Water-based gels containing HDP had less extent of texture hardening, lower degree of crystallinity, and more homogeneous microstructure during 4°C aging. However, for the starch gels in sucrose syrup or coconut milk, HDP induced greater gel hardening, higher degree of crystallinity, and denser gel microstructure during chilled storage. This could be due to the crystallization of sucrose or lipid/amylose-lipid complexes. Nevertheless, HDP enhanced freeze-thaw stability of the gels, regardless of the liquid media used (p ≤ 0.05). According to the consumer test of the model desserts subjected to a single freeze-thaw cycle, the sample containing 50% HDP gel in sucrose syrup or 25% HDP gel in coconut milk gained the highest hedonic score of texture and overall acceptance (p ≤ 0.05).Effects of starch formulation, highly concentrated sucrose solution, and coconut milk on the stability of starch gels kept under chilled and frozen conditions were determined. Gels containing rice starch (RS), tapioca starch (TS) (RS:TS of 1 : 0.85), and hydroxypropyl distarch phosphate  were prepared from 15% starch suspension using water, 45°Brix sucrose syrup or coconut milk as liquid media. After aging at 4°C for 21 days, starch gels had higher hardness and chewiness, with lower cohesiveness and springiness ( Rice-tapioca starch blend has widely been used in ASEAN traditional dessert recipes. In comparison with rice starch gel, the gels containing rice-tapioca starch blends had a lower degree of starch retrogradation, slower changes in the gel texture during chilled and frozen storage, and better freeze-thaw stability , 2. NeveStabilized or substituted starches have generally been used as a texture modifier in food products. These modified starches were shown to reduce hardness with enhancing the elasticity of the starch-based foods. The main mechanism underlying such texture modification was decreasing degree of retrogradation via the bulky substituted groups, including an esterified acetyl group and etherified hydroxypropyl group . In thisRice starch (33.75% amylose) and tapioca starch (31.4% amylose) were obtained from Cho Heng Rice Vermicelli Co., Ltd.  and Thai Tham Factory , respectively. Hydroxypropyl distarch phosphate (HDP) derived from tapioca starch was supplied by Siam modified starch . Food grade sucrose  and coconut milk  were purchased from local market.w/w of starch, dry basis), the blended starch sample was mixed with potable water, 45°Brix sucrose solution, or coconut milk and continuously stirred at 250 rpm for 5 min. The suspension was then heated to 80°C, held for 20 min under continuous stirring at 200 rpm, and subsequently poured into a rectangular mold (4 × 5 × 1 cm3) or 150 ml centrifuge tubes for gel formation. The gel samples in the rectangular molds were kept at 4°C for 3 hours to obtain the freshly prepared gels or stored at 4°C for 21 days before further analyses. The starch gels in the centrifuge tubes were kept frozen (-18°C) before evaluating freeze-thaw stability and microstructure.Composition of the starch blends is shown in 3) was placed at room temperature until its temperature reached 25 ± 3°C. Texture profile analysis was performed using P/100 cylindrical probe, 40% strain of compression level, and 1 mm/s of test speed. Crystallinity pattern found in the aged starch gels was evaluated from the X-ray diffractogram. Samples were prepared with the similar method explained in Arlai and Tananuwong [Hardness of the freshly prepared gels and the aged starch gels was determined by Texture analyzer , using the method of Arlai and Tananuwong . BrieflyFreeze-thaw stability of the gel samples was determined by the method listed in Arlai and Tananuwong . Single Microstructure of aged starch gels, kept chilled or frozen, was determined by a scanning electron microscope (SEM) , under 50x magnification and 15 kV accelerated voltage. Sample preparation was previously explained .2 solution at the weight ratio of 0.8 : 1. The dough was steamed for 5 min, kneaded for 10–15 min, and rested at room temperature for 30 min. The dough was extruded through the perforated plate (0.5 cm pore diameter) into boiling water, hot sucrose solution (45°Brix), or hot coconut milk. The noodle-like thick starch strands were heated in the selected liquid medium for 5 min. The temperature of water, sucrose solution, or coconut milk was maintained at 100 ± 2°C during the heating step. The weight ratio of the liquid medium to starch strands was 3 : 1. The gelatinized starch strands were then removed from the heating medium, packed with additional water, 45°Brix sucrose solution or coconut milk, corresponding to the liquid medium used in the heating step. Appearance of the final products is shown in Model dessert based on the traditional Thai dessert (Plakrim-Kaitow) was prepared using the starch blend, having the starch composition as listed in The sensory evaluation for consumer acceptance testing was performed by 30 untrained panelists, 20–55 years old, composed of 5 men and 25 women. All panelists were familiar with this kind of traditional Thai dessert. The 9-point hedonic scale questionnaire  was used to evaluate the consumer preference of the freshly prepared samples and freeze-thawed samples. Test attributes were appearance, color, flavor, texture (softness), and overall acceptance of the dessert. Temperature of the samples used for sensory evaluation was 35 ± 2°C.A completely randomized design was applied to experiments related to the physical properties of the starch gels. Randomized complete block design was used for the sensory test. The analysis of variance was performed. Difference among means was evaluated by Duncan's new multiple range test at the confidence level of 95%. SPSS software version 23  was used to conduct all statistical analyses.p ≤ 0.05). However, the cohesiveness of both freshly prepared and aged gels was not affected by HDP and the type of aqueous medium used (p > 0.05). Springiness of the freshly prepared gels was not influenced by these two factors (p > 0.05). Nevertheless, the springiness of the aged samples prepared with concentrated sucrose solution was the lowest, regardless of the HDP addition (p ≤ 0.05). Softer texture of the blended starch gels containing HDP could be due to the lower degree of starch retrogradation. Application of HDP neither increased brittleness nor elasticity  of the gels. In addition, long-term aging at chilled temperature induced an increase in hardness and chewiness, with a decrease in cohesiveness and springiness of the gels. The degree of change in hardness and chewiness of the gels largely depended on the level of HDP used.Textural parameters of the aged starch gels in different aqueous media are shown in p ≤ 0.05). This could result from the poor retrogradation ability of HDP. The steric hindrance of the hydroxypropyl functional group was shown to lessen the interaction between amylose/amylopectin chains; thus, the starch retrogradation was retarded [p ≤ 0.05). This could be due to the different mechanisms, apart from starch retrogradation, that induced the hardening process of aged starch gels in the presence of sugar and emulsified fats [For the gels prepared from starch-water suspension, starch retrogradation was proposed as the major phenomenon causing an increase in gel hardness and chewiness during storage at 4°C. It was clearly seen that HDP effectively retarded gel hardening during chilled storage. Lower percentage of the increase in those textural parameters was evidenced in the formula containing greater proportion of HDP p ≤ 0.0. This coretarded . Howeveried fats .θ of 17° and 20°, representing B-type crystallinity of the retrograded starch and amylose-lipid complex V-type crystallinity [θ of 11.8°, 12.8°, 18.9°, 19.7°, 20.9°, 24.8°, 25.2°, 31.2°, and 32.1° [θ of 17° and 20° were also found  showed the exothermic peaks of fat crystallization, having peak temperature of 5°C and 2°C, upon cooling of the homogenized coconut milk from 30°C to -15°C [θ of 19°-20° in the X-ray diffractogram. Therefore, apart from starch retrogradation, sucrose and fat crystallization could play an important role on the hardening of starch gels incorporated with sucrose and coconut milk, respectively, during chilled storage.According to our previous study, multiple phenomena could occur during chilled storage of rice-tapioca starch gels prepared with concentrated sucrose syrup or coconut milk, including starch retrogradation, sucrose recrystallization, and amylose-lipid complex formation . Based oallinity , were obso found . Howeverk et al.  reportedp ≤ 0.05) could be due to less extent of starch retrogradation occurred during chilled storage. Nevertheless, HDP seemed to provide smaller effects on the microstructure of the starch gels prepared with concentrated sucrose solution or coconut milk.The microstructure of the aged starch gels in various aqueous media is shown in p ≤ 0.05), indicating that more sucrose crystals were formed. These gel samples thus had a larger increase in gel hardness and chewiness (p ≤ 0.05). Blended starch gels containing recrystallized sucrose became more brittle and less elastic.The extremely dense structure of the starch gels in sucrose syrup is shown in Figures hewiness . NeverthAs for the starch gels prepared with coconut milk, a denser gel network with less porous structure was obtained Figures , in compThe overall results from this section indicated that the addition of HDP in rice-tapioca starch gels might not effectively retard textural changes during prolonged storage at chilled temperature. The type of aqueous media used to prepare gel samples played an important role in such changes. Starch retrogradation might not be the only phenomenon that induced these quality changes. In the system containing high solute concentration, particularly sugars used in traditional dessert formula, crystallization of solutes during aging could be the main mechanism underlying the changes in gel texture. For the complex gel system containing emulsion, both fat crystallization and amylose-lipid complex formation could highly impact the textural quality of the aged starch gels .p ≤ 0.05). Starch modification via hydroxypropylation and crosslinking was shown to reduce the syneresis in taro starch [Syneresis of the different gel systems after freezing and thawing is presented in o starch , rice sto starch , and sago starch . This coth freeze-thaw cycle was seen in all samples . Hence, it was interesting to evaluate if HDP addition could help improve consumer preference of the desserts containing those starch gels. In the model dessert, preparation of starch gels was modified to minimize the effects of sucrose and emulsified lipids on the gel texture. Dough of the blended starch was prepared and subsequently cooked in water, sucrose syrup, or coconut milk. According to this approach, sucrose and lipids could mainly migrate into the exterior part of the gels and might not thoroughly distribute within the starch gels. Textural modified effects of sucrose and lipids could thus mainly occur at the outer part, while the inner core could maintain the characteristics of blended starch gels. This approach could help mitigate the undesirable effects of sucrose and lipid crystallization on the starch gel texture during storage.HDP was clearly shown to reduce hardness and chewiness but minimally influenced springiness and cohesiveness of the starch gels in different aqueous media  and imprp ≤ 0.05). An increase in the overall acceptability scores of these HDP added samples (p ≤ 0.05) could mainly result from a greater preference in the gel texture. Softness enhancing ability of HDP, without losing the elasticity of the gels, was thus desirable in the unaged desserts prepared with sucrose syrup.Results from the consumer acceptance test of the fresh and frozen model dessert comprising starch gels with HDP are shown in p ≤ 0.05). This could be due to the lower degree of starch retrogradation in the samples containing HDP, which could retard the changes in gel texture during freezing and thawing. Interestingly, for the dessert samples containing sucrose syrup, the gels containing 50% HDP gained the highest hedonic scores of softness and overall acceptability (p ≤ 0.05). However, the desserts with 25% and 50% HDP gels in coconut milks obtained similar hedonic scores of softness and overall acceptability (p > 0.05). Therefore, a higher level of HDP might be required to enhance the consumer acceptability of the dessert samples prepared with sucrose syrup.Effects of HDP on enhancing consumer preference of the model desserts were evidenced in the frozen samples . For anyHDP has long been used as a texture modifier in starch-based products, particularly those requiring long-term cold storage. For freshly prepared gels in water, sucrose syrup, and coconut milk, the formula with a greater level of HDP had lower hardness and chewiness, with the slight difference in springiness and cohesiveness. When considering the stability of normal starch gels, especially those containing low concentration of solutes and lipids, retrogradation was the major mechanism inducing the changes in texture-related quality of the gels during cold storage. Therefore, HDP could be applied successfully to retard those quality changes. However, in complex gel systems containing high concentration of solutes and/or emulsified lipids, stability of the gels could be affected by various phenomena. According to the samples aged under chilled temperature, this study indicated that the crystallization of sucrose and lipid/amylose-lipid complexes greatly influenced the texture of rice-tapioca starch gels incorporated with sucrose syrup and coconut milk, respectively. In those circumstances, HDP could not effectively retard the gel hardening during storage. However, HDP could enhance freeze-thaw stability of all samples, regardless of the aqueous media used in the gel preparation. The ability of HDP to improve the sensory quality of the rice-tapioca starch gels in different aqueous media was also determined in the model dessert. HDP was shown to enhance consumer acceptance of the freshly prepared starch gels in sucrose syrup. Additional benefits of HDP were reflected in the frozen dessert prepared with sucrose syrup and coconut milk. Addition of 25% HDP in the gel formula was sufficient to maximize the overall acceptability of the freeze-thaw samples containing coconut milk. However, for the dessert prepared with sucrose syrup, 50% of HDP was required to achieve the highest overall acceptability. Overall results from this study could expand the viewpoint for the application of HDP in complex starch-based food systems, including the ASEAN traditional desserts.


