Karyomegalic interstitial nephritis (KIN) is a rare cause of chronic interstitial nephritis characterized by enlarged renal tubular epithelial nuclei. The first case of KIN reported in a kidney graft was in 2019. Here, we report the first case of KIN in 2 brothers receiving kidneys from 2 different unrelated living donors. A male kidney transplant recipient with focal segmental glomerulosclerosis as the original kidney disease presented with graft impairment and proteinuria, and graft biopsy revealed KIN. This patient had a brother who was also a kidney transplant recipient and had one episode of graft impairment and was diagnosed with KIN as well. Karyomegalic interstitial nephritis (KIN) is a rare cause of chronic interstitial nephritis characterized by enlarged renal tubular epithelial nuclei, and its incidence is less than 1% of renal biopsies . The karIn December 2021, A 37-year-old male Egyptian kidney transplant recipient was referred to our kidney transplant follow-up outpatient clinic. During his follow-up, he was presented with graft impairment (an increase in serum creatinine from 0.6 to 1.2 mg/dl). He was diagnosed with focal segmental glomerulosclerosis (FSGS) Fig.  showed kKaryomegalic interstitial nephritis, a type of chronic interstitial nephritis, is described when there are enlarged renal tubular epithelial nuclei, interstitial fibrosis, and tubular atrophy. The exact cause of KIN is unknown; however, viral infections, toxic agents, and genetic causes are proposed. Toxic causes include exposure to alkylating agents, heavy metals, and mycotoxins, especially ochratoxin A –10. KIN Karyomegalic interstitial nephritis is a slowly progressive chronic interstitial nephritis. Clinically, patients present with mild to moderate renal impairment, reaching ESKD in the fourth or fifth decade of life; however, children may be diagnosed with KIN , 18. PatRegarding management, there is no specific treatment for slowing the progression of kidney disease in patients with KIN. Corticosteroids were used in trials but without improving renal outcomes . HoweverThe original kidney disease in our reported cases was nephrotic syndrome. According to the available slides and data, the eldest brother had FSGS as an original kidney disease, but the details of tubulointerstitium were not available. His graft biopsy showed podocytopathy, which may delineate the early recurrence of FSGS. There are reported cases of coexistence of FSGS and KIN in the literature , 22. TheProteinuria in KIN, as mentioned earlier, is usually less than 1 g/day, but here in the eldest brother, the proteinuria was in the nephrotic range. This can be explained by the presence of glomerular basement membrane (GBM) abnormalities detected by an electron microscope with a diagnosis of collagen IV abnormality. Collagen IV abnormality was linked to Alport syndrome, thin basement membrane disease, or genetically mediated FSGS . The preThe presence of karyomegalic cells in the renal graft has many differential diagnoses. They present as KIN, BK nephropathy, or T-cell-mediated rejection. In KIN, the nuclei are large, pleomorphic, hyperchromatic, and diffuse in both proximal and distal tubules across the biopsy without intranuclear inclusion and tubulitis. In BK nephropathy, the nuclei are not pleomorphic and contain intranuclear inclusion bodies with focal distribution across proximal and distal tubules and tubulitis. In addition, viral stains, like SV40, are positive. In cell-mediated rejection, the nuclei are not pleomorphic or hyperchromatic and are present in areas of tubulitis without intranuclear inclusion bodies.The occurrence of KIN in the renal allograft may be due to recurrence, donor-associated disease, or de novo. One of the limitations of this case report is the absence of genetic studies on two brothers, the unavailability of their native biopsies, and the non-performance of SV40 to exclude the diagnosis of BK nephropathy.


International Journal of Impotence Research 10.1038/s41443-021-00503-x, published online 2 December 2021Correction to: For this article parts of the text have been converted to footnotes. They read as follows:The Brussels Collaboration on Bodily Integrity [10] defines ‘medically necessary’ in the following way: ‘(1) the bodily state poses a serious, time-sensitive threat to the person’s well-being, typically due to a functional impairment in an associated somatic process, and (2) the intervention, as performed without delay, is the least harmful feasible means of changing the bodily state to one that alleviates the threat’.By ‘our’, ‘we’, and ‘us’, I mean all embodied people.


The mechanical response of a contractile cell anchored to the substrate through focal adhesions is studied by means of an asymmetric pre-strained tensegrity structure obeying a neo-Hookean stress–strain law. The aim is to assess the influence of overall asymmetric contraction on the cell durotaxis and on the growth of the focal adhesion plaque. The asymmetric kinematics of the system is obtained in two ways, that is by assuming a gradient of the substrate stiffness and through asymmetric buckling. Equivalent springs are purposely considered to represent the stiffness of the ensemble formed by the substrate, the focal adhesion plaque and the integrin ligands. Then, contraction results from elastic strains induced by competing polymerization and actomyosin contraction. The cell mechanical response in terms of durotaxis and its coupling with focal adhesion plaque growth is finally analysed with respect to the effects of asymmetry, gaining some insights into how this asymmetry could participate to redirect cell migration, both in terms of durotaxis and mollitaxis. Particularly, the interplay among cell contractility, asymmetric buckling and substrate stiffness gradients and their influence on cell mobility is investigated. The forces exerted by the cell on the substrate are shown to induce durotaxis and influence the growth of the focal adhesion plaque-like structure linking the cell to the extracellular matrix.The internal organization and the overall shape of the cell are controlled by both the cytoskeleton system and the extracellular matrix. Their interaction stimulates fibronectin fibril assembly and fibres orientation ,2, whichThe growth and development of the focal adhesions is affected by the substrate stiffness. Focal adhesion complexes that grow on a stiffer substrate are more stable and elongated than those grown on soft substrates ,7. Cell Both cytoskeleton and substrate stiffness play a relevant role in cell locomotion ,14. Whenin vitro by Lo et al. 0, +∞ and different stiffness of the substrate, while setting the average stiffness of the substrate to the value kav = 100 pN nm−1. Furthermore, pre-contraction of the fibres On the other hand, both the displacements a shows the profiles of the displacements of the system at the soft substrate side, η. Three different values of the ratio χ = ks/kh have been used, so that the smaller χ the higher the stiffness gradient. Remarkably, for a fixed value of the average substrate stiffness, the maximum displacements of the system occur at η = 1.In this regard, figure 4b. It can be observed that η, i.e. ηcr,1 and ηcr,2, which also make vanishing both the values of a. These critical values of η are referred to as critical asymmetry ratios, because they imply a transition from positive to negative of the sign of the net displacement. Particularly, positive values of η belonging to the interval  in figure 4b, elicit positive durotaxis, that is, displacements towards the stiff side, while negative values of η outside the aforementioned interval of asymmetry ratios. For instance, for η and the substrate stiffness ratio χ can be appreciated in the inset figure of figure 4b. Particularly, the surface of χ = 0.01 for a large stiffness gradient, the red plane, for χ = 0.1 when the maximum displacement for positive durotaxis decreases, and the violet plane, for χ = 1, when the profile of χ coincides in fact with that previously shown in figure 4b. For fixed pre-contraction and pre-polymerization levels, however, the critical η values remain constant, hence, the durotaxis domain in terms of η remains unaltered. The ηcr,1 and ηcr,2 for variable χ. Thus, the question arises of what is the main factor governing these critical values. Preliminary tests indeed suggest investigating how the values of the critical values ηcr,1 and ηcr,2 change for variable pre-strains. This issue is commented on in §5.The system durotactic attitude can be drawn from the profiles of the net displacement . 5ηcr,1 and ηcr,2 on the pre-strains, the equilibrium problem has been reformulated to take into account that the critical condition at which the system switches from positive to negative durotaxis also corresponds to η turns out being unknown.To systematically study the dependence of the critical asymmetry ratios equation . Hence, equation  and the a in terms of η, i.e. ηcr , versus the pre-strain values. The solution obtained at λf*, belonging to the plane in red in figure 5a, is then plotted in figure 5b. This latter figure highlights the two critical values that define the relevant interval of positive durotaxis. The profile is not significantly affected by the value of The numerical solution of the system of the aforementioned nonlinear equations in the four unknowns ηcr,1 = 0.775 and ηcr,2 = 1.289 are expectedly found at λf* = 0.882. However, it can be further observed that while ηcr,2 increases for higher pre-contraction levels, ηcr,1 reduces. Hence, the interval of positive durotaxis enlarges for increasing pre-contraction levels, while negative durotaxis requires higher or lower values of η to manifest. On the other hand, for values larger than 0.89, buckling does not occur and durotaxis is not triggered. However, when a small pre-contraction level is assumed, the system is on the verge of negative durotaxis for small perturbations of the system geometrical symmetry. Pre-contraction and pre-polymerization, indeed, produce cross-current tendencies as the system will exert pushing or pulling forces on the plaque depending also on the balance between these competing pre-strains. Hence, in case of combinations of the pre-strains with prevailing pre-polymerization, negative durotaxis may emerge, otherwise, positive durotaxis will manifest.The aforementioned values . 