To study the effect of different surface treatments on the micro-shear bond strength and surface characteristics of zirconia. µm Al2O3 particles (A), etching with Zircos–E Etching solution for 2 hours (E), and a combination of air abrasion and etching (AE). After the surface treatment, all specimens were ultrasonically cleaned and 10 resin cement cylinders were attached to the zirconia discs in each group. A micro-shear bond strength test was performed in a universal testing machine at a crosshead speed of 0.5 mm/min. The fracture surfaces were assessed under a compound microscope. SEM, EDAX, and AFM analyses were done for the zirconia specimens after being subjected to surface treatment. Statistical analysis for the bond strength test was done using the Shapiro–Wilk test, one-way analysis of variance (ANOVA), and Post hoc Tukey test.  Two types of zirconia ceramics were tested: opaque (O) and translucent (T). Each type of zirconia was further allotted into four groups based on the type of surface treatment method. The four groups were: control (C), air abrasion with 110  The micro-shear bond strength values for the groups were as follows in megapascals (MPa): OC 18.96 (5.54), OA 22.66 (2.51), OE 28.48 (4.50), OAE 28.63 (4.53), TC 22.82 (5.46), TA 25.36 (5.17), TE 28.12 (4.76), and TAE 32.00 (3.47). The one-way analysis of variance (ANOVA) and post hoc Tukey HSD tests were done which showed significant results in the groups. In opaque zirconia, significant differences were seen in the etching and air abrasion with etching groups when compared with the control and air abrasion groups. There was no difference between the etching and air abrasion with etching groups. For translucent zirconia, the only significant difference was seen in the air abrasion with etching group in comparison with the control and air abrasion groups. The mode of failure was majorly adhesive. The surface topography and surface roughness showed significant differences between the groups. The EDAX results showed material loss that occurred due to sandblasting in the air abrasion groups.  Etching with Zircos–E Etching solution significantly increased the bond strength of zirconia to resin cement when compared with other surface treatment methods. In translucent zirconia, the best results can be achieved by combining etching with air abrasion. Ceramics are available in three forms: crystalline, partly crystalline, or non-crystalline. [2) have been known to have many important applications in the medical field. [Ceramic, which originates from the Greek word talline.  Ceramicsl field. .Out of all the zirconia-based materials, yttrium cation-doped tetragonal zirconia polycrystals (Y-TZP) have remarkably higher mechanical properties due to the stabilization of the tetragonal phase.  Zirconia3Y-TZP is mainly used in dentistry for the fabrication of dental crowns and fixed partial dentures. The opaque nature of zirconia is one of its major drawbacks.  A solutiClose adaptations of restorative margins along with bond durability are important for the success of any ceramic restoration clinically. , 13 ReceWhile hydrofluoric acid (HF) etching has been successful in roughening the surface of silica-based ceramics and in increasing their wettability, –25 HF do2O3 particle air abrasion, tribochemical silica coating, Er: YAG laser, primer application, and silica nanofilm deposition have been used to achieve higher and stable bonding to resin cement. 10-methacryloyloxyidecyl-dihydrogenphosphate (MDP) is a phosphate ester monomer that forms a bond with densely sintered zirconia by chemically reacting with it [2O3) particles followed by the application of MDP in the form of a primer or as a constituent of the cement is generally considered the gold standard for surface modification of zirconia dental restorations. [Hence, zirconia surface treatment such as Al with it . The userations.  However,rations. , 29 Thesrations. .2SO4), nitric acid (HNO3), and phosphoric acid (H3PO4). [Solutions for etching zirconia have been developed, including a solution composed of multiple acids, which can increase zirconia's surface roughness.  The zirc(H3PO4).  Zircos–E(H3PO4). , 33 AlsoμSBS) of zirconia to resin cement after various surface treatments are very scarce.The adhesive strength of the bonding systems has been routinely evaluated using shear bond strength tests in the laboratory.  Studies Therefore, this study evaluates the effect of various surface treatments on the surface microstructure, surface topography, and micro-shear bond strength of opaque and translucent zirconia with resin cement.The null hypothesis was that there would be no effect of various surface treatments on the surface microstructure, surface topography, and micro-shear bond strength of opaque and translucent zirconia with resin cement.Zirconia discs of 3Y-TZP and 5Y-TZP with dimensions 18 mm diameter and 4 mm thickness were milled from fully sintered, isostatically pressed zirconia blanks . The types of zirconia used in the study are described in The sample size for this study was calculated based on an earlier study. Cho et al.  in theirN = Sample sizeZ = Z scorea = Alpha errorZ(1 − (α/2)) = Z score for the alpha error chosenβ = Power1-Z(1 − β) = Z score for the power chosenσ = Average standard deviationd = The minimum difference in the values which will make clinically relevant impact.Based on the results of this study, which showed a shear bond strength of 16.15 MPa and 7.09 MPa with a standard deviation of 1.35 in the air abrasion and etching groups, respectively, the required sample in each group is was calculated to be 10 with 5% alpha error, 90% power of the study, and a clinically significant difference of 2 units using the following equation:For the Control Groups (OC and TC): No surface treatment was done.For the Air Abrasion Group (OA and TA): Each disc was subjected to air abrasion using 110 µm aluminium oxide particles  for 15 seconds at a distance of 10 mm at 4 bar pressure in the sandblasting machine . [ermany).  After thFor the Etching Group (OE and TE): The discs were kept immersed in the Zircos–E Etching solution for 2 hours according to the manufacturer's instructions and thereafter rinsed under cold tap water. [p water. .For the Air Abrasion and Etching Group (OAE and TAE): The discs are first subjected to air abrasion using 110 µm aluminium oxide particles for 15 seconds at a distance of 10 mm at 4 bar pressure in the sandblasting machine. [machine.  After thAfter the surface treatments of the groups, zirconia discs were cleaned in an ultrasonic cleaner  with distilled water for 10 minutes.Two discs from each group were chosen randomly for microstructure analysis. For each sample, sputtering was carried out with a gold layer of 10 nm. Following sputtering, the scanning electronic microscope  was used for surface analysis in all four groups. SEM was supplemented with Energy Dispersive Analysis of X-rays , Abingdon, United Kingdom) to analyze the elemental composition of the discs subjected to different surface treatments.μm). The average or arithmetic surface roughness (Ra), root mean square value roughness (Rq), and peak height/maximum roughness  of the discs were noted as numeric values in nanometers.Two discs from each group were selected randomly for surface roughness measurement using Atomic Force Microscopy . Specimens were tested under a non-contact mode utilizing an AFM cantilever with magneto-resistive sensors incorporated in its tip. The measurements were made at three random locations on each disc using a standardized rectangular spot  was applied to all the surface-treated discs with a micro-brush and gently air-dried followed by 2 coats of adhesive  which was applied, gently agitated, and dried with a stream for the evaporation of the solvent. Then, according to the manufacturer's instructions, the adhesive was light-cured for 10 seconds. The disc dimensions would only permit 4 samples to be attached per disc. Hence, a total of three discs were used for a total of 10 samples. 4 Tygon tubes with an internal diameter of 0.8 mm and a height of 5 mm were cut for each disc (4 Tygon tubes per disc). Resin cement  was dispensed on a pad and mixed. Each Tygon tube was loaded with cement and placed on the surface of the disc. The four Tygon tubes per disc were positioned at a distance of 5 mm from each other. The samples were light-cured for 40 seconds according to the manufacturer`s instructions. Similarly, three more Tygon tubes were attached to the zirconia disc. A sharp blade was used for the removal of the Tygon tubes from the disc. This resulted in each disc having 4 resin cement cylinders as seen in 2) and the shear bond strength in megapascals (MPa) was obtained.The micro-shear bond test involves the application of a loading force using a blade from a universal testing machine to a resin cylinder bonded to a substrate disc. To position the zirconia disc perpendicular to the blade, a heat cure PMMA block of dimension 40 mm × 20 mm × 25 mm was fabricated. It also stabilized the zirconia disc so that the disc would not move during the test. This was then positioned in the universal testing machine . A customized blade , Mangalore, Karnataka, India) was positioned at an angle of 90° at the junction of the resin/ceramic interface. A shear load was applied to each resin cement cylinder, at a crosshead speed of 0.5 mm/min, until the specimen fractured as seen in A compound zoom microscope  was used at 40x magnification to classify the failure mode as adhesive (at the resin cement/zirconia interface), cohesive (within the resin cement or the zirconia), or mixed (with both adhesive and cohesive failures). Two samples for each type of failure in the opaque and translucent zirconia groups were selected and subjected to the scanning electronic microscope (SEM) for analysis of the surface microstructure after testing.α = 0.05). Statistical analysis was done with IBM SPSS Statistics software (version 20), Chicago, USA. The distribution of data was assessed using the Shapiro–Wilk test. An independent t-test was performed to compare the opaque and translucent zirconia groups in terms of the micro-shear bond strength values. Statistical analysis of the results for the micro-shear bond strength test was performed by one-way analysis of variance (ANOVA) and post hoc Tukey HSD test. A chi-square test was done to analyze the mode of failure for all groups.95% confidence interval was used for all statistical tests (μSBS) and standard deviation with the F value of 8.431, P-value < 0.001 using one-way ANOVA. The same is represented in µSBS values were highest for the OAE group, followed by the OE group, OA group, and lastly the OC group. In the translucent group, the µSBS values were highest for the TAE group, followed by the TE group, TA group, and lastly the TC group.In the opaque group, the A compound zoom microscope was used at 40x magnification to classify the failure mode as adhesive or mixed. None of the samples showed cohesive failures.Figures Z) are given in Figures P-value ≤ 0.05 was considered. If the calculated P-value was less than the threshold, which was chosen for the statistical significance, the null hypothesis was rejected and if the P-value was greater than the threshold chosen, then the null hypothesis was accepted.The distribution of data was found to be normally distributed using the Shapiro–Wilk test. For all the tests, a significance level of μSBS) using the one-way ANOVA test showed that the difference is statistically significant with a P-value of <0.001. Comparison of the micro-shear bond strength , particularly with the development of dental computer-assisted design/computer-assisted manufacturing (CAD/CAM) systems.  