IVC), IVC resection along with liver resection may be needed to effect a cure. Furthermore, if the IVC defect is large, IVC reconstruction with vascular graft after resection is required. There are limited reports of cases of IVC reconstruction using a graft. By reviewing data from the literature of previous studies, the present study was aimed at investigating the surgical outcomes of liver resection with IVC resection and reconstruction using an artificial vascular graft. PubMed was searched for previous articles reporting cases with the combined surgery. The search was limited to articles in English, and cases with exceptional surgeries such as in situ cold perfusion, and ante situm and ex vivo techniques were excluded from this study. Surgical outcomes of the extracted cases were investigated. Cases dealt only with primary closure after IVC resection, and those in which the IVC tumor thrombus was treated by opening the IVC wall, removing the thrombus and then closing the IVC without wall excision were not included in this study. The literature search identified 13 studies, including 111 cases. Operative mortality in the reported cases was 8.1% (9 out of 111 cases). Thrombus in the artificial vascular graft was observed in two cases, and patency of the graft during the follow‐up period was confirmed in 109 of the 111 cases (98.2%). These results suggested that the surgical outcomes of liver resection combined with IVC resection and reconstruction using the artificial vascular graft were favorable.In cases where liver tumors invade the inferior vena cava ( These tumors sometimes directly invade the inferior vena cava (IVC). In cases with IVC invasion, liver resection combined with IVC reconstruction provides the only chance for a cure. Furthermore, if the area affected by the IVC resection is large, a vascular graft is required, although the graft is not necessarily needed in cases with a smaller defect area.2In December 2017, PubMed was searched for previous articles reporting cases with liver resection combined with IVC resection and reconstruction using artificial vascular graft. The search was carried out using the terms “liver resection”  in combination with “inferior vena cava”  and “reconstruction” . The searched articles were first screened by title of the articles. Then, the search was limited to articles in English. “Case report” articles and duplicate articles were excluded from this study. Cases dealt only with primary closure after IVC resection, and those in which the IVC tumor thrombus was treated by opening the IVC wall, removing the thrombus and then closing the IVC without wall excision were not included in this study. Cases that received associated procedures such as in situ cold perfusion, and ante situm and ex vivo techniques were also excluded from this study. The remaining potentially relevant articles were further assessed for their relevance based on content of the articles. Thus, cases with liver resection combined with IVC resection and reconstruction using an artificial vascular graft were extracted from the final selected articles. Flowchart representing the selection of articles for this study is shown in Figure 3Through the literature search, no systemic reviews were found and there were 13 study articles that met the inclusion criteria  graft , or bovine or horse pericardium were used. This review did not include cases with pericardium for patch repair. For a larger degree of IVC involvement, the entire circumference of the IVC was replaced with an artificial vascular graft. As the artificial vascular graft for the circumferential replacement of IVC, although Dacron graft  was used in limited cases, ePTFE graft was used in most cases. There were two patterns of clamping reported based on the extent and location of IVC tumor involvement Figure . The firSurgical outcomes in each extracted study are also shown in Table 4http://www.ncd.or.jp), which is a nationwide prospective registry linked to the surgical board certification system,The literature review showed that, in previous studies, operative mortality rate of liver resection combined with IVC resection and reconstruction using artificial vascular graft was 8.1%. Incidence of postoperative complications was undetermined because of unclear definitions of complications. In the previous studies, patency of the artificial vascular graft was 98.2%. We also experienced 12 cases of combined surgery, and summary of these cases showed that rates of mortality and postoperative complications defined as ≥grade III of the Clavien‐Dindo classification were 0% and 16.7%, respectively, and that the patency of the artificial vascular graft was 100% (unpublished data),Several concerns have been discussed regarding the procedure of liver resection combined with IVC resection and reconstruction using an artificial vascular graft. First, there is a problem regarding the necessity for IVC reconstruction after its resection. Although it is well known that IVC blood flow is responsible for renal insufficiency and lower extremity edema, IVC reconstruction does not seem feasible in cases with infrarenal IVC invasion with adequate collateralization of blood flow. However, this is not the case with suprarenal IVC invasion, which is the most common in cases with IVC invasion by liver tumors. Determining which patients will tolerate IVC resection without the previously mentioned problems is difficult. Reconstruction would be considered when suprarenal invasion is noted. The second problem is the necessity for antithrombotic therapy after the reconstruction. In more than half of the previous studies, antithrombotic therapy was given after the reconstruction, but no clear criteria for giving antithrombotic therapy were commented upon in each study. Antithrombotic therapy was carried out mainly by using heparin, although there were several cases where nafamostat mesilate was used with heparin, and those where aspirin was used after the heparin dose. This prevalence of antithrombotic therapy might be as a result of a study showing that, in two cases where thrombus was identified in the graft, antithrombotic therapy had been discontinued. However, necessity for antithrombotic therapy after the reconstruction has not been examined in previous studies with high level evidence such as prospective studies comparing outcomes between cases with and without antithrombotic therapy. Furthermore, this concern should be discussed considering also the abovementioned necessity for IVC reconstruction after the resection. Thus, this problem seems complicated, although it will ideally be solved in the future. Last, there have been other proposed procedures such as in situ cold perfusion,In summary, surgical outcomes of liver resection combined with IVC resection and reconstruction using an artificial vascular graft were investigated based on a literature review of previous studies. The results of the investigation showed that the outcomes were favorable, suggesting feasibility of the combined surgery.Conflicts of interest: Authors declare no conflicts of interest for this article.


The analysis of the different samples by FTIR-ATR spectroscopy showed the presence of the polyoxometalates (and the cationic starch) on the top surface of the paper. In addition, the determination of surface energy parameters, namely the polar component (σsp) and the dispersive component (σsd) of the surface energy, by contact angle measurements revealed that, for the new samples, the polar component level was much higher than that of the uncoated base paper. The quality of inkjet printing, evaluated by parameters such as the gamut area and the optical density, was considerably improved by these surface treatments.A new approach for the chemical modification of the surface of paper based on the application of colloidal mixtures containing cationic starch and polyoxometalates on uncoated base paper is presented. Polyoxometalates with the Keggin-type structure and physical properties similar to those presented by coating pigments, namely H The treatment of the paper surface in order to control surface properties relevant to printing, such as surface energy, smoothness, porosity, and chemical properties, has been thoroughly explored. In this context, the modification of the paper surface properties should lead to a good paper-ink interaction (in inkjet or offset printing) in order to fix the ink colorants on the paper surface (without spreading) and to allow rapid absorption or evaporation of the ink vehicle. Therefore, a good affinity between the dyes (or pigments) used as colorants and the chemical compounds used in the paper surface treatment is desirable.Two general approaches for the treatment of the paper surface are usually considered: surface sizing and coating. Surface sizing is usually performed by the application of an aqueous suspension of cationic starch alone or combined with a synthetic polymer, such as poly(styrene-co-acrylate) or poly, among others ,2. The cet al. m- or [XzMxOy]n-, where z ≤ x, and M stands for Mo, W, or V, and X is a variable element. They present unique structural and electronic properties which make them appealing in several fields, ranging from molecular architecture and catalysis to materials science [12O40]p− −  are metal-cluster anions with the general formulae p− , where X12O40]p−  are wellg others ,23,24,250](p+4)− .3PW12O40·23H2O, H4SiW12O40·24H2O, and K7PW11O39·9H2O) with properties similar to those of coating pigments such as high reflectance in all