Pre-harvest sprouting (PHS) severely reduces rice grain yield, significantly affects grain quality, and leads to substantial economic loss. In this study, we aimed to characterize the physicochemical properties and processing quality of the Garumi 2 flour rice variety under PHS conditions and compare them with those of the Seolgaeng, Hangaru, Shingil, and Ilpum rice varieties and the Keumkang wheat variety. Analysis of the molecular structure of starch revealed uniform starch granules, increased proportions of short-chain amylopectin in DP 6–12 (51.0–55.3%), and enhanced crystallinity (30.7–35.7%) in rice varieties for flour compared with the Ilpum cooking rice variety. PHS significantly altered the starch structure and gelatinization properties of Garumi 2. It also caused surface pitting and roughness in Garumi 2 starch granules and decreased their crystallinity. Collectively, the findings of this study provide important novel insights into the effects of PHS on the physicochemical properties of Garumi 2 floury rice for flour. Oryza sativa L.) is one of the three most important crops worldwide. It is a dietary staple for over 34% of the global population. Per capita rice consumption has declined by 2.3% annually over the past 30 years [Rice (30 years . In contflo7)” that is easily ground in small-scale mills [N-methyl-N-nitrosourea (NMU) [To use rice flour as a raw material in processed foods, the source rice material must first be milled by the wet or dry method. The former entails soaking the rice in water and minimizes starch damage. It generates rice flour granules of uniform size and enhances product quality . Howeverle mills . Seolgaele mills ,7,8. Hanea (NMU) ,7,8. Shiea (NMU) ,10,11. INon-glutinous rice used in cooking is transparent and hard because its starch is compact. In contrast, rice varieties for flour are categorized as soft or floury. Floury rice starch granules are sparsely arranged throughout the endosperm, whereas soft rice starch granules are fine and arranged along the external region. In fact, soft and non-glutinous rice starch granules have similar appearance in endosperm cross-sections ,13.In the present study, Garumi 2 is a type of floury rice, while Seolgaeng, Hangaru, and Shingil are types of soft rice. Although these rice varieties for flour have excellent processing quality, little is known about their milling and other properties. The starch in rice varieties for flour has a molecular structure resembling that of wheat. However, polygonal rice varieties have unique starch structures amenable to dry milling and conducive to active pre-harvest sprouting (PHS).PHS refers to seed germination before harvest. PHS occurs mainly under sustained high temperature and high relative humidity. PHS causes yield losses, adversely impacts grain quality, and results in considerable economic loss. Shingil has the lowest PHS ratios; those for Seolgaeng, Hangaru, Shingil, and Garumi 2 were 2.4%, 5.5%, 0.5%, and 23.2%, respectively. PHS significantly varied with transplanting date, nitrogen application, and climate . Korea iThe aims of this study were to elucidate the physicochemical properties of flour prepared by dry milling for five different rice varieties and to contribute to the development of novel rice utilization strategies in the processed food industry.2O5-K2O fertilizers were applied at the rates of 9–4.5–5.7 kg/10 a. Split fertilizer application was performed at basal (50%), tillering (25%), and panicle initiation (25%). When the grain moisture reached 15%, rice husks and impurities were removed with a laboratory rice huller , Garumi 2 was divided into Garumi 2-P (under PHS) and Garumi 2-N (non-PHS) by segregating samples with rice germs blackened by PHS. They were milled along with the other varieties, passed through a 100-mesh sieve, and used as test samples.The rice varieties for flour included Seolgaeng, Hangaru, Shingil, and Garumi 2. They were selected for comparison with the cooking rice variety Ilpum and the wheat variety Keumkang cultivated in Korea. In 2019, all five varieties were planted after tillage of a designated experimental plot at the Gyeongsangbuk-do Agricultural Research & Extension Services. The planting distance was 30 cm × 15 cm, and N-Pw/v) NaOH. The alkaline treatment was repeated until the yellow color disappeared and there was no further biuret reaction. The precipitates were collected, washed with deionized water, neutralized with 1 N HCl, washed again with deionized water, and centrifuged  at 1300× g and room temperature for 10 min. Isolated starch was dried at room temperature and passed through a 100-mesh sieve.Rice grains were soaked in water, and their starch was isolated by alkaline treatment . Soaked * (lightness), ±a* (redness/greenness), and ±b* (yellowness/blueness) values three times to estimate the color difference. The cylindrical container was filled firmly to remove all air for sample measurement. The measurement was made at approximately <1 s, and the wavelength was 400–700 nm. A standard white plate was used after calibration. The color difference ΔE was calculated as To measure the color difference of a rice variety in its flour state, we put sample rice flours in a 30 mm transparent container and then used a colorimeter  to measure Hunter’s LGranule size distribution of the flour from each experimental variety was measured using a laser diffraction particle size analyzer . The samples of flour were immersed in ethanol for 30 s after ultrasonic treatment as previously described .w/v) sulfuric acid was added to terminate the reaction, and the mixture was centrifuged at 1000× g and room temperature for 5 min. Thereafter, 0.1 mL of each supernatant was transferred to a separate conical tube containing 0.1 mL amylose–glucosidase enzyme mixture (20 U/mL). The reaction proceeded at 40 °C for 10 min. Then, 4 mL GOPOD solution was added before a 20 min chromogenic reaction and absorbance was measured at 510 nm. The proportion of damaged starch was calculated as follows:Damaged starch is the portion of starch that is mechanically disrupted during the processes used to extract or refine starch . DamagedRice endosperm amylose and protein were measured in a Cervitec Grain Inspector 1625 . The Cervitec 1625 Grain Inspector used in our experiment is a device system that measures the amylose and protein contents of rice in a non-destructive manner. Milled rice samples were determined as previously described .Starch pasting properties were measured with a Rapid Visco Analyzer . For each sample, 3 g rice flour and 25 mL deionized water were added to the sample canister and spun at 960 rpm for 10 s to produce rice flour suspensions. The mixtures were centrifuged at 160 rpm thereafter until the analysis was complete. The samples were heated to 50 °C for 1 min, and the temperature was increased at 12 °C/min until 95 °C, maintained at 95 °C for 2.5 min, cooled to 50 °C, and maintained at 50 °C for 2 min. The RVA viscogram plotted initial pasting temperature, and peak, trough, final, breakdown, and setback viscosities.v/v) ratio, and the suspension was injected into an aluminum pan with a micro-syringe. The pan was sealed for 1 h and heated from 30 °C to 100 °C at 10 °C/min in a DSC . Onset temperature (To), peak temperature (Tp), conclusion temperature (Tc), and gelatinization enthalpy (ΔH) were measured in triplicate.The gelatinization properties of starch were analyzed by differential scanning calorimetry (DSC) as previously described . BrieflyStarch granule and transverse section morphologies were compared by scanning electron microscopy (SEM). Grains were broken naturally and observed in cross-section by SEM  as previously described . Starch The amylopectin branch chain length distribution was analyzed in a high-performance anion-exchange chromatography system fitted with pulsed amperometric detection (HPAEC-PAD) according to a previously described method , with ceThe crystalline structure of the starches was analyzed by x-ray diffractometry . Crystallinity and crystal strength were compared against the peak position and height measured at diffraction angles (2θ) in the range of 5–40° with a Cu-kα target. The scanning speed was 0.04° 2θ/s, the voltage was 40 kV, and the current was 20 mA. Relative crystallinity was divided into amorphous (Aa) and crystalline (Ac) regions and calculated with the formula  = Ac/(Aa + Ac) × 100) in Origin v. 7.0 .−1 intervals in the range of 3500–1000 cm−1.Fourier transform infrared (FT-IR) was used to examine the external regions of the starch granules . The FT-p < 0.05). Duncan’s multiple range test was conducted to separate treatment means. The software used was Statistical Analysis System (SAS) v. 9.2 . Data are presented as means ± standard deviation (SD) and all tests were conducted in triplicate.One-way ANOVA was performed to identify significant differences among treatment means (Rice flours color was reported in L* (lightness) for Hunter value, ±a* for red/green, and ±b* for yellow/blue . Rice fl50) >50%. In contrast, the other rice flours had granule sizes in the range of 40–73 μm and were ranked in the following order: Shingil > Seolgaeng > Keumkang > Hangaru > Garumi 2. The size of rice flour granule suitable for the production of Paeksolgi, a Korean traditional rice cake, is 60 mesh or 100 mesh [Ground rice flour granule size distribution directly affects final product (bread and noodle) quality as it influences gelatinization properties  and gel 100 mesh . Hence, The main rice endosperm components related to palatability are amylose and protein contents. Palatability increases when the protein content is <7% and the amylose content is low. Protein content is correlated with rice hardness ,37. The The protein and amylose contents were measured with a Cervitec grain inspector. The protein content differed by ≤0.5% among Ilpum, Seolgaeng, Hangaru, and Shingil. Garumi 2 showed a significantly lower protein content than the other varieties but the protein content was higher in Garumi 2-P than Garumi 2-N. Shingil and Garumi 2 had the highest and lowest protein content, respectively. The amylose content was similar in Shingil and Keumkang. Both varieties displayed the highest amylose content among all six strains tested. All rice varieties for flour except Shingil contained lower amylose content than Ilpum. Garumi 2-P exhibited less amylose than Garumi 2-N . This rePasting is the phenomenon following gelatinization in the dissolution of starch. It involves granular swelling, exudation of molecular components from the granule, and eventually, total disruption of the granules . Rice stDSC thermodynamically describes gelatinization by measuring the enthalpy of the endothermic starch gelatinization reaction. DSC measures heat absorption or release by chemical reactions during phase changes such as melting . DSC chaThe starch samples from 5 rice and Keumkang wheat varieties were morphologically