6We investigate the response of the plaque to a simultaneous lack of symmetry and stiffness gradient presence in terms of the forces exerted on the focal adhesion complexes and the related plaque growth flux.a,b displays the force on the plaque, Fp, and the net plaque growth flux, J defined by equation , positi. 6.1b and 6b highlight three main stages of the durotaxis and the net growth flux for variable asymmetry ratio. In particular, the curves of Fp intercept the abscissa at two critical η, ηcr,1 and ηcr,2, whose values turn out being independent of the stiffness gradient χ.Figures 4J, since it vanishes at values of η which are different from the aforementioned ηcr,1 and ηcr,2 discriminating positive from negative durotaxis. Nevertheless, the critical η values of J are key to the plaque stability, as they correspond to equilibrium states of the plaque where neither assembly nor disassembly takes place, any other η corresponding instead to either positive or negative fluxes. On assuming that the system can leverage both Lp and η to reach equilibrium states of the plaques, the observed findings can be clarified as follows.η < ηcr,1. The flux J is negative. The plaque is not in equilibrium, and will expectedly disassemble up to disappear or reach a new stable length, at which neither assembly nor disassembly takes place. Alternatively, the system can leverage asymmetric buckling and increase the asymmetry ratio η to approach the closest equilibrium position.ηcr,1 ≤ η ≤ ηcr,2. The flux J is part positive and part negative. The plaque will accordingly tend to a new length at which neither assembly nor disassembly is allowed. Alternatively, the plaque may reach an equilibrium state by moving the position of the hinge.η > ηcr,2. The flux J is negative, thus the plaque is not in equilibrium. The plaque will either depolymerize up to a stable length or disappear; alternatively, the system can decrease η to tend to the closest equilibrium state.As previously mentioned, the same remarks do not strictly apply to . 7Lp and the parameter kav is investigated in §§7.1 and 7.2, respectively. In the case of symmetric buckling and stiff homogeneous substrates, the system reportedly requires larger stable plaque lengths with respect to the case of soft substrates [kav and variable Lp. In §7.2, besides the influence of kav for variable χ and η is studied, because the average substrate information is not per se sufficient and must be complemented by the knowledge of the stiffness ratio to fully characterize the substrate stiffness.When addressing durotaxis, state-of-the-art experimental studies often report parameters related to the mechanical behaviour of adherent cells detected at variable average substrate stiffness . Therefobstrates . In §7.1. 7.1η = 1, two other values of η were taken, η = 1.2, 1.4, which are pre-critical and post-critical values according to the evaluations performed. The results show that both asymmetry and stiffness gradient strongly affect the values taken by both Fp, shown in figure 7a, and the flux J, plotted in figure 7b.Besides the symmetric reference solution for the symmetric case at kav = 100 pN nm−1 and assuming the stiffness ratio χ = 1, 0.1, 0.01, where χ = 1 corresponds to the uniform substrate, and smaller values of χ refer to higher substrate stiffness gradients. On an observational basis, from figure 7b, it can be inferred that the stable plaque length decreases for increasing asymmetry on a uniform substrate, χ = 1. Analogously, in symmetric or slightly asymmetrical systems, with η = 1 and η = 1.2, respectively, the stable lengths decrease for enhancing stiffness gradients, namely, decreasing χ. In the case of markedly asymmetrical systems, corresponding to the profile with η = 1.4 in the figure, the growth flux is always negative and the plaque can never reach equilibrium, and this independently of the stiffness ratio χ characterizing the substrate.Both the figures have been plotted at fixed . 7.2a,b, respectively, for the same χ and η values adopted in the previous section and increasing substrate average stiffness kav. The magnitude of the force exerted by the system on the adhesion plaque is an increasing function of the substrate average stiffness kav and plateaus beyond a critical average stiffness. An analogous trend is exhibited by J, the only difference being dictated by the shift of the zero-crossing points due to the threshold For the purpose of assessing the role played by both the average substrate stiffness and stiffness gradient on the plaque force and growth law, these have been plotted in a semi-logarithmic scale in figure 8Fp versus kav is in line with literature experimental and analytical results [The obtained trend of  results ; however results .Fp on the average stiffness must also be discussed in light of the values taken by η and χ. Based on the obtained profiles, it can be inferred that Fp cannot overcome its asymptotic value. Moreover, symmetric systems on homogeneous gradients exert the maximum absolute value of the force Fp on the plaque.The dependency of the force on the plaque a confirm what is already highlighted in figure 7a.Moreover, the trends shown by figure 8η larger than 1.It also shows that pushing forces decrease their value for increasing stiffness ratio so that plaques placed on high soft-to-hard substrate gradients are pushed to a lesser extent than their homologous located on homogeneous environments. On the other hand, pulling forces exerted on the plaque during positive durotaxis are smaller in asymmetric systems, that is, for J, there is no direct correspondence between pulling and pushing forces and the plaque assembly and disassembly. Nevertheless, pushing forces always come along with plaque disassembly, and, analogously to what is detected for the force Fp, the disassembly flux is weaker on high stiffness gradients than on homogeneous substrates. An opposite tendency emerges in the case of positive flux, where the assembly process intensifies on homogeneous substrates up to a certain value of kav, beyond which J asymptotically tends to a constant value independently from χ, and attenuates in asymmetric systems.With regard to the plaque flux, due to the constant term The present comparative analysis suggests that asymmetric buckling might be regarded as a weakening of the plaque assembly, while the same remark does not strictly apply to the disassembly process. Furthermore, the plaque disassembly tendency is lower in the presence of strong stiffness gradients as well as of soft average substrate stiffness. With respect to asymmetric buckling, symmetric buckled configurations on homogeneous substrates display the highest assembly and disassembly forces for increasing average stiffness.. 8In a previous contribution , it has The present study further suggests that, in the positive durotaxis regime, asymmetric systems solicit smaller stable lengths of the adhesion plaque than symmetric ones.Though a non-monotonic relationship between focal adhesion size and cell speed was experimentally detected, suggesting that cell motility might both increase or decrease with the plaque length based on the considered cell length range , the argMoreover, the higher the stiffness gradient of the substrate, the shorter the focal adhesion complex. Thus, higher substrate gradients also affect cell mobility.In the present model, the lack of symmetry of the mechanical system generally delays buckling, which, in turn, requires higher pre-contraction or pre-polymerization than those necessary to activate buckling in symmetric systems.J net of the constant The force acting on the focal adhesion plaque is tensile or compressive for positive and negative displacements, respectively. This aspect holds only for the flux The model suggests that, in general, both the substrate stiffness and its gradient are key to focal adhesion plaques’ stability. In particular, focal adhesion plaques developing on soft substrates possibly undergo smaller growth fluxes than those located on both hard and uniform substrates. This remark adds further insight into the durotaxis mechanism as well as offers inspiration for experimental validation.. 9Cell locomotion is an inherently asymmetric, polarized, process. It is also influenced by substrate stiffness gradients.In the present contribution, two sources of asymmetry were introduced, that is an internal geometrical source through asymmetric buckling of the cell microtubule-like component and an external source accounting for gradients of the substrate stiffness at the cell ends. Particularly, the cell–focal adhesion–substrate complex has been simulated using a nonlinear tensegrity model capable of asymmetric buckling and connected to springs with graded stiffness.In a nutshell, the system durotaxis, whether positive or negative, turns out to be influenced by the plaque geometry, the substrate stiffness ratio and average stiffness, and, finally, by the asymmetric contractility latent in the tensegrity geometry.η to stabilize focal adhesion plaques, while exhibiting both durotaxis and mollitaxis. Particularly, when negative durotaxis takes place, the magnitude of the pushing force on the plaque decreases with decreasing average stiffness. Associating compressive forces with depolymerization [Remarkably, for certain stiffness gradients and suitable combinations of the contraction and polymerization pre-strains, the system can leverage the change of configuration associated with a change of rization , comparaAn ongoing extension of the present model accounts for the elastic-brittle behaviour of the springs representing the focal adhesion–ECM complexes so that, once a critical displacement is reached, the substrate anchorage is released.