the visible wavelength range, brightness, solubility (or good mixing) in water, compatibility with the other components of the formulation used in the surface treatment (only cationic starch in this study) were chosen with the objective of increasing the quality of inkjet printing.A new methodology consisting in the application of a colloidal suspension (with adequate rheology) containing cationic starch and a polyoxoanion at the paper surface, is presented. Polyoxometalate compounds (H°C) of cationic starch with solids contents of 10 wt % and 12 wt % have not very different viscosities. At the solids content of 14 wt % a great increase of the viscosity to values near 70 mPa.s is observed. Given the reference value for some industrial applications (around 35 mPa.s), suspensions having 12 wt % of cationic starch were used in all subsequent experiments +) . Considering the relative amounts of cationic starch and polyoxometalate solids used in each formulation for the treatment of the paper surface, 80/20 w/w, respectively , νas(W-O), νas(W-Ob-W) and νas(W-Oc-W) (Ob and Oc denote oxygen atoms from octahedra sharing edges and corners), respectively 3- and [SiW12O40]4- anions have similar structure, but the latter anion has a higher charge density.The contact angles of the five papers with water as well as the surface energy parameters computed by using the Owens and Wendt model  are presn energy . The papsd was obtained by using the Fowkes model +) at the paper surface, provide regions of high interaction with the sulfonic groups (anionic) of the inks. On the other hand, the polyoxoanions, located close to the quaternary ammonium groups of modified starch are not directly attracted to the anionic dyes structures. However, when the polyoxoanions are immobilized at the paper surface the counter-cations of the compounds used for the surface treatment are also available. For the heteropolyacid compounds used in the surface treatment of paper samples 2 and 3, there is also a concomitant release of protons to the medium  due to their strong acidity (as shown above in 2.1). These protons should be close to the polyoxoanions structure, being attracted by the polyoxoanion and simultaneously attracting the sulfonic acid groups, the latter of which can be protonated  are pleasantly surprising since a high gamut area was achieved, although the optical density (namely of cyan and yellow) was not as high as that of papers 2 and 3. In this case, potassium cations together with the polyoxoanions are released to the paper surface. It is unlikely that potassium ions have the same affinity to the anionic dyes as the protons, although present in higher number (seven cations per one anion). However, paper sample 4 proved to have the most polar surface of all paper samples  which can facilitate the retention of some colorants.The results obtained with cationic starch and K2) produced with a Eucalyptus globulus Kraft pulp was used as the base paper for the surface treatments. Cationic starch solid (with a nitrogen content of 0.37%) and α-amylase solution were supplied by the industry. Cationic starch suspensions having solids content of 10%, 12% and 14 wt %  were previously analysed using a Rheometer Rheostress (Haake), with the objective of selecting the one with the most suitable viscosity for further experiments. For the improvement of inkjet printing quality, the polyoxometalate compounds considered were H3PW12O40·23H2O, H4SiW12O40·24H2O, and K7PW11O39·9H2O. The former two were supplied by Sigma-Aldrich and the latter was prepared as described by Haraguchi et al. [A commercial calendered uncoated paper  was added to 10 g of cationic starch. After heating the mixture to 65 °C with vigorous stirring, more 20 mL of hot water (60 °C) was added and the heating was extended to 70 °C. Then, 3.3 μL of α-amylase was added and the mixture was heated again up to about 80 °C and left at this temperature for 5 min. The starch enzymatic conversion was stopped by the addition of 1.7 mL of a ZnSO4 solution (30 g/L). The colloidal suspension obtained was heated up to 90–92 °C, and left stirring at this temperature during 15 min [°C, 2.5 g of solid polyoxometalate compound was added in small portions and the resultant suspension left stirring for about 10 min. The cationic starch content was finally adjusted to 12.0 ± 0.1 wt % by the addition of hot water. The suspension was maintained at approximately 50 °C before its application on the paper surface.Water  of each paper sample, and the corresponding polar (σsp) and dispersive components (σsd) by the Owens and Wendt method [The contact angles of the paper samples with four distinct liquids  were measured with a DataPhysics OCA20 using the sessile drop method, in order to determine the surface energy , gamut area, print-through and some line and dot quality parameters in a specified mask printed with a HP Deskjet F370-target IJ12  on the paper samples.Both the optical density, gamut area, and print-through, whose definitions may be found elsewhere ,40,41 we7PW11O39.In the present work a new methodology for the functionalization of paper surface, aiming a better printability, was developed. This methodology is based on the use of colloidal suspensions containing cationic starch and polyoxometalates for the treatment of the paper surface. Several new paper samples were obtained, and characterized by FTIR-ATR spectroscopy and contact angle measurements. The results of these studies showed that, for the new samples, the polyoxometalates (and cationic starch) are present on the paper surface and modify the surface energy parameters at a significant extent, in comparison to the base paper. In fact, after the surface treatments, the paper surface became much more polar, being the most polar surface that of the paper treated with the suspension containing cationic starch and the polyoxometalate Ksp > 50 mN/m; σsd < 3 mN/m) enables the rapid diffusion of the ink solvent, which may contribute to an easier separation of the ink colorants from the solvent helping on their immobilization at the paper surface.The new paper samples were evaluated with regards to inkjet printing quality, and very interesting results were obtained. In general, the gamut area and the optical density increased in comparison to the base paper, while the print-through and intercolor bleed decreased. This accounts for the significantly higher polarity of the surface of the new samples in comparison to the base paper. The highly polar surface of all treated papers  and the paper sample treated only with cationic starch (sample 1). The better results of gamut area and optical density of cyan, magenta and yellow for the paper samples 2, 3 and 4 when compared to those of paper 1 were interpreted in terms of the chemical interaction of the components of the dyes-based ink with the compounds used to modify the paper surface. It is proposed that the polyoxoanions and the corresponding counter-cations, by increasing of the electrostatic interactions with the anionic dyes  provide a better retention of the colorants at the paper surface. Work is in progress to better understand the processes occurring at the paper-ink interface for these new paper samples functionalized with cationic starch and polyoxometalates.Comparison was made between the paper samples treated with mixtures of cationic starch and polyoxometalates (Hi.e., with low pick-up. Finally, considering the compounds that were used, it is expected that the method presented in this study for the modification of the paper surface has not any negative impact in terms of paper recycling and environment when compared with the conventional methods of surface sizing and coating used at industry. Additional specific studies will have to be carried out to access the possibility of paper recycling without significant losses of the cationic starch and polyoxometalate compounds during the deinking process.Overall, the use of polyoxometalate compounds together with cationic starch represents a novel approach for the treatment of paper surfaces. The formulation has two components: cationic starch and polyoxometalate, and the alteration of the surface properties, as demonstrated, is possible with the application of only small amounts of the chemicals at the paper surface,


In “Empathy and its discontents” Bloom  argues tI am often touched by things that I see happen”; , the same cannot be said for compassion. In the Trend in Cognitive Sciences article [1] it is “positive feelings toward others, a desire that others do well and do not suffer…”, but elsewhere it is defined by Bloom similarly as to what Batson  refers t” Bloom, . Adding Keltner, . This co” Bloom, , p. 24 aindividuals who encounter the suffering of large numbers of people (e.g. starvation in Sudan) are more likely to respond with sadness, distress or even apathy as a result of their inability to cope with suffering of such a large magnitude”  but is more in line with his definition of compassion. For instance, the work cited by Bloom on the identifiable victim effect  as the motivator of moral decisions  when making moral decisions. However, some form of emotion (regardless of how biased it is) may be a necessary, though not sufficient, condition for action by both individuals and societies. Zaki  points tAll authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.This research was funded by grants from National Science Foundation, Vetenskapsrådet, and Riksbankens Jubileumsfond.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.