ComparedTo resolve this bonding problem of zirconia restorations, alternative surface treatment methods have been tried using mechanical and chemical approaches. , 37 It hRecently, a zirconia etching solution (Zircos–E Etching solution) has been introduced containing (HF), hydrochloric acid (HCl), sulfuric acid (H2SO4), nitric acid (HNO3), and phosphoric acid (H3PO4).  The use µSBS) of resin cement with opaque and translucent zirconia was evaluated after subjecting the zirconia specimens to different surface treatment methods: no treatment, air abrasion, etching, and a combination of air abrasion and etching.In the present study, the micro-shear bond strength (µSBS values were the highest for the OAE group, followed by the OE group, OA group, and lastly the OC group. Statistical analysis shows that the difference between all the groups is significant except between the OC and OA groups and between the OE and OAE groups. This leads us to assume that air abrasion does not increase the bond strength of opaque zirconia significantly as compared to an untreated surface. Also, etching the opaque zirconia surface will lead to similar bond strength values when compared to air abrasion followed by etching. Hence, etching alone can improve the bond strength of opaque zirconia to resin cement without the need for air abrasion before it.In the opaque group, the µSBS values were the highest for the TAE group, followed by the TE group, TA group, and lastly the TC group. However, statistical analysis shows only two significant results. The statistically significant results were seen between the TC and TAE groups and between the TA and TAE groups. So, air abrasion with etching of translucent zirconia surface can significantly improve the bond strength as compared to no treatment, or air abrasion or etching surface treatments.In the translucent group, the The surface microstructure analysis was done using SEM supplemented with EDAX and AFM. The SEM images at 200x and 1000x revealed significant differences between the groups.The surface of the OC and TC groups showed a smooth surface interspersed with grooves on the surface. The grooves represent a machined surface. The surface of the OA and TA groups showed irregular roughening of the surface which can be attributed to aluminium oxide sand particles hitting the surface of the zirconia randomly and creating large peaks and valleys with sharp edges on the surface. Grooves were not seen on the surface due to the action of the sand particles.The OE and TE groups showed a much more uniform treated surface. The surface has no peaks or valleys but had an erosive roughened appearance. The presence of grooves showed that the surface of the zirconia was not aggressively roughened. This is because of the etching solution which chemically treated the zirconia to create micro-porosities on the surface. The surface of the opaque etching group showed multiple pits on the surface along with the erosive surface. The translucent group showed no such surface pitting. The translucent zirconia is more susceptible to etching with acids and the zirconia crystals get leached out uniformly. In the opaque zirconia, since the zirconia crystals are denser, the etching solution leaves pits on the surface of the zirconia due to the non-uniform dissolution of the zirconia.The OAE and TAE groups showed characteristics of both air abrasion as well as etching groups. Peaks and valleys can be seen on the surface but with an erosive texture as seen in the etching groups. Hence, it has a wave-like appearance.The EDAX results show that all the specimens consisted of zirconia, oxygen, and hafnium. In the OA and TA groups, the presence of aluminium was also detected which can be due to the remnants of the aluminium oxide sand particles. In OA and TA groups, the weight percentage of zirconia is relatively lesser as compared to the other groups. This further supports the fact that air abrasion aggressively roughens the surface, leading to material loss and damage to the surface of the zirconia.The AFM analyzes the surface roughness values of the groups. The OC and TC groups had the least values and all the treatment methods increased the surface roughness of the zirconia marginally. The OA and TA groups had the highest roughness values among all the groups. This can be correlated to the SEM analysis; it can be concluded that even though the roughness is higher in the air abrasion group, it is irregular unlike the OE and TE groups and OAE and TAE groups where the roughness is more uniform even if the average roughness value is lesser. Extreme roughness can lead to the weakening of the structure. This is supplemented by the EDAX values which show a lesser zirconia content in the air abrasion groups. Keeping in mind the micro-shear bond strength values, it can be stated that adequate roughness can be achieved with etching which is enough to improve the bond strength. Beyond a certain roughness value, the micromechanical adhesive bond of resin cement to zirconia will not be affected.The comparison between the opaque and translucent zirconia roughness values shows greater values in the translucent groups. This can be attributed to the fact that translucent zirconia is less dense and more susceptible to surface treatment.Previous studies assessing the effect of the Zircos–E Etching solution on the bond strength of zirconia showed varying results.Cho et al.  in theirAnsari et al.  comparedRecently, a study done by Zadeh et al.  gave conIn shear tests, it is very important to understand that the values obtained are mainly useful in comparing and ranking various materials and methods. Hence, the presence of a control group is essential to analyze the results of any laboratory test. None of the previous studies which evaluated the shear bond strength of zirconia after etching with Zircos–E included a control group for reference.The results of the present study proved that various surface treatments have an effect on the micro-shear bond strength of opaque and translucent zirconia to the resin cement and also affect the microstructure and the surface topography of the opaque and translucent zirconia. Hence, the null hypothesis was rejected.Etching with the Zircos–E Etching solution significantly increased the bond strength of zirconia ceramics to resin cement when compared with other surface treatment methods. In translucent zirconia, the best results can be achieved by combining etching with air abrasion.There are certain limitations to the present study. The effect of thermocycling on the bond strength of zirconia after various surface treatments was not assessed. The roughness values were assessed only for two specimens per group. Analysis of all the samples would enable us to establish a better correlation between roughness and bond strength.Further studies with variations in etching time, the temperature of the etching solution, types of cement, size of the air abrasion sand particles, effect of thermocycling, and the effect of zirconia primers would give us a more detailed insight into the effect of surface treatment of zirconia to improve bond strength.2O3 particle air abrasion, tribochemical silica coating, Er: YAG laser, primer application, and silica nanofilm deposition can also be tested and the results can be compared to the conventional treatment methods. Previous studies have evaluated the role of post treatment cleansing on the micro-shear bond strength of lithium disilicate [Other zirconia surface treatments such as Alsilicate . The effThe present report evaluated the bond strength of zirconia. However, as previously conducted for composites, future studies testing other mechanical properties such as hardness  and flexZircos–E Etching solution can significantly increase the bond strength of opaque zirconia to resin cement as compared to other surface treatment methods.Etching of translucent zirconia with the Zircos–E solution after air abrasion results in a significantly higher bond strength value with resin cement as compared to other surface treatment methods.SEM and AFM analyses revealed significant differences between different surface treatments. Even though air abrasion provided a rougher surface, it did not necessarily improve the bond strength. Etching creates uniform roughness on the surface and improves bonding.EDAX results further proved that air abrasion aggressively roughens the surface and caused material loss.With the limitations of the present study, the following conclusions can be drawn:


Conclusions: The presence of low serum 25(OH)D concentrations after LDLT suggested that post-transplant low serum 25(OH)D concentrations may develop with the homogenous phenomenon of VDR rs2228530 and CYP2R1 rs10741657 genetic modifications in recipients regardless of graft pathology.Background: Most cases of advanced liver diseases are associated with low serum 25-hydroxyvitamin D and vitamin D deficiency. This phenomenon may occur in living donor liver transplantation (LDLT). Aims: We conducted this study to explore the interplay between VDR and CYP2R1 in liver graft and compared our findings with the pathological interpretation of serum 25(OH)D concentration. Methods: In total, 60 patients received liver graft biopsy after LDLT and were separated (1:1) into two groups: graft rejection group and graft non-rejection group. We extracted both of the recipients’ and donors’ serum DNA to investigate the vitamin D receptor (VDR) rs2228530 and CYP2R1 rs10741657 single nucleotide polymorphisms (SNPs) using real-time polymerase chain reaction. We also extracted DNA from liver graft tissues to explore the genetic alleles of VDR rs2228530 and CYP2R1 rs10741657 after LDLT. Serum biochemistry profile and 25(OH)D concentrations were measured before and after LDLT. Results: There were no significant differences in serum VDR rs2228530 and CYP2R1 rs10741657 genetic alleles between recipients and donors. The percentage of genetic modification was 33.4% (10/30) for the rejection and non-rejection groups in VDR rs2228530, and 66.7% (20/30) for both groups in CYP2R1 rs10741657. Serum 25(OH)D concentrations were significantly lower after LDLT D30 than that before LDLT in the rejection ( Because activated vitamin D is predominantly synthesized