Newcastle disease (ND) is a major threat to the poultry industry, leading to significant economic losses. The current ND vaccines, usually based on active or attenuated strains, are only partially effective and can cause adverse effects post-vaccination. Therefore, the development of safer and more efficient vaccines is necessary. Epitopes represent the antigenic portion of the pathogen and their identification and use for immunization could lead to safer and more effective vaccines. However, the prediction of protective epitopes for a pathogen is a major challenge, especially taking into account the immune system of the target species.In this study, we utilized an artificial intelligence algorithm to predict ND virus (NDV) peptides that exhibit high affinity to the chicken MHC-I complex. We selected the peptides that are conserved across different NDV genotypes and absent in the chicken proteome. From the filtered peptides, we synthesized the five peptides with the highest affinities for the L, HN, and F proteins of NDV. We evaluated these peptides in-vitro for their ability to elicit cell-mediated immunity, which was measured by the lymphocyte proliferation in spleen cells of chickens previously immunized with NDV.Our study identified five peptides with high affinity to MHC-I that have the potential to serve as protective epitopes and could be utilized for the development of multi-epitope NDV vaccines. This approach can provide a safer and more efficient method for NDV immunization. Newcastle disease (ND) is a viral infection that affects various systems of birds, including the neurological, gastrointestinal, reproductive, and respiratory systems. Clinical signs of ND include snoring, difficulty breathing, watery discharge from the nostrils, facial swelling, paralysis, tremors, and neck twisting, which is a sign of central nervous system involvement. ND is caused by paramyxovirus serotype 1 (APMV-1), also known as Newcastle disease virus (NDV), which has a genome comprising 15Kb of negative-sense single-stranded RNA (-ssRNA). NDV is highly contagious and can infect more than 200 bird species, significantly affecting poultry production worldwide .ND is typically controlled using inactivated, live, or attenuated vaccines of genotypes I and II, including lentogenic, Hitchenner-B1-LaSota, V4, NDW, I2, and F strains. Inactivated vaccines are inoculated subcutaneously or intramuscularly, inducing a high level of humoral response with no side effects, although they induce a weak cellular response. In contrast, live and attenuated vaccines are typically inoculated via the ocular-nasal route, stimulating a mucosal immune response. Some genotypes, such as genotype II-LaSota, can induce a strong immune response without reverting to virulence or inducing recombination, although in some cases, it can be reactive in young birds, causing post-vaccination respiratory reactions , 5, 6.ex-vivo assays where the ability of a peptide to evoke lymphocyte stimulation is measured [Currently, there is growing interest in developing vaccines based on immunogenic subunits of the pathogen (epitopes), which are theoretically safer and potentially more effective. Epitopes are peptides that can bind to major histocompatibility complex type I (MHC-I) or type II (MHC-II) and be recognized by CD8/CD4 cells via the T-cell receptor (TCR), inducing a cellular (MHC-I) or humoral (MHC-II) response –9. The pmeasured , 13.The development and use of multi-epitope vaccines against diseases in the poultry industry are currently limited –22, and Out of the candidate epitopes that passed the filters, we selected five epitopes with the highest affinity score for MHC-I. Two of these epitopes are part of the large polymerase protein (L), two are from the haemagglutinin-neuraminidase (HN), and one is from the fusion protein (F) Table .Table 1In Table ex-vivo assay demonstrated significant differences in IFN-γ production between the groups tested with different peptides. The vaccinated group showed a higher number of spots than the control group. Additionally, in the vaccinated group, the samples stimulated with the epitopes exhibited a smaller spread in the number of spots than the control group. The number of INF-γ spots of epitopes 02 and 05 and the pool showed a negative asymmetric distribution, mostly clustering in the second quartile. The number of spots of epitopes 01, 03, and 04 showed a positive asymmetric distribution in the third quartile. Kruskal–Wallis, Poisson, and negative binomial tests confirmed significant differences in the number of spots between the vaccinated and control groups for epitopes 01, 02, 03, and 05. Epitope 04 was only significant with the Poisson test. The pool of the five peptides showed significant differences in all three tests  , InfectiIn the present study, the selection of 8- and 9-mer epitopes was aimed at evoking a cell-mediated immune response by T lymphocytes. Therefore, the selected epitopes were not only limited to NDV surface proteins (F and HN) but also included non-exposed proteins –22, 26. Gallus gallus alleles. In our study, we developed an ad-hoc tool based on artificial intelligence (AI), specifically an artificial neural network. We trained the AI with experimental data on the affinity of the majority MHC-I allele of the Cobb-500 chicken and a diverse set of peptides (data not shown). We refined the peptide selection by conservation and tolerance filtering of the epitopes, where conservation filtering aimed to find peptides present in as many NDV sequences as possible and tolerance filtering aimed to exclude peptides with potential autoimmune responses.Generally, freely available tools are used to predict avian T-type viral epitopes, including NDV –19 viralThe importance of peptide affinity for the MHC complex as an epitope should not be overestimated, as it may not necessarily be recognized by the TCR receptor of CD8 T cells , 28, 29.In-silico peptides have been designed and evaluated in Sudan, Iran, and Uganda [wet lab evaluations. While in-silico predictions are improving over time, they cannot replace ex-vivo or in-vitro experimentation. Wet lab experiments are necessary to have a closer reference to reality. In our study, we limited ourselves to evaluating only the top 5 epitopes in a small number of SPF chicken spleens. Further in-vitro and in-vivo evaluations are still required before applying the peptides in farms. However, this is a first step towards turning our epitopes into NDV vaccines.d Uganda –22, 26 uOur results suggest that the tested epitopes could serve as a booster vaccine against virulent variants, such as genotype XII Table , as theyex-vivo evidence that MHC-I-like T-type epitopes against NDV can evoke cellular responses. The immunoinformatics tools employed are suitable for the design of immunogenic T-type epitopes. Before this work, our team developed an artificial intelligence algorithm based on artificial neural networks to predict the affinity of an arbitrary peptide for the most frequent MHC-I allele of Cobb-500 chicken farms in Peru. The convolutional neural network was trained with experimental MHC-I/peptide affinity data (to be published).These findings motivate us to design new immunogenic epitopes against other pathogenic viruses of poultry interest. In the future, we will evaluate their use in synthetic multi-epitope proteins, which would protect against a variety of viral pathogens with the same vaccine. This study provides the first In conclusion, our study demonstrates the feasibility of using a combination of bioinformatics and experimental approaches to identify and evaluate potential vaccine candidates against NDV. The selected epitopes showed the ability to evoke a cell-mediated immune response in vaccinated animals, which is essential for protecting against NDV infection. Further studies are needed to evaluate the efficacy of these epitopes as vaccine candidates in field trials.The design and evaluation of MHC-I cognate NDV epitopes involved the use of bioinformatics tools, animal handling, and ex-vivo evaluation Fig. .Fig. 2Flad-hoc artificial neural network developed by our group (to be published) was utilized to predict MHC-I cognate epitopes. The epitopes were selected based on their highest affinity score and then filtered for conservation using tblastx  and tolerance against the Gallus gallus proteome (UniProt: UP000000539). To evaluate the conservation of the top five peptides, we performed a BLAST search  against complete genomes of genotypes II (live vaccines) and XII (highly virulent). Finally, the selected epitopes were synthesized at a concentration of 10–14 mg and with a purity of greater than 75% by GenScript (USA).We downloaded 21,374 genomic  and protein sequences of NDV from various geographic regions, strains, and genotypes from GenBank as of the 2017 publication. The multifastsat.py v.1.4.9 script  was used8.0 TCID 50%) on days 1 and 10. The vaccine was diluted in 30 ml of distilled water and administered by eye drop in doses of 0.05 ml per bird. At week 8 (with an average weight of 595g), birds in both groups were sacrificed by cervical dislocation, and spleens were collected in DMEM medium  with antibiotics and transported cold.The study involved two groups of hens (2 vaccinated and 2 control) obtained from the hatchery of the FARVET SAC company in . The birds were specific pathogen-free (SPF) Hyline female chicks and were reared in the Poultry Pathology Laboratory of the Universidad Nacional Mayor de San Marcos (UNMSM). The handling of the birds followed protocols approved by the Animal Welfare Ethics Committee (CEBA) of the UNMSM. The birds were housed in cages at the experimental facility, where conditions were carefully controlled, maintaining temperatures between 20°C to 22°C and a 12-h light–dark cycle. They had ad libitum access to food (crushed corn) and water throughout the study period. The vaccinated group received NDV LaSota vaccine  supplemented with 10% fetal bovine serum , filtered at 70 µm and washed at 300 × g for 10 min. Lymphocytes were obtained by gradient centrifugation with Histopaque 1077 . The cells were then assessed for viability and concentrated to 5 × 102 for 48 h. INF-γ secretion was detected with 0.5 µg/ml of biotin-conjugated anti-chIFN-γ and 100 µl of Streptavidin-HRP. The plate was read with the AID EliSpot Reader System ELR04 , and spots were counted manually using ImageJ v.1.53k software [The cellular response was measured by ELISpot assay , 35 in tsoftware . The dif


Stepping down after a decade of service as editor of this journal, this brief testimonial recognises the pivotal contributions made by Professor David Skuse and highlights his stellar career achievements as an academic. BJPsych International. I cannot claim that I know David well, but perhaps I can say I know him well enough, as I have certainly known of him for a while through my dealings with the Royal College of Psychiatrists and its journals, and everyone who does know David well speaks of him highly and wishes him well, as do I.After a decade of success, David Skuse is stepping down as Editor-in-Chief of the BJPsych International, he is described as conscientious, considerate and caring, and if you add to this (and I would certainly elect to do so) courteous and competent, then you have a ‘full house’ – a hand that few editors genuinely hold – although many would bluff otherwise. Perhaps the best way of summing up these attributes and the many others that he is in possession of is that David is quintessentially a gentleman. Fortunately for the journal and indeed the College, over the years, he has imbued his role as editor with many of these qualities.Achieving such favour in any walk of life is no mean feat, even at the best of times, but this is especially the case in relation to the role of Editor-in-Chief of a psychiatric journal, a position that seldom increases one's circle of friends. Speaking to those who have had much more to do with David in his role as Editor-in-Chief of the Science and Nature, not to mention The Lancet and Nature Genetics. It is therefore no surprise that his work has accrued nearly 30 000 citations and an H-index that would be the envy of most academics. And yet, a significant part of his mission in the journal has been to encourage research by those new to the art of research and for the journal to capture mental science across cultures to ensure it has a truly international presence.Another reason why the journal has succeeded academically, as exemplified by its CiteScore, is by attracting contributions to its pages from the four corners of the world. However, possibly David's most winning qualities as an editor are that he is a quiet achiever and that he puts others before him, as evidenced by his self-effacing final editorial, in which he generously highlights the work of others and credits the journal's success to the collective efforts of those around him rather than claiming them as his own. It is this magnanimity that sets him apart, along with his scholarly excellence. I say this with some caution as it is important not to misconstrue the latter as magniloquent aggrandisement. For instance, I doubt many would be aware that David has published both in alma mater, and underwent psychiatric training in London, at the Maudsley Hospital and Institute of Psychiatry – bastions of excellence that I too had the good fortune of experiencing. Thus, at some subliminal level, I feel we have a connection that is further strengthened by being editors of College journals. It is therefore with great admiration that I thank David for his outstanding service to the global community of the BJPsych International.Although I began this testimonial and expression of gratitude by stating that I do not know David personally, and this is still true, I feel I do know him somewhat by virtue of a shared medical provenance and commonality of training. David graduated in medicine from Manchester University, my