Significant positive correlation also exists among activities of AGPase, SSS, GBSS, and SBE, except for GBSS-SBE. In summary, our study has revealed that the reduced conversion of sucrose to starch in the late grain filling stage is the main cause for the low kernel weight and total starch accumulation of the waxy wheat. The reduced conversion also appears to be a factor contributing to the lower grain yield of the waxy wheat.Waxy wheat has unique end-use properties; however, its production is limited due mainly to its low grain yield compared with non-waxy wheat. In order to increase its grain yield, it is critical to understand the eco-physiological differences in grain filling between the waxy and non-waxy wheat. In this study, two waxy wheat and two non-waxy wheat cultivars were used to investigate the differences in starch-associated enzymes processes, sucrose and starch dynamics, yield components, and the final grain yield. The results indicated that the mean total grain starch and amylose content, the average 1000-kernel weight and grain yield of the waxy wheat were lower than those of the non-waxy wheat at maturity. The amylose content was significantly and positively correlated with the activity of GBSS (r = 0.80,  Common wheat cultivars have three homeologous waxy genes, Wx-A1, Wx-B1 and Wx-D1, and waxy wheat mutants lack all three Wx proteins. The Wx proteins are also known as granule-bound starch synthases, which have a role in the synthesis of amylose5. However, due to low grain yield, the waxy wheat has not been widely cultivated in recent years. To improve the current situation, there is an urgent need for improved understanding of the differences in yield performance between the waxy and non-waxy wheat cultivars at the field level.With the growing in health-conscious consumers, there has been an increase in demand for high-quality wheat flour. The endosperm starch content and composition are two critical parameters for evaluating the quality of wheat flour. For conventionally cultivated wheat, namely non-waxy wheat, starch is a mixture of about 75% amylopectin and 25% amylose. To further improve the quality of wheat flour products such as reducing staling in flour products, keeping baking goods fresh for a longer period of time, and improving the palatability of noodles, the replacement of regular wheat flour with waxy wheat flour may provide a promising solution, as waxy wheat grains contain almost 100% amylopectin in their starch7, but also reflects the capacity of the sink tissues. Regarding the source-sink relationship, the photoassimilates produced in source tissues as leaf, stem and root were transported into the amyloplast in the form of sucrose, which is the main carbohydrate transported in higher plants. Sucrose is degraded to fructose and uridine diphosphate glucose (UDPG) which are the main precursors of starch synthesis by sucrose synthase (SUS: EC 2.4.1.13)9. The ability of the sink tissues to accept and convert photoassimilates can be affected by the sink strength, which is an important limiting factor to wheat grain yield, and the activity of SUS can be considered as an indicator of sink strength11.The endosperm starch content (approximate 70% of the dry weight) not only influences the grain weight and quality12, including ADPG pyrophosphorylase (AGPase: EC 2.7.7.27), starch branching enzyme (SBE: EC 2.4.1.18), starch debranching enzyme (DBE), granule bound starch synthase (GBSS: EC 2.4.1.21) and soluble starch synthase(SSS: EC 2.4.1.21). GBSS and SSS are the two forms of starch synthase14. For catalyzing the first unique step in starch synthesis, AGPase is the rate-limiting enzyme and is the most important determinant of seed sink strength16. Starch synthases catalyse the elongation of the linear glucan chains17, moreover the different genetic characterization assigns preferential functions for individual isoforms to synthesize amylose or amylopectin. It is generally known that in the endosperm, amylose is synthesized by AGPase and GBSS13. SBE that was formerly known as Q-enzyme plays an important role on amylopectin synthesis, which is the only plant enzyme that can introduce α-1,6-glucosidic linkages into α-polyglucans19.A coordinated series of enzyme-catalyzed reactions in wheat endosperm result in starch synthesisWx genes22. It is reported that the seed weight of inferior spikelets of rice can be improved by increasing the starch-synthesizing enzymes activities23. In this study, we compared two waxy wheat cultivars with two non-waxy wheat cultivars to investigate the starch content and accumulation, the activities of enzymes responsible for starch biosynthesis in developing grain, 1000-kernel weight and grain yield with the objective to better understand the differences between the wheat varieties and elucidate factors affecting the 1000-kernel weight and grain yield of the waxy wheat.Previous researches on waxy wheat have focused on the end-use quality, composition of the grain and the expression of the −1 organic matter, 100.01 mg kg−1 alkali hydrolysable N, 50.11 mg kg−1 Olsen-P, and 149.08 mg kg−1 exchangeable K at 0–20 cm soil depth in 2012, while 19.57 g kg−1 organic matter, 107.97 mg kg−1 alkali hydrolysable N, 33.92 mg kg−1 Olsen-P, and 124.23 mg kg−1 exchangeable K at 0–20 cm soil depth in 2013.Field experiments were conducted at the Agricultural Experiment Station 32°39′N, 119°25′E) of the Agricultural College of Yangzhou University in China during the winter wheat growing seasons in 2012–2013, and 2013–2014. The field site is situated in a humid subtropical climate zone, with average air temperature of 13 °C to 16 °C, total precipitation of 800 to 1200 mm, total sunshine of 2000 to 2600 h, and a frost-free season of 220 to 240 d. The meteorological data during the wheat growing seasons across the two study years, including temperature and precipitation are shown in Fig. 19°25′E oWx-A1, Wx-B1, Wx-C1 alleles based on a PCR test (data not shown)] and two non-waxy wheat cultivars Yangmai20 (Y20) and Yangfumai4 (YF4), .A total of four wheat cultivars were tested, including two types of waxy wheat cultivars Yangnuomai1 (YN1), Ningnuomai1 (NN1), [which lack functional −1, applied with the ratio 5:1:2:2 at four stages: pre-sowing, four-leaf stage, jointing stage and booting stage, respectively. Application rate of P2O5 (P) and K2O (K) were both 144 kg ha−1: half the amounts of P and K were applied before sowing and the remaining half were applied at the jointing stage. The seeds were sown at a planting density of 225 × 104 ha−1 with 30 cm row spacing on 3 November of 2012 and 28 October of 2013. Seedling number was maintained at 225 × 104 ha−1 by removing or adding seedlings at the three-leaf stage. Plants were harvested on 3 June of 2013 and 28 May of 2014. Weeds, insects and diseases were controlled as required to avoid yielding loss; other production management practices were based on those used by local farmers.All plots were supplied with 240 kg N ha24.At maturity, an area of 1.2 × 1 m (4 rows included) was harvested manually for the determination of the grain yield. Grain yield was adjusted to 13% moisture. Number of spikes was counted manually for two rows within 1 m. The number of kernels per spike was counted manually to calculate the mean value from 50 continuous spikes. For 1000-kernel weight, 1000 kernels were randomly selected and weighed. The average seed volume was determined via the displacement method26. Wheat grains that were marked flowering on the same day were first ground using a mortar, and the powder was then degreased twice with anhydrous ether. A 100 mg fraction of each sample was used to determine amylose and amylopectin contents. A calibration curve was derived using pure amylose from potato  and pure amylopectin from potato . The sum of amylose and amylopectin contents was designated as the total starch content.The total starch, amylose, and amylopectin contents were determined via the dual wavelength iodine binding method28. SSS and GBSS activities were assayed according to Nakamura and Cheng28. SBE activity was assayed according to Li et al.29.Twenty kernels weighed at different filling stages were tested. AGPase activity was assayed according to Nakamura and Cheng31. Ten dried spikes those were flowered on the same day were selected and killed at 105 °C for 30 min and then dried at 80 °C for measurements of grain weight and starch content. And then they were weighed, and then the dried endosperm was ground into powder for analysis. Samples of the powdered endosperm prepared as mentioned above (100 mg) were extracted with 8 mL of 80% ethanol at 80 °C for 30 min, followed by two extractions with 8 mL of 80% ethanol. The supernatants were combined and purified by 10 g activated carbon overnight, fixed to a constant volume at 50 ml, and purified by filtering. One-hundred microliters of 2 M sodium hydroxide was added to 0.9 ml filtrate, boiled at 100 °C for 10 min. The mixture was cooled to room temperature, then 1 ml of 0.1% resorcinol and 3 ml of 10 mol/L hydrochloric acid were added. The reaction mixture was boiled at 80 °C for 60 min, cooled to room temperature, and the sucrose content was determined by measuring OD at 500 nm.The sucrose content was measured by the resorcinol technique34.The SUS activity was assayed according to Rufty, Doehlert and WardlawP = 0.05. Pearson’s correlation analysis (SAS V.9.1) was performed to establish the relationships among all measured properties. The ANOVA (α = 0.05) was performed to identify significant differences between treatments, multiple comparisons between means (SAS V.9.1) were made using the least significant difference (LSD0.05) test if the F-tests were significant.All data were checked for normality based on the Kolmogorov–Smirnov test with SAS proc mixed (SAS V.9.1). The effects of year, cultivar and their interaction on test parameters were tested with year, cultivar as fixed effects, year as repeated measurements, and block as a random effect. Differences among least square means (LSMEANS) for all treatment pairs were tested by the LSD procedure at a significance level of The test found that grain sucrose content was significantly affected by cultivar, year and their interactions Fig. . Averageth–25th days after