in the liver, low 25(OH)D is a predictor of and is closely associated with severe liver disease, particularly in cases with poor prognosis ,2,3,4,5.n = 18/n = 12, n = 17/n = 13, and n = 29/n = 31, respectively. The underlying diseases were hepatitis B virus (HBV)-related liver diseases in 33 cases (included 11 cases with end stage liver diseases (ESLD) and 22 with hepatomas); hepatitis C virus (HCV)-related ESLD in 9 cases (included 6 with hepatomas and 1 HBV plus HCV); alcoholic liver disease in 9 cases; primary biliary cirrhosis in 3 cases; and cryptogenic cirrhosis in 6 cases. In addition, recipients who did not undergo liver graft biopsy (n = 30) were also evaluated as normal control group in this study. The mean age of the recipients in the normal control group was 57.6 years old. There were 16 males and 14 females. The primary underlying liver diseases were HBV-related liver diseases in 13 cases (included 6 cases with ESLD and 7 with hepatomas); HCV-related liver diseases in 14 cases (included 5 cases with ESLD and 19 with hepatomas); alcoholic liver disease in 2 cases, and cryptogenic cirrhosis in 1 case. The mean graft cold ischemic time and warm ischemic time were 51.9 ± 64.6 min and 37.9 ± 6.7 min. Graft-to-recipient-weight ratio was >0.8%. Recipients with unexplained abnormal liver functions as clinically required will undergo graft biopsy to evaluate etiology according to pathology result. In the rejection group, 76.7% (23/30) of patients had mild degree acute rejection, and 20% (6/30) had moderate degree acute rejection . The mean age of the recipients in the rejection group and non-rejection group and the donors were 56.1, 55.7 and 34.6 years old, respectively. Genders  of the recipients in the rejection group and non-rejection group and the donors were ejection . One casIn the non-rejection graft pathology group, 40.0% (12/30) had fatty liver disease, including 20.0% (6/30) with mild degree (<5%), 6.7% (2/30) with moderate degree (6–20%), and 13.3% (4/30) with severe degree (>2%). All of those donors were within normal limits for body mass index and had not been diagnosed with a non-alcoholic fatty liver disease during donor evaluation before LDLT. Notably, 20.0% (6/30) of patients had recurrent hepatitis C infection who had not received directed active anti-viral agent or inferno plus ribavirin treatment before LDLT. Furthermore, 20.0% (6/30) of patients were diagnosed with a non-specific reactive change during the study. One case was diagnosed with a post-transplantation lymphoproliferative disorder and survived after optimal chemotherapy.p = 0.414). By contrast, in CYP2R1 rs10741657, the genotyping allele frequencies were greater in AA and GG alleles and smaller in AG alleles between the recipients and donors before LDLT, but this different was also not significant (p = 0.143).The serum study on recipients and donors of the VDR rs2228530 and CYP2R1 rs10741657 is shown in p value = 0.371). By contrast, 66.7% (20/30) of patients did not have modified genotyping, with 53.3% AG and 13.3% GG alleles in the rejection group, and 40% AG, 20% GG, and 6.7% AA alleles in the non-rejection graft pathology group (p = 0.260).In the liver graft biopsy tissues study , the VDRp = 0.212). On the other hand, only 33.4% of patients did not have genetic allele modification in both groups, including 26.7% for AG and 6.7% for AA alleles in the rejection group, and 26.7% for AG and 6.7% for GG alleles in the non-rejection graft pathology group (p = 0.226).For the modifications of CYP2R1 rs10741657 genotyping after liver graft biopsy, 66.7% (20/30) of patients showed modification in both rejection groups, including 20% with GG to AA, 20% with AA to AG, 13.3% with GG to AG, 6.7% with AG to AA, and 6.7% with AG to GG modifications in the rejection group, and in the non-rejection graft pathology group, 33.3% with GG to AA, 13.3% with GG to AG, 13.3% with AA to AG, and 6.7 with AA to GG modifications (n = 30). The mean serum 25(OH)D level was 44.59 ± 36.93 ng/mL, which was statistically significantly higher than both rejection  and non-rejection with graft pathology groups  after LDLT. In the graft rejection group, low serum 25(OH)D concentrations were significantly lower after LDLT compared with before (p = 0.0001). The same was also true after LDLT (p = 0.0017) in the non-rejection graft pathology group  serum 25(OH)D level in the non-biopsy group of recipients (gy group .Serum 25(OH)D concentrations have been suggested as predictors for advance end-stage liver disease and liver cirrhosis with severe fibrosis, particularly in cases of non-alcoholic steatohepatitis or chronic C hepatitis infection ,3,5. In As shown in n = 28/60) of the recipients enrolled in this study were transplanted with the indication of hepatomas rather than that of acute decompensated liver cirrhosis or acute liver failure; thus, these recipients had pre-LDLT normal serum 25(OH)D levels due to relatively preserved liver function. In our study group, 6 of the 28 hepatomas developed in an HCV-related ESLD conditions; of course, these 6 recipients also presented reduced serum 25(OH)D levels. It is well-established in the literature that hepatomas are associated with reduced 25(OH)D serum levels, mainly because most of them developed in a cirrhotic/fibrotic liver. Actually, all of the remaining 22 hepatomas of our series developed in a non-cirrhotic/fibrotic liver. The association of vitamin D deficiency with disease severity, infection, and worse prognosis was previously reported in patients with liver disease. For liver transplant recipients, preoperative serum vitamin D status was correlated with disease severity and highly associated with invasive infection in the first 28 post-transplant days (PODs). Furthermore, the value on POD 28 had a strong association with graft function . In Tabln = 60) in our study. According to a previous report, vitamin D deficiency correlated with liver insufficiency, and 25(OH)D concentration would significantly improve after liver transplantation; however, still more than 50% of recipients were significantly deficient after liver transplantation . In our In our current study, all of the 60 recipients who underwent biopsy had abnormal graft function. The timing at which the serum 25(OHD) concentration was measured prior to LDLT and after LDLT was the same as the timing at which the liver biopsy was performed. Notably, their serum 25(OH)D concentrations were significantly lower after LDLT than before LDLT, regardless of liver graft pathology. Moreover, in recipients who did not undergo liver graft biopsy because of normal liver function, we additionally examined the post-LDLT D30 serum 25(OH)D level in the non-biopsy group of recipients as a normal control. The post-LDLT D30 serum mean 25(OH)D level in the non-biopsy group of recipients was statistically significantly higher than both rejection and non-rejection with graft pathology groups after LDLT. The result suggests that normal liver graft function will not cause the decrease of serum 25(OH)D level.Evidence from the research of Zhou Q et al. suggests that vitamin D deficiency is an important independent risk factor for acute rejection; vitamin D supplementation may reduce the risk of acute cellular rejection and infection in vitamin D deficient liver allograft recipients . The mecIn this study, low serum 25(OH)D levels after LDLT were measured in recipients undergoing graft biopsy for the reason of post-transplant abnormal liver function, and the low 25(OH)D levels in serum after LDLT were observed in rejection and non-rejection groups  . FurtherThere are some limitations in our current study. Because protocol liver biopsy after LDLT is not permitted in our liver transplant program, we only evaluated 60 recipients who had received liver graft biopsy as clinically required for evaluation of post-LDLT abnormal liver function in our liver transplantation program.p = 0.0001) and non-rejection graft pathology (p = 0.0017) groups.The strength of our current study is the clinical findings following our previous research, firstly suggesting the homogenous phenomenon of sequence changes in graft liver CYP2C19 from the different genotypes between the donors and the recipients . Based oIn conclusion, we would like to emphasize that the development of low serum 25(OH)D concentrations occurred after LDLT in both acute rejection and non-rejection group with abnormal liver function and graft pathology such as fatty change, recurrent hepatitis, acute cholangitis, non-specific pathological change and PTLD. Additionally, from the viewpoint of genetic variants in the VDR gene for regulating vitamin D levels, this finding also suggested that post-LDLT serum low 25(OH)D level would occur in recipients with homogenous phenomenon of genetic modifications of VDR rs2228530 and CYP2R1 rs10741657 polymorphisms, regardless of graft pathology.(n = 108), biliary atresia (n = 21), pediatric LDLT due to glycogen storage disease, Wilson’s disease (n = 16), and diseased donor liver transplantation (n = 6). The inclusion criteria were an adult liver transplantation who received a selective post-LDLT liver graft biopsy as clinically required. Finally, a total of 60 recipients was enrolled in our study. In addition, recipients who did not undergo liver graft biopsy (n = 30) were also evaluated in this study as normal control group. In this 2-year research period, there were 190 patients receiving liver transplantation in our hospital. The exclusion criteria were acute issues on chronic liver failure due to sepsis, primary bacterial peritonitis, esophageal variceal bleeding with shock n = 30), and graft non-rejection group (n = 30).Because protocol liver biopsy after LDLT was not permitted in our liver transplant program, all of the enrolled cases underwent liver graft biopsy for clinically being suspicious of acute rejection-related graft dysfunction or to evaluate the causes resulting in post-LDLT abnormal liver function, defining as biochemistry test with “one” or “both” of the following condition: (1) elevated AST and ALT tripled than normal level (2) Total bilirubin level > 2 mg/dL without evidence of biliary complication. All of the liver graft biopsy procedures were performed 1–4 weeks after LDLT. The biopsy tissue was analyzed by two independent liver transplant pathologists. Depending on the histological diagnosis, the patients were divided into two groups: graft rejection group (n = 30) as a normal control. The optical density of each well at 405 nm was determined within 30 min with a microplate reader. Each sample was assayed in duplicate by a single operator to assess interassay precision. By definition, serum 25(OH)D levels of greater than or equal to 30 ng/mL (75 nmol/L) were considered within normal limits in our study.Serum 25(OH)D concentrations were measured in recipients before and after LDLT 30-day (D30) and the same time of graft biopsy by use of a 25(OH) vitamin D enzyme-linked immunosorbent assay kit . Serum