compared by SEM . In geneX-ray diffraction (XRD) patterns of the rice varieties were compared from each other in terms of starch granule crystallinity. This parameter was also used to analyze amylopectin crystallinity. The diffraction width was narrow and sharp as crystallinity increased. Hence, the starch granule clusters were regular . Both wh−1/1155 cm−1 ratio. There was also OH stretching at 3200 cm−1/3500 cm−1, OH bending at 1580 cm−1/1640 cm−1, and CH bending at 1330 cm−1/1400 cm−1 [−1/1022 cm−1 is employed to determine the degree of crystallinity of the starch granule surfaces [−1/1022 cm−1) than Garumi 2-N. This result was consistent with the XRD data , smaller granules (40–73 μm), and more uniform starch granule surfaces than the cooking rice variety Ilpum. The RVA analysis revealed that Shingil had the lowest viscosity. The thermal enthalpy of gelatinization was lowest for Shingil and highest for Hangaru, whereas the short-chain amylopectin ratio was highest for Shingil. The XRD analysis demonstrated that the relative degree of crystallinity for Shingil closely approached that of Keumkang. In contrast, Ilpum had the lowest degree of crystallinity. The FT-IR analysis showed that the 1047/1022 cmThe results of this study suggested that Garumi 2 had superior processing quality as characterized by uniform starch granules, high heat transfer resistance, low starch damage rates, and starch granules similar in size to that of wheat flour. Moreover, this research revealed that PHS caused severe starch damage, an excess of small granules, an increase in pasting properties, alterations in short-chain amylopectin composition, changes in starch structure, and a reduction in the degree of crystallinity in Garumi 2. Overall, the findings of this study provide useful insights into the effects of PHS on rice starch physicochemistry and may contribute to the development of novel rice strains suitable for the food processing.


TCR repertoire diversification constitutes a foundation for successful immune reconstitution after allogeneic hematopoietic cell transplantation . Deep TCR Vβ sequencing of 135 serial specimens from a cohort of 35 allo-HCT recipients/donors was performed to dissect posttransplant TCR architecture and dynamics. Paired analysis of clonotypic repertoires showed a minimal overlap with donor expansions. Rarefied and hyperexpanded clonotypic patterns were hallmarks of T cell reconstitution and influenced clinical outcomes. Donor and pretransplant TCR diversity as well as divergence of class I human leukocyte antigen genotypes were major predictors of recipient TCR repertoire recovery. Complementary determining region 3–based specificity spectrum analysis indicated a predominant expansion of pathogen- and tumor-associated clonotypes in the late post–allo-HCT phase, while autoreactive clones were more expanded in the case of graft-versus-host disease occurrence. These findings shed light on post–allo-HCT adaptive immune reconstitution processes and possibly help in tracking alloreactive responses.  The success of allogeneic hematopoietic cell transplantation  strictly depends on the ability of a donor’s engrafted immune system to both secure effective graft-versus-leukemia (GvL) responses and overcome infectious complications resulting from transplant-related immunodeficiency. While the spectrum of specificities recognized by the transplanted donor’s immune system constitutes a theoretical baseline, graft-versus-host disease (GVHD), infections, and tumor surveillance reactions affect the dynamics of recovery and direct a significant portion of the TCR repertoires toward antigenic triggers inherent to these processes , 2. Thus18 complementary determining region (CDR) sequences (TCR diversity is the result of a stochastic process involving rearrangements of the V(D)J segments within the TRA, TRB, TRD, and TRG genes , which, equences . Within equences . CDR3-enequences . These iequences .Over the past 2 decades, several methods have been implemented for the characterization of the T cell recognition spectrum. Early investigations, exploiting spectratyping strategies, were consistent in showing the long-lasting skewing of post–allo-HCT TCR repertoires , 15. HigTaking advantage of lessons learned from the previous attempts to recursively analyze T cell reconstitution dynamics after transplant , 21, 23,We prospectively collected 135 blood samples from 35 consecutive recipients and respective related donors  using thP = 0.035, P = 0.001), accounting for a generalized contraction of the TCR repertoire , while T cell content in the graft, intensity of conditioning regimen, graft source, T cell depletion, type of donor, and donor/recipient CMV status did not affect any of those parameters (We found that donor and recipient pre-HCT ISIs were predictive of a broader TCR repertoire diversity in both early and late time points post-HCT (rameters .P = 0.032) as a result of a direct correlation with a higher number of unique clonotypes . Donor class I HED was also directly correlated with late TCR richness , although no impact was seen on the resulting ISI , as an indirect measure of the extent of immunopeptidome presented and, hence, as an immunogenetic predictor possibly influencing post–allo-HCT repertoire diversification. We found that recipient mean class I HED impacted day +180 ISI . More specifically, shared CDR3 sequences recognizing epitopes from CMV, EBV, and influenza A infections were the most represented in the recipient cohort across the whole posttransplant period. In contrast, autoimmune-associated specificities were the most expanded, especially in the first phase after HCT, while clonotypes related to antitumor adaptive immune responses were mostly identified in the latest phases . Indeed, known specificities accounted for only 8%–10% of recipient TCR Vβ repertoires. Known clonotypes were grouped according to a t phases . Of noteIn an effort to track specific immune responses, we characterized the proportion and the clonal expansion of all the clonotypes with recognizable specificities present in our samples. Interestingly, we found different patterns of expansion across the post–allo-HCT period. Specifically, while clones associated with response to infection and tumor surveillance were more expanded in late posttransplant phases, somehow measuring the antigenic challenges of a more immunocompetent adaptive system, autoreactive clonotypes were instead inflated at day +30 and particularly expanded at GVHD onset .P = 0.043) and lower risk of relapse  while the presence of a higher proportion of pathological expansions negatively affected OS . Moreover, a higher overlap at day +30 was associated with an increased risk of chronic GVHD , implying a possible role of donor-imprinted specificities in fostering this complication.Univariable Cox regression analysis was performed to assess how diversity (ISI) and donor-recipient overlap (overlap coefficient), along with the other quantitative metrics  influenced the probability of survival, acute and chronic GVHD, and relapse see . Overall+ clonotypes may be identified as mono- or oligo-expansion of specific Vβ families in blood samples and tissues from patients developing acute GVHD , previous studies have indicated that patients’ TCR repertoires are severely altered in terms of diversity and skewing in relationship to clinical outcomes and D/R clonotype sharing , 21, 23.In the case of acute GVHD, both in a cross-sectional and serial analysis, we showed that an increased size of a few clonotypes, likely associated with alloreactive responses, accounted for the majority of the expanded clonal spectrum, thus diminishing the overall diversity of the TCR repertoire and impairing competent immune surveillance. Consistent with this conclusion, patients with higher TCR diversity had better survival outcomes and a lower risk of relapse, while a higher median size of pathological expansion and an increased overlap with donor repertoires was associated with risk of chronic GVHD.Based on the theory that CDR3 clonotypes can serve as markers of specific antigenic drives, we have also performed a comprehensive analysis of the spectrum of TCR targets using databases of clonotypes with defined specificities and disease associations. Although most of the clonotypes recognized unknown epitopes, we were able to identify a variety of CDR3 sequences associated with responses to pathogens, autoimmunity, allergy, and tumor surveillance. Of note is that our analysis included an annotation of all the CDR3 sequences described to date in the context of anti-CMV infection. Using this reference, we unveiled the dynamics and the impact of CMV serostatus and infection on diversity and pathological expansion across different patient groups. Although recent studies focused on posttransplant immune dysfunction after CMV reactivation , 21, herThe specificity spectrum overlap analysis clarified the origin of the recipient repertoire; most of the clonotypes in recipients were newly generated or present at frequencies of less than 1/5000 , and only less than 3% were composed by clonotypes overlapping with the respective donor repertoire. Indeed, only a small fraction of pathologically expanded recipients’ specificities originated from expanded donors’ clonotypes, suggesting that either individual recipient’s antigenic drivers or very-low-frequency donor-derived clones expand in order to maintain the TCR complexity. These findings highlight the prevailing role of individual patients’ antigenic challenges and posttransplant thymic rebound in shaping the new TCR spectrum , 32.