anthesis, but decreased after the 25th day after anthesis for both types of wheat  increased during early grain filling period and decreased during the late period after anthesis. The peak activities of the AGPase and GBSS occurred on the 25day Fig. . The AGPpy = 0.0072, pc < 0.0001), number of spikes  and the 1000-kernel weight ; however, the interaction between year and cultivar was not significant. For the grain yield, a significant cultivar effect was detected (pc < 0.0001), but not for year and year-cultivar interaction.The year and cultivar significantly affected the number of kernels per spike , while the amylose content did not significantly correlate with other enzyme activities except for GBSS  and sink activity as indicated by activities of the enzymes such as SUS and AGPase in endosperm, which is closely associated with carbohydrate utilization and storage41. Moreover, early studies have found that waxy wheats generally have higher sucrose content and lower transforming ability compared with non-waxy wheat42. The sucrose content is significantly correlated with the starch synthesis in the early period of grain filling (before 25th days after anthesis) in wheat43 and rice44. The SUS plays an important role in sucrose synthesis and degradation in plants, especially in wheat grains; It is generally believed that the main function of the SUS is to catalyze the degradation of sucrose in grains46. The decrease of SUS activity leads to lower rate of conversion from sucrose to starch and a high residual sucrose content43, and further resulting in a lower dry matter accumulation characteristic of rice grains48. In this study, we recorded significant differences in the sucrose content and SUS activity in grains among waxy and non-waxy wheat cultivars after anthesis. Throughout the grain filling, the grains of waxy wheat had higher sucrose content, but SUS activity in grains was different between the earlier and later stage after anthesis. Our results suggested that waxy wheat had higher sucrose degradation and bioavailability during early grain filling stages, but was lower at the later stage compared with non-waxy wheat. More sucrose and less grain starch synthesis ability could be characteristic of waxy wheat in comparison to non-waxy wheat especially in the late period after anthesis. These results were in agreement with some previous studies conducted in waxy42 and non-waxy46 wheats.The conversion of sucrose to starch in wheat grain was closely related to starch accumulation, for example, high starch accumulation was observed when the conversion of sucrose to starch was high49, which is the major factor influencing both grain yield and quality12. The first unique step of the starch synthesis is catalyzed by AGPase, which is considered to be the rate-limiting enzyme because of the positive correlation between the activity and starch accumulation rate51. In this study, AGPase activity increased at the early grain filling stage, leading to increased accumulation of the total starch, amylose and amylopectin, as well as their accumulation rate. We also found that the AGPase activity in waxy wheat was lower than that in non-waxy wheat in the whole grain filling period, resulting in lower total starch content in waxy wheat seeds compared with non-waxy wheat. At 30d after anthesis, the AGPase activity started to decrease, resulting in a reduction in the rate of starch, amylose and amylopectin accumulation. During the later grain filling period, the starch accumulation and accumulation rate of waxy wheat were lower than that of non-waxy wheat. Similar findings have also been reported in previous studies: in other crops higher AGPase activity resulted in higher crop starch biosynthesis and grain yield including maize52, rice53 and wheat54.Starch is synthesized and accumulated during the grain-filling process17. The absence of waxy protein resulting from waxy mutations in wheat and other cereals could lead to amylose-free starch in endosperm, with low activity of GBSS in waxy wheat55. GBSS activity determines amylose content in the endosperm of rice56 and wheat57. In this study, the GBSS activity of waxy wheat was significantly lower than that of non-waxy wheat throughout the grain filling period; the amylose accumulation and accumulation rate in waxy wheat were also lower than that in non-waxy wheat. These results indicated that waxy wheat had lower grain starch and amylose synthesis capacity compared with non-waxy wheat, particularly in the case of amylose. Moreover, GBSS is a critical component in the process of amylose formation58; some studies have also reported that reduction of amylose content and GBSS activity has been observed in many species that lack amylase and all of these specifically lack GBSSI activity14. GBSS is a critical enzyme in the process of amylose biosynthesis58 and its activity is closely associated with amylose content as observed in many species that lack amylase and GBSS, specifically the GBSSI activity14.GBSS is exclusively involved in the synthesis of amylose, where it catalyzes the extension of long glucans within the amylopectin fraction59. Amylose, amylopectin and total starch accumulation rate in wheat grains were significantly and positively correlated with activities of SBE, SSS and GBSS60, and a reduction in enzyme activity appears to be the main factor affecting decreased starch synthesis43. In this study, enzymatic activities of SSS and SBE were higher in waxy wheat than in non-waxy wheat, while amylopectin accumulation and amylopectin accumulation rate was significantly higher in waxy wheat compared with the non-waxy wheat during early grain filling stages. During the late grain filling stage, activities of both enzymes were lower, while the amylopectin accumulation and accumulation rate decreased more quickly in waxy wheat compared with non-waxy wheat. These results were similar to those of Tan et al.61 who reported that lower activities of AGPase, SSS, GBSS and SBE and the lower accumulation of amylose, amylopectin and starch in waxy wheat compared with non-waxy wheat and also in lower starch accumulation cultivar had lower enzyme activities in non-waxy wheat58.The SBE activity did not significantly influence the amylopectin content but significantly positively related to the amylopectin accumulation rate, it is postulated that SBE might not play an important role in the formation of amylopectin, but to its fine structure modification, it is stated in maize for the unique function of SBEI in modulating the branching pattern in normal starch by decreasing local clustering of amylopectin branch points62.The SSS and SBE play an important role in the formation of the branched structure of amylopectin molecules42, and also lower starch, amylose and amylopectin accumulation and accumulation rate than non-waxy wheat in the grain filling period61. It is possible that higher soluble sugar content and lower starch content were due to a low conversion efficiency and the absence of GBSS at the period of grain filling because these two factors have been shown to influence grain weight in waxy rice63 and in waxy wheat20. Higher activities of SBE, APGase, SSS and GBSS are generally attributed to increased kernel weight in wheat, while starch accumulation rate is significantly correlated with activities of SBE, AGPase, SSS and GBSS in both waxy wheat61 and non-waxy wheat41. Seed sink strength and seed yield would all be expected to be enhanced by increasing the AGPase activity16. The activities of AGPase, SSS, GBSS and SUS play important roles in determining individual grain weight in wheat by regulating starch synthesis in grain endosperm64. Further, many studies show that there is a coordinating action in starch biosynthesis among these enzymes60. The four important enzymes for starch biosynthesis played the important role but not the whole starch biosyntheses net.Waxy wheat was found to have lower starch content and higher sugar content than non-waxy wheatIn this study, grain yield appeared to be influenced by sucrose and starch factors. The total starch and amylose content at maturity in grains of waxy wheat were 13.29 and 88.27% lower respectively than those of non-waxy wheat. The 1000-kernel weight of waxy wheat was 7.05% lower than non-waxy wheat, and the grain yield of waxy wheat was 12.33% lower than that of non-waxy wheat. Reduced activities of AGPase, SSS, GBSS and SBE and a lower accumulation of amylose, amylopectin and starch were also observed during the later stage of grain filling in waxy wheat compared with non-waxy wheat. The amylose content was significantly and positively correlated with GBSS activity, while amylose was significantly and positively correlated with the 1000-kernel weight. The total starch content and amylopectin content were significantly and positively correlated with AGPase, SSS and SBE activities. Among the activities of AGPase, SSS, GBSS and SBE, there were significant correlations, except between the activities of GBSS and SBE.Our study suggests that low AGPase activity could be the cause of low total starch content in waxy wheat grain, and low GBSS activity could be the contributing factor to insufficient amylose content. During the early grain filling period, no significant difference was found in sink size between the waxy and non-waxy wheat, while higher sucrose degradation and bioavailability and activities of SSS and SBE in grains of waxy wheat were found. Low activities of SSS and SBE in waxy wheat are likely the cause of low amylopectin accumulation in the later stage of grain filling. We speculate that the four enzymes, AGPase, SSS, GBSS and SBE, may have a complementary relationship with regards to starch biosynthesis; thus, the absence of GBSS can reduce the activity of the other three enzymes, leading to low total starch content in grains of waxy wheat. The low activity of SUS during later period of grain filling may have caused the reduced degradation of sucrose and the absence of the precursor of starch in grains of waxy wheat. It appears that weakened starch synthesis in the later stage of grain filling of waxy wheat is likely to be the main cause of low kernel weight and total starch accumulation, which ultimately leads to lower grain yield in waxy wheat.


