that influences and can modify their mechanical and gas-barrier properties. w < 0.8 . Above 0Xm, C, and k) and the correlation coefficients (R2) for each biodegradable TPS:CH composite film. The parameter Xm corresponds to the moisture content present as the first layer of water that interacts directly with the material, called the monolayer, and is the optimal water content for the conservation and storage of dry products. An increase in the amount of water present in the monolayer of the material was observed as the chitosan ratio increased in the starch films  of chitosan and OH– from starch, which promote the formation of intermolecular bonds and improve the TS behavior of materials. It has been reported that incorporation of chitosan improves the mechanical behavior of starch films when the concentration of chitosan is at least 40% [east 40% . The valeast 40% . When coeast 40% . Similareast 40% . Thus, seast 40% .w/w) for mechanical properties. The higher content of plasticizer tends to increase plasticity, allowing for higher deformation; however, the TS decreases [In general, the “rule of mixtures” is not fulfilled according to the results found in this study, despite incorporating a constant plasticizer content on the total base of the polymers. This does not exert a compatibilizing effect or homogeneous interaction with each polymer. Consequently, the interactions between glycerol and starch are stronger than those between glycerol and chitosan, as shown by the increased TS a value (ecreases . A signiecreases .Several techniques were used to develop and characterize modified corn starch films mixed with different proportions of chitosan and plasticized with glycerol. Composite films with good workability and good characteristics were obtained to be used as food packaging material. Chitosan interacts effectively with starch, as evidenced by FT-IR, XRD, DSC, TGA, and mechanical results, which revealed a strong interaction between the two polymers. Biodegradable films based on starch–chitosan mixtures exhibited an improved water-vapor barrier compared to other starch-based materials. The TPS25-CH75 composite had the lowest permeability to this gas, although a weaker mechanical behavior than other mixtures.The acetylation of starch was characterized using the sessile drop method, FT-IR, and XRD, and the results showed that the amylose and amylopectin double helices had been modified due to the substitution of OH groups by bulkier acetyl groups. In terms of mechanical performance, the samples showed higher rigidity as the chitosan ratio increased, except for the TPS75-CH25 blend, which showed a ductility similar to that of TPS100-CH0 but with better resistance. Furthermore, the thermal behavior of this material, in terms of the degradation temperature, was higher than that of the other formulations. These thermal transitions show miscibility between the components of the blends, with high degradation temperatures, making them susceptible and a good option for processing on a larger scale in plastic-materials production equipment.Based on the performance of the materials obtained in this study, although their behavior is not yet on par with conventional synthetic plastics, their gas-barrier properties were found to be similar to those presented by some low-density polyethylenes. However, further research is necessary concerning the continued development of these biodegradable materials in order to bring entirely environmentally friendly materials to the market in the future with a satisfactory operation for use in the food packaging industry.


Polyomavirus-associated nephropathy (PVAN), mainly caused by the BK virus, is one of the most important infectious complications of kidney transplantation. The leading histopathologic characteristics of PVAN is viral cytopathic effects, such as nucleomegaly with smudged or clumped chromatin and intranuclear ground-glass inclusion, mostly in tubular epithelial cells. Moreover, tubular necrosis, tubulitis, interstitial inflammation, atrophy, and fibrosis have been noted. Positive immunohistochemistry (IHC) staining for SV-40 highlights the infected epithelial cells of renal tubules.A total of 85 core needle biopsies of transplanted kidneys were evaluated histologically and were stained for SV-40 using the IHC method. In addition, a follow-up of graft failure was performed.Our findings revealed that the frequency of polyomavirus infection in kidney transplant patients in the Northeast of Iran is 4.7%. There was no significant correlation between PVAN and graft rejection. Although a higher rate of graft loss was observed in PVAN patients, in comparison with non-PVAN patients (25% vs. 14.8%), the difference was not statistically significant. Moreover, patients with immunohistochemically confirmed PVAN and those with histopathologic features of viral-like cytopathic effects had significantly lower graft survival in the follow-up period .The frequency