+ and CD8+ TCR repertoires on post–allo-HCT immune reconstitution and complications, our study provides a systematic and in-depth overview of TCRβ repertoire, taking into consideration the multidimensional nature of TCR diversification, expansion, and antigen recognition, together with the interactions dictated by posttransplant complications and pretransplant immunogenetic determinants.Despite the limited sample size and the absence of cell sorting, which may help to better understand the relative contribution of CD4Our analysis of post–allo-HCT TCR reconstitution demonstrates that NGS-based clonotyping of CDR3 tags can be used for analytic purposes to assess the status of T cell immune reconstitution as well as to detect and monitor specific T cell clones as markers of pathological immune processes.Genomic DNA was isolated directly from fresh or cryopreserved unfractionated peripheral blood mononuclear cells with the Nuclei Lysis Solution (Promega) according to manufacturer’s instructions.Immunosequencing of the CDR3 regions of human TCRβ chains was performed using the ImmunoSEQ Assay (Adaptive Biotechnologies), as previously described –35. In bRearrangement details from HCs (age matched with donors recruited in our cohort) derived from the Emerson and DeWitt study  within the normalized downsampled data set. Details concerning the bioanalytic workflow are reported in Sequencing results are accessible through the publicly available repository of the immuneACCESS platform (Adaptive Biotechnologies).https://clients.adaptivebiotech.com/pub/pagliuca-2021-jcii). All other remaining data (including rearrangement details of the downsampled TCR data sets) are available within the article and supplement.Genomic data that support the findings of this study have been deposited in the immuneACCESS platform  in a univariable setting  and Excel (Microsoft 365).All metrics in the study were generally treated as continuous variables and categorized when needed. Median, IQR, mean, and 95% CIs were used where appropriate. Frequency and distribution of categorical variables were expressed as a percentage. For all relevant comparisons, after testing for normal distribution, comparative analyses between 2 groups were performed by 2-sided paired or unpaired Student’s  setting . OS was ectively . All staThis is a prospective noninterventional monocentric study, conducted at our institution, under the institutional review board of the Cleveland Clinic Foundation (IRB 5024 and 4927). All consecutive patients undergoing a first allogeneic HCT for myeloid malignancies and who gave their consent to this research were included. Patient recruitment was conducted between September 2015 and January 2017; blood samples were collected prospectively from recipients  and respective related donors  for TCRβ sequencing see .All patients had been regularly followed until September 2020 (or death). Pertinent clinical data, including age, sex, disease diagnosis, type of transplant, HLA typing, conditioning regimen, GVHD prophylaxis, acute and chronic GVHD, disease relapse, and other complications, were collected. Objective medical data, including ancillary testing, laboratory results, medical complications, and medication profiles, were abstracted through standardized chart review after each visit. All patients provided written informed consent before enrollment, in accordance with the Helsinki Declaration of 1975, revised in 2008 .SP collected, analyzed, and interpreted the data; performed the bioinformatic and statistical analysis; and wrote the manuscript. CG and SH performed clinical data collection and participated in the analysis interpretation. RZ helped with statistical methodology development. SK, CMK, LT, MZ, AK, HA, BKJ, and YG helped in sample and data collection. TL and VV edited the manuscript, helped in data interpretation, and gave helpful intellectual insights during the study. HEC, BJP, BKH, and NSM actively participated in patient recruitment, management, and follow-up and critically revised the manuscript. JPM designed and conceptualized the study, interpreted the data analysis, and edited the manuscript. First and last authors took responsibility for the integrity and the accuracy of the data presented. All authors reviewed and approved the final version of this manuscript.


However, during the last few years, we have observed robust clinical and pre-clinical scientific investigations on zirconia both as a prosthetic and an implantable material. At the same time, we have witnessed consistent technical and manufacturing updates with regards to the applications of zirconia which appear to gradually clarify points which until recently were not well understood. Methods: This critical review evaluated the “state of the art” in relation to applications of this biomaterial in dental components and its interactions with oral tissues. Results: The physico-chemical and structural properties as well as the current surface treatment methodologies for ZrO2 were explored. A critical investigation of the cellular response to this biomaterial was completed and the clinical implications discussed. Finally, surface treatments of ZrO2 demonstrate that excellent osseointegration is possible and provide encouraging prospects for rapid bone adhesion. Furthermore, sophisticated surface treatment techniques and technologies are providing impressive oral soft tissue cell responses thus leading to superior biological seal. Conclusions: Dental devices manufactured from ZrO2 are structurally and chemically stable with biocompatibility levels allowing for safe and long-term function in the oral environment.Background: Dental components manufactured with zirconia (ZrO Additionally, they should not affect or interfere with the recipient’s physiology and general health. Prosthetic components and implants made from zirconia  and baddeleyite (ZrO2) minerals which are the major sources of zirconium. Zirconate is comparatively more abundant, but less pure, and hence requires significant processing in order to obtain zirconia [2) resulting from baddeleyite, which is also known as zirconia, is a coarse oxide and presents as a monoclinic crystal structure at room temperature. However, it can be purified and processed at high temperatures and under high pressure to obtain a tetragonal or a cubic structure. The cubic structure is hard, optically flawless and translucent in appearance and is mostly used for making precious stones or gas sensors [Regarding the material properties, zircon is a grey-white metal mined from the earth, it occurs in nature and is a precursor of zirconium; whereas zirconia is an oxide of zirconium which is a ceramic. Zirconia does not occur naturally as a pure oxide, therefore it needs to be extracted from minerals such as zirconate (ZrOzirconia . Baddele sensors .2O3), calcium oxide (CaO) or magnesium oxide (MgO) are added to zirconia to stabilize it. The addition of different amounts of stabilizers also allows the formation of partially or fully stabilized zirconia which, when further combined with the changes in processes, may result in ceramics with high flexural strength/fracture toughness and excellent chemical resistance. A fully stabilized zirconia can be obtained by adding a sufficient number of stabilizing oxides, such as 16 mol% magnesia (MgO), 16 mol% limestone (CaO) or 8 mol% yttria (Y2O3). Since the partial stabilization of zirconia is also obtained with the same oxides, but in smaller proportions (like 2–3 mol% yttria), a multiphase structure is created, which usually consists of tetragonal and cubic zirconia in majority along with the monoclinic form precipitated in small amounts [Discrete crystallographic structures are seen in the spatial arrangement of atoms in zirconia, thereby representing a property called polymorphism. Zirconia has been classified based on specific geometry and dimensional parameters into three phases or crystal structures, namely, the monoclinic, tetragonal and cubic phases . Pure zi amounts . The tra amounts . Thanks 2 (hafnium oxide) which has very similar structural and chemical properties to zirconia. Zr4+ ions have a coordination number of seven for the O2− ions that occupy the tetrahedral interstices, thus making the average distance between the Zr4+ ion and three of the seven O2– ions, 2.07 Å. As the average distance between the Zr4+ ion and four O2– ions in the structure is 2.21 Å, one of the angles (134.3°) differs considerably from that of the tetrahedral value (109.5°). Therefore, the structure of the oxygen ion is not planar but a curve is seen in the plane of the four oxygen ions, whereas a completely erratic plane is seen in case of the other three oxygen ions [The natural form of zirconia, i.e., baddeleyite, contains approximately 2% HfOgen ions .4+ ions show a coordination number of eight, but the shape once again gets distorted because of the fact that four oxygen ions are present at a distance of 2.065 Å in a tetrahedron plane, whereas the other four are at a distance of 2.455 Å in another tetrahedron plane which is elongated and rotated by 90° [In the tetragonal phase, zirconia appears as a straight prism with rectangular sides. Zrd by 90° .The structure of cubic zirconia is represented as a simple cubic lattice with eight oxygen ions, surrounded by a cubic arrangement of cations, which is known as fluorite, i.e., the oxygen ions are present at the tetrahedral interstices of a cubic lattice structure (CFC) of cations .Sandblasting, which is also known as airborne particle abrasion, produces a surface topography that has micro-roughness. Various parameters affect the roughness that is created on the implant surface, this includes the size, shape and kinetic energy of the particles used in the sandblasting process . During The process of acid etching is performed with either hydrofluoric acid, nitric acid or sulfuric acid. Acid etching treatment can also be used to overcome alumina contamination as it has been proven to remove the alumina residues . Heat trThis technique involves coating the surface of the material with a specific infiltration glass and then heating it at a temperature higher than its glass transition temperature. This is followed by the infiltration of molten glass that occurs between the material grains . This tePolishing gives a comparatively smoother surface than acid etching and sandblasting . It is kIn contrast with sandblasting and acid etching techniques, laser treatment exerts zero risk of surface contamination as there is no direct contact between the laser and the biomaterial . The las−) and oxygen (O2−) groups are formed on the outermost layer. The formation of hydroxylated oxide surface improves the surface reactivity with the surrounding ions, amino acids and proteins in the tissue fluid. When compared to hydrophobic surfaces, osteoblasts cultured on hydrophilic surfaces have shown to exhibit higher levels of differentiation markers, including alkaline phosphatase and osteocalcin [Various studies have shown that bone implant contact of the implants treated with ultraviolet (UV) light was deeply enhanced because of the effect of superhydrophilicity . A matereocalcin ,50. The eocalcin ,52.4)(CP)-based coatings are generally fabricated using plasma-spraying techniques. Despite its numerous drawbacks, this technique is said to provide low cost and a high deposition rate [Coating of yttrium stabilized zirconia (YSZ) with reinforced hydroxyapatite (HA) has shown positive results in the enhancement of adhesive strength and coating stability . Becauseion rate ,54  has been proposed as an alternative to traditional techniques. An approach that combines both plasma electrolytic oxidation and electrophoretic deposition has been made to fabricate and coat zirconia or hydroxyapatite film on zirconium ,58,59. G implant .Biomimetic surface modification, also called biofunctionalization, involves immobilization of biomolecules on the surface to change their biochemical properties and biological responses . BiofuncAn autonomous process by which the components are organized into patterns or structures without any external intervention is called self-assembly  Table 1Table 1. 