All of them underwent an uneventful DMEK as a single procedure performed by one experienced surgeon (MAG) with a mean unfolding time of 7.2 ± 4.9 min (range 4 to 15). The essential steps, including patient preparation as well as DMEK graft implantation, orientation, unrolling and centering are detailed. At 6 months, BCVA was 0.6 ± 0.2, ECD was 1945.0 ± 455.5 cells/mm2 and CCT was 497.0 ± 19.7 microns. Conclusions: We hereby present the keys to overcome tightly scrolled grafts of very young donors, which prove perfectly suitable for DMEK surgery. The graft shape tends towards a double-roll and specific maneuvers are strongly recommended.Purpose: To report on the surgical maneuvers recommended for a successful unfolding of very young donors in order to accomplish an uneventful Descemet Membrane Endothelial Keratoplasty (DMEK) surgery. Methods: Five patients  with Fuchs endothelial cell dystrophy who underwent DMEK with very young donors (between 20 and 30 years old) were included. The following demographic data were assessed: donor’s age, donor’s endothelial cell density (ECD), preservation time, recipient’s age and sex and unfolding surgical time. Best-corrected visual acuity , ECD and corneal central thickness (CCT) were assessed preoperatively and at 6-month follow-up. Results: Donors’ mean age was 23.6 ± 3.6 years (range 21 to 30) and the mean ECD was 2748.6 ± 162.6 cells/mm Descemet Membrane Endothelial Keratoplasty (DMEK) has become the most popular type of corneal transplant since Melles et al. described it in 2006. It allows for decreased rates of graft rejection, better visual acuity outcomes and faster recovery compared to Descemet stripping automated endothelial keratoplasty (DSAEK) . HoweverThe number of DMEK surgeries is growing worldwide as it has become the surgery of choice for corneal endothelial diseases, which has resulted in an increasing demand for tissue. In this regard, theoretically, accepting younger donors with better endothelial cell density (ECD) would be preferable for longer expected graft survivals and would increase the availability of tissue. Over the last two decades, corneal donor characteristics have been extensively studied. Postoperative results of young  and older  donors have been compared considering preoperative ECD, preservation time, death-to-preservation time and donor diabetes status, but no differences in 6-month ECD or rebubbling rates have been found . Schaub Although younger donors have been described as providing safe tissue for DMEK surgery, they represent a surgical challenge as their grafts tend to result in tighter rolls with a longer intraoperative unfolding time. There is still limited data available on the outcomes and complication rates following the use of younger donor grafts. Few techniques have specifically addressed tighter rolls in the literature, but none have described the particular behaviour of twenty-year-old donors’ DMEK grafts ,5.The purpose of this paper is to report on the surgical maneuvers recommended for a successful unfolding of very young donors in order to accomplish an uneventful DMEK surgery. To the best of our knowledge, this is the first paper describing a specific surgical technique with this kind of young donors’ corneal grafts.In this case series, we present the patients’ characteristics and the DMEK surgical techniques on five very young corneal donor endothelial grafts performed at Hospital Clinico San Carlos in Madrid, Spain. The paper was conducted in compliance with the tenets of the Declaration of Helsinki. No Ethics Committee approval was required.The five eligible donor corneas were from healthy donors between the ages of 20 and 30 years old and were preserved in Eusol-C preservation media  at 4 °C for 3 to 4 days after enucleation. Corneal donor characteristics were analysed with EKA-10 .The following demographic data were assessed: donor’s age, donor’s ECD, preservation time, recipient’s age and sex and unfolding surgical time (from the moment after injection until the gas bubble was inserted under the graft). BCVA , ECD and corneal central thickness (CCT) were also evaluated preoperatively with a 6-month follow-up in all cases. Cases where a 12-month follow-up was available were also registered.All patients underwent DMEK surgery under general anesthesia . Prior tDescemet membrane graft dissection of the young donor corneas was performed with a modified SCUBA technique using blunt dissection and starting at the scleral spur. No difficulties were found in any of the cases, such as tight adhesions or radial tears. Three donor corneas were split for Deep Anterior Lamellar Keratoplasty (DALK) and DMEK surgeries, so the endothelial graft was positioned on a bandage contact lens  and punched as previously described by Melles’ group .Once the graft was free in all 360 degrees, it was stained with trypan blue 0.06%  for 2 min. Given the availability of intraoperative optical coherence tomography (OCT), the graft was not marked. In all 5 cases, the tissue rolled into a perfect double-roll without the need for Balanced Salt Solution (BSS) bursts . The graPreparing the host cornea was similar to any other DMEK procedures. There was no need to remove the epithelium in any of the patients. Three incisions were made—the main wound with a 2.75 mm keratome at 12 o’clock and two side ports at 2 and 10 o’clock with a 23 G MVR blade. The descemetorhexis was performed with a reverse Sinskey hook under air. A 2 mm peripheral rim of host Descemet was left to avoid peripheral corneal oedema and bullae postoperatively.® 700, ZEISS, Oberkochen, Germany; ® glass injector , which was rotated in order to introduce the graft in the AC with the correct orientation. After this, the AC was shallowed immediately after graft insertion and prior to injector removal to maintain the orientation of the graft. Then, a 10/0 nylon single suture was placed in the main wound to seal the AC.In all cases, a double scroll orientation was confirmed by direct visualization of the graft inside the injector under the microscope before insertion . In 4 ofOnce the graft was inserted, the next step was performing centering maneuvers to ensure that the graft was properly positioned prior to unscrolling the tissue , which wFive patients  who underwent DMEK due to Fuchs Endothelial Cell Dystrophy (FECD) with no evidence of haze or posterior stromal fibrosis were included . One of 2 (Donors’ mean age was 23.6 ± 3.6 years (range 21 to 30), and the mean ECD was 2748.6 ± 162.6 cells/mm2 . All of 2 and 497.0 ± 19.7 microns of CCT. Cases 4 and 5 were performed 6 months ago, so data from the 12-month follow-up are not available (NA) yet. No cases of rebubbling or primary graft failure were observed. For up to 12 months, no cases of graft rejection or immune reaction have been noted. None of the patients had increased intraocular pressure, and macular OCT was normal in all follow-ups. No association between donor characteristics and the recipient outcome was noted.Postoperative outcomes are collected in DMEK has become the superior form of endothelial keratoplasty in routine cases of corneal endothelial diseases in many countries worldwide. Although DMEK presents multiple advantages over DSAEK, such as less graft rejection and faster visual recovery, graft preparation and the learning curve are important limitations of DMEK. Successful DMEK surgery depends on a range of factors, including surgical skill, tissue processing technique and donor selection criteria. While younger donor corneas may pose a challenge during DMEK surgery due to tighter rolls, younger donors also tend to have higher ECD counts, which could thus improve transplant outcomes. Hence, it is essential to describe useful maneuvers in this type of donor grafts, which we have proven to have a strong tendency towards forming double rolls.Several techniques for graft stripping have been described, including the “standard no-touch technique,” which was described in detail by Melles in 2008 and then further modified by hydrodissection or air dissection. It has previously been published that young donors have strong adherences, which may increase the risk of tears. However, there is no clear evidence in the literature that one of these techniques is more or less suitable for DMEK graft preparation in young donor corneas. In our case series, the graft preparation, starting at the scleral spur, was uneventful in all five cases with no tears or any other event ,8.Graft orientation during the surgery is the first critical step for a successful outcome, especially when using young donors and tight rolls. In all of our cases, the double scroll graft was visually inspected before insertion and set so that the scrolls were facing upwards. It is not that easy with older donors when a single roll is formed in the injector. For those cases, the Veldman Venn technique, which was first described by Peter Veldman, is very useful . It is aWhen it comes to dealing with extreme young donors , despite being a “supertight” roll, we have hereby demonstrated that they tend to fold in a “double-roll” configuration, which is a remarkable and new finding. The unfolding of double-roll grafts is known to be easier and faster. In fact, Odkell et al. recently described a technique for forming a double-scroll graft from a single-scroll graft without causing additional damage to the graft . Their tTo date, there are only a few papers published in the literature that have described DMEK with young donors . None ofp = 0.049) [The disparity of results regarding young donors has been described in the literature. Rodriguez-Calvo et al. reported more complications with young donor corneas (>38 years), including spontaneous free-floating DMEK grafts, graft detachments or rebubbling, which were statistically significant compared to older donors (= 0.049) . In thei= 0.049) . Accordi= 0.049) .There are other challenging patient’s and graft’s characteristics we can encounter when doing DMEK surgery, such as severe corneal edema, posterior stromal fibrosis, small eyes, big grafts, very deep or shallow AC, release of fibrin in AC if strong tapping, moderate manipulation or previous phacoemulsification surgery, dark irises, high vitreous pressure or no vitreous pressure in vitrectomized eyes. Many authors around the world have described several techniques or ways to overcome these limitations, including debriding epithelium to improve visibility and endo-illumination to see the edges of the graft if there is poor visualization . We do nOlder donors (over 55 years old) are normally preferred as they are looser and unfold easier in DMEK surgery. Young donors’ corneas are not commonly selected due to the tight-roll behaviour and the longer unfolding times . HoweverIn summary, we hereby present possible keys to overcome the tightly scrolled grafts of very young donors. These corneas are perfectly suitable for DMEK surgery without technical difficulties peeling off the graft. The graft shape tends towards a double-roll, and it is important to ensure the correct orientation when injecting into the AC. Shallowing the AC and placing a small bubble between the graft and the iris are strongly recommended maneuvers. Therefore, although most surgeons may prefer using corneas from older donors for DMEK because younger donors tend to have tighter rolls that may be more challenging to unfold, proper training, and technique refinement and instrumentation may help unfold tight young donor grafts and improve patients’ outcomes.