biochemistry profiles were measured after LDLT with the same timing at which the liver biopsy was performed. We have additionally examined the post-LDLT D30 serum 25(OH)D level in the non-biopsy group of recipients . Genotyping was performed to detect the single nucleotide polymorphisms (SNP) VDR rs2228530 and CYP2R1 rs10741657 with a ready-to-use, manufacturer-validated, predesigned allele-discriminating TaqMan SNP assay for polymerase chain reaction (PCR) amplification in clear optical 96-well plates on a 7500 Fast Real-Time PCR system , according to the manufacturer’s instructions. The VDR SNP rs2228570 and CYP2R1 rs10741657 genotypes were also studied in liver tissues via selective graft biopsies after LDLT on the same 7500 Fast Real-Time PCR System with Custom TaqMan SNP genotyping assays (Applied Biosystems) for allele discrimination. Both the serum and liver graft biopsy tissue SNPs were selected according to VDR rs2228530 AA/AG/GG and CYP2R1 rs10741657 AA/AG/GG allele frequencies and functional clinical implications. All genotypes were assayed in duplicate to assess intraassay precision. Genetic modification is defined as the difference of the SNP of VDR rs2228570 and CYP2R1 rs10741657 genotypes before and after LDLT.The study was approved and authorized by the hospital’s ethical committee . Our research was performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all participants or their legal guardians in accordance with the Declaration of Helsinki. All of liver tissue in our study were procured from the liver transplantation center in Kaohsiung Chang Gung Memorial Hospital. No liver tissues were procured from prisoners.t-tests were used to compare serum 25(OH)D concentrations in the rejection and non-rejection graft pathology groups before and after LDLT. P values less than 0.05 were considered to indicate a statistically significant difference.Statistical analyses were performed using SPSS statistical software . Fisher’s exact test with a two-sided model was used to compare VDR rs2228530 and CYP2R1 rs10741657 genetic polymorphisms in donors and recipients before LDLT, and the SNP allele modifications in VDR rs2228530 and CYP2R1 rs10741657 genotypes of the liver graft biopsy tissues. Student’s


Films performed at 120 rpm (S120 and S120-TiO2NP) showed completely processed starch and homogeneously distributed nanoparticles, leading to much more flexible nanocomposites than those obtained at 80 rpm. The incorporation of TiO2NP led to an increase in storage modulus of all films and, in the case of S120-TiO2NP, to higher strain at break values. From the Kohlrausch–Williams–Watts theoretical model (KWW), an increase in the relaxation time of the nanocomposites was observed due to a decrease in the number of polymer chains involved in the relaxation process. Additionally, S120-TiO2NP showed effective protection against UV light, greater hydrophobicity and faster biodegradation in compost, resulting in a promising material for food packaging applications. Biodegradable polymers have been strongly recognized as an alternative to replace traditional petrochemical plastics, which have become a global problem due to their long persistence in the environment. In this work, the effect of the addition of titanium dioxide nanoparticles (TiO According to ISO 17088 standards, one of the conditions that plastics must meet to be considered biodegradable under industrial composting conditions is to reach 90% degradation in less than 6 months [Plastics are one of the main sources of solid waste in large cities. In particular, the consumption of single-use plastics from food packaging has been identified as a global environmental pollution crisis since they can take up to hundreds of years to decompose ,2,3. Com biomass . BiodegrThe need to reduce the excessive use of single-use plastics that damage the environment generated a great demand for new biodegradable and/or compostable materials with specific properties and easy production. The capabilities of many types of nanocomposites based on biodegradable polymers are currently being explored to try to meet this demand ,8,9. StaAmong processing methods, extrusion is a technique that has been extensively used to produce plastic materials. However, starch extrusion involves multiple chemical and physical reactions, such as gelatinization, decomposition, melting and crystallization ,15. Diff2NP) have shown bactericidal capacity against E. coli and S. aureus and the ability to reflect and absorb UVA (320–400 nm) and UVB (290–320 nm) rays, protecting against sunburn, photoaging and extending product’s shelf life after harvesting [The addition of nanofillers, such as silver, zinc oxide and titanium dioxide nanoparticles, has been widely used in science to obtain starch-based nanocomposites for external packaging due to their outstanding properties and antimicrobial characteristics ,21,22. Prvesting ,24,25. Irvesting .2NP, Arezoo et al. and Goudarzi et al. stated that these fillers can efficiently protect products against UV light, being useful as UV-shielding packaging materials [2NP into thermoplastic starch films prepared by casting led to more flexible materials with higher mechanical resistance while Oleyaei et al. reported a slight increase in the hydrophobicity of potato starch with 1 wt.% of TiO2NP nanocomposites [Regarding starch/TiOaterials ,28. Yousmposites ,30.2NP on thermoplastic starch films produced by an industrial technique such as extrusion. Xiong et al. used different concentrations of TiO2NP in films from potato starch (60%) with glycerol (40%) prepared by blending and obtained nanoparticle agglomeration [2NP.However, to date, there are few investigations on the effect of TiOmeration . However2NP on the morphology, crystalline structure, mechanical properties, UV-vis absorption and biodegradability under industrial composting conditions of cassava starch-based nanocomposites obtained by extrusion and thermo-compression technology at different processing conditions.This work aims to evaluate the effect of the incorporation of TiO2NP, mixture of rutile and anatase, purity > 99%) were purchased in Sigma-Aldrich . TiO2NP diameter sizes were smaller than 100 nm, according to the manufacturer.Cassava starch with about 18 wt.% amylose was obtained by C.A.I.S.A. , analytical grade glycerol and titanium dioxide nanoparticles (TiO2NP (1 wt.%) using extrusion at 80 rpm or 120 rpm of screw speed and thermo-compression . These parameters were chosen from previous studies, in which starch-glycerol films not very sticky and very easy to handle were achieved [Thermoplastic starch films were developed using cassava starch (60%), glycerol (20%) and distilled water (20%) with and without TiOachieved . A co-roachieved ,33. Fromachieved . Samplesachieved ,36,37.−1 with a resolution of 0.4 cm−1 and an average of 64 scans. Experiments were carried out in triplicate and no significant differences were observed.ATR-FTIR spectra of all the developed film were recorded in a Jasco FT-IR 4100 spectrometer  with attenuated total reflectance (ATR) in the range 4000−600 cmIn order to investigate the cross-section morphology of the films, SEM analysis was performed using field emission scanning electron microscopy . Samples were fractured under liquid nitrogen and then coated with platinum by sputtering before observation., UK) using Cu (K-α) radiation . Samples were scanned over a diffraction angle range of 2θ = 5–35° at a step size of 0.026° and a scan speed of 200 s. A smooth curve connecting the peak baselines was plotted on the diffractograms, and the relative crystallinity was estimated by the ratio of the area above the smooth curve (upper diffraction peak area) over the total diffraction area [XRD patterns of starch films were evaluated by a PANalytical Empyrean  and stored at room temperature to simulate a humidity-controlled chamber. The sealed cell was weighed every 24 h for 7 days. Linear models of the results were performed, and the slope G (g/s) was obtained. Then WVP (g/msPa) was calculated using Equation (1):e (m) is the film thickness, A (m2) is the test area, DP (Pa) is the saturation vapor pressure of water at room temperature, and HR is the relation of the difference in relative humidity between the cell and the desiccator.Water vapor permeability (WVP) of developed films was determined according to ASTM E-96 procedure with the following modifications ,40: FilmThree replicates were performed for each sample. The mean value and standard deviation are reported.MC) was determined in accordance with the standard method of the AOAC [im∼0.5 g), dried in an oven at 100 °C for 24 h and then weighted (fm). Moisture content was calculated as Equation (2):Moisture content (the AOAC . SamplesAssays were carried out in triplicate.The contact angle (θ) was reported as an indicator of the hydrophobicity of the materials. It was determined using an Optical Tensiometer  at 25 °C, from the angle formed by the tangent of a water drop (2 μL) deposited on the surface of the film and the film surface. Tests were performed in triplicate.b), strain at break (εb) and tensile toughness (T) values were obtained from them. Young’s modulus was calculated as the slope of the stress-strain curves in the initial linear region and tensile toughness was determined as the area under the curves up to the point of fracture. At least, 10 replicates were performed for each system.Uniaxial tensile properties were studied using Brookfield CT3 Texture Analyzer  at room temperature according to ASTM D882-02 (2002) recommendations at a crosshead speed of 4 mm/min and room temperature . Films wτ> through a known mathematical model, the semi-empirical function Kohlrausch–Williams–Watts (KWW). This model presents the stress σ = σ(ε) for viscoelastic materials as Equation (3):E0 is the modulus of the relaxed state (for long times), E0 + E1 is the initial modulus and β is an empirical parameter that takes values between 0 and 1 and quantifies the width of the spectrum of relaxation times. The characteristic time τ* is an average relaxation time that characterizes molecular motions in amorphous systems [τ* = <τ> × β. The stress (σ)–strain (ε) curves can be analyzed to consider them as a spectroscopy technique  from est systems , and canτ* and β were determined.The KWW model on the films was evaluated from the fit of σ-ε curves with Equation (3), from which the parameters UV–vis spectra was studied employing a spectrophotometer . Film samples (1 × 3 cm) were cut and put into a quartz cell, and the absorbance spectrum between 250 and 900 nm was registered. All tests were studied in triplicate presenting no significant differences.2 with a CO2 non-dispersive infrared gas analyzer . DMR was composed of 4 parallel series  to remove water vapor from the outflow gas stream before reaching the NDIR sensor. The reactor content  is the only component of the system that is modified between each line. The first line only contains compost (blank), the second