2, as well as innovations in the base-material can induce individualized cellular responses promoting a better performance of the device once it is implanted [2 have attracted the attention of researchers and clinicians due to their biocompatibility properties  when compared to the unmodified surfaces [2. In addition, in vitro tests showed that the expression of genetic factors such as osteopontin, osteocalcin and BMP-2 was much greater for the treated surface [Animal studies demonstrate more conclusive results with regards to osseointegration. In rabbits, Mostafa and Aboushelib developed a bioactive surface (coated with nano-hydroxy apatite particle or platelet-rich plasma) on ZrOsurfaces . Likewissurfaces  revealed surface . Moreove surface   and the Periotest. It should be noted that none of those techniques and modalities measure osseointegration per se, they rather assess implant stability. The Periotest assesses stability by measuring the amount of micromovement of the implant and the RFA measures the frequency returning from an implant. In both cases, values are given to the measurements and correspond to a certain level of implant stability. Torque removal forces are used as a biomechanical measure for anchorage or osseointegration in which the greater forces required to remove implants may be portrayed as an increase in the strength of osseointegration  Table 3Table 3.In the study conducted by Sennerby et al. , it was Hence, it can be concluded that the torque removal value for zirconia implants was surely increased after the process of surface modification but could not exceed that of titanium implants.To test the biocompatibility of zirconia, various in vitro tests were conducted on osteoblasts, fibroblasts, lymphocytes, monocytes and macrophages where it was observed that zirconia had no cytotoxic effect on the bone forming cells and rather made them capable of elaborating the extracellular matrix by synthesizing various essential and structural proteins . Zirconi2O3 were inserted into the femurs of monkeys, zirconia was found to be biocompatible to hard tissue when tested in vivo. As compared to alumina, no difference in bone reaction was seen in case of zirconia [During in vivo biocompatibility tests of zirconia, it was found that when it was implanted in the soft tissue, a thin layer of fibrous tissue encapsulated it, like what is seen with alumina. Furthermore, no cytotoxicity was observed in the soft tissue in relation to wear products of zirconia ,131. Acczirconia . In a stzirconia .Titanium implants have been recognized for many years as the gold standard in implantology. However, currently, a range of studies have been comparing the new possibilities of implants made from zirconia . Tetè etThese comparisons between the different biomaterials for implant development show the intense evolution of materials derived from zirconia with promising results to achieve the results observed with the gold standard.2 have seen increased application in dental clinics, largely due to technological developments in relation to treatments and changes in the properties of this material. One of the main restrictions on the use of zirconia was its high difficulty in being superficially treated in order to have a period of rapid osseointegration [2-based surfaces [2 after the period of osseointegration or cementation are extremely difficult due to their hardness and the possibility of creating cracks with the use of specific burs [In recent years, application of dental components derived from ZrOegration ,14. Howesurfaces ,9,140. Csurfaces ; technolsurfaces . Finallyfic burs . Thus, t2. Many methodologies have evolved towards the design of micro- and nano-topographies in attempts to elicit optimal cellular response and behavior. In addition, many in vitro and in vivo studies reveal advantages for ZrO2 with regards to the interaction with soft tissue cells such as fibroblasts, blood cells and epithelial cells; the same applies to proteins adsorption, cells alignment and biocompatibility.This critical review explored the main physico-chemical and biological aspects that influence the healing process and adaptation of dental components manufactured with ZrO2 surfaces showed excellent bone apposition and elicit better, or at least, similar, soft tissue responses comparing with other recognized dental materials for oral rehabilitation, demonstrated over time under appropriate conditions and when undergoing similar or identical surface treatment. Advantages in relation to periodontal seal are demonstrated by better attachment and alignment of collagen fibers with components developed in ZrO2. Less bacterial adhesion on smoother zirconia ceramic components, mainly prosthetic components, has been demonstrated and proven by a large range and number of reports. The prospects for a broader range of components made from ZrO2 have become increasingly promising for all areas of oral rehabilitation such as intra-bone, transmucosal and supra-gingival components.Clinically, ZrONevertheless, the constant technological evolution of the surface treatment of materials as well as in their macro, micro and nanoscopic design, reveal the need for intense research with clinical controls for a safe and predictable application of innovative materials in the market. At this stage it is not possible to clearly determine which combination of surface properties of zirconia performed the best at all cell levels, it is still unclear and remains a challenge for researchers and clinicians. To solve this challenge there is promise of multifunctional surfaces . Thus, more studies are needed to improve bioactive surfaces for supra and subgingival implantable ceramics.


Bifidobacterium animalis subsp. lactis (Bifido). Ethyl cellulose (ETC) was used as a shell material with different core compounds . The core–shell microcapsules have an average diameter between 3 µm and 15 µm depending on the core compounds, with a distinct interface that separates the core and the shell structure. The ETC microcapsules displayed relatively low water activity (aw below 0.20) and relatively high values of viable cells (109–1011 CFU/g), as counted post-encapsulation. The effect of different core compounds on the stability of probiotics cells over time was also investigated. After four weeks at 30 °C and 40% RH the electrospray encapsulated samples containing Bifido–glycerol in the core showed a loss in viable cells of no more than 3 log loss CFU/g, while the non-encapsulated Bifido lost about 7.57 log CFU/g. Overall, these results suggest that the viability of the Bifido probiotics encapsulated within the core–shell ETC electrosprayed capsules can be extended, despite the fact that the shell matrix was prepared using solvents that typically substantially reduce their viability.Electrosprayed ethyl cellulose core–shell microcapsules were produced for the encapsulation of probiotic  Probiotics are live microorganisms that are intended to have health benefits when consumed or applied to the body. Probiotics are generally sensitive to harsh environmental and processing conditions such as low pH, digestive enzymes, heat treatment, water activity, and molecular oxygen. To ensure probiotics’ healthy benefits, their metabolic activity has to be preserved in foods and during the complex digestive process until they reach the target site of the gastrointestinal tract (GIT) ,2.6–108 CFU of live probiotics per gram of foodstuff [Probiotics’ health benefits can be ensured by supplying a minimum of 10oodstuff . Thus, poodstuff .Several technologies are available for the encapsulation of probiotics in foods  . Among aAlternatively, electrospray stands out as a promising encapsulation technology of probiotics, producing dried nano-microcapsules from the breakup of a flowing liquid by means of an electrical field, at room temperature without heat, and without affecting the viability of the living cells . FurtherPolysaccharides such as pullulan , alginatBifidobacterium animalis subsp. lactis Bb12 was encapsulated within electrosprayed capsules consisting of whey protein concentrate (WPC) and pullulan [Lactobacillus acidophilus cells were encapsulated within electrosprayed core–shell alginate–zein microcapsules [Lactobacillus plantarum [Bifidobacterium longum subsp. infantis CECT 4552 [For instance, pullulan . Lactobacapsules . In anotECT 4552 .Ethyl cellulose (ETC) is a linear polysaccharide produced by the replacement of the cellulose’s hydroxyl end groups with ethyl end groups. ETC is non-water soluble and more hydrophobic than other commercial cellulose ethers , and it Furthermore, it has been demonstrated that ETC can be used to produce electrospun fibers and electrosprayed capsules ,20,21,22Bifidobacterium animalis subsp. lactis (Bifido) probiotic cells. Different compounds  were included within the microcapsule core. ETC microcapsules’ functionality to preserve the viability of the encapsulated Bifido probiotics was evaluated.Based on the potential of ETC to be used as encapsulating material for bioactives, in this study electrosprayed core–shell ETC microcapsules were developed for the encapsulation and protection of w/w) ethoxy groups, degree of substitution of 2.5 [All chemicals and materials used in this study, including ethyl cellulose  was obtBifidobacterium animalis subsp. lactis (Bifido) (strain deposit number DSM 33443) was selected as the model strain supplied by Chr Hansen A/S . The strain was subjected to freeze pelletization, and stored at −80 °C.w/v of ETC in ethanol 96%  or 8% w/v of ETC in acetone. The core solutions were prepared after thawing the Bifido cells at ambient conditions and mixing them with the different compounds  two syringe pumps , each one feeding the shell and the core solution at the flow rate of 0.42 mL/min and 0.21mL/min to the co-axial needle, respectively (concentric needles with an inner diameter of 1.42 mm for shell solution and 0.48 mm for core solution), and (ii) a high voltage generator  was utilized to provide an applied voltage of +30 kV at the co-axial needle and an applied voltage of −5 kV at the collector. The distance from the co-axial needle to the collector was 10 cm. The electrospraying process was performed in a fume hood to keep the process under 30% relative humidity using nitrogen. The collected capsules were them removed from the collector and stored at −80 °C prior further analysis.v/v in 0.1 M phosphate-buffered solution, pH 7.4) for 1 h. The samples were then dehydrated in a graded ethanol series  followed by immersion in hexamethyldisilazane (HMDS). Samples (free Bifido and ETC core–shell capsules loaded with Bifido) were coated with 6 nm of gold for better conductivity using a sputter coater  and imaged in a Quanta FEG 3D SEM . The microcapsule diameter was calculated using Image J analysis software , by measuring 50 different capsule diameters for each sample. The average diameter was calculated from these measurements for each microcapsule.The size and morphology of the electrosprayed ETC core–shell capsules and free Bifido were investigated using a scanning electron microscope (SEM). Free Bifido samples were fixed with glutaraldehyde  solution  at 37 °C for 10 min protected from light was performed prior to visualization using an Axioplan Imager Z1m microscope .