This study was conducted to investigate the effect of subpressure on the bond strength of resin to zirconia ceramic. The subpressure would create a pressure gradient which could clean out the bubbles in the adhesives or bonding interface.Twenty-eight pre-sintered zirconia discs were fabricated. Half of them were polished , and the rest were sandblasted . After sintered,the surface roughness of the zirconia discs was measured. Then, they were randomly divided into two subgroups (n = 7). The groups were named as follows: PC: P + no additional treatments; PP: P + 0.04 MPa after application of adhesives; SC: S + no additional treatments; and SP: S + 0.04 MPa after application of adhesives. Resin columns were bonded to the zirconia specimens to determine shear bond strength (SBS). The bonding interfaces were observed and the fracture modes were evaluated. Statistical analysis was performed on all data.The surface roughness of group S was significantly higher than that of group P (P<0.05). The SBS values were PC = 13.48 ± 0.7 MPa, PP = 15.22 ± 0.8 MPa, SC = 17.23 ± 0.7 MPa and SP = 21.68 ± 1.4 MPa. There were significant differences among the groups (P<0.05). Scanning electron microscopy (SEM) results showed that the adhesives of group SP and PP were closer and denser to the zirconia ceramic than that of group PC and SC. The proportion of the mixed fracture mode significantly increased after adding subpressure (P< 0.05).Subpressure can improve the shear bond strength of resin to zirconia ceramics and increase micro-infiltration between the adhesives and the zirconia ceramics, especially on the rough surfaces. With the increasing demand for esthetics in prosthodontic treatments, various all-ceramic restorations have been widely used in clinical settings . ZirconiMany surface treatments have been used to improve the bond strength of resin to zirconia ceramics over the past two decades, including air abrasion , diamondThe subpressure infiltration technique is based on the vacuum infiltration theory , 29, whi2O3 particles at 2 bar blasting pressure from a distance of 10 mm for 10 sec. All zirconia discs were densely sintered  at a heating speed of 5°C/min to 1450°C, holding for 2 h, and then naturally cooling to room temperature. All zirconia discs were ultrasonically cleansed in deionized water for 10 min to remove surface debris prior to use,dried with clean air flow and kept in a desiccator.Twenty-eight discs  were fabricated from pre-sintered yttria-stabilized tetragonal zirconia polycrystal (Y-TZP) ceramic blocks  with a diamond saw  under copious water. Half of the discs were polished (P) using 1200 grit water-proof silicone-carbide paper, the other half were sandblasted (S)  using 120 μm AlThe surface roughness of the zirconia ceramic was measured after being densely sintered with a surface roughness measuring instrument , and the zirconia ceramic surfaces were observed by 3-D roughness reconstruction with scanning electron microscopy .In this study, the novel subpressure device consists of a subpressure box, a vacuum pump, a vacuum gauge, and a three-way valve. The schematic diagram of the subpressure infiltration technique is shown in Prior to the application of subpressure, group P (polished) and S (sandblasted) were randomly divided into two subgroups, respectively, labeled PC: P + no additional treatments, PP: P + 0.04 MPa after applying adhesives, SC: S + no additional treatments, and SP: S + treated with 0.04 MPa after applying adhesives.2. 3). The resin column  was made of composite resin  with a stainless steel mold in advance. Then, the resin column was put on the zirconia surface, and covered over the hole of transparent tape exactly. 20 N of force was loaded on the resin column for 1 min before curing from three directions for 10 sec with a light curing unit  and holding for 3 min. All specimens were stored in distilled water at 37°C for 24 hours. Finally, zirconia substrates of each specimen were embedded in methyl methacrylate resin.A piece of transparent tape (20 μm in thickness) with a circular hole (4 mm in diameter) was placed on the zirconia surface to define the bonding area. The primer and luting agents  were applied to the zirconia surface according to the manufacturer’s instructions. Group PP and SP were placed in the subpressure box under 0.04 MPa for 3 min after applying the primer and luting agents, respectively , and S is the cross-section surface area (mm2).Shear bond strength (SBS) test was performed using a universal testing machine  at a cross-head speed of 0.5 mm/min until the resin column was separated from the zirconia substrate. The SBS values were calculated according to the following formula:The debonded surfaces of the specimens were inspected by SEM (500× magnification) to assess the fracture modes, which were classified as the following:Cohesive fracture: A fracture within the resin layer, adhesives or zirconia.Interface fracture: A fracture at the adhesive-zirconia or resin-adhesive interface.Mixed fracture: Both cohesive and interface fractures were observed in the same disc.Student’s t-test was performed to evaluate the differences in roughness between group P and S. Two-way ANOVA was performed to study the contributions of the subpressure, the sandblast and the interaction of these factors on the SBS. A chi-square test was used to examine the difference in the distribution of the fracture modes . The statistical significance level was set at α = 0.05.The surface roughness values of group P and S were 0.92 ± 0.81 μm and 3.13 ± 1.01 μm, respectively, and there was a significant difference between them (P<0.05). Representative 3-D images are shown in The mean values and standard deviations of the SBS values were PC = 13.48 ± 0.7 MPa, PP = 15.22 ± 0.8 MPa, SC = 17.23 ± 0.7 MPa, and SP = 21.68 ± 1.4 MPa. There was a significant difference in the SBS among the groups (P<0.05) . Two-wayAs At present, there are amounts of cases where the retention of restorations is reliant on bonding. Poor bonding may result in restorations becoming loose or dislodged, or it may produce micro-leakage, resulting in plaque accumulation, which can lead to secondary caries, marginal gingivitis, etc. . Hence, Previous studies have suggested that roughness affects the bond strength , 33, 34.In this study, the subpressure infiltration technique was as follows. First, the zirconia surface was covered with adhesives. When the coated zirconia specimens were held in the subpressure chamber, the bubbles between the zirconia and the adhesives or in the adhesives were exhausted due to the pressure gradient. When recovering to atmospheric pressure, the pressure on the adhesives pushed them into the voids or fissures of the zirconia surface, which made the adhesives close to the zirconia substrates.As the subpressure infiltration technique mechanism mentioned, SEM results  verifiedThe current theories of mechanics suggest that the adhesive must penetrate into the voids within the adherent surface, and the air bubbles on the interface should be cleared out as possible to promote adhesion. The subpressure could effectively remove air on the interface and amplify the penetration of the adhesives into the micro-pits and micro-hollows of the zirconia surface, which would consequently increase the bonding area and the mechanical locking function , 36. In Furthermore, the viscosity of the adhesives materials, the solvent in the primer or the luting agent could affect the infiltration of the zirconia. Low viscosity of the adhesives is conducive and will allow faster infiltration of the gap. Usually, the strength of low-viscosity adhesives is lower than that of high-viscosity adhesives , 40. AddFinally, compared to other treatments on the zirconia, the subpressure infiltration technique had the advantages of lower technical sensitivity, low cost, short processes and a wide application to other materials. Additionally, the subpressure infiltration technique was not limited to the shape of the restorations.This in vitro study suggests that the subpressure is an efficient method of improving the bond strength of resin to zirconia, and it is more effective on a rough surface.S1 Text(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


An ever growing number of reports on graft rejection and/or failure even with good HLA matches have highlighted an important role of non-HLA antigens in influencing allograft immunity. The list of non-HLA antigens that have been implicated in graft rejection in different types of organ transplantation has already grown long. Of these, the Major Histocompatibility Complex class I chain-related molecule A (MICA) is one of the most polymorphic and extensively studied non-HLA antigenic targets especially in the kidney transplantation. Humoral response to MICA antigens has repeatedly been associated with lower graft survival and an increased risk of acute and chronic rejection following kidney and liver transplantation with few studies showing conflicting results. Although there are clear indications of MICA antibodies being associated with adverse graft outcome, a definitive consensus on this relationship has not been arrived yet. Furthermore, only a few studies have dealt with the impact of MICA donor-specific antibodies as compared to those that are not donor specific on graft outcome. In addition to the membrane bound form, a soluble isoform of MICA (sMICA), which has the potential to engage the natural killer cell-activating receptor NKG2D resulting in endocytosis and degradation of receptor–ligand interaction complex leading to suppression of NKG2D-mediated host innate immunity, has been a subject of intense discussion. Most studies on sMICA have been directed toward understanding their influence on tumor growth, with limited literature focusing its role in transplant biology. Furthermore, a unique dimorphism  at position 129 in the α2 domain categorizes MICA alleles into strong (MICA-129 met) and weak  binders of NKG2D receptor depending on whether they have methionine or valine at this position. Although the implications of MICA 129 dimorphism have been highlighted in hematopoietic stem cell transplantation, its role in solid organ transplantation is yet to be explored. This review summarizes the currently available information on MICA antibodies, soluble MICA, and MICA-129 dimorphism in a setting of solid organ transplantation. The Major Histocompatibility Complex (MHC) class I-related chain genes A and B (MICA and MICB) are a new family of proteins encoded within the human HLA class I genes, first described in 1994 by two independent groups of researchers . While tMIC genes are located within the MHC class I region of chromosome 6 p21.3. A total of seven genes, designated as MICA to MICG, have so far been described, of which MICA and MICB are the only functional genes, while MICC to MICG are essentially pseudogenes , 6. MICAThe domain structure of MICA is much like that of the classical HLA class I molecules with 30% sequence homology and three extracellular domains. Of these, the α1 domain is encoded by exon 2, α2 by exon 3, and α3 by exon 4. The transmembrane (TM) region is encoded by exon 5, while the carboxy-terminal cytoplasmic tail is encoded by exon 6. There