a known reference material (native starch powder) and the other two the samples to be measured. In addition, a temperature-controlled system was made by covering the reactors with heating jackets and using a thermosensor to set the temperature at 58 °C, in order to simulate industrial composting conditions.A Direct Measurement Respirometric (DMR) system based on ISO 14855-1 set up was developed to evaluate the aerobic biodegradation of the films under industrial composting conditions . The DMRl series  in which2 present in the air outflow from the reactors. Since water condensed after exiting the environmental chamber can damage the NDIR, a water filter  was installed.The NDIR was used to measure the concentration of CO2 to mass, Equation (4) was used [2mCO is the mass of evolved CO2 (g), C is the CO2 concentration (ppm), F is the flow rate, T is the time between each measuring sequence, 22,414 is the volume of 1 mol of gas in cm3 at STP, 44 is the molecular weight of carbon dioxide (g/mol), and 106 is the ppm conversion factor.To convert the concentration of COwas used (4)mCO2=%M is the percentage of mineralization, 2mCO (g) is the amount of evolved CO2 in the sample, 2bmCO (g) is the amount of evolved CO2 in the blank, and m2thCO (g) is the theoretical amount of carbon dioxide. The carbon content of the samples was 41%, 42% and 41.2% for starch, S120 and S120-TiO2NP, respectively, determined by Carbon Determination in Soil  and it was used to calculate 2thmCO.The percentage of mineralization (biodegradation) can be determined following Equation (5) and Ref. :(5)%M=mCFor this test, 250 g of industrial compost based on agricultural residues (Orgánicos de Argentina S.A.) with pH 6.8, volatile solids 18 ± 2% and 59 ± 2% humidity was used in each reactor. The compost was sieved on a 2 mm sieve and preconditioned at 58 °C for 3 days before using. Deionized water was incorporated to adjust the moisture content to about 50%. Twenty-five grams of perlite  was mixed with the compost and a 3 cm column of perlite was added at the bottom of the reactor to provide better aeration. Twenty grams of samples cut into 2 × 2 cm were mixed with the compost and perlite. On the other hand, native starch powder used as reference was mixed with the compost and perlite. In both cases, a proportion of 1:6 parts (on dry basis) compost was used, as ISO 14855-1:2012 recommends.2 evolved from the reactors was recorded every 6 h.During the test, the reactors were incubated in the dark. The compost was stirred and compost humidity was adjusted every day. The CO2 assessment.To observe the macroscopic physical changes occurring during composting conditions, three films of the 20 g of sample were enclosed in plastic grids and mixed with the rest of the samples, compost and perlite. At 1.5, 3 and 5 days of the test, samples were carefully removed from the grid and photographed. Then, they were taken back into the reactor in order to not interrupt the cumulative COp < 0.05 de confidence. The reported values are the mean and the error of the mean.Data analysis was made using Python with −1 attributed to the O–H stretching vibration due to free and inter- and intra-molecular hydrogen bonding of the films [−1 associated the symmetric and asymmetric C-H stretching; and a peak at 1640 cm−1 assigned to the water adsorbed by starch molecules, among others. There are no changes in the wave number of the bands nor the appearance of new bands due to the different processing or the nanoparticle’s addition. However, after the incorporation of TiO2NP, slight broadening in the 3300 cm−1 band was observed. This behavior suggests an increased degree of hydrogen bonding since oxygen atoms in TiO2 formed hydrogen bonding with H atoms of the OH groups in the starch [The main normalized ATR-FTIR spectra of different films are shown in he films ; a double starch .120). This suggests that the higher mechanical energy produced by the increase in the screw speed contributed to the improvement in the complete process of the starch grains.FE-SEM micrographs of the cryogenic fracture surface of the films showed some broken starch grains in the matrix made at 80 rpm e, which 2NP behaved markedly differently depending on the nanocomposite manufacturing process. While in the case of S80-TiO2NP an inhomogeneous distribution of nanoparticles was observed with the presence of some holes, in the nanocomposite prepared at 120 rpm a great improvement in the distribution of the nanofiller and prevention of holes generation was found  of the films were observed when starch was processed at different rpm. The relative intensity of the peak at 18° decreased while that of the peaks corresponding to the Vh-type structure at 19.6° increased in Sd to S80 . These rhis film ,53. As a2NP incorporation, the characteristic peak of Vh-type crystalline structure at 19.6° decrease in S120-TiO2NP, which demonstrates that the nanoparticles could have restricted the recrystallization of molecular starch, as has already been reported in the literature [2 nanoparticles [With TiOterature , since tterature . Furtherarticles , and cau80 compared to S120 is observed, which could be due to the more tortuous path for water due to the presence of broken starch grains. The incorporation of TiO2NP decreases WVP of S80-TiO2NP and increases it in S120-TiO2NP. In the first case, the behavior could be associated with the presence of some holes in S80-TiO2NP that favored the diffusion of water molecules through the film, leading to increases in WVP. In the case of S120-TiO2NP, it would be due to the greater distribution of TiO2NP in this nanocomposite with respect to S80-TiO2NP. The presence of well-dispersed nanoparticles could increase the tortuous path by dispersing water molecules and thus decreasing the diffusion rate.WVP values did not show significant differences between the films, as can be shown in 80 compared to S120 perhaps as a result of its higher crystallinity since, as it is known an increase in crystallinity leads to a decrease in MC [120-TiO2NP and was explained by the stable hydrogen bonds between TiO2NP and matrix components leading to a decrease in the number of water molecules with the possibility of being captured by starch. Thereby, the penetration of water molecules is more difficult, increasing the water resistance of the nanocomposites, as shown in the literature on starch-TiO2NP nanocomposites prepared by casting [se in MC ,56. The  casting ,30.80 and S120, nor with the addition of TiO2NP. A decreasing trend of Θ in S80-TiO2NP can be observed compared to S80 –strain (ε) curves of all films, which exhibit the typical behavior for thermoplastic starch materials [σb), strain at break (εb), Young’s modulus (E) and toughness (T) values obtained from the σ-ε curves, as well as the parameters from the theoretical KWW model are shown in 80 presented higher E and σb values but lower strain at break than S120 and no significant differences in T. The increase in E was expected due to the presence of starch granules in S80, as shown in SEM images  was higher than that of Sh powder ,62. Briah powder . In powdh powder .2 nanoparticles noticeably accelerated the biodegradation rate and increased the final biodegradation percentage of S120-TiO2NP compared to S120 and the reference. This behavior has not been previously observed in starch nanocomposites. Luo et al., obtained higher biodegradation rates under industrial composting conditions of (Poly-lactic) acid films when incorporating TiO2NP [2NP may accelerate some hydrolytic reactions. Similarly, Del Campo et al. observed that the incorporation of ZnO in a biodegradable commercial polymer (Ecovio) significantly accelerates the disintegration process under a composting environment [2NP delayed the starch film biodegradation since it is well-known that these nanoparticles provide antimicrobial activity to polymeric nanocomposites [The incorporation of TiOg TiO2NP , suggestironment ,67,68, imposites ,70. Howemposites .120-TiO2NP, S120, and powder starch reached 106 ± 5%, 83 ± 7% and 83 ± 6% biodegradation, respectively. S120-TiO2NP showed greater mineralization than 100%, which according to the literature is an indication of the priming effect attributed to the over-degradation of the indigenous organic carbon present in the compost when glucose and its polymers are tested [120-TiO2NP can be identified as biodegradable since its percentage of mineralization was greater than 90% and that of the positive control (powder cassava starch) was greater than 70% [At the end of the test, after 42 days, Se tested ,72,73,74than 70% ,75.120-TiO2NP, where more pieces were disintegrated compared to S120. On the 3rd day, only a very few pieces of S120-TiO2NP were observed, while on the 5th day a total degradation was noticed and S120 still presented many pieces.2. These results demonstrate the relevance of determining materials biodegradability by employing respirometric assays, since macroscopic analysis could underestimate the required time for the complete mineralization, especially in the case of fast-degrading polymers such as starch-based films.Even though the films had almost completely disappeared by the third day and fifth day, their biodegradation process had not been completed, as can be seen in 2NP significantly accelerates the disintegration process of starch-based nanocomposites under an industrial composting environment.The results of biodegradation tests show that TiO2NP in thermoplastic starch-based nanocomposites were evaluated. Increasing the extrusion screw speed from 80 rpm to 120 rpm led to materials with lower crystallinity that can withstand greater deformations before breaking and nanocomposites with TiO2NP homogeneously dispersed in the starch matrix. Based on the results, greater hydrophobicity in the nanocomposites prepared at 120 rpm was observed due to the destruction of the hydrogen bond formed between the O in H2O and the H in C-O-H of the starch by the addition of TiO2NP, which also led to higher Young’s modulus and relaxation time values. The characteristics of TiO2NP were reflected in the effectiveness of the UV light protection of the films for their possible use in light-blocking packaging and in their greater crystallinity. This investigation revealed that TiO2NP used in starch films initiated or advanced some hydrolytic reactions under industrial composting conditions, accelerating the disintegration and biodegradation of the material. It was concluded that the use of TiO2NP in cassava starch-based materials could be promising for its use as fast degrading material under industrial composting conditions and light-blocking packaging with improved mechanical resistance.The investigation of biodegradable polymers as an alternative to replace traditional petrochemical plastics for environmental preservation is relevant nowadays. In the present study, the extrusion processing conditions of cassava starch films and the incorporation of TiO