−1 to 650 cm−1 at room temperature (20 ± 2 °C). Each measurement was derived an average from 4 scans.FTIR spectra of the core–shell microcapsules loading Bifido and free Bifido cells were obtained using a FTIR spectrometer . The FTIR spectra were acquired at a wavelength ranging from 4.000 cmThe water activity (aw) of the ETC–Bifido microcapsules was measured using a water activity analyzer , at 25 °C.Bifidobacterium animalis were stored in a desiccant cabinet, , containing a saturated solution of potassium carbonate at a relative humidity (RH) of 40% at 30 °C. The viability of encapsulated and non-encapsulated Bifido cells was determined by colony-forming unit (CFU) analysis. The samples were diluted 1:100 with Maximum Recovery Diluent ) at 37 °C.Electrosprayed samples containing w/v % L-cysteine hydrochloride monohydrate by pour plating, aiming for colony counts between 30–300 CFU/plate. Plates were incubated at 37 °C under anaerobic conditions for 3 days, and the results were calculated as the average of 2 plates. Cell viability was measured at the time points of 0, 2, and 4 weeks.The suspension was homogenized by stomaching  at intermediate speed for 2 min, and further dilutions were performed in MRD. Appropriate volumes of appropriate dilutions were plated in de Man Rogosa Sharpe (MRS) agar supplemented with 0.05 The encapsulation efficiency (EE) of the microcapsules was calculated by determining the number of viable cells inside the capsules (N) divided by the number of viable cells in the initial solution (N0), as expressed in (1) : (1)EE .Student’s The Bifido cells used in this study displayed a typical rod-like structure, with an average length of about 1–1.5 µm as observed by SEM , which iThe size and the morphology of the electrosprayed ETC core–shell capsules loaded with Bifido are shown in The electrosprayed core–shell capsules that contain only Bifido in the core (S1), displayed an average diameter of 3.33 ± 1.18 µm . HoweverAn increase in the average diameter of the microcapsules was also observed when acetone was used as ETC solvent (S4), in comparison to the capsules prepared using ethanol (S3) . FurtherTo study the distribution of the encapsulated cells within the microcapsules, the cells were stained and analyzed using confocal microscopy. The composition of the capsule, the permeability of oxygen, and the aw are key parameters to preserve the viability of probiotic cells . In respWhen ethanol was used as the ETC solvent for the microcapsule shell, the water activity increased in the order S1 < S2 < S3 . The slightly higher water activity of the sample S2 comparatively to S1 could be attributed to the presence of hydrophilic maltodextrin, which increases the affinity for water binding. The highest value of aw was found for S3, most likely due to the hydrogen bonding and molecular dynamics of the water of the aqueous dispersion of Bifido–glycerol mixtures in the core , which i−1 corresponding to amide groups of the existing proteins at the cell membrane. Furthermore, the bands at 2968, 2973, and 2878 cm−1 are mainly attributed to the asymmetric stretches of methyl and methylene groups of the lipid components of the membrane, mainly fatty acids, as also suggested elsewhere [−1 could be related to the (C-O) vibrations of COO- groups from esterified lipids and fatty acids [−1 are provided by the (P=O)s of PO2− groups [The FTIR spectra of free Bifido  shows th− groups .−1 was observed, due to the -OH groups of the polysaccharide backbone [−1. Other relevant vibration peaks around 1054–1011 and 1275 cm−1 were mainly attributed to C-O-C stretch and C-H bending, respectively [The shell compound of the microcapsules was ethyl cellulose, and consequently a distinct peak at 3464 cmbackbone . The intectively . It is iFor the samples prepared using ethanol as a shell solvent (S1–S3), S1 displayed the lowest value of viable probiotics at 9.62 log CFU/g. The number of viable encapsulated probiotics for S2 and S3 was about 10.90 log CFU/g and 10.01 log CFU/g, respectively. The increase in the initial viable cells counts of the sample S2 in comparison to S1 suggests that maltodextrin has some protective effect on Bifido (note the similar encapsulation efficiency of these samples). Sample S4 (ETC shell electrosprayed using acetone), showed slightly higher cell viability counts (11.28 log CFU/g) in respect to S3 (ETC shell electrosprayed using ethanol). The higher number of viable encapsulated probiotics detected in S3 and S4, in respect to S1, could be due to the higher initial cells number, the higher EE, and also due to the protection of the glycerol core compound. Overall, these results suggest that the probiotic cells encapsulated within the core-shell ETC electrosprayed capsules preserved their viability after electrospraying, despite the fact that the shell matrix was prepared using solvents that typically substantially reduce their viability. This further confirms that the solvent of the shell matrix (ETC) did not migrate into the core of the microcapsules during electrospray processing, where the probiotics were mostly located .Due to the higher encapsulation efficiency obtained for samples S3 and S4, their stability over time was also assessed using plate counting (30 °C and 40% RH) and compared with non-encapsulated Bifido cells (free Bifido) . In the The non-encapsulated Bifido cells lost about 3.84 log CFU/g of their viability, within two weeks, and showed a log loss of 7.57 CFU/g after four weeks at 30 °C and 40% RH. The electrospray encapsulated Bifido cells only lost about 3 log CFU/g of viable cells B. Previo9–1011 CFU/g) counted post-encapsulation. After four weeks at 30 °C and 40% RH, the encapsulated samples contained Bifido–glycerol in the core, showed a loss in viable cells no more than 3 log CFU/g, while the non-encapsulated Bifido lost about 7.57 log CFU/g. Overall, the present study clearly demonstrates the potential of electrosprayed ETC core–shell microcapsules to encapsulate probiotic cells and to extend their viability over time.Electrosprayed ethyl-cellulose core–shell microcapsules were produced for the encapsulation of Bifido probiotic cells. The average diameter of the core-shell capsules was between 3 µm and approximately 15. A distinct shell and core microstructure were observed, indicating that the shell matrix of ETC and the solvent did not migrate into the core of the capsule, where the probiotics were mostly located. The core–shell microcapsules of ETC displayed a relatively low aw (below 0.20) and relatively high values of viable cells (10


Lung transplant patients have the lowest long-term survival rates compared to other solid organ transplants. The complications after lung transplantation such as primary graft dysfunction (PGD) and ultimately chronic lung allograft dysfunction (CLAD) are the main reasons for this limited survival. In recent years, lung-specific autoantibodies that recognize non-HLA antigens have been hypothesized to contribute to graft injury and have been correlated with PGD, CLAD, and survival. Mounting evidence suggests that autoantibodies can develop during pulmonary disease progression before lung transplant, termed pre-existing autoantibodies, and may participate in allograft injury after transplantation. In this review, we summarize what is known about pulmonary disease autoantibodies, the relationship between pre-existing autoantibodies and lung transplantation, and potential mechanisms through which pre-existing autoantibodies contribute to graft injury and rejection. Lung transplantation (LTx) is the only viable option for many chronic end-stage pulmonary diseases (CPD). The number of LTx performed annually in the US is rapidly increasing, and the demand for donor lungs far exceeds availability. Despite the improved surgical techniques and post-transplant management, the long-term survival has not significantly improved over the last decade and remains the lowest compared to other solid organ Tx . AccordiArguably, organ transplantation was made possible through optimization of surgical techniques and subsequent manipulation of the immune system. Significant improvements have been made in controlling the recipient’s immune system post-transplant, largely by modulating T cell immune responses. While modulation of adaptive immunity is essential for graft survival other factors occurring earlier in the transplant process also play a role in graft injury. Initial immune-mediated graft injury occurs upon reperfusion, referred to as ischemia reperfusion injury (IRI). Following IRI, activation of innate and adaptive immune response drives targeted graft damage. This damage manifests as post-Tx complications such as PGD, acute rejection, and CLAD, all of which predispose to increased risk of mortality. The generally accepted model of immune-mediated graft damage proposes a feed-forward mechanism starting with donor graft injury, IRI-mediated immune activation and graft damage, thereby promoting subsequent adaptive immune activation and T cell specific graft damage. However, this model does not take into account the recipients’ lung specific pre-Tx immune system. Patients with end-stage lung diseases, such as chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD) often have autoimmunity, which is increasingly being recognized as a potential driver of graft injury post transplantation , 3.The presence of a lung-specific autoreactive immune system pre-transplant may well predispose targeted attack of the donor lung upon implantation, and further exacerbate the alloimmune effector mechanisms activated post-transplant. Immune-mediated graft damage remains a major obstacle, and manipulation of the recipient immune system currently only occurs upon transplantation. Therefore, gaining a better understanding of the immune factors present within the LTx recipient prior to transplantation and how these factors contribute to shaping post-transplant graft outcomes can have a significant impact on the patient outcome. In this review, we will focus on the impact of pulmonary disease-associated autoantibodies and pulmonary disease-specific autoantibodies in LTx, and postulate on the clinical significance of autoantibodies identified in patients.Over the last 20 years, accumulated data proports a role for autoimmunity in pulmonary disease pathogenesis and progression of certain lung pathologies . How pulAutoantibodies are antibodies produced by the immune system with reactivity to self-antigens. Multiple mechanisms can render host molecules antigenic, interestingly, those same mechanisms