are five introns of which the first is the largest intron . The gen2-microglobulin (β2-m)  Figure .Unlike the ubiquitous expression of classical HLA class I molecules, MIC proteins have limited tissue distribution being expressed constitutively on epithelial cells especially in the gastrointestinal tract , endotheIn a study involving total body tissue scan of both MICA and MICB transcription using Northern blot assay, Schrambach et al. reported that both the genes are transcribed in virtually all body tissues except the central nervous system . The surMICA is the most polymorphic non-classical class I gene known so far with 105 alleles having already been reported and new alleles being continuously identified.In contrast to MICA, the genetic polymorphism of MICB is limited with a total of 45 alleles reported so far.Despite the highly polymorphic nature of MICA genes, only one functional site has been identified that appears to affect the binding of MICA ligands to its receptor NKG2D. Accordingly, a non-synonymous Methionine to Valine change at position 129 of the α2 domain categorizes MICA alleles into “MICA-129 met,” which is a strong binder of NKG2D receptor and “MICA-129 val” having weak binding ability. This dimorphism is identified on a single SNP rs1051792 A>G polymorphism at position 454 in exon 3 of MICA gene, corresponding to amino acid position 129 of the MICA protein. It has been shown that MICA-129 met has a 10- to 50-fold greater capacity to complex NKG2D than those with MICA-129 val . The funval/val genotype with increased risk of chronic graft-versus-host disease development in patients undergoing hematopoietic stem cell transplantation (HSCT). Furthermore, the same study revealed that the serum levels of soluble MICA isoform and the presence of antibodies to MICA were associated with cGvHD, which is a major complication following HSCT . Among patients of primary renal grafts, survival was even lower (87.8%) compared with 93.5% for those who did not have MICA antibodies (p = 0.005). Interestingly the association of MICA sensitization with GS was observed in patients well matched for HLA. Independent analysis of 326 patients with 0 or 1 HLA-A, -B, or DR mismatches also showed that recipients with MICA antibodies had poorer GS of 83.2% compared to 95.1% of those without MICA antibodies. However, the study did not investigate the impact of possible confounding factors that are likely to influence graft loss.The first major study to evaluate the potential association of MICA antibodies with overall allograft survival was conducted by Zou et al. . It was Subsequently, Lemy and colleagues  studied It is important here to debate on the results of two important studies—one carried out by Zou et al.  and the Our experience with live donor renal transplantation  is very de novo occurrence of MICA antibodies posttransplantation has been a subject of intense investigation. In a study involving 185 consecutive live related donor renal transplant patients, we analyzed posttransplant serum samples at varying time periods and reported a significant decline in 2-year GS if both HLA and MICA antibodies were detected (The issue of detected . The surp < 0.0001) and 72% for 69 patients with the presence of MICA antibodies (p = 0.02). Among those with living donor grafts, 4-year GS was 78% for 275 patients without HLA antibodies, 62% for 90 patients with HLA antibodies (p = 0.0008), and 80% for 21 patients with MICA antibodies (p = NS). In the 14th workshop, 1-year GS for deceased donor recipients without MICA antibodies was 96.8% as compared to 82.7% for 33 patients with MICA antibodies alone (p = 0.0005). However, the same was not observed with living donor recipients as 19 patients with MICA antibodies had 100% 1-year GS. Multivariate analysis at both time points revealed that MICA antibodies were significantly and independently associated with reduced GS in deceased donor grafts, providing strong evidence for the involvement of these antibodies with graft rejection. It may be mentioned that these studies did not consider previous AR episodes or other confounding factors that are also likely to influence graft loss. The explanation for the lack of significance of MICA antibodies in living donor transplants was attributed to the limited number of patients in the study group.Simultaneously, a large collaborative international study coordinated by Paul Terasaki tested sera for both HLA and MICA antibody production from 1,329 recipients of renal grafts (964 from deceased donors and 365 living donors) from 21 participating centers as a part of the 13th International Histocompatibility and Immunogenetics Workshop Conference (IHIWC) and 22 centers as a part of the 14th IHIWC . Only thde novo MICA antibodies and those that did not.Up until around 2009, the specificity of MICA antibodies and epitopes recognized by them had received very little attention. Gautier et al.  performep < 0.05). Using epitope mapping with a synthesized library of overlapping peptides from the extracellular domains of MICA molecules, the investigators determined nine antigenic regions reactive with MICA antibodies in patient’s serum. Four of these regions were mapped to variable sites in the molecule with polymorphic amino acids while five antigenic regions located in constant region had shared epitopes found in all MICA alleles.At around the same time, Suarez-Alvarez and colleagues  combinedde novo HLA and MICA antibodies in 15 patients following renal transplantation  2 years following transplantation, as were those with HLA-DSA alone who showed significantly reduced eGFR after 3 years. Thus the kinetics of antibody response in this study pointed toward an accelerated graft dysfunction in the presence of MICA antibodies.Whether there is an influence of MICA allele mismatching on antibody production and graft rejection is not fully clear? Cox and coworkers  screenedp = 0.28). By Cox multivariate analysis, graft loss was found to be independently associated with the number of HLA-DR mismatches, AR within the first year posttransplantation, 1-year serum creatinine ≥1.5 mg/dl, and the presence of HLA antibodies at 1 year, but not the presence of MICA antibodies. Another study comprising of 84 renal allograft recipients with a follow-up of 4 years reported that more than one-third of the recipients developed antibodies to HLA and/or MICA and the percentage of recipients who developed de novo antibodies increased with time after transplantation elapsed. Recipients positive for these antibodies had higher serum creatinine levels and worse allograft function than those without antibodies . Analysis by Luminex revealed 55.5% of AR patients as compared to only 6% without rejection had MICA antibodies (p = 0.0020). They also performed MICA allele DNA-typing for donors and recipients where the recipient was positive for MICA antibodies. All patients with MICA antibodies and AR had MICA-DSA although five patients also had autoantibodies. This study was limited by the small number of patients; nevertheless it was the first study to show a possible correlation between MICA-DSA and AR. Additionally, they determined MICA mRNA levels in endomyocardial biopsies obtained from 10 cardiac transplant recipients and found these to be higher in biopsies showing rejection than those without it. In majority of the cases, MICA expression was higher immediately following transplantation, independent of the rejection event, suggesting an upregulated antigen expression due possibly to cellular stress.A number of studies have shed light on the correlation of MICA antibodies to cardiac allograft rejection episodes. Suarez-Alvarez  demonstrp = 0.06). Others however demonstrated a significant association of anti-MICA antibodies with AR and CAV  derived from the proteolytic shedding of membrane bound molecule appears in the serum. MICA, a ligand for NKG2D receptors, forms a complex with ERp5, a disulfide isomerase/chaperone and induces a conformational change enabling proteolytic cleavage of MICA by ADAM proteases. The interaction of NKG2D by the sMICA results in the endocytosis and degradation of receptor–ligand complex and thus suppresses NKG2D-mediated host innate immunity Figure .p < 0.03). On combined analysis of MICA antibodies and sMICA, the authors found tendency for MICA antibodies to occur in the absence of sMICA in the AR group of patients. Conversely, the sMICA levels were detected in patients without MICA antibodies and in absence of AR. These authors published another paper in the same year, where they monitored sMICA levels in pretransplant serum samples and at 15 days, 3 months and 1 year posttransplantation, in 34 heart transplant recipients (p = 0.0001), whereas 9 of the 13 patients, in whose serum sMICA was not detected, developed AR. These observations are in conformity with the previous study suggesting that presence of sMICA contributes to better graft acceptance. Recent experiment conducted on an animal model (rat-to-mouse cardiac transplantation) also demonstrated a negative association of sMICA with AR. The investigators reported that xenografts having anti-MICA antibodies and experiencing AR tended to develop in the absence of sMICA  suggesting that dynamic changes in these levels are associated with BCS development.Zou et al. , in an aClearly, there is a general dearth of published literature evaluating a possible correlation between circulating levels of sMICA and graft outcome in solid organ transplantation. Studies involving larger cohorts and diverse ethnic groups are needed to determine the applicability of sMICA as a potential biomarker of prognostic importance in solid organ transplantation.Despite clear indications of MICA antibodies impacting graft outcome adversely, a definitive consensus on this relationship is yet to be arrived. Furthermore, only a few studies have dealt with the impact of MICA-DSA as compared to those that are NDSA on graft outcome. Two factors are important while analyzing the role of MICA antibodies: (i) currently employed pretransplant crossmatch procedures are not sensitive enough to detect MICA DSA and (ii) the currently used immunosuppressants for induction and maintenance may not be effective in suppressing the immune response against MICA antigens because they are all directed at suppression of T cell response albeit through different mechanisms. Data so far suggest that circulating levels of soluble MICA could well prove to be a potential biomarker of prognostic importance in the assessment of patients after renal transplantation. At the present moment, there is scarcity of published literature evaluating a possible correlation between production of sMICA and their titers with graft outcome in renal transplantation. Further studies involving larger cohorts and diverse ethnic groups could help to reinforce the current findings. Our data on MICA-129 dimorphism adds another dimension in defining its exact role and influence following solid organ transplantation.AKB and NKM designed and wrote the paper. NKM provided excellent inputs and advice.The authors declare that the review article has been prepared independent of any commercial or financial relationships and without any potential conflict of interest.