of polyomavirus infection in kidney transplant patients in the Northeast of Iran is 4.7%. There was no significant correlation between PVAN and graft rejection. Furthermore, we observed that polyomavirus infection accelerates the course of graft loss. Human polyomaviruses are the members of the polyomaviridae family, which includes ubiquitous viruses, such as the BK virus and JC virus. These agents cause asymptomatic infections among immunocompetent people. However, immunosuppression can lead to the activation of these viruses. Polyomavirus-associated nephropathy(PVAN), mainly caused by the BK virus, is one of the most important infectious complications of kidney allograft . In the In the current study, core needle biopsies of transplanted kidneys were obtained from the archives of the pathology departments of three major medical centers of Ghaem Educational Hospital, Imam Reza Educational Hospital, and Mashhad Pathobiology Laboratory, Mashhad, Iran during 2009-2019. Eighty-five cases with complete clinical data and sufficient tissue in paraffin blocks were included in the study. The specimens devoid of the medullary portion were excluded. All hematoxylin and eosin-stained slides were reviewed by two independent pathologists. Histopathologic data, such as nuclear changes imply viral-like cytopathic effects, namely nucleomegaly, smudged/clumped chromatin, and intranuclear ground-glass inclusion with or without a halo in the epithelium of renal tubules. The histopathologic evidence of graft rejection (classified according to Banff classification 2018), histopathologic features of recurrent or de novo glomerulopathies, and calcineurin inhibitor nephrotoxicity features were recorded. Demographic and clinical data, including age, gender, and serum creatinine level at the time of biopsy were obtained from medical records. In the follow-up, the detection of any graft failure was defined as the loss of graft function and permanent need for dialysis any time after the transplantation. The IHC was performed on formalin-fixed paraffin-embedded tissue sections using the SV40 antibody  to detect large T-antigen of a polyomavirus in infected cells. The slides were evaluated by two pathologists and the results were considered positive in the case of at least one cell with nuclear staining of SV40 antibody.P<0.0001). Additional histopathological findings are listed in In the present investigation, 85 needle biopsies of transplanted kidneys were evaluated. Most patients were male  and the mean age of the patients was 35.87±13 years. In histopathologic evaluation, viral-like cytopathic changes were observed in 18 (21.2%) biopsies , howeverP=0.554) (P=0.049) (P=0.027)  patients lost their graft function and permanently needed dialysis. Out of four SV40-positive patients, only 1 (25%) showed graft failure during this period. This case had BK nephropathy class B according to the Banff Working Group classification . From thP=0.554) . The meaP=0.049) . MoreoveP=0.027) . It shouPolyomavirus infection has a high frequency in the general population as seropositivity for BK virus and JC virus in different populations and various age groups reaches up to 99% and 78%, respectively -13. Alth et al. reviewed 11 studies showing the mean prevalence of 4.91% for BK nephropathy, which is almost similar to our findings (et al., the incidence of PVAN was 5.1 (et al. reported a higher frequency of 13.1% in the Iranian population (et al. studied 247 renal transplant patients in the Northeast of Iran and found only two cases with positive PCR results for BK virus in the urine. Although their reported frequency is much lower than ours, the methodology is different and histopathological evaluation was not performed in their study (Hirschfindings , 8. In t was 5.1 . In our pulation . Among tir study . et al. revealed that BK nephropathy is a risk factor for donor-specific antibody formation which in turn results in antibody-mediated graft rejection (Graft loss following PVAN is estimated as 10%-80% in different centers . In the ejection . Based oejection . In histejection .The current study indicated a complete agreement between the PCR method for the evaluation of BK virus plasma viral load and SV40 IHC staining in paraffin blocks. This study was conducted for the first time in the Northeast of Iran using the IHC method for renal biopsies to confirm PVAN in transplanted kidneys and analyze graft survival. The limitation of this retrospective study was the low sample size, which was inevitable due to the insufficient tissue material and incomplete medical records.In conclusion, the results of the present study revealed that the frequency of polyomavirus infection in kidney transplant patients in the Northeast of Iran is 4.7%. Patients with PVAN and those with viral-like cytopathic effects had significantly lower graft survival in the subsequent period. Consequently, we conclude that polyomavirus infection accelerates graft loss.