can occur during or as a result of pulmonary disease onset. For example, environmental exposure to pollutants, such as those found in cigarette smoke, a major risk factor for COPD development, can drive mutations and post-translational modifications such as oxidation, carbonylation, and citrullination to pulmonary peptides rendering them antigenic . FibrotiChronic obstructive pulmonary disease (COPD) is a group of progressive pulmonary disorders characterized by chronic pulmonary inflammation and damage to small airways and alveolar airspace that results in airflow limitation. The specific pathogenesis of COPD remains unknown, but it is widely accepted that cigarette smoke (CS) exposure is one of the leading causes. CS promotes innate and adaptive immune cell infiltration into the lung and airways of COPD patients , 11. IncAutoantibodies and autoantigens have been successfully identified in COPD patients and animal models of disease. A systematic review of all clinical studies investigating autoantibodies in the context of COPD was published in 2019 by Byrne et al. . The revMany of the most well-studied autoantibodies, such as the ones listed above, are common to other autoimmune diseases such as rheumatoid arthritis, systemic sclerosis, and lupus . While tThe most glaring gap in the literature, as discussed in Duncan 2012, is the functional characterization of autoantibodies in COPD pathogenesis . Thus faInterstitial lung disease (ILD) is a group of multiple pulmonary diseases characterized by progressive scarring or fibrosis of the lungs. ILD is an extremely complex disease because it is commonly associated with connective tissue diseases (CTD-ILD) such as rheumatoid arthritis, lupus, and sclerosis, or non-connective tissue disease origin referred to as interstitial pneumonia with autoimmune features (IPAF). In this context of CTD-ILD, ILD can be one of many manifestations of an established CTD or may be the only manifestation of the CTD. Interestingly, many CTDs can be categorized as autoimmune disorders suggesting that ILD may also comprise an autoimmune component. For example, ILD manifests and is the leading cause of mortality in Systemic-Sclerosis, an extensively studied autoimmune disease with anti-nuclear autoantibodies (ANA)  and anti-endothelial cell antibodies (AECA) in greater than 90% of patients with potential pathogenic roles . AdditioWhether or not the majority of autoantibodies identified in CTD-ILD patients are involved in disease pathogenesis remains unknown, but their clinical significance as diagnostic markers that shape patient is growing in significance. For example, a subset of clinically amyopathic dermatomyositis (CADM) patients with rapidly progressive ILD have been shown to present with circulating anti-CADM140/MDA5 autoantibody . StudiesIn 2011, guidelines from the American Thoracic Society/European Respiratory Society/Japanese Respiratory Society and Latin American Thoracic Association highlight ANA, anti-CCP, and RF as necessary autoantibodies to test for in all potential ILD patients . Not surPulmonary disease autoimmunity and lung transplant can be seen as a ‘perfect storm’ for graft injury and rejection. While several studies have investigated autoantibodies in context of lung transplantation few have attempted to determine the clinical significance or the mechanism of action of pre-existing autoantibodies, present prior to transplantation, on post-transplant outcomes. It is important to note that many of the autoantigens identified in pulmonary disease patients are not lung-specific, but rather have a broad biodistribution. While pulmonary disease-associated autoantigens that are not lung-specific may still be clinically relevant, it is possible that lung-restricted self-antigens may also be clinically relevant with regard to LTx outcomes because they could represent biomarkers of graft health/damage or contribute pathogenically to graft damage.In this context, pre-existing autoantibodies that recognize lung-restricted self-antigens can be produced prior to LTx . Lung-reAlthough there are not any studies directly comparing the tissue damage that occurs in pulmonary disease to graft damage, it is reasonable to hypothesize that similar enzymatic degradation of extracellular matrix occurs early post-transplant. The innate immune cells associated with injury in both pulmonary disease and graft damage are essentially the same, and can induce post-translational modifications to ECM proteins through secretion of proteases and production of ROS. Along this line of thinking, the LTx recipient’s pre-existing autoantibodies could recognize self-antigens that are present and exposed in the new graft. Therefore, the recipient’s immune system is primed to target and attack the graft prior to transplant which could manifest as severe pre-existing autoantibody-mediated graft damage once the graft is implanted. Potential mechanisms by which pre-existing autoantibodies mediate graft damage could be increased complement activation and/or FcyR binding, which we discuss later in this review.Although it is enticing to hypothesize about the potential pathogenicity and clinical relevance of pulmonary disease autoantibodies in LTx, definitive experimental and clinical data supporting potential mechanisms are lacking. The presence of pre-existing autoantibodies in transplant patients and their association with poor clinical outcomes has been well established in other solid organ transplants, specifically in renal and heart transplants; driving the hypothesis that pre-existing autoantibodies may also play a role in LTx outcomes , 31. As Primary graft dysfunction (PGD) is a progressive acute lung injury syndrome that manifests within the first 72 hours after lung transplantation and affects 25-30% of LTx patients . The ISHRecently, the presence of pre-existing autoantibodies has been associated with increased risk of PGD in multiple retrospective clinical studies. The most well studied pre-existing autoantibodies correlated with PGD onset are Collagen V (Col V) and K-alpha1-tubulin (KAT). Col-V is a minor fibrillar collagen found in the lungs where it is normally sequestered from recognition by collagen I and collagen III overlays. Exposure to Col-V is hypothesized to occur upon matrix metalloprotease (MMP) degradation of the extracellular milieu during ischemia reperfusion injury, and immunogenic Col-V fragments can be found in BAL fluid –41. KAT de novo anti-Col-V antibody development post transplantation, and vice versa. Taken together these data suggest that autoantibodies to either self-antigen are capable of inducing lung injury that leads to epitope spreading post transplantation  development post-LTx  in cystic fibrosis patients both pre- and post-Tx . A seconpost-LTx . De novosurvival –78. Intesurvival .Overall, more research into pre-existing autoantibodies and PGD is needed. Large screenings, such as those used to discover autoantibodies in COPD and ILD patients, would help identify and measure pre-existing autoantibodies before and after LTx. Following identification, functional analyses of pre-existing autoantibodies are needed to determine if they are biomarkers, pathogenic, both, or neither. This information could hold the key to novel pre-Tx or perioperative treatments that reduce the risk of PGD development. Reducing the incidence of PGD is essential step to overcome poor survival. PGD is also a major risk factor for chronic lung allograft dysfunction (CLAD), the main cause of late-stage mortality , 80.1) from baseline FEV1 following LTx. There are three CLAD phenotypes referred to as bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome , and neutrophilic reversible allograft dysfunction (NRAD). However, NRAD was not included in the most recent ISHLT consensus report. BOS, the most common CLAD phenotype, accounts for approximately 60-75% of CLAD cases  remains the largest obstacle to long term allograft survival. ISHLT defines CLAD as the consistent decline in forced expiratory volume in 1 second  and the heterogeneity of antibody characteristics . Nonetheless, functional characterization could lead to the development of patient risk stratification strategies if the autoantibodies prove to be biomarkers. Further, a better understanding of the type and function of autoantibodies could be leveraged to develop novel antibody-targeted therapeutics designed to reduce graft damage if autoantibodies are found to be pathogenic.The inherent heterogeneity among antibodies and their structure gives them the ability to mediate different immune responses. Antibodies, or immunoglobulins, are composed of two heavy and two light chains. The heavy chain, or constant domain (Fc), specifies effector function and the light chain, or variable domain (Fab), binds to antigen. There are five main Fc immunoglobulin (Ig) isotypes; IgM, IgG, IgA, IgE, and IgD. The IgG and IgA isotypes can be split into IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2 subclasses, respectively.The Fc immunoglobulin classes differ in various properties such as structure, complement fixation, and FcyR binding [For detailed review see: Schroeder and Cavacini ]. HistorIgA. IgA has been well studied in the context of mucosal immunity and renal function (IgA nephropathy), however, little is known about IgA and solid organ transplantation. A singular report has analyzed pre-existing IgA autoantibodies in LTx patients. Kaza et al.  play crucial roles in transplantation outcome. However, significantly less research has focused on the influence and importance of different IgG subclasses. The four IgG subclasses, IgG1, IgG2, IgG3, and IgG4, have different physical properties, abundance, and effector functions. For example, IgG1-IgG3 are complement fixing with IgG3 having highest binding efficiency to C1q followed by IgG1 and IgG2  and subclass  have all been shown to interact with different Fc receptors and have different complement fixation capabilities. Structural changes to the Fc portion of immunoglobulins such as glycosylation can also greatly impact the antibody-mediated inflammatory response. Unfortunately, little is known with regard to these antibody features and as such the pathogenic mechanisms and corresponding antibody characteristics of disease specific pre-existing autoantibodies is largely still unknown. Gaining a better understanding of the epitopes, Ig-sub class and glycosylation state may facilitate the development of novel personalized pharmacotherapeutics that can be leveraged to augment immunosuppression and improve the outcomes of lung transplant recipients.All authors contributed to the conceptualization, writing, and editing of the manuscript. All authors contributed to the article and approved the submitted version.and NIAID U01 AI132894-01 to CA), TL1 TR001451 to AM, U01CA242096 to PMA (multi-PI), .These studies were supported by grants from the NIH (NHLBI R1R01HL 