Type 1 diabetes (T1D) is thought to be an autoimmune disease driven by anti-islet antigen responses and mediated by T-cells. Recent published data suggests that T-cell reactivity to modified peptides, effectively neoantigens, may promote T1D. These findings have given more credence to the concept that T1D may not be solely an error of immune recognition but may be propagated by errors in protein processing or in modifications to endogenous peptides occurring as result of hyperglycemia, endoplasmic reticulum (ER) stress, or general beta cell dysfunction. In the current study, we hypothesized that diabetes-associated epitopes bound human leukocyte antigen (HLA) class I poorly and that post-translational modifications (PTM) to key sequences within the insulin-B chain enhanced peptide binding to HLA class I, conferring the CD8+ T-cell reactivity associated with T1D.www.iedb.org), 138 published HLA class I-restricted diabetes-associated epitopes reported to elicit positive T-cell responses in humans. The peptide binding affinity for their respective restricting allele(s) was evaluated in vitro. Overall, 75% of the epitopes bound with a half maximal inhibitory concentration (IC50) of 8250 nM or better, establishing a reference affinity threshold for HLA class I-restricted diabetes epitopes. These studies demonstrated that epitopes from diabetes-associated antigens bound HLA with a lower affinity than those of microbial origin (binding threshold of 500 nM for 85% of the epitopes). Further predictions suggested that diabetes epitopes also bind HLA class I with lower affinity than epitopes associated with other autoimmune diseases. Therefore, we measured the effect of common PTM  on HLA-A*02:01 binding of insulin-B-derived peptides, compared to native peptides. We found that these modifications increased binding for 44% of the insulin-B epitopes, but only 15% of the control peptides.We first identified, through the Immune Epitope Database  contains supplementary material, which is available to authorized users. Type 1 diabetes (T1D) is characterized by the presence of autoantibodies and islet beta cell loss leading to metabolic dysfunction and hyperglycemia. Beta cell loss is, in part, mediated by T-cells that are reactive to insulin-derived epitopes . RespondThe polymorphisms of HLA class II genes are major risk factors for T1D and CD4 T-cells are widely studied and continue to be of high interest. Human CD4 T-cells recognizing a modified epitope from the insulin-A chain were first described in the context of HLA DR4 . AnotherWe hypothesized that CTLs recognizing diabetes epitopes could escape negative selection in the thymus by having only weak HLA class I binding capacity. The present study was designed to systematically address the issue of HLA binding affinity of human class I-restricted epitopes derived from diabetes-associated antigens. Accordingly, we experimentally determined the HLA class I binding capacity of epitopes from diabetes-associated proteins that were reported to illicit positive CTL responses in humans with T1D. Further, utilizing bioinformatic predictions, we compared the class I binding patterns of diabetes epitopes with those of non-diabetes autoimmune disease and viral epitopes. Finally, we experimentally tested whether PTM modification by citrullination, chlorination, deamidation, and oxidation of insulin epitopes could increase peptide binding to HLA-A*02:01, the HLA class I allele most commonly studied in the context of T1D is HLA-A*02:01, and thereby potentially leading to a stronger in vivo T-cell response.www.iedb.org) and generates reference sets of validated epitopes from various disease indications. Here, the tool was applied to epitopes in the IEDB that are derived from a set of human proteins associated with diabetes [To identify diabetes-associated CTL epitopes from the published literature, we employed a bioinformatics tool recently developed in the Sette laboratory . This todiabetes , further51Cr-release assays. Responses induced following either in vitro or ex vivo stimulation were allowed. Finally, only epitopes between 8 and 11 residues in length were considered, agreeing with the most canonical peptide sizes bound by class I molecules. This generated a set of 138 epitopes  in classical competition assays based on the inhibition of the binding of high affinity radiolabeled ligands to purified major histocompatibility (MHC) molecules, as described in the Methods. In instances where the precise restriction was not available, binding to the most common representative subtype from the same allele family was assayed . The affinity of each epitope to its reported HLA restricting allele(s) is shown in Table In total, 56 (48.7%) of the 115 A*02:01-restricted epitopes bound with high affinity (IC50 < 500 nM); another 34 (29.6%) bound with intermediate affinity (IC50 in the 500–5000 nM range). Of the 38 non-A*02 restrictions, only 3 (7.9%) were associated with an affinity of 500 nM or better and another 10 (26.3%) bound with only intermediate affinity. Thus, overall, 103 of 153 (67.3%) of the HLA/epitope combinations were associated with binding at the 5000 nM, or better, level; that is, 59 (38.6%) were associated with high affinity, and another 44 (28.8%) with intermediate affinity.In terms of thresholds, 75% of the epitopes bound with an IC50 of 8250 nM or better, and 90% of the epitopes bound with an IC50 of 48,000 nM Fig. . These rThe binding threshold identified above is notable in that it is somewhat higher  than the binding threshold (500 nM) previously found to be associated with the vast majority (> 80%) of HLA class I-restricted T-cell epitopes derived from various pathogens –13. To mThese observations were not entirely surprising, as it has been hypothesized that self-epitopes might bind with reduced affinity, and that this is likely characteristic of autoimmunity in general . HoThe measured and predicted lower affinity of diabetes epitopes could be explained if the epitopes recognized by diabetes-associated T-cells are often PTM products, and the modification is associated with increased HLA binding. To test this hypothesis, which was also previously suggested by McGinty and James , we analIn terms of oxidation, Strollo et al.  using soStrollo et al. also reported that the tyrosines (Tyr) in positions 16 and 26 in the insulin B-chain (positions 40 and 50 in the unspliced sequence) were chlorinated. Therefore, another 29 peptides were synthesized to evaluate chlorination of both tyrosine residues.Further, deamination of asparagine (Asn) and glutamine (Gln) residues have been described . DeaminaIn addition to the various published PTMs of the B-chain, we also investigated the effect of citrullination throughout the entire unspliced insulin sequence, which required the synthesis of 70 peptides. Collectively, a total of 276 peptides were synthesized, comprising 181 modified peptides and the corresponding 95 unmodified 9- and 10-mers. All peptides were evaluated for binding to HLA-A*02:01, as described in the Methods. The HLA-A*02:01 binding of each modified peptide is listed, along with its cognate unmodified  peptide, in Additional file We next evaluated the impact of these PTMs on the HLA-A*02:01 binding capacity of insulin-derived peptides, including nine that were previously reported as A*02:01 restricted T cell epitopes .In addition to these previously published PTM epitopes, another 13 peptides (Table p = 0.05).Additional analyses evaluated the effect of 166 modifications of 86 control non-epitopes . For these control peptides, improved binding  was found for only 14 of the 166 modifications (8.4%); this corresponded to only 13/86 (15%) unique control peptides acquiring higher affinity following modification. The difference in rate of improvement, compared to the A*02-restricted insulin-B-derived epitope set, was significant  of native epitopes that are weakly to non-immunogenic in the thymus could induce unanticipated T-cell activation in the periphery. This would imply that T1D is less an error of the immune system and more the result of peripheral tissue dysfunction. Recent publications demonstrating the presence of hybrid and modified peptides, as well as reactive T-cells, in T1D+ subjects provides additional evidence to support this idea .The present analyses assessed the capacity of 138 diabetes-associated CTL epitopes identified in the published literature to bind their reported HLA class I restricting allele(s). The data generated established a binding affinity reference threshold of 8250 nM that captures 75% of HLA class I-restricted diabetes epitopes, representing an affinity threshold somewhat lower than observed for pathogen-derived epitopes and predicted for epitopes from non-T1D autoimmune diseases. Notably, only 38.6% (59/138) of the diabetes epitopes studied bound at least one of its reported restriction elements with high affinity (IC50 < 500 nM) and another 28.8% (44/138) bound at least one allele with intermediate affinity (IC50 in the 500–5000 nM range). A generally lower rate of binding was associated with non-A2 alleles, possibly related to the fact these alleles have been less studied, and therefore the dataset might be inherently less representative and associated with reduced accuracy in terms of HLA restriction determination. It is also possible, as reported in a previous study [Intriguingly, from our data it appears that the lower overall affinity of diabetes-associated epitopes might be unique to this indication, as lower binding affinity thresholds do not extend to other autoimmune epitopes. The reasons for these apparent differences are not clear, but might be related to inherent differences in the immune