Background: Arterial stiffness was the pathological basis and risk factor of cardiovascular diseases, with chronic inflammation as the core characteristic. We aimed to analyze the association between the arterial stiffness measured by cardio-ankle vascular index (CAVI) and indicators reflecting the inflammation degree, such as count of leukocyte subtypes, platelet, and monocyte-to-lymphocyte ratio (MLR), etc.Methods: The data of inpatients from November 2018 to November 2019 and from December 2019 to September 2020 were continuously collected as the training set  and the validation set (700 cases), respectively. A retrospective analysis of gender subgroups was performed in the training set. The association between inflammatory indicators and CAVI or arterial stiffness by simple linear regression, multiple linear regression, and logistic regression was analyzed. The effectiveness of the inflammation indicators and the CAVI decision models to identify arterial stiffness by receiver operating curve (ROC) in the training and validation set was evaluated.Results: The effect weights of MLR affecting the CAVI were 12.87% in men. MLR was the highest risk factor for arterial stiffness, with the odds ratio  of 8.95 (5.04–184.79) in men after adjusting the covariates. A cutpoint MLR of 0.19 had 70% accuracy for identifying arterial stiffness in all participants. The areas under the ROC curve of the CAVI decision models for arterial stiffness were >0.80 in the training set and validation set.Conclusions: The MLR might be a high-risk factor for arterial stiffness and could be considered as a potential indicator to predict arterial stiffness. Arterial stiffness is characterized by degradation in the extracellular matrix of the mediator layer, while atherosclerosis is caused by the accumulation of lipids in the intimal layer initiating the migration of inflammatory cells . Arteriahttps://doi.org/10.7910/DVN/SGHUV2. This was a retrospective, cross-sectional study, and all samples do not contain personal identification. We were unable to obtain written consent from all patients. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki.All materials have been made publicly available at the HARVARD Data verse and can be accessed at We evaluated the arterial stiffness risk of the indicators and constructed CAVI decision models in the training set. The predictive capability for arterial stiffness of the multiple regression models was constructed in the training set, and these indicators were validated in the training set and the validation set.To construct the decision model of CAVI, the data of patients who were hospitalized in the General Medical and Geriatrics Department of Wuhan Union Hospital from November 2018 to November 2019 and accepted arterial elasticity measurements were collected as a training set. And data from December 2019 to September 2020 were applied to verify the identifying validity for arterial stiffness of the indicators and the constructed models. Samples where the leukocyte subtype counts or ratio may be severely affected by diseases or medicine were excluded. The specific exclusion criteria are as follows: (1) definite or suspicious blood system diseases, such as infectious mononucleosis, leukemia, and lymphoma, etc.; (2) currently or recently used immunosuppressive agents, such as cyclophosphamide, cyclosporine, etc.; (3) suffering from autoimmune diseases that seriously affect the number of white blood cells, such as systemic lupus erythematosus and Graves disease; (4) severe hypersplenism; (5) taking medication that may affect the white blood cell count or infusing blood; (6) suffering from other malignant tumors and are undergoing radiotherapy or chemotherapy.The participants' smoking and alcohol history and clinical diagnosis including HTN and diabetes mellitus (DM) were collected simultaneously. Ten milliliters of venous blood in the fasting state in the morning after the patients were admitted to the hospital were obtained, and the samples were sent to the laboratory immediately. Blood counts were evaluated by BC-3000 auto hematology analyzer . Then, the NLR, MLR, and PLR as the ratio of neutrophils to lymphocytes, monocytes to lymphocytes, and platelets to lymphocytes were calculated, respectively. The levels of uric acid, triglycerides (TGs), cholesterol (CHOL), low-density lipoprotein, and fasting blood glucose were determined by Union Hospital's standard biochemical index laboratory measurement procedure. The NLR, MLR, and PLR were calculated as the ratio of neutrophil count to lymphocyte count, monocyte count to lymphocyte count, and platelet count to lymphocyte count, respectively.The CAVI adopts the pulse-wave algorithm and utilizes the stiffness parameter β to reduce the influence of blood pressure fluctuations on the detection of vascular elasticity. The arterial elasticity of the patient was measured with the VS-1000 AS tester of Japan Foton Company. The patient rested quietly for about 15 min before the examination and then measured in the supine position. The blood pressure of the extremities at the position of the upper arm and ankle arteries