Cancer Med. 2019; 8: 783–794. doi:10.1002/cam4.1950Li, J, Xu, H, Wang, Q, Wang, S, Xiong, N. 14‐3‐3ζ promotes gliomas cells invasion by regulating Snail through the PI3K/AKT signaling. wileyonlinelibrary.com), has been retracted by agreement between the journal's editor in chief, Dr Stephen Tait, and John Wiley & Sons Ltd. The retraction has been agreed due to concerns regarding image duplication. A detailed investigation confirmed substantial inconsistencies and errors. As a result, the journal no longer has confidence in the results and conclusions reported.The above article, published online on 18 January 2019 in Wiley Online Library ( Cancer Med. 2019; 8: 783–794. doi:10.1002/cam4.1950Li, J, Xu, H, Wang, Q, Wang, S, Xiong, N. 14‐3‐3ζ promotes gliomas cells invasion by regulating Snail through the PI3K/AKT signaling. wileyonlinelibrary.com), has been retracted by agreement between the journal's editor in chief, Dr Stephen Tait, and John Wiley & Sons Ltd. The retraction has been agreed due to concerns regarding image duplication. A detailed investigation confirmed substantial inconsistencies and errors. As a result, the journal no longer has confidence in the results and conclusions reported.The above article, published online on 18 January 2019 in Wiley Online Library (


Various modification treatments have been carried out to improve the physicochemical and functional properties of various types of starch and flour. Modification by acetylation has been widely used to improve the quality and stability of starch. This review describes the effects of acetylation modification and its dual modifications on the physicochemical properties of starch/flour and their applications. Acetylation can increase swelling power, swelling volume, water/oil absorption capacity, and retrogradation stability. The dual modification of acetylation with cross-linking or hydrothermal treatment can improve the thermal stability of starch/flour. However, the results of the modifications may vary depending on the type of starch, reagents, and processing methods. Acetylated starch can be used as an encapsulant for nanoparticles, biofilms, adhesives, fat replacers, and other products with better paste stability and clarity. A comparison of various characteristics of acetylated starches and their dual modifications is expected to be a reference for developing and applying acetylated starches/flours in various fields and products. Native starches/flours generally have several drawbacks related to functional, pasting, and physicochemical properties, which can limit their use in various applications. These limitations include low swelling ability, absorption capacity, solubility, starch clarity, and freeze stability ,2,3,4. MThe modification of acetylation in starch/flour has been reported to increase swelling ability, clarity of starch paste, and stability of starch against retrogradation ,13,14. AModifying acetylation combined with cross-linking or hydrothermal treatment can improve the clarity of the paste, texture, and thermal stability ,22. SeveChanges in the characteristics of starch/flour due to the modification of acetylation are highly dependent on the degree of substitution (DS) and treatment conditions such as the source of starch, type of reagent, pH, temperature, and time ,15,24. TModifying a starch affects its characteristics, and the changes that occur depend on the type of modification applied. Chemical modifications such as acetylation weaken the starch’s structure, thereby increasing its hydration capacity and reducing its tendency to retrograde ,26. MeanAcetylation modification can increase the functionality of starch and its applications, especially in foods. The high hydration ability of acetylated starch has the potential to be used as a thickening agent. Several studies also reported that acetylation modification has good stability and resistance to retrogradation and syneresis, so it has the potential to be applied as a stabilizer in products that require low-temperature storage ,45. In aAcetylated modified starch, especially in nanocrystal form, has been effectively applied for encapsulation and as a delivery system for various drugs and other active compounds . de OlivAcetylated modified starch, especially in nanocrystal form, has been applied to improve the quality of bioactive films. Promhuad et al.  applied Acetylated modified starch and its dual modification can also be used as a stabilizer in various emulsion products and can act as a fat replacer. Yao et al.  applied Acetylated starch can also be applied directly in the manufacture of various food products such as bread and noodles. For some food products, such as bread, retrogradation causes bread stalling, where the quality of the bread decreases in the form of a harder texture . AcetylaAcetylated modified starch and its dual modification can also be used in the chemical field, namely as a waste treatment coagulant and as an adhesive, especially to increase its water resistance and shear strength . Gu et aThe characteristics of starches and flours could be improved by chemical, physical, and combination or dual modifications. These modifications aim to change some of the properties of starch by altering its original structure through physical treatment or changing the hydroxyl groups in starch through chemical reactions such as oxidation and esterification ,51,52,53Chemical modification can change the significant characteristics of starch and flour. Chemical modifications can be carried out through acid hydrolysis, oxidation, cross-linking, and the addition of functional groups such as acetylation. In general, chemical modification adds new functional groups to the starch, which then affect the physicochemical properties of the modified starch ,56,57,582, and Na2CO3 as catalysts [Acetylation is a chemical modification technique conducted through the esterification of starch using acetic anhydride, acetic acid, and vinyl acetate reagents and alkali atalysts ,62. The atalysts ,64. Acetatalysts ,65,66,67The degrees of substitution (DS) affect the physicochemical and functional characteristics of acetylated modified starch/flour ,72. The 2, NaOH, and KOH 1 N in the acetylation of waxy cornstarch produced DS of 0.077, 0.081, and 0.085, respectively [The acetylation method involving reactant types and concentrations can affect the acetylation reaction’s efficiency. The DS of starch acetate is greater when the reactant concentration is high . Generalectively . The DS ectively . Several−1, which interprets C–O carbonyl stretching vibrations, CH3 symmetry deformation vibrations, CH3 antisymmetric deformation vibrations, and carbonyl C=O, respectively [−1, which interprets the reduction of hydroxyl groups. Additionally, the spectrum of acetylated starch also shows that the anhydroglucose units tend to shift towards high wave numbers [Infrared spectroscopy or FT-IR is also frequently used to determine the DS value of acetylated modified starch/flour. An example of the effect of acetylation modification on the FTIR spectra of starch can be seen in ectively ,99,100. 1H NMR, 13C NMR, or 13C–1H COSY. NMR spectra can show changes in anhydroglucose units due to the substitution of hydroxyl groups by acetyl groups. An example of the effect of acetylation modification on the NMR spectra of starch can be seen in The DS value of acetylated starch can also be determined based on nuclear magnetic resonance (NMR) spectra, which generally use 2, C3, and C6 show a proton signal from the anhydroglucose unit between 4.4 and 5.6 ppm; the signal at 5.10 ppm corresponds to the anomeric proton of the link , whereas the anomeric proton of the junction  exhibits a small signal at 4.86 ppm [3) and the ester group (C=O). The longer the reaction time, the greater the area of the two signals, which means that the DS value of the acetylation increases [The hydroxyl (-OH) groups of C4.86 ppm ,102. Figncreases .Some of the advantages of acetylation modification include increasing starch clarity, lowering the gelatinization temperature, increasing the stability of frozen storage, and being more resistant to retrogradation ,103. TheFunctional characteristics are the properties of starch/flour that affect the usability of starch when applied. The functional characteristics of starch/flour can be viewed through several parameters, including amylose leaching/solubility, oil absorption capacity (OAC), water absorption capacity (WAC), freeze-thaw stability (FTS), and swelling power (SP). Swelling power is related to the amount of water absorbed, where the greater the swelling power value, the more water is absorbed. Starch granules will swell when heated in water, which is caused by the breaking of hydrogen bonds between starch molecules so that starch molecules will bind with water ,107.The swelling of granules is closely related to the release of starch molecules from granules or what is known as amylose leaching. As gelatinization proceeds, the water present in the starch suspension enters the outer amorphous and crystalline regions (located near the amorphous lamellae) ,109. TheWater absorption capacity (WAC) is defined as the ability of granules to absorb water. WAC determines the amount of water available for starch gelatinization during cooking . The gelStarch paste stored at freezing temperatures will cause the water contained in the paste to form ice crystals, and these ice crystals will melt during thawing and release of water from the granules, which is called syneresis . Freeze-Retrogradation occurs when starch components that have been gelatinized re-associate. This association causes the starch structure to become more compact, making the paste more turbid. The modification of acetylation in corn starch was reported to increase starch’s stability against retrogradation and increase the clarity of starch paste ,87. BaseStarch with high solubility and hydration ability is needed in several processed food industries, such as in the manufacture of noodles, bakery products, jelly, and many more ,125. AceModification by acetylation could increase the swelling power, solubility, and WAC as the DS increases. The modification of acetylation causes an increase in SP in sweet potato starch , potato Improvements in starch hydration and water absorption capacity after modification by acetylation were found in sweet potato starch , potato The modification of acetylation can also affect the solubility of starch and flour. Acetylation will generally increase solubility, mainly due to the addition of acetyl groups that disrupt the interactions between starch molecules, which in turn increases the affinity of starch molecules to dissolve in water . The incSeveral studies reported that the combination