140470-01A1, NHLBI 3RO1HL140470-03S1 The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The intensive distribution of catalytic center on the starch surface and the hydrophobic microenvironments derived from the OSA chains furnished the Se-starch80 with a high GPx-like catalytic activity . This value was about 1.5 × 105 times higher than that of a typical small-molecule GPx mimic (PhSeSePh). In addition, the Se-starch80, without any cytotoxicity, showed a saturated kinetic catalytic behavior that is similar to a typical enzyme. This work exemplifies a biodegradable selenium-functionalized polymer platform for the high-performing GPx mimic. Selenium-functionalized starch (Se-starch80) is one of the main functional foods used for selenium supplementation. In traditional agriculture, Se-starch has some deficiencies such as long growth cycle and unstable selenium content that prevent its antioxidant performance. In this study, Se-starch was prepared by the nucleophilic addition between NaSeH and carbon-carbon double bond of octenyl succinic anhydride waxy corn starch ester (OSA starch). Some techniques such as  Selenium (Se) is an important trace element that participates in the synthesis of 25 Se-containing proteins in the human body. Glutathione peroxidase (GPx), with the –SeH catalytic center, is a particularly important antioxidant enzyme that scavenges the excess free radicals in human body. GPx and therefore protects human against oxidative damage , inflammStarch, one of the most important foods for human diet, is expected to be an ideal food source of selenium supplements. However, due to the limited metabolic process, the selenium-containing starch from natural plants is rarely found so far. Compared with protein, starch reveals a more stable structure in the chemical modifications. Sun et al.  synthesi1H NMR, XPS, SEM-EDS, etc. The catalytic mechanism of Se-starch as a GPx mimic was also studied in vitro. In addition, a MTT assay was used to investigate the cytotoxicity of Se-starch. This study may provide a new method for preparing the Se-functionalized starch with high GPx-like catalytic activity.It is well-known that the octenyl succinic anhydride starch ester (OSA starch) is a typical modified starch with wide applications in foods and medicines ,27. In t2O2), selenium powder, sodium borohydride (NaBH), and 4-nitrothiophenol (NBT) were purchased from J&K Scientific, Ltd., San Jose, CA, USA. Ethanoland sodium hydroxidewere of analytical grade and purchased from Xilong Chemical Co., Ltd., Shantou, China. 3-Carboxy-4 nitrothiophenol (TNB) was synthesized according to the previously reported method , in which the intercept is 1/vmax and the intercept is the absolute value of 1/Km. The [S] is the concentration of substrate  is the concentration of enzyme.The reaction constant  at 490 nm. The relative cell viability was depicted as the percentage relative to the optical density derived from the control test.MTT  assay was used to measure the cytotoxicity of Se-starch80 toward HepG2 cells according to the previously reported literature . The den(NaSeH):n = 1.5:1 and reaction time 6 h revealed the highest catalytic activity. Such Se-starch with the Se content of 0.033 mg/kg was selected for the characterizations. In this work, as shown in 1H NMR spectroscopy was used to characterize the changes of OSA starch during the reaction. As shown in 1H NMR spectrum showed a proton signal peak at 5.25 ppm, which was ascribed the proton of −HC=CH− in the OSA chains [1H NMR spectrum of Se-starch80, suggesting the −HC=CH− participated in the reaction. In this reaction system, NaSeH is a strong nucleophile that can undergo a reaction similar to the −SH and −HC=CH− groups [3/2, 5/2) signal peak appeared at about 54 eV, indicating the negative bivalent selenium (Se2−) in the Se-starch [2 to form the red nano Se0. However, the Se-starch was white, which further proved that the −SeH was covalently bonded to the starch skeleton, rather than being presented as elemental selenium, which is usually gray or red [−6 wt%). These results suggested that the anchored −SeH was mainly distributed on the surface of starch, which would profit the exposure of the active center for catalytic reaction.In order to determine the selenization reaction mechanism, the A chains . This pe− groups . While te-starch . Generaly or red . Further−1 (O−H stretching), 2930 cm−1 (C−H stretching) [−1 (C=O stretching), 1021 cm−1 (C−O stretching) [The OSA starch granule showed a smooth surface C, while etching) ,39, 1652etching) . These petching) . In addietching) . (NaSeH):n(-HC=CH-), and volume ratio of ethanol (V%) on the Se content and catalytic activity  were investigated. The NBT and CUOOH were used as the substrates to test the υ0. υ0 under the conditions of reaction temperature 40 °C, n(NaSeH):n(−HC=CH−) = 1:1 and volume ratio of ethanol 30%. The υ0, similar to the Se content, increased with the prolongation of reaction time in the initial stage of 6 h. Due to the equilibrium of reaction, the further increase of reaction time did not promote the υ0 and the Se content significantly. Therefore, the reaction time of 6 h was selected for preparing Se-starch.Similar to natural enzymes, the catalytic activity of GPx mimic may be greatly affected by structural changes in their skeleton, catalytic center, and peripheral recognition ,24. For υ0 under the conditions of reaction time 6 h, n(NaSeH):n(−HC=CH−) = 1:1, and volume ratio of ethanol 30% was conducted by altering the temperature from 30 to 70 °C :n(−HC=CH−) from 1.0 to 2.2 at the conditions of reaction time 6 h, reaction temperature 30 °C, and volume ratio of ethanol 30%. As shown in υ0 did not change significantly as the molar ratio of NaSeH and −HC=CH− increased. This result might be ascribed to the partial gelatinization of starch by alkaline NaSeH, which coated the Se active center and hindered its catalysis. Although the high content of NaSeH could promote the Se content of Se-starch, the excessive NaSeH resulted in a troublesome purification of the product. Therefore, the molar ratio of NaSeH and −HC=CH− was set to be 1.5 for the further experiments. The effect of reaction temperature on the Se content and to 70 °C B. The Seυ0 under the conditions of reaction time 6 h, reaction temperature 30 °C, and n(NaSeH):n(−HC=CH−) = 1.5:1. At the low volume ratio of ethanol , the OSA starch was completely emulsified and dispersed in the solution, resulting in a difficulty for separating the products from the solution  revealed a high catalytic activity (υ0 = 3.64 μM/min). This value is 1.5 × 105 times higher than the catalytic activity of a typical small-molecule GPx mimic (PhSeSePh) at the reaction system of NBT and CUOOH.solution E. The in2O2 and CUOOH were used as the hydroperoxide substrates. H2O2 is more hydrophilic than the CUOOH for the absence of the p-cumyl group in H2O2. These substrate combinations were employed to test the catalytic activity of Se-starch80 (υ0 at the reaction systems with various substrates. In the system containing both hydrophobic substrates (NBT + CUOOH), Se-starch80 exhibited the highestυ0, while it showed the minimumυ0 in the system of TNB and H2O2, two hydrophilic substrates. These results suggested that the hydrophobic interaction played an important role in the catalysis. The hydrophobic microenvironments in the Se-starch80 were testified by the pyrene fluorescence probe experiment [−6 M) revealed a fluorescence intensity ratio  of 1.81 at peak 1 (372 nm) and peak 3 (383 nm). However, the solution containing pyrene (concentration = 1.0 × 10−6 M) and Se-starch80 with a lower I1/I3 of 1.66, indicating the formation of hydrophobic microenvironments in the Se-starch80 for gathering pyrene molecule [υ0 of Se-starch80 was comparable to that of Micellar Catalyst , the reaction constant , Michaelis‒Menten constant , and the catalytic efficiency  were calculated and listed in Km, an index of the affinity between catalyst and substrates, designates the concentration of substrate at v = 0.5 vmax, where v is the reaction rate. A lower Km indicates a stronger affinity between catalyst and substrates. Taking the reaction system of CUOOH + TNB and the system of CUOOH + NBT as the comparison, the Km of Se-starch80 at the system of CUOOH + TNB is inferior to the Km derived from the system of CUOOH + NBT, indicating that Se-starch80 revealed a higher affinity to NBT than TNB. The higher affinity for TNB could be due to the bearing of –COOH, which could interact with the hydroxyl of starch via hydrogen bond. However, the catalytic activity (υ0) and the catalytic kinetic parameters including υmax, Kcat, and Kcat/Km of Se-starch80 in the system of CUOOH + TNB were smaller than that in the system of CUOOH + NBT. A possible explanation was that the strong affinity of Se-starch80 toward TNB resulted in a poor dissociation of the substrate from catalyst after catalytic reaction. The active centers in the catalyst were occupied by the bonded substrates, which hindered the subsequent reaction and consequently decreased the catalytic activity of Se-starch80.The catalytic behavior of Se-starch80 was further investigated by changing the concentration of hydroperoxide substrates at the system containing the thiophenol substrates (0.15 mM). As shown in The exposure of Se-starch80 to the proliferation of HepG2 cancer cells was conducted to test the cells growth in a concentration-dependent manner . For allv0 = 3.64 μM/min). The high catalytic activity of Se-functionalized starch was attributed to the intensive distribution of catalytic center on the surface of starch and the hydrophobic microenvironments. The Se-functionalized starch did not require any bioconversion to perform the GPx-like function and showed a typical saturated kinetic catalytic behavior. In addition, the biodegradable starch skeleton did not change significantly during the modification and such Se-functionalized starch had no toxicity to cells. Therefore, a non-toxic Se-starch was prepared by a chemical modified method, which is expected to become a novel supplement with GPx-like activity for solving the nutrient deficiency of selenium. This work may provide a potential for the preparation of Se-functionalized starch.In this paper, a new strategy for producing the Se-functionalized starch with GPx-like catalytic activity was developed by the nucleophilic addition between NaSeH and OSA waxy corn starch. The covalently linked −SeH on the starch surface provided it with catalytic centers, while the OSA chains supplied the hydrophobic microenvironments for gathering substrates. The optimal technological conditions for preparing Se-functionalized starch were that reaction time 6 h, temperature 30 °C, molar ratio of NaSeH to −HC=CH− 1.5 and volume ratio of ethanol 80%. Such Se-functionalized starch with a Se content of 0.033 mg/kg revealed a high capacity for catalyzing the reaction between CUOOH and NBT (