was measured, and the electrocardiogram, heart sound graph, and pulse-wave waveform were recorded at the same time. The bilateral CAVI (m/s) value was calculated, and the average of the measured values on both sides was employed in the study. Arterial stiffness is considered as CAVI ≥9 (m/s); otherwise, it is non-arterial stiffness (non-AS).U test and t test were used to compare the difference between non-AS and AS in non-normal distribution and normal distribution quantitative data, respectively. A χ2 test was used to compare the difference of categorical data between the groups. Analysis of covariance (ANCOVA) is used to compare continuous variables between groups after adjusting for confounding factors. The inflammatory indicator difference was tested between AS and non-AS on the sample set after implementing confounding variables by individually pair-matched case–control analysis. The scatter plots were drawn between CAVI and each inflammation indicator, and the fitted linear lines for each inflammation indicator were calculated. The multiple regression analyses were performed using the stepwise backward method to determine the contribution size of inflammation indicators and clinical variables to arterial stiffness. The odds ratios (ORs) and 95% confidence intervals for arterial stiffness were calculated with logistic regression after adjustment for covariates that may affect arterial elasticity. A subgroup analysis of inflammatory indicators to AS risk in people with different clinical pathophysiological characteristics was carried out. Finally, the receiver operating characteristic (ROC) curves were determined to assess discrimination of these indicators and final multiple regression models to diagnose arterial stiffness. p < 0.05 was considered statistically significant. All calculations and graphs were done using R (version 3.6.3-Mac OS X 10.11).Quantitative data of non-normal distribution and normal distribution were expressed as medians with interquartile range and mean ± SD, respectively. Categorical data were expressed as amounts with percentages. The Mann–Whitney The training set and the validation set included 1,089 and 700 sample materials, respectively. The materials stratified by arterial stiffness are summarized in p < 0.001), NE (p < 0.001), MLR (p < 0.001), NLR (p = 0.003), and PLR (p = 0.034) remained significant differences in the group of AS and non-AS, whereas PLT (p = 0.063) and LY (p = 0.914) were not significantly different between the groups , monocytes (MO), NLR, MLR, and PLR of participants with arterial stiffness were significantly higher than those of patients without arterial stiffness, whereas lymphocytes (LY) and platelets (PLT) were lower in the AS group. Then, with age, sex, BMI, TGs, CHOL, HTN, and DM as covariates, which show a significant difference between the AS and non-AS groups, we further performed the ANCOVA to test the differences of leukocyte counts and their ratios between patients with and without AS. The results indicated that after adjusting the confounding variables, MO . The results indicated that only NLR and MLR showed the significant risk for AS in all subgroup analyses, and the risk value of MLR  was much higher than NLR (OR = 1.29–1.93) .p > 0.05), and their AUCs were all 0.87 in the training set and 0.80–0.84 in the validation set  in men, but it was non-significant in women (p > 0.05); the efficacy of MLR in suggesting arterial stiffness in men is better than in women (AUC = 0.72 vs. 0.67). The intimate association between MLR and CAVI in men may confirm the fact that men are more susceptible to ischemic heart disease of large vessels than women  can be found below: Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.BQ: conceptualization. RW, YW, YL, LB, LL, and LH: methodology and data collection. YW: data curation, formal analysis, visualization, and writing—original draft. RW: supervision and writing—review & editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This article aims to present all aspects regarding patient selection, planning, and implantation technique for a new off-the-shelf pre-cannulated multi-inner branch stent graft. The stent graft comes in 4 different versions with proximal diameters of 33 and 38 mm and distal diameters of 26 and 30 mm. The 4 inner branches are located in the middle segment, which has a diameter of 24 mm.With inner branch technology, the field of application for the treatment of thoracoabdominal aortic aneurysms (TAAA) has been further extended. In addition to routine use in elective cases the pre-cannulation of the inner branches predisposes especially for emergencies. Pre-cannulation is intended to reduce the time to cannulation and the radiation dose. All steps of planning, stent-graft deployment, and cannulation of the inner branches are described in detail.The E-nside stent graft represents a promising new endovascular therapy in the treatment of acute and elective TAAA. By using inner branch technology, this endograft combines the advantages of fenestrated and branched stent grafts. Indication, planning, and implantation require experience in branched and