During the in vitro digestive stage of the stomach and small intestine, the particle size of the curcumin emulsion continued to increase, and the absolute potential continued to decrease. Our work showed that OSA-pre-treatment ultrasonic rice starch could improve curcumin bioavailability by increasing the encapsulation efficiency with stronger stability to avoid the attack of enzymes and high intensity ion, providing a way to develop new emulsion-based delivery systems for bioactive lipophilic compounds using OSA starch.The emulsification of ultrasonic-assisted prepared octenyl succinic anhydride (OSA) rice starch on curcumin was investigated in the present study. The results indicated that the encapsulation efficiency of curcumin in emulsions stabilized by OSA-ultrasonic treatment rice starch was improved, from 81.65 ± 0.14% to 89.03 ± 0.09%. During the in vitro oral digestion, the particle size and Zeta potential of the curcumin emulsion did not change significantly ( Curcumin is a natural small molecule polyphenol compound with anti-inflammatory, anticancer, and antioxidant functions ,2. It alNatural starch has poor solubility in cold water, thermal instability, easy coagulation, and poor film forming and emulsification. In order to better meet the requirements of industrial production, starch is usually modified by physical, chemical, or biological methods. Octenyl succinate starch ester (OSA starch) is a widely used chemically modified starch. It is prepared by esterification of octenyl succinic anhydride (OSA) and starch under alkaline conditions. The introduction of hydrophobic groups (olefin long chain) and hydrophilic groups (carboxyl group) makes OSA starch amphiphilic . AttribuUltrasonic treatment influences the starch particle morphology and molecular structure through microjet, mechanical shear, and local heating generated by the cavitation mechanism . At the In order to explore the effects of different properties of OSA rice starch assisted by ultrasound on the efficiency of curcumin delivery, we used ultrasonic-assisted OSA rice starch as the emulsifier to construct the curcumin emulsion carrier. The loading rate and bioavailability of different emulsions were measured, and the particle size, Zeta potential, apparent morphology, and free fatty acid release of the emulsion at different in vitro digestive stages were observed and further established the way and mechanism of how ultrasonic-assisted OSA rice starch affects the release of curcumin during in vitro digestion Therefore, the present study might provide as a reference for the development of the potential application of ultrasonic-assisted OSA rice starch on the encapsulation of bio actives.p < 0.05), indicating that higher intensity ultrasonic treatment had more serious damage to the starch structure, and the amorphous area in the starch and even part of the crystallization area was also affected. Therefore, OSA substitution not only occurred in the amorphous area on the surface of the starch particles, but also reacted with the active site in the crystallization area, resulting in an increase of DS and RE [As shown in S and RE .The encapsulation efficiency of curcumin in the OSA rice starch emulsion system increased by 0.97–1.15 times, while the loading capacity increased from 1.36 µg/mg to 1.81 µg/mg , indicatp < 0.05). After simulated oral digestion, the particle sizes of all emulsions increased slightly. Compared with the initial emulsion, the particle sizes of NRS, ORS, UORS-150W, and UORS-600W increased by 33%, 25%, 8.1%, and 7.7%, respectively. However, compared with other digestive stages, the change of particle size in oral digestion was not obvious , but a significant difference in the particle size between the two components. The results showed that although the increase of the surface charge of the emulsion droplets could enhance the electrostatic repulsion between the droplets, the absolute value of the Zeta potential of the emulsion samples at the stomach digestion stage was at a low level, which was not enough to maintain the stability of the emulsion, and the stability of the emulsion depended on the steric hindrance. The results could explain why the ultrasound-assisted OSA starch emulsion maintained higher stability at the stomach stage: on the one hand, the ultrasound-assisted OSA starch had larger DS, which could increase the surface charge density of the emulsion drops and enhance the electrostatic repulsion between the emulsion drops. On the other hand, the starch molecules are connected with more OSA groups, and the OSA groups are more evenly distributed in starch, which is conducive to the formation of the emulsified layer, with strong rigidity and a tight structure, and can better protect oil droplets.In the gastric digestion stage, the absolute value of emulsion Zeta potential decreased significantly, indicating that the charge density on the droplet surface decreased. It was noteworthy that there was no significant difference in the Zeta potential between NRS and ORS in the gastric stage . It was speculated that the absolute potential of the OSA starch group with ultrasonic assistance was higher, which might be because more free fatty acids were generated by hydrolysis during intestinal digestion [After 120 min of intestinal digestion, the absolute potential value of all OSA starch emulsions increased and even exceeded the level before simulated digestion, and the absolute potential value of OSA starch emulsions assisted by ultrasound was significantly higher than that of other components  pancreatic lipase hydrolyzed the oil; (4) the hydrolysates of oil were compounded with particles, vesicles, and micelles in the emulsion system [Previous studies also found that after OSA starch emulsion was digested in the small intestine, the number of oil droplets decreased, which might relate to the following factors: (1) the small intestine digestive liquid added would dilute the emulsion system; (2) Can system . At the n system .p > 0.05). The above results indicated that the digestion rate and degree of oil in OSA starch emulsion prepared by ultrasound were improved, which might have attributed to the smaller particle size of OSA starch emulsion prepared by ultrasound. A large number of studies have confirmed the relationship between the oil digestion rate and oil droplet interface area. The smaller the droplet diameter was, the larger the effective interface area of oil and enzyme contact would be, thus improving the oil digestion rate [As shown in ion rate ,32,33. BOnly when the lipid soluble functional factors in the emulsion system were released from the carrier matrix into mixed micelles or vesicles could they be absorbed and transported by the small intestinal epithelial cells. The proportion of lipid soluble functional factors transferred to micelles, namely bioavailability, is an important indicator that is widely used in in vitro digestion experiments.p > 0.05).As can be seen from A positive correlation was found between the bioavailability of the curcumin emulsion and the final release of free fatty acids (FFA) . StudiesRice starch (CAS: S7260), curcumin (CAS:458-37-7), pepsin (CAS: 9001-75-6), porcine pancreatic α-amylase (CAS: 9000-90-2), α-amyloglucosidase , invertase , and bile (CAS: 8008-63-7) were purchased from Sigma-Aldrich. Soy oil was bought from Yihaikerry Co., Ltd. . Octenyl succinic anhydride and other chemicals of analytic grade were purchased from Aladdin Co., Ltd. .w/w) starch slurry was treated with ultrasonic powers 150 W and 600 W with an ultrasonic device  under 25 °C for 20 min and then dried at 40 for 24 h. Native rice starch, rice starch with ultrasonic powers 150 W and 600 W in 25 °C were labeled as RS, URS-150W, and URS-600W, respectively. Ultrasonic pre-treated rice starch was esterified with OSA using the method of Zhang et al. [w/w) and the pH was adjusted to 8.5. Then, OSA  was added slowly within 1 h, with continuous stirring at 35 °C, while maintaining a pH at 8.5 and reacting for 2 h. The reaction was terminated by adjusting the pH to 6.5 and the mixture was centrifuged at 8000 rpm for 10 min. The sediment was washed twice with distilled water and then twice with ethanol to remove any free OSA. The OSA-starch samples were oven-dried at 40 °C for 24 h and passed through a 100-mesh standard sieve. For reference, native rice starch without ultrasonic treatment was also modified with OSA, as described above. OSA-starch samples prepared by the conventional aqueous method and by ultrasonic pre-treatments under different powers were named as ORS, UORS-150W and UORS-600W, respectively. The native rice starch was also labelled as NRS.The ultrasonic rice starch was prepared according to Yang et al. . Brieflyg et al. . The staw/w) of NRS, ORS, UORS-150W, and UORS-600W was gelatinized for 20 min and then cooled to room temperature for the water phase. A total of 0.2% (w/w) curcumin was dissolved in soy oil and stirred in room temperature for 1 h for the oil phase. Subsequently, the mix ratio of the water phase and the oil phase was 4:1 (w/w), and then the crude emulsion was prepared by high-speed shearing for 3 min with a high-speed disperser  at 15,000 rpm/min. The final curcumin carrier emulsion system was obtained by using a high-pressure homogenizer  with a pressure of 150 MPa. The degree of substitution (DS) and reaction efficiency (RE) of OSA-starch were measured using the titration method [A total of 5.0%  were prepared with ethanol. The absorbance of all curcumin solutions was read with 425 nm with a spectrophotometer  and the standard curve was made. One mL emulsion with 5 mL dichloromethane was stirred thoroughly for 2 min, and the downward layer was collected, and the absorbance was read at λ = 425 nm. The free curcumin in the emulsion was calculated using Equation (1).The simulated digestion process containing the oral, gastric, and intestinal phases was used to monitor the potential digestion of all samples and the corresponding fluid was prepared as described previously—with minor modifications . The dro2PO4 (0.888 g), NaHCO3 (1.694 g), Na2SO4 (0.57 g), KSCN (0.2 g), and (NH4)2CO3 (0.2 g) were dissolved in 1000 mL deionized water and the pH was adjusted to 6.8. Emulsions were mixed with SSF at a volume ratio of 1:1, and the pH was adjusted to 6.8. The mixtures were continuously stirred at 37 °C for 3 min at 100 rpm.Stimulated saliva fluid: NaCl (0.298 g), KCl (0.896 g), NaH3 (2.1 g), NaCl (2.758 g), MgCl2 (0.1 mM), KCl (0.515 g), KH2PO4 (0.122 g), and (NH4)2CO3 (0.048 g) were dissolved in 1000 mL deionized water, and the pH was adjusted to 2.5 with HCl. Before use, pepsin  was added to the solution. The sample from the oral phase was mixed with the SGF at a volume ratio of 1:1, and the pH was adjusted to 2.5. The mixtures were continuously stirred at 37 °C for 2 h at 100 rpm.Stimulated gastric fluid: NaHCO2 (0.244 g), bile salt (44 g), and pancreatin (2.2 g) were dissolved in 1000 mL deionized water and the pH was adjusted to 7.0. Before