response associated with T1D progression compared to multiple sclerosis and rheumatoid arthritis, the two indications from which the other autoimmune epitopes evaluated in the current study were derived.To evaluate the potential impact of common PTMs on the HLA class I binding capacity, we synthesized a large set of insulin-derived A*02:01-restricted T-cell epitopes, and also control peptides derived from human insulin but not associated with T1D, incorporating various modifications, including citrullination, chlorination, deamidation, and oxidation. We found that these modifications increased binding for 44% of the known T-cell epitopes, but only 15% of the control peptides. This data supports the hypothesis that the epitopes recognized by diabetes-associated T-cells are often PTM products, and these modified epitopes are associated with increased HLA binding in peripheral tissue. Further study is required to understand the exact contribution of PTMs in T1D disease onset and to ascertain if they initiate disease, are a by-product of disease, or play a role in progression, and also to evaluate if they can be developed diagnostically to identify pathogenic T cells or clinically as a therapeutic.It will be important for future studies to establish the generality of these findings by testing epitopes restricted by other HLA molecules. Query of the IEDB revealed that at least a dozen insulin-derived epitopes are restricted by other, non-A*02, alleles. The analyses could also be expanded to include HLA class II molecules, since posttranslational modifications (and citrullination in particular) have been discussed as a potential factor in modulating autoreactivity in rheumatoid arthritis and other autoimmune pathologies. Further, the present study provides a model to extend a similar analysis towards other antigens associated with T1D, such as GAD, HSP-70, IA-2, and IGRP.+ PBMC from healthy, at-risk, and T1D+ donors for functional assessments, including T-cell proliferation and cytokine production. Parallel experiments could address similar analyses of insulin peptides, both native and post-translationally modified, that bind the Kd and Db class I expressed by NOD mice to allow for a detailed in vivo evaluation of disease relevance. Further experiments could also start to address whether similar findings can be extended to insulin peptides that bind HLA class II, and in particular utilizing the diabetes associated DQ8 molecule.It will also be crucial to test the modified epitopes associated with increased binding for recognition using PBMCs from at-risk, diabetic, and non-diabetic individuals. We have herein identified modified peptides that are high affinity A*02:01 binders, and could represent novel epitopes. It is of obvious interest to include these potential new epitopes in experiments using HLA-A*02:01Clearly, our understanding of the functional role of T-cells and HLA in T1D development and progression is continually evolving. Overall, these findings provide further evidence that human T1D is not solely the result of autoimmunity and may be driven by immune responses to neoantigens, generated from protein processing defects and metabolic dysregulation leading to cell stress.The present study experimentally and bioinformatically assessed the MHC binding capacity of HLA class I restricted T cell epitopes to demonstrate that T1D-associated may have lower overall affinity than epitopes associated with other pathological indications. These observations lend credence to the hypothesis that T1D-associated epitopes may be products of posttranslational modification, and that these modified epitopes are associated with increased HLA binding in peripheral tissue. Indeed, assessment of the binding capacity of PTM versions of known HLA A*02:01 restricted T1D-associated epitopes found increased binding capacity for 44% of the known T-cell epitopes, but only 15% of control peptides. Overall, these findings provide further evidence that human T1D is not solely the result of autoimmunity and may be driven by immune responses to neoantigens, generated from protein processing defects and metabolic dysregulation leading to cell stress.d. Each competitor peptide is tested at six different concentrations covering a 100,000-fold range, and in three or more independent experiments. As a positive control, the unlabeled version of the radiolabeled probe is also tested in each experiment. Utilizing a previously defined threshold [Peptide-MHC affinities were measured using classical competition assays based on the inhibition of binding of a high affinity radiolabeled ligand to purified HLA class I molecules, as previously described . Brieflyhreshold , 12, pephttp://www.iedb.org) [50), the SMM algorithm provides a percentile score expressing the relative capacity of each peptide to bind each specific allele, compared to a universe of potential sequences of the same size.Binding predictions were performed using the command-line version of the SMM prediction tool available on the Immune Epitope Database website (edb.org) , 20. BesPeptides were synthesized by A and A (San Diego) as crude material on a 1 mg scale, or purified (> 95%) by reverse phase HPLC. PTM of individual residues, including deamidation, citrullination, chlorination and oxidation of cysteine was performed as part of the standard Fmoc synthesis. Because of the unavailability of Fmoc-Oxo-His, oxidation of histidine was performed post-synthesis. As a result, histidine cannot be individually oxidized, and in all corresponding peptides all other oxidizable residues  are similarly oxidized. Throughout, in the corresponding peptide sequences, the respective PTMs are identified as follows. B: Cysteic acid; J: chloryl tyrosine; O: iso-aspartic acid; Z: iso-glutamic acid; U: citrulline. The human insulin sequence utilized was UniProt accession number P01308.Additional file 1:Diabetes-associated proteins. Table  listing antigens associated with T1D, pre-diabetes, and diabetes mellitus studies. (DOCX 56 kb)Additional file 2:CTL epitopes associated with previously defined diabetes-associated proteins. Table  listing identified following IEDB query structured as described in the text. (DOCX 131 kb)Additional file 3:Measured binding affinities. Table  listing the modified and native peptides studied and their measured HLA A*02:01 binding capacity. (DOCX 135 kb)Additional file 4:Location of modified residues in full length insulin sequence. Figure (.pdf) highlighting specific insulin residues targeted for modification, as described in the text. (DOCX 103 kb)


There was an error in the Data Summary in the published article. PacBio assembly numbers beginning with CP0119XX should be CP0179XX, as shown below.https://www.ncbi.nlm.nih.gov/nuccore/CP017928; https://www.ncbi.nlm.nih.gov/nuccore/CP017929; https://www.ncbi.nlm.nih.gov/nuccore/CP017930; https://www.ncbi.nlm.nih.gov/nuccore/CP017931; https://www.ncbi.nlm.nih.gov/nuccore/CP017932; https://www.ncbi.nlm.nih.gov/nuccore/CP017933).CAV1015 CP017928–CP017933 .CAV1016 CP017934–CP017937 (The author apologizes for any inconvenience.


Journal of Applied Clinical Medical Physics announces that the article “Reducing motion artifacts in 4D MR images using principal component analysis (PCA) combined with linear polynomial fitting model,” by Juan Yang, Hongjun Wang, Yong Yin, and Dengwang Li has been retracted.The JACMP has issued the following retraction statement:The Journal of Applied Clinical Medical Physics retracts the article “Reducing motion artifacts in 4D MR images using principal component analysis (PCA) combined with linear polynomial fitting model,” which was published in this Journal in the March/April issue, Volume 16, Number 2 (2015). This retraction comes at the request of the authors, following an admission that the scientific data was improperly obtained and reported. The request signed by the authors can be viewed here:The http://www.jacmp.org/index.php/jacmp/article/view/5165/pdf_300JACMP was proper in every respect. There was no indication within the manuscript that the data had its origin in an institution other than that of the four authors.The Editor‐in‐Chief reviewed all aspects of the submission, and the peer‐review process, and found the review by the The Editor‐in‐Chief wishes to acknowledge the cooperation of the corresponding author in this matter, and commends him for his commitment to the scientific process.Supplementary MaterialClick here for additional data file.


CP) with protein from cheap sources such as soybean could potentially improve its nutritive value but may alter its functional and sensory properties. This study was therefore conducted to determine the effect of soy protein isolate (SPI) inclusion (0%–20%) on some functional and sensory properties of cassava starch‐based CP. Functional, pasting, and sensory acceptability were determined using standard methods. Increase in soy protein isolate significantly (p < .05) decreased dispersibility, packed bulk density, swelling power, peak, trough, breakdown, final, and setback viscosities, but increased least gelation concentration, water absorption capacity, and solubility index. This study further showed that despite increasing addition of SPI up to 20%, sensory acceptability of the cassava starch‐based CP formulations did not differ significantly, and most of them had very similar acceptability when compared to that of corn starch‐based CP.Fortification of custard powder ( It is a rich source of energy and it serves as food security and income‐generating crop for millions of people in the developing countries  are inexpensive source of protein; they are used as protein supplement in different forms in food combinations. Soy