fenestrated stent graft technology. The introduction of fenestrated and branched stent grafts has significantly reduced these perioperative risks in the treatment of TAAA. The duration of stent graft production was also often a limiting factor, as urgent cases could not be treated due to the custom design of the grafts.Endovascular therapy for aortic aneurysms has become a common alternative to open surgical therapy in recent years. Especially in the treatment of thoracoabdominal aortic aneurysms (TAAAs), open surgical therapy represents a high-risk intervention due to the risks of paraplegia, organ failure, or mortality. Only vascular morphology was a limiting factor for this prosthesis in a certain percentage of cases.With the introduction of the first off-the-shelf multibranch endograft system in 2012, a large number of urgent TAAAs could be treated endovascularly.57 Due to its outer branch design, the renovisceral segment should have a diameter of at least 30 mm according to the planning sheet, although diameters of 25 mm are possible in some cases with suitable morphology. Therefore, its use in pararenal or juxtarenal aortic pathologies is often not feasible.Both the short- and long-term outcomes of these patients showed excellent results.With a new off-the-shelf inner branch stent prosthesis, it became possible to treat even these pathologies due to the 4 pre-cannulated inner branches. According to its instructions for use (IFU), renovisceral segments with a diameter of at least 24 mm can be treated. In addition, inner branches can combine the advantages of outer branches and fenestrations. While the advantages of outer branches are flexibility, improved sealing and fixation for bridging stents, and less critical positioning of the branches, the advantages of fenestrated prostheses are that they require less space (narrow aortas) and less aortic coverage.This paper aims to describe all aspects of planning, stent graft deployment, and cannulation of the inner branches.The E-nside prosthesis (JOTEC GmbH) is a multibranched endograft with 4 antegrade oriented inner branches for the renovisceral vessels . The proThe 24 F delivery system comes with a nonhydrophilic atraumatic tip [8.2 mm outer diameter (OD)] and a hydrophilic outer sheath . The stent graft is released by a so-called “squeeze to release mechanism,” where each click corresponds to a 4 mm step.Different radiopaque markers are used for identification. A total of 5 tubular markers were used for the proximal and 3 ring markers for covering the distal end of stent graft. The inlet of each inner branch is marked with 1 ring marker, while the outlet is marked with 3 tubular markers. A total of 2 “E” markers display the maximum and minimum overlap for the proximal landing zone and provide rotational orientation [anterior/posterior (A/P) projection in the coronal plane]. An “8” marker (platinum–iridium) marks the distal overlapping line  and that the patency of the inner branches is guaranteed throughout the cannulation phase .The diameters of the celiac trunk (CT) and superior mesenteric artery (SMA) inner branches are 8 mm and those of the renal arteries are 6 mm, respectively. All inner branches have an antegrade orientation and a length of 20 mm. All 4 branches present with enlarged and oval-shaped outlets to allow for greater variability of the bridging stents. The design of the outlet of the inner branches results in flexibility and range in the axial plane of α=50° for the CT and SMA and β=70° for the renal arteries. In the sagittal plane, the degree of freedom is 0°<γ<45°.All 4 inner branches were pre-cannulated with polyimide (PI) tubes with a length of 1465 mm, which extended to the proximal end of the stent graft. The inner diameter is 0.5 mm (0.018 in) and the OD is 0.7 mm (0.035 in). All tubes are loaded with a 0.018 in disposable transportation wire that has to be removed to use the pre-cannulation tubes.According to the IFU, the following requirements must be met:Adequate iliac/femoral access compatible with an 8.5 mm OD delivery systemAngle ≤75°, 40 mm proximal to the CTAngle ≤30°, 20 mm distal to the lowest renal arteryLength of proximal landing zone ≥30 mmLength of distal landing zone ≥30 mmNo angulation in the proximal and/or distal landing zoneNo severe angulation in the thoracovisceral segment of the aorta ranging from 40 mm proximal to the CT to 20 mm distal to the lowest renal arteryAccording to the structural specifications of the E-nside endograft, the following requirements exist for the renovisceral vascular anatomy:The distance in height between the outlet of the inner branches of the stent graft and the ostia of the target vessels must be between 10 and 50 mm.Longitudinal angulation  between the outlet of the inner branches and the ostia of the target vessels can be up to 45° for all visceral vessels.Lateral variability  between the outlet of each inner branch and the ostium of its target visceral vessel can be up to 50° for CT and SMA and up to 70° for the renal arteries.All target vessels must be suitable for antegrade cannulation.The current IFU requirements are subject to change as