use, bile salts (10 mM) and pancreatin (1:3000) were added to the solution. The sample from the gastric phase was mixed with the SIF at a volume ratio of 1:1, and the pH was adjusted to 7.0. The mixtures were continuously stirred at 37 °C for 2 h at 100 rpm.Stimulated intestinal fluid: CaClg, 4 °C, for 40 min. One mL raw digestive solution from the intestinal phase was mixed well with 5 mL chloroform/ethanol . the organic layer containing curcumin was collected, and the mixture was extracted twice. The extraction solution was combined and diluted with ethanol. The UV absorbance value was measured at 425 nm and the concentration of curcumin was calculated according to the standard curve. The formula for calculating the bioavailability of the emulsion system is as follows:After simulated in vitro digestion, the digestive solution was centrifuged at 10,000 The particle size and Zeta potentials of the starch sample were characterized using the static light scattering instrument . Before analysis, the initial, oral, stomach, and small intestinal (5 min and 120 min) diluted with deionized water was used as dispersant, and the suspension was sonicated for 5 min to disperse the starch granules before testing. The refractive index of water and starch was set as 1.33 and 1.50, respectively. The results were characterized by mean particle size, D10, D50, and D90 from the three replicates.In a process simulating intestinal digestion, the oil is hydrolyzed to free fatty acids under the catalysis of pancreatic lipase. Referring to the previous research with some modifications , oleic aConfocal laser scanning microscope  was used to observe the micromorphology of droplets in the original emulsion sample, the sample after simulated oral digestion, the sample after simulated gastric digestion, and the sample after simulated intestinal digestion for 5 min and 120 min. Nile red solution (0.01 g/mL) and Nile blue solution (0.01 g/mL) were prepared in propanol, then vortex-mixed for 30 s, respectively. 1 mL sample was mixed with 400 μL Nile red and 400 μL Nile blue solution to label the oil and starch particles on the surface of the droplet, respectively. After staining for 40 min, 10 μL emulsion drops were placed on the slide, and the cover slide was covered for observation and image collection. CLSM observation was carried out at room temperature using Ar/Kr and He/Ne visible light laser single mode, with laser wavelengths of 488 nm and 638 nm.p < 0.05. All statistical analyses and figures were conducted using Origin 2018 .The data was expressed as mean ± SD. The analysis of variance model (ANOVA) was used to test significant difference between means at In summary, an emulsion-based delivery system of curcumin was successfully developed using ultrasonic treatments of rice starch-OSA as the carrier. The particle size and potential charges of OSA rice starch emulsion assisted by ultrasound were smaller than native rice starch. The particle size and potential charges did not change during in vitro oral digestion. The addition of UORS-150W and UROS-600W in the curcumin potentially increased the bioavailability of curcumin and the rate of release of fatty acids during in vitro digestion. These results have important implications for the design of emulsion-based delivery systems, which enhance the health benefits of curcumin. However, the loading function needs to be verified by in vivo experiments.


This Special Issue in renal transplantation covers a variety of clinical and research areas in kidney transplantation. The recent decade is associated with an ongoing shortage of organs for transplantation with efforts to increase the organ pool with DCDs and extended criteria donors. However, with the increasing success rate of kidney transplants, there is also a growth in the candidate list because of removal of the age barrier and transplantation of high risk patients with other comorbidities. The future seems promising with the development of innovative non-invasive technologies introducing biomarkers for diagnosis of rejection and ischemic reperfusion injury, use of cell therapy for tolerance induction, development of artificial organs, and overcoming immune and non-immune barriers in xenotransplantation. This Special Issue will touch some of these topics that are in the frontiers of the modern era of kidney transplantation.On the clinical side, there are two papers covering the effect of age and other demographics criteria on long-term outcome after transplant.The first paper, by Dr Yemini et al., is from my group, and it discusses the “Long-Term Results of Kidney Transplantation in the Elderly: Comparison between Different Donor Settings” . Our papIn a “Systematic Review and Meta-Analysis on The Impact of Recipient Demographics on Outcomes from Living Donor Kidneys” , Dr. BelAs for the effect of immunosuppression on malignancy Dr. Imamura et al. preformed a long term multi-center study showing that “Everolimus Reduces Cancer Incidence and Improves Patient and Graft Survival Rates after Kidney Transplantation” . Two other papers focus on pretransplant sensitization. The first paper is by Righini and his colleagues on the “Impact of the Type of Dialysis on Time to Transplantation: Is It Just a Matter of Immunity?” . In thatUse of biomarkers in transplantation is another innovative area of interest in recent years. In this Special Issue there are two papers looking at correlation of biomarker and outcome after transplant. The first one is “Pretransplant Serum Uromodulin and Its Association with Delayed Graft Function Following Kidney Transplantation” . In that2) in the renal allograft after reperfusion and compared the rSO2 between recipients of a deceased donor and a living donor. They showed that rSO2 remained significantly lower in the DDRT group compared to the LDRT group throughout the 50 min after reperfusion and that reperfusion rates were significantly faster in the LDRT group during the first 5 min post-reperfusion. Interestingly, intraoperative rSO2 strongly correlated with allograft function up to 14 days post-transplantation. They concluded that NIRS may be a useful intra-operative tool to assess the degree of preservation/reperfusion injury and predict early allograft function.The last few years have seen the emergence of many new technologies designed to examine organ function, including new imaging techniques, transcriptomics, genomics, proteomics, metabolomics, lipidomics, and new solutions in organ perfusion, which has enabled a deeper understanding of the complex mechanisms associated with ischemia-reperfusion injury (IRI), inflammatory process, and graft rejection. This issue includes “A Review of Current and Emerging Trends in Donor Graft-Quality Assessment Technique”  written Lastly, future technologies to develop organs to replace the current source of human organs for transplant are in the focus of many research groups around the world. Aiming to achieve future generation of a new kidney Dr. Garcia-Dominguez and his colleagues studied “The effect of Sildenafil Citrate in enhancing renal organogenesis following metanephroi allotransplantation” . Sildena


Humoral immunity is crucial for protection against invading pathogens. Broadly neutralizing antibodies (bnAbs) provide sterilizing immunity by targeting conserved regions of viral variants and represent the goal of most vaccination approaches. While antibodies can be selected to bind virtually any region of a given antigen, the consistent induction of bnAbs in the context of influenza and HIV has represented a major roadblock. Many possible explanations have been considered; however, none of the arguments proposed to date seem to fully recapitulate the observed counter-selection for broadly protective antibodies. Antibodies can influence antigen presentation by enhancing the processing of CD4 epitopes adjacent to the binding region while suppressing the overlapping ones. We analyze the relative positioning of dominant B and T cell epitopes in published antigens that elicit strong and poor humoral responses. In strong immunogenic antigens, regions bound by immunodominant antibodies are frequently adjacent to CD4 epitopes, potentially boosting their presentation. Conversely, poorly immunogenic regions targeted by bnAbs in HIV and influenza overlap with clusters of dominant CD4 epitopes, potentially conferring an intrinsic disadvantage for bnAb-bearing B cells in germinal centers. Here, we propose the theory of immunodominance relativity, according to which the relative positioning of immunodominant B and CD4 epitopes within a given antigen drives immunodominance. Thus, we suggest that the relative positioning of B-T epitopes may be one additional mechanism that cooperates with other previously described processes to influence immunodominance. If demonstrated, this theory can improve the current understanding of immunodominance, provide a novel explanation for HIV and influenza escape from humoral responses, and pave the way for a new rational design of universal vaccines. Antibodies are a fundamental component of human immunological defense, and one of their most important functions is to confer protection against viruses and exogenous microorganisms upon primary infection or vaccination ,2,3. AntThe repertoire of naïve B cells and BCRs is extremely diverse thanks to BCR rearrangement during B cell development, resulting in the potential to produce antibodies against virtually any epitope of a given antigen ,10,11. DThe goal of most vaccines is to induce the vigorous and long-term production of nAbs with the ability to prevent any future infection. However, some viruses deploy diverse strategies to escape antibody neutralization, including the mutation of viral antigens  ,3,13. AnPoor or defective bnAbs induction following vaccination in the context of HIV and influenza has been extensively studied. The following potential explanations for defective bnAbs production  for influenza) have been proposed: poor epitope accessibility ,17,18, hAround 30 years ago, antibody binding was shown to influence antigen processing and presentation, with CD4 T-cell epitopes either inhibited or boosted based on their relative positioning to the antibody epitope ,23,24. HIn recent decades, several hypotheses have been proposed to explain the molecular mechanisms underlying the inconsistent induction of bnAbs, especially in the context of HIV and influenza vaccination studies.The variation of immunodominant epitopes due to the accumulation of random mutations in the viral genome represents one of the most successful strategies for viruses to escape the host immune system ,13. ThisEpitope accessibility has long been regarded as a fundamental requirement to effectively mount humoral responses ,30,31,32The frequency of germline B cell precursors shapes the humoral response to immunogenic antigens by impacting GC occupancy ,3. A lowThe high mutational load required to develop bnAbs can also represent a major challenge . Indeed,A variety of host genetic factors can influence the outcome of viral infections, most notably polymorphisms within the HLA class I and II loci ,42,43,44Antigen processing and presentation were initially investigated using macrophages or dendritic cells as a model of antigen presenting cells (APCs) . In suchPioneer work by Berzofsky and Celada indicated that antibody binding could differentially boost antigen presentation to some T cell clones, either through the modulation of antigen uptake or proteolytic processing ,52,53,54Given the impact of BCR and antibody binding on antigen processing, we postulated that the relative positioning of B and CD4 T cell epitopes shapes immunodominance. To test this hypothesis in the absence of studies that map B and CD4 T cell immunodominant epitopes within single patients, we analyzed the 3D positioning of published B and T cell epitopes in immunogenic antigens known to elicit strong and neutralizing humoral responses, such as measles HA, diphtheria toxoid, vesicular stomatitis virus glycoprotein (VSV-GP), and SARS-CoV-2 spike protein. To restrict our focus to highly immunodominant and HLA-independent regions, we gathered published data for B and T cell epitopes and selected determinants found in multiple patients or host species . This approach allowed us to select determinants that were likely to be dominant in the presence of different HLA class II alleles. In addition, we decided to focus our analysis on experimentally validated rather than predicted epitopes . Across Overall, these data suggest that immunodominant and neutralizing B-cell epitopes are mostly not overlapping with and often adjacent to dominant CD4 T-cell epitopes, increasing the chances of an antigen presentation boost rather than suppression upon BCR binding. Additionally, antigens that are associated with a rapid nAb response contain immunogenic CD4 determinants scattered in several portions of the viral proteins. Scattered CD4 T cell epitopes increase the likelihood for dominant B cell clones targeting multiple regions of the antigen to emerge, facilitating a neutralizing humoral response. The presence of conserved and dominant viral CD4 epitopes that support nAb production raises the intriguing question about why such viruses have evolved to maintain these immune determinants. Some of these epitopes may be part of protein regions that do not allow for a high mutational load, as it would result in a loss of viral fitness. However, this possibility would not explain why such conserved regions contain dominant CD4 epitopes. A second possibility is that these viruses maintained these conserved and dominant CD4 epitopes to obtain long-term fitness advantages. Indeed, this may be particularly the case of aggressive and fast replicating cytopathic viruses (including VSV and measles), which are highly infectious and induce widespread cell damage. The early and potent neutralization of such viruses is key for reaching a balance in the host–pathogen interaction, as hosts incapable of neutralizing such viruses are likely to succumb to the infection. In addition, these viruses usually escape Ab responses by infecting a new host prior to the generation of antiviral adaptive immunity and are thus not highly affected by neutralization. For these reasons, these cytopathic viruses were likely not selected to evolve a protective mechanism against the pressure of the adaptive immune system.In contrast, we postulated that the clustering of dominant CD4 T cell epitopes could reduce the immunogenicity of specific antigen portions, suppressing T cell help to B cells targeting those regions. To test this hypothesis, we modeled the relative positioning of B and T cell epitopes in well-studied antigens that efficiently escape broad Ab neutralization, such as HIV gp120 and influenza HA. To minimize the effects of viral antigen variability and HLA class II polymorphisms, we focused on experimentally validated and highly immunodominant CD4 determinants presented by multiple HLAs  and conserved B cell epitopes targeted by bnAbs.The CD4 binding site in HIV gp120 represents, among others, one of the most promising targets to achieve broad anti-HIV protection. Indeed, second-generation anti-CD4 binding-site antibodies  broadly neutralize HIV-1 primary isolates and suppress infection upon intravenous injection in chronically infected patients, representing a potent clinical tool. Intriguingly, immunodominant CD4 epitopes are not scattered throughout the whole gp120 protein; rather, they are clustered in the outer domain  Figure . In addiCrystal structures of HIV gp120 and 3BNC117 bnAb (PDB 4jpv) are presented in Similarly, immunodominant CD4 T cell epitopes (401–430aa)  largely Χ2 = 23.0485, n = 66, p < 0.0001, To quantify the observed differences in the relative positioning of B and T cell epitopes, we grouped together antigens that were strong or poor inducers of long-lasting humoral responses and measured the frequency of B epitopes that were distant from (>15aa), adjacent to (<15aa), or overlapping (0aa) with CD4 T cell epitopes. In support of our hypothesis, the relative positioning of B and T cell epitopes is highly associated with the immunogenicity of the antigens analyzed mutation of viral antigens. Antibodies able to neutralize multiple viral variants are defined as broadly neutralizing, and they represent the ultimate target of most vaccination strategies. BnAbs represent a fundamental tool to mount effective protection against highly mutating viruses, such as influenza and HIV. Despite the prolonged efforts to induce such humoral responses upon vaccination, bnAbs are generally highly subdominant when compared to other strain-specific antibodies.Understanding the rules defining immunodominance is of paramount importance to improve the design of future vaccination strategies. In recent decades, several hypotheses have been suggested to explain the molecular mechanisms underlying the inconsistent induction of bnAbs, including antigen mutation, epitope accessibility, high BCR mutational load required, B-cell precursor frequencies, and HLA class II polymorphisms. Nevertheless, none of these hypotheses fully recapitulates the counter-selection of bnAbs, especially in the context of isolated antigens in vaccination studies. Pioneering work by Berzofsky and Celada demonstrated that BCR or antibody binding to antigens could boost or inhibit the presentation of specific CD4 epitopes based on their relative positioning. Indeed, antibodies can both sterically hinder and inhibit the presentation of CD4 T cell epitopes located within the Ab-bound region or stabilize adjacent epitopes to facilitate their mounting on HLA molecules, thus enhancing their presentation.In the present work, we propose the theory of immunodominance relativity, according to which the relative positioning of B and T cell epitopes within an antigen shapes immunodominance. Indeed, we found that subdominant conserved regions targeted by bnAbs  often overlap with clusters of highly immunodominant CD4 epitopes. In support of this observation, anti-CD4 binding site and anti-HA stem Abs inhibit antigen presentation upon binding, reinforcing the idea that overlapping B and T cell epitopes can lead to the inhibition of antigen presentation. Our analysis suggests that bnAb B cell precursors specific for conserved regions of HIV and influenza inhibit the presentation of immunodominant CD4 determinants upon BCR binding, resulting in poor T cell help during the GC reaction and consequent counter-selection in favor of other dominant B cell clones. On the contrary, non-neutralizing or strain-specific immunodominant B cell precursors may boost the presentation of adjacent immunodominant CD4 epitopes, resulting in increased T cell help and selective advantage during GC reactions. It is worth highlighting that the mechanism we proposed in this study might have different impacts on B cell immunodominance depending on the B epitopes analyzed. Indeed, specific bnAb-targeted B cell determinants may be subdominant as a function of other immunosuppressive mechanisms, which are extensively discussed above. Interestingly, the potent anti-HIV bnAb antibody can eventually be isolated from infected patients years after the infection. A possible explanation for this event in light of our proposed theory is that extensive T–B crosstalk and BCR mutational load in GCs over the years might overcome defects in antigen presentation by bnAb-bearing B cell clones, as the impairment in antigen presentation is significant but not absolute. We would also like to point out that a conformational (rather than linear) epitope analysis might impact epitope interactions and will be of great interest to obtain in this context. Nevertheless, our analysis likely comprises both linear and conformational epitopes, as the immunodominant B regions provided likely contained both linear and structural determinants.This working hypothesis currently lacks formal experimental demonstration; however, we built the model gathering data from a multitude of independent publications over the last four decades and found supportive experimental evidence published by different groups. Finally, we propose that the relative positioning of B–T epitopes may be one additional mechanism that cooperates with the other above-mentioned processes to influence immunodominance. If demonstrated, this theory can improve the understanding of the immune responses against current and future pandemics and will indicate a rational way to design antigens for effective vaccination strategies.To focus the analysis on HLA-independent immunodominant determinants, experimentally validated CD4 and B epitopes that were found to be dominant in at least two different species/mouse strains or multiple patients were selected for further testing. B-cell epitopes were arbitrarily classified as distant from (>15aa), adjacent to (<15aa), or overlapping (<0aa) with dominant CD4 epitopes based on their distance and positioning within the antigenic linear sequence. As such, peptides distant less than 15aa were considered as likely to be near enough for the Ab to suppress the CD4 epitope.To highlight the inter-positioning of B- and CD4-dominant epitopes in 3D structures, the above-mentioned PDB files were modified using Pymol (2.3.5). CD4-dominant epitopes were labeled in blue, B-dominant epitopes in red, and overlapping regions in magenta. Amino acids essential for bnAb binding to antigens are listed in the indicated figures, and those overlapping with CD4-dominant epitopes are highlighted in magenta.p < 0.05 was considered significant. In the summary graphs, the points indicate samples and horizontal lines are the means. Error bars indicated standard error mean (SEM).Prism software (GraphPad 9.0.1) was used for all statistical analysis. A chi-squared test was performed to evaluate the relationship between immunogenicity and the relative distribution of B- and T-cell epitopes within antigens.