of modification by esterification (acetylation) + hydrothermal treatment was able to increase SP, WAC, and starch solubility ,23,97. TThe acetylation modification can also reduce retrogradation tendencies and increase the clarity level of starch paste ,129,131.Modification by acetylation can lead to an increase in the hydration ability of starch granules. This is due to the disorganization of the intragranular structure followed by the following events: (1) disruption of the intermolecular hydrogen bonds of the starch and increased penetration of water into the amorphous regions ,88, (2) The acetyl group can have two properties, namely hydrophilic and hydrophobic. Acetylation at low DS makes starch tend to be hydrophilic, while acetylation at high DS makes starch tend to be hydrophobic ,134. TheSeveral studies have reported that acetylation can reduce the tendency of starch to experience retrogradation and syneresis and shows a better level of clarity and transparency of starch than natural starch ,85,89. TAcetylation can reduce the occurrence of syneresis in starch. A decrease in the percentage of syneresis indicates an increase in the stability of the paste against frozen storage ,15. SyneAcetylation modifications can also be combined with other modifications. The combination of acetylation with hydrothermal treatments (ANN and HMT) can increase the hydration and solubility of starch ,97. A siThe substitution of ester groups (acetyl/hydroxypropyl) can weaken the crystalline structure of granules so that water penetration into the granules becomes easier and SP increases ,23. NoneEgodage  reportedPasting properties are indicators that determine starch properties during processing, which affect the cooking quality and functionality. Pasting properties described as an amylographic curve can be used to determine the application of starch in food ingredients ,139. StaBased on the amylographic curve, starch is classified into four types, namely types A, B, C, and V ,144. StaIn general, acetylation modification causes a decrease in starch gelatinization temperature so that it facilitates product application in terms of energy saving and its utilization in products that are susceptible to heat . SeveralThe substitution of acetyl groups causes weak intermolecular forces, which decreases the gelatinization temperature of starch acetate. This statement is supported by several researchers who reported a decrease in gelatinization temperature after the modification of acetylation, including sweet potato starch , potato The substitution of the acetyl group weakens the intermolecular bonds between amylose and amylopectin, thereby facilitating the penetration of water into the amorphous region, which is followed by an increase in the ability to absorb water during gelatinization ,85,88,90Acetylation modification can reduce the setback viscosity (SB), which indicates an increase in starch storage stability, especially at cold temperatures. This statement is reinforced by several studies which reported a decrease in SB after modification of acetylation ,127,129.The modification of acetylation with different materials and types of starch can produce different pasting properties as well. Acetylation with vinyl acetate and acetic anhydride in starch can cause a decrease in the gelatinization temperature due to the weakening of the intermolecular interactions of starch ,150. HowHydrothermal modification can increase the starch gelatinization temperature followed by a decrease in peak, BD, and SB viscosity, while acetylation causes a decrease in PT and SB followed by an increase in PV and BD ,151,152.The shape and size of starch granules vary depending on the starch source. Most of the tuber starches have oval-shaped granules, but some have round, polygonal, and spherical ones, and some are irregular; the size varies from 1 to 110 µm depending on the type of starch . For exaChanges in the morphology of starch granules are commonly found in modified starches. Several studies have stated that acetylation modification causes granule damage  ,88,94. HIn general, acetylation can cause the fusion and aggregation of starch granules ,88,147. In addition, several studies have stated that acetylation reactions can also cause the formation of holes or pores so that the surface of the granules becomes rough ,90,158. Changes in the morphology of starch granules due to acetylation modifications can have different results. Several factors affect the morphological diversity of acetylated starch, namely internal factors such as the content of amylose-amylopectin and external factors such as type of reactant, concentration of reactant, reaction time, temperature, type of alkali, and concentration of alkali. Morphological changes will increase with the greater substitution of acetyl groups ,160. ThiThe combination of acetylation with other modifications , such as hydrothermal treatment, also produces a variety of granule morphology. Acetylation modifications generally cause granule aggregation and fusion, whereas hydrothermal modifications (HMT and ANN) are more likely to maintain their integrity ,161,162.The dual modification of ANN-acetylation can produce starch with morphological characteristics resembling acetylated starch. This indicates that the modification of esterification/acetylation has a dominant effect . Egodagec). The linear line is made by connecting two intensity points at 4° and 37° (2θ); the area that lies between the curve and the linear line is known as the amorphous region (αa). The ratio of the upper area (αc) to the total diffracted area (αc + αa) is known as the degree of crystallinity or relative crystallinity [The crystallinity of starch can be determined by observing the X-ray diffraction pattern. The X-ray diffraction pattern is related to the formation of semicrystalline regions during modification so that the amorphous and crystalline areas can be identified ,165. Theallinity ,167,168.A (close to type A) and CB (close to type B). Type C starch showed strong diffraction peaks at 17° and 23° (2θ) and some small peaks around 5.6° and 15° (2θ). CA-type starch showed a shoulder peak at 18° (2θ), while CB type showed two shoulder peaks at 22° and 24° (2θ) [Based on the intensity peaks formed, the starch crystallinity is divided into three types, namely types A, B, and C. The type A diffraction pattern has a distinctive pattern with peaks of 23°, 18°, 17°, and 15° (2θ), commonly found in cereal starch (rice) ,170, and24° (2θ) . Type C 24° (2θ)  V-type c24° (2θ) , in theiThe type of starch crystallinity due to modification treatment can change, as indicated by the change in diffraction intensity. The acetylation treatment conditions and the dual modifications applied can change the polymorphic properties of starch. In general, acetylation at low DS can weaken the diffraction intensity, but no change in the crystalline diffraction pattern was found. This is supported by several studies, which stated that starch acetate weakened diffraction intensity but still retained its type of crystallinity ,86,175. The modification treatment of both acetylation and dual modification can increase, decrease, or not change the relative crystallinity (RC) of starch. The rearrangement of the double helix structure in starch granules can increase starch crystallinity, while the decrease occurs due to the partial gelatinization of starch granules ,176. TheThe effect of acetylation on crystallinity depends on the type of starch and the treatment conditions. Differences in starch crystallinity are affected by several internal components of starch, including the interaction of double helices in crystals, the arrangement of double helices in the crystalline area, and the number of crystalline areas, which is affected by amylopectin content and chain length, and crystal size ,165. TheThe substitution of hydroxyl groups with acetyl groups can weaken the hydrogen bonds that connect between starch molecules, which then causes a decrease in starch crystallinity . This stAcetylation modification can be combined with several other modifications, especially hydrothermal modification, to obtain the desired crystallinity characteristics. In general, the combination of acetylation and hydrothermal modification  did not cause a change in the crystalline diffraction pattern, but a change in the RC did occur. The combination of ANN + acetylation modification caused a decrease in crystallinity in waxy  starches . NonetheThe modification of acetylation and the combination of ANN + acetylation did not cause any changes in the starch crystalline diffraction pattern. However, the modification of acetylation could cause a decrease in RC and increases in DS. This was due to the substitution of acetyl groups weakening the formation of intermolecular hydrogen bonds, causing a weakening of the crystalline structure . The duaThe modification of acetylation in starch/flour has several advantages, including a relatively easy modification process that can produce starch/flour, which has a high swelling ability, clear starch paste, and good stability against retrogradation ,100,102.The modification of acetylation in starch/flour generally causes the fusion of starch granules, increasing the granule hydration ability, solubility, paste viscosity, storage stability and decreasing the gelatinization temperature and retrogradation stability. Acetylation generally does not change the crystalline structure of starch because it takes place in the amorphous areas of the granules, and some of them reduce the degree of crystallinity. Changes in starch/flour characteristics due to acetylation are strongly affected by the degree of acetyl group substitution. Dual acetylation modification with hydrothermal treatments such as HMT/ANN or cross-linking can produce starch/flour with better cooking and storage stability than native starch or single-modified starch and broaden its application to various products.The modification of acetylation in starch/flour is continuing to develop, especially to increase the efficiency of the modification process and the application of acetylated starch in various fields and products. The efficiency of the modification process is being improved, including via pre-treatment through the formation of porous starch, such as by ultrasonication, partial hydrolysis, or oxidation. Acetylated starch/flour was also developed through dual modifications, including cross-linking and hydrothermal treatments, to increase thermal stability. The application of acetylated starch has also been developed more broadly to produce starch nanoparticles, which can then be used for encapsulation, starch-based composite, biofilms, drug delivery systems, and other applications.


Neohesperidin (NH), a natural flavonoid, exerts multiple actions, such as antioxidant, antiviral, antiallergic, vasoprotective, anticarcinogenic and anti-inflammatory effects, as well as inhibition of tumor progression. In this study, the NH–taro starch complex is prepared, and the effects of NH complexation on the physicochemical properties, structure and in vitro digestibility of taro starch (TS) are investigated. Results showed that NH complexation significantly affected starch gelatinization temperatures and reduced its enthalpy value (ΔH). The addition of NH increased the viscosity and thickening of taro starch, facilitating shearing and thinning. NH binds to TS via hydrogen bonds and promotes the formation of certain crystalline regions in taro starch. SEM images revealed that the surface of NH–TS complexes became looser with the increasing addition of NH. The digestibility results demonstrated that the increase in NH  could raise RS (resistant starch) from 21.66% to 27.75% and reduce RDS (rapidly digestible starch) from 33.51% to 26.76% in taro starch. Our work provided a theoretical reference for the NH–taro starch complex’s modification of physicochemical properties and in vitro digestibility with potential in food and non-food applications. According to the International Diabetes Federation (IDF), approximately 537 million adults are suffering from diabetes, and this number is predicted to rise to 783 million by 2045. Natural compounds, such as polyphenols, are considered starch-modifying agents, which can potentially produce modified starches without the use of any undesirable chemicals ,2,3. PlaDuring co-gelatinization, one of the mechanisms by which polyphenols and starches interact is via the hydroxyl and carbonyl groups in polyphenols, which can interact with starches through Van der Waals forces and hydrogen bonds between their hydroxyl and carbonyl groups, which interferes with the reassociation of starch chains; another is that the inner hydrophobic helix of the amylose can accommodate polyphenols stabilized by hydrophobic interactions, which results in the formation of a V-type inclusion complex . TherefoIn recent years, due to their lack of apparent toxicity, citrus flavonoids (a type of polyphenol) have attracted wide attention in the food industry as natural food additives . NeohespColocasia esculenta (L.) Schott), the fifth largest tuber crop in the world, is widely grown in tropical and subtropical regions [Taro ; 2930 cm−1 (C–H); 1648 cm−1 (H2O); 1085 cm−1 ; 1022 cm−1 ; 927 cm−1 (the 1-type absorption band of D-pyran glucose); 854 cm−1 (the 2α-type absorption band of D-pyran glucose); and 765 cm−1 ( the type 3 absorption band of D-pyran glucose) [−1 had a wider range with the increase in NH  . Comparese in NH c–h, whicIt has been shown that the addition of flavonoids with multi-hydroxyls to starch can promote the attachment of flavonoid molecules to the surface of amyloid chains via hydrogen bonding and thus increase the intensity of absorption peaks for O–H . In thiso, Tp and Tc showed an upward trend, from 46.90 to 54.92 °C, from 60.04 to 63.46 °C and from 69.09 to 69.40 °C, respectively, with the increase in ratio of the NH addition to taro starch. These results suggested that polyphenols increased the thermal stability of starch. Similar to our results, previous studies reported that the addition of caffeic acid increased the To, Tp and Tc values of maize starch [o, Tp and Tc) of rice starch and potato starch [o, Tp and Tc). On the contrary, polyphenols reduce the thermal stability of starch [o, Tp and Tc) [o, Tp and Tc values of the proso millet starch [o, Tp and Tc) of potato starch [The gelatinization of starch is a physical transition process. During this process, the amylopectin double helices disassociate, and the crystalline structure is transformed to amorphous. The amount of energy required for the disintegration of double helices is defined as the change in enthalpy . The theo starch . Howeverf starch . The fort starch ; and proo starch . TherefoGelatinization enthalpy depicts the energy required for destroying the double-helix structure and eliminating crystallinity within the starch granules, which is an indicator of the loss of molecular order structure based on interactions between the particles ,14,16,56The steady rheological curves of the TS–NH complexes are shown in 2) of all curves were above 0.99  and n (rate index) < 1 further indicate that the pastes formed by native taro starch and TS–NH complexes are typical pseudoplastic fluids and show shear-thinning behavior [K) values indicate higher viscosity [K and viscosity show a significant upward trend  of the samples are depicted in At the first stage of digestive reaction, α-amylase can randomly and indiscriminately excise on α-1,4 glycoside bonds in the dextran molecules and produce glucose, maltose and extreme dextrin ; then, aTaro was sourced from Fenghua Research Institute, NingBo University of Technology, and taro starch was extracted according to the method described in our previous report after first cutting the taro tubers into small pieces . NH (CASw/w) were combined and dispersed in 100 mL 40% ethanol and boiled for 20 min with continuous stirring until completely gelatinized. The suspensions were cooled down to room temperature and then centrifuged at 5000 rpm for 15 min. The sediments were washed with 40% ethanol twice, and then some of them were used for rheological analysis and digestibility experiments, and the remainder were lyophilized and crushed into powder (through a 100 mesh) for other routine analyses. All supernatants collected after washing were further used for analysis of binding ability by HPLC.NH–taro starch complexes were prepared as per Zheng et al. with some modifications . Taro stv/v) were used as the mobile phase of HPLC at a velocity of 1 mL/min. The detection wavelength was 280 nm, and the column temperature was set to 30 °C. The amount of NH was determined based on the retention time and the chromatographic peak area. The concentrations of NH were obtained based on the standard curve. The binding amount and loading efficiency were calculated with the following equations [1, W2 and W3 represent the weight of initial NH, free NH and taro starch, respectively.The concentrations of NH in the supernatants and washed solutions were analyzed according to a previous report . Brieflyquations :Binding The microstructure of the gelatinized starch was observed using a scanning electron microscope . The starch powder was dispersed and uniformly placed on the carrier platform before conductive coating in a sputter coater  was performed, and then the sample was then firmly fixed by a rubber ear syringe. The images were taken under an acceleration voltage of 2 kV and were photographed at 3000× magnification according to a previous report .X-ray diffraction patterns were analyzed using a Siemens D5005 X-ray diffractometer  equipped with a copper tube, producing Cu-Kα (1.54 Å) radiation. The diffractograms were obtained by scanning at the angle ranging from 4° to 45° at a rate of 1°/min (2θ) and a step size of 0.02° (2θ), and the accelerated voltage and current were set to 40 kV and 40 mA .−1 [The ordered structure of the complexes of NH and taro starch was investigated using a Thermo Scientific Nicolet IS50 spectrometer equipped with an attenuated total reflectance (ATR) sampling accessory . The powder samples (2 mg) were pressed into KBr pellets (200 mg), and Milli-Q water was used as the blank control. Spectra were automatically baseline-corrected by OMNIC 8.0 software and deconvoluted over the range from 4000 to 400 cm−1 .2O was then added. The samples in DSC pans for gelatinization were equilibrated at 4 °C for 48 h before testing. The samples were measured during run heating from 25 to 95 °C at a rate of 10 °C/min under 20 mL/min flowing nitrogen, and an empty sealed pan was used as a reference. The onset temperature (To), peak temperature (Tp), conclusion temperature (Tc) and enthalpy of melting (ΔH) were calculated from the DSC endothermic curves.The gelatinization properties of NH–TS complexes were determined by using a differential scanning calorimeter  following our previous method . The samw/v) in distilled water were prepared using screw cap centrifuge tubes. The shear rates were increased from 0.1 to 1000 s−1 at 25 °C. The Herschel–Bulkley equation was used to fit the experimental data in regression as follows:σ, 0σ, K, γ and n represent shear stress (Pa), yield stress (Pa), consistency coefficient (Pa·sn), shear rate (s−1) and rate index (dimensionless), respectively.Static rheological properties of taro starch with different ratios of NH were determined using a Discovery Hybrid Rheometer  equipped with a parallel plate geometry . NH–star2) were incubated at 37 °C together with the enzyme solutions . Then, 1 mL mixed enzyme solution was added to the samples while being constantly vibrated in a 37 °C water bath. The hydrolysates (1 mL) were sampled at 20 min and 120 min by transferring into 20 mL 80% ethanol (to inactivate the enzymes) and then centrifuged at 4000 rpm for 10 min. The glucose content from the supernatant was determined using a glucose assay kit described in 20 and G120 are the glucose contents of the digestive solutions at 20 min and 120 min, respectively; 0.9 is the conversion factor from starch to glucose.Enzyme hydrolysis of NH–TS complexes was performed according to a previous method . The gelectively . G20 andp < 0.05.All experiments were performed at least three times. All data were analyzed by one-way analysis of variance (ANOVA) using SPSS 19 statistical software, and Duncan’s test was used to compare the means at a significance level of The amount of binding between NH and TS increased with the ratio of NH. NH significantly influences the gelatinization parameters of TS, and NH enhances the thermal stability and decreases the enthalpy value of taro starch in facilitating gelatinization. The viscosity and shear stress of TS–NH complex pastes increased with the ratio of added NH. NH alters the granule morphology and crystal structure of TS remarkably. FT-IR spectra confirmed that the binding modes of TS–NH are based on non-covalent bonding (hydrogen bonding). TS–NH complexes showed lower digestion efficiency and increased the RS fractions in starch. The effects are dependent on the amount of NH added. Our results demonstrated that NH can be employed to improve the health effects and edible value of TS by modifying its quality and provided a theoretical reference with NH–taro starch complex for modification of the physicochemical properties and in vitro digestibility with potential in food and non-food applications.


In case of need for esthetical improvement of zirconia restorations, an individualization using extrinsic staining can be applied. This study aimed to evaluate the surface roughness and fatigue strength  of high-translucency zirconia  with extrinsic characterization and/or glaze. n = 20) according to the surface finishing protocol: C (control), C + G (extrinsic characterization followed by a glaze layer), and G (glaze layer). The surface roughness (Ra) was analyzed with a contact profilometer. Subsequently, the specimens were subjected to a fatigue load profile starting at 120 N during 20,000 cycles at 4 Hz frequency, with a 5% increase at each step until failure. The failed specimens were evaluated under a stereomicroscope. Surface roughness analysis was evaluated by using one-way ANOVA and post hoc Tukey tests (95%); while fatigue survival probability was analyzed with Kaplan–Meier and Mantel–Cox .  Sixty (60) zirconia discs (12 × 1.2 mm) were obtained, sintered, and randomly distributed among three groups (µm)A followed by G (0.30 ± 0.10 µm)B, and C (0.19 ± 0.02 µm)C. While for fatigue strength, the G protocol presented a higher mean value A, followed by C + G (192.75 and 186.61–198.88)B and C (172.50 and 159.43–185.56)C.  One-way ANOVA revealed that surface roughness was affected by the finishing protocol, where C + G showed the highest mean value (0.46 ± 0.18  Surface finishing protocols modify the surface roughness and fatigue strength of high-translucent zirconia. Regardless of the surface roughness, both glazing protocols improved the ceramic fatigue strength, favoring the restoration's long-term survival. Polishing  was performed with silicon carbide sandpapers with decreasing granulation . After cleaning in an isopropyl alcohol ultrasonic bath for 5 min, the samples were sintered  to obtain final dimensions of up to final dimensions of 12 mm in diameter and 1.2 mm in thickness [High-translucent zirconia blocks , were machined into a cylindrical shape (14 mm diameter) and then cut into discs (hickness .http://www.openepi.com). The sample size was determined based on a two-sided comparison of means, with a significance level (α) of 0.05 and a power (1−β) of 0.80. The formula used for the calculation was based on the standard deviation (σ) observed in a pilot study and the desired minimum detectable difference (δ) between the groups. Considering these parameters, a total of 12 samples per group were determined to be necessary to achieve adequate statistical power for the study. However, 20 samples per group have been prepared and considered in this investigation.The calculation of the sample size was performed using the online calculator OpenEpi (n = 20) according to the finishing protocol: C, control, G, glazed, and C + G, characterized and glazed. In the C group, the discs did not receive any finishing protocol after sintering. Group G received a thin layer of glaze  in one surface of the disc with a 1 : 2 powder/liquid dilute ratio  [Discs were then randomly assigned into three groups (Germany) . The glaSurface analysis of the C (control), G (glaze), and C + G (extrinsic characterization followed by glaze) discs was conducted using a contact roughness meter . Three readings were taken at various angles on each specimen, and the average roughness parameter (Ra) was evaluated according to ISO 4287-1997 standards, utilizing a Gaussian filter and a 0.8 mm cutoff wavelength . By calcTo define the fatigue test loading profile, three specimens from each group were tested under the monotonic biaxial flexural strength test. For that, the discs were positioned on a circular metal base with 3.2-mm diameter balls equidistant from each other, forming a plane (ISO 6872:2015) , with thFatigue strength analysis was performed using the stepwise test . From thFor this, the specimens were tested  using a mechanical fatigue machine , with the same device configuration and disc position as used in the monotonic test . A hundrα = 0.05. The number of cycles and the load to fail during the fatigue test were analyzed by the reliability software using the Kaplan Meier and Mentel–Cox (log rank) (p < 0.05) survival analysis function. All analyses were performed using SPSS statistics software program . The images obtained were qualitatively evaluated.After confirming the assumptions of normality using the Kolmogorov–Smirnov test (95%), roughness data were tabulated for descriptive statistics for each group and then subjected to one-way ANOVA, followed by post hoc Tukey test, both with p < 0.001). C + G showed the highest mean Ra value (0.46 ± 0.18 µm)A followed by G (0.30 ± 0.10 µm)B, and C (0.19 ± 0.02 µm)C.One-way ANOVA revealed that the surface roughness of the groups was affected by the surface finishing protocol (α = 0.05) tests, specimens that were only glazed showed the highest fatigue strength and the number of cycles required to fracture; while polished (C) specimens showed the lowest mean fatigue strength, and the lowest number of cycles until fracture.The means and 95% confidence interval (CI) of the strength of flexural fatigue and the cycles of fracture are presented in The survival graphs of the groups as a function of time (number of cycles) and load are presented in The fractographic analysis pointed out defects that possibly originated the fracture in the tensile side of the specimens  on a high-translucent zirconia surface roughness, fatigue strength, and survival probability. Even with translucency improvement of zirconia, it still presents opacity and a high value as characteristics. Clinicians should strive to offer restorative solutions that exhibit the lowest possible roughness and the highest achievable gloss, regardless of whether it involves a direct restoration or an indirect restoration –24. TherSeveral studies have indicated that the application of a characterization layer can significantly increase surface roughness, leading to the accumulation of bacterial biofilm , which iμm in order to be undetectable by the patient. In the present study, the C + G group exhibited the highest mean roughness value (0.46 µm), followed by the G group (0.30 µm) and the C group (0.19 µm). Although there were statistically significant differences between the groups, it is important to note that all groups fell within the clinically acceptable range for the surface roughness. Therefore, based on the maximum roughness threshold recommended by the previous research of Jones et al. [In a previous study by Jones et al. , particis et al. , the finµm [By choosing materials with lower surface roughness, the dentist and the dental technician can reduce the risk of bacterial plaque retention and improve the long-term success of the dental treatments , 21, 30.µm .It is worth to mention that zirconia with a characterization layer and glaze firing would be indicated for a better replica of the appearance and the optical properties of natural teeth. However, the final shade and translucency of ceramic restorations can be significantly influenced by the type of material, cement, and substrate . The preA previous in vitro study by Zucuni et al.  comparedµm), suggesting that the evaluated zirconia after characterization followed by a paste glaze layer falls within the desired roughness threshold [In this study, the glaze layer was applied by the layering technique; however, spray or paste glaze can also be used as previously mentioned. Zirconia treated with spray glaze is suggested to have a rougher surface compared to the control one, presenting isolated “islands” of glaze on its surface , 34. WhiDespite the roughness assessment, the effect of a glaze layer on the fatigue performance of stained high-translucency zirconia is still scarce . For thaIt is important to mention that the results of fatigue cycling tests can assist dental professionals in selecting the most suitable restorative material for a particular patient and evaluating the long-term performance of dental restorations. While fatigue cycling can provide valuable information about the mechanical properties of Yttria-stabilized zirconia high translucent and other materials, it is only one aspect of the overall evaluation of a dental restoration. Other factors such as esthetics, biocompatibility, and handling must also be considered when selecting the restorative material for each case –9, 36. AThis study focused on three missions to evaluate the zirconia performance in different load situations. For an anterior area, represented by 150 N , both C One potential concern with C + G restorations is their resistance to wear, particularly in the case of stained zirconia restorations. When zirconia restorations are stained, a layer of colored glass is added to the surface of the material , 39. ThiThe choice of the surface parameter Ra, or the arithmetical mean roughness, is justified in restorative dentistry due to its widespread acceptance and relevance in assessing roughness . While pAs study's limitations, the absence of thermocycling tests would be necessary for investigating the mechanical behavior of the present zirconia in a more dynamic scenario , 40. Theµm [μm in another study by Dal Piva et al. [The thickness of the finishing layer plays a significant role in influencing the outcomes, as observed in the C + G group where the layer is thicker. This discrepancy likely impacted the results. In the context of Yttria-Stabilized Tetragonal Zirconia Polycrystal (YTZP), a prior study demonstrated a glaze layer with a thin and uniform surface, measuring between 24 and 28 µm , 46. In It is crucial to exercise caution when interpreting the results of this study, considering the in vitro nature of the setting and the need for further clinical trials to confirm the findings . As limiThe results of the study demonstrated that polished zirconia exhibited the lowest survival probability compared to glazed zirconia, indicating that glazing promotes higher long-term fatigue strength. Therefore, the application of either an extrinsic characterization followed by a glaze layer or a glaze layer as surface finishing protocols can enhance the survival probability of high-translucent zirconia, irrespective of the increase in surface roughness. These findings suggest that the additional steps of characterization and glazing contribute to the improved durability and longevity of zirconia restorations, outweighing the potential impact of increased surface roughness. Clinicians can therefore consider both surface finishing protocols to enhance the survival probability of high-translucent zirconia restorations while achieving satisfactory esthetic outcomes.