clinical experience with this novel stent graft increases.A total of 4 different paper sheets are available for planning and measuring . The firAfter all measurements have been taken, they can be entered on the second sheet (branch position sheet) . For tarThe third sheet  can be used to determine the proximal and distal landing zones . Here, iOn the fourth sheet (comments + product selection sheet), comments on anatomic characteristics can be noted, and the corresponding product can be selected based on the calculated dimensions .If thoracic aortic stent graft implantation is required, it should be performed first to ensure sufficient proximal stability. In principle, the implantation procedure should be from proximal to distal. Due to the length of the covered aorta, a spinal catheter should be considered in a 1-stage procedure to reduce the risk of spinal ischemia. For elective cases, so-called “Temporary Aneurysm Sac Perfusion” may be considered to achieve spinal vessel conditioning through residual perfusion of the aneurysm. Retrograde cannulation and completion of the inner branches via femoral access is performed according to the technique published by Makaloski et al. This technique offers a lower risk of access site-related complications than the brachial approach and is the preferred technique at our institution.Systemic full heparinization is performed according to body weight, with an activated clotting time of 250 to 300 seconds. The common femoral arteries are selected as access vessels via a surgical cut-down or percutaneous approach. A brachial or femoral approach can be selected for cannulating the inner branches. For the brachial approach, as reported in other studies, there are no differences in complication rates concerning side selection and should be adapted to the individual patient aortic arch configuration and conditions of the operating room/hybrid room.In the case of a previously implanted thoracic aortic stent graft, it is important to verify that its distal end is compatible in diameter (for at least 30 mm in length for overlap) with the proximal diameter of the selected E-nside stent graft. It must be positioned 20 mm  proximal to the ostium of the CT. The E-nside delivery system is advanced in A/P projection via an extra stiff 0.035 in guidewire through the access vessel into the abdominal aorta. Tactile elevation of the gray handpiece of the delivery system must always point toward 12:00 and ensure correct alignment of the stent graft when it is advanced into the thoracoabdominal aorta. The 2 proximal “E” markers are used for fluoroscopic verification of the correct rotation of the stent graft. These markers have to face 12:00 in AP projection. Angiography is used to visualize the renovisceral segment. Still in A/P projection, alignment of the prosthesis in relation to the renal arteries is now performed under fluoroscopy control. For this purpose, the centers of the outlets of the renal inner branches are aligned according to the calculations on the “branch position sheet”. If necessary, a rotation adjustment must also be made according to the planning. After verifying the correct orientation and longitudinal position of the “E” markers and renal inner branches, the orange controller has to be brought on position “D”, and the deployment of the stent graft can be started by the “squeeze to release” mechanism. After deployment of the first few centimeters of the proximal stent graft portion, gentle adjustment of the stent graft position by slowly rotating the outer sheath and the black knurled cap with both hands can be performed if necessary until the intended orientation is confirmed once again. Proceed with the “squeeze to release” mechanism until the whole stent graft is deployed. To fully deploy and detach the stent graft from the delivery system, proximal tip capture must be released by rotating and pushing the knot at the distal end of the delivery system.After full deployment, the cannulation process of the inner branches can be started. The sequence of the pre-cannulated PI tubes is shown on a sketch on the handlebar . The CT After all intended renovisceral vessels have been supplied with bridging stent grafts and connected to the inner branches, the orange lever at the handlebar of the delivery system must be brought into the “N” position. Now, the white “squeeze to release” handle must be held, and at the same time, the gray part of the delivery system must be slowly retracted under fluoroscopic control until the nose cone sits back onto the outer sheath. The entire delivery system can then be removed.If necessary, the aorto bi-iliac E-tegra stent graft (JOTEC GmbH) with active fixation can be used for distal extension.Final angiography is performed to assess the correct position of the stent prostheses and bridging stent grafts. If desired and technically possible, an on-table rotational or postoperative computed tomography scan can also be performed for better assessment .The E-nside multi-inner branch stent prosthesis represents a new off-the-shelf device that attempts to combine the advantages of branched and fenestrated prostheses in the field of complex endovascular aortic repair. This is ensured by the inner branch design and the pre-cannulation technique. These results are comparable to the anatomical feasibility study by Bisdas et al for the Zenith t-Branch endograft . It should be noted that these problems could be overcome by additional procedures, such as creation of an iliac conduit or additional proximal implantation of a tapered stent prosthesis; therefore, the actual clinical suitability should be higher.The feasibility of this new device was tested in a retrospective analysis of Crawford I–IV TAAA and reported suitability of 43%. However, causes for exclusion mainly involved the iliac access vessels and proximal aortic diameters, while suitability regarding the renovisceral segment was given in 79% of patients. In a recently published meta-analysis on the Zenith t-Branch prosthesis, good technical success rates and satisfactory mid-term results with a 30 day mortality of 5.8% were achieved in elective and urgent scenarios. Two studies demonstrated a 30 day mortality of 9%–14% for urgent and emergent treatment of TAAAs.12 This shows that these stent grafts are a safe alternative due to the off-the-shelf availability, thereby enable a rapid and precise therapy.The fact that good clinical results can be achieved with off-the-shelf multibranched endografts has already been demonstrated for the Zenith t-Branch. Early results showed both technical success and reintervention-free survival of 100% at a follow-up time of 5 months. In addition, in very narrow anatomies, bridging stent grafts should be lined with a bare metal stent to avoid compression.Due to the design of the E-nside inner branched stent graft and the minimum diameter of 24 mm in the renovisceral segment, it could also play a role in the treatment of urgent juxtarenal and pararenal aortic pathologies. Although an increased risk of paraplegia would have to be accepted due to the increased aortic coverage, the advantage lies in the less invasive nature of the treatment of these aneurysms, which can often only be treated with complex open surgery as an alternative in emergency situations.Although the inner branch design would offer the possibility of reducing the supraceliac coverage of the aorta and thus the risk of spinal ischemia, a comparison with the Zenith t-branch shows a similarly long distance from the beginning of the covering to the outlet of the CT branch (E-nside: 93 mm vs Zenith t-branch: 99 mm) because the E-nside is principally designed as a thoracoabdominal stent graft. However, the different proximal diameters of the stent graft, 33 and 38 mm, allow a wider range of thoracic aortas to serve as native landing zones, thus avoiding the need for an additional thoracic stent graft in some cases.The restriction of the angle in the proximal  and distal  portion of the stent graft in the IFU is worth mentioning as it can have a limiting effect. This is mainly to avoid endoleaks (type I and III) in the landing or overlap zone, if proximal thoracic endovascular aortic repair (TEVAR) or distal extension is necessary. In addition, compression of the inner branch of the CT by excessive kinking in the proximal portion should be prevented. This is not the case with the E-nside stent graft, where all inner branches are pre-cannulated with a PI tube off-the-shelf. Due to pre-cannulation, the delivery system of the E-nside stent graft, similar to fenestrated stent grafts, must remain in the vessel until all branches and target arteries are completed. This may increase the risk of peripheral ischemia or even spinal cord ischemia. However, due to the high variability, pre-cannulation and low susceptibility to kinking of the inner branches, the time to cannulation of the target vessel can usually be kept very short.In a first study, promising results with a thoracoabdominal stent prosthesis with an inner branch design could already be demonstrated. Advantages of the inner branch design were particularly noted for patients with complex/narrow aortas, postdissection TAAA, failed previous FEVAR cases, and for all those cases with an “awkward take-off of the viscerals” unsuitable for fenestrations or outer branches. It was found that inner branches that were not pre-cannulated were often difficult to cannulate.One of the shortcomings of the current study is that at present, the first clinical results with the E-nside stent graft are still pending, and data on the durability and resistance of the inner branches, especially in anatomies with long bridging stent grafts, are lacking. Based on the first positive experiences in a premarket release and the already implanted numbers, these results should be available soon.The E-nside multi-inner branch endograft represents a new off-the-shelf device that attempts to combine the advantages of branched and fenestrated prostheses in the field of complex endovascular aortic repair. Although pre-cannulation of the inner branches simplifies implantation, this new aortic device involves a complex procedure, and advanced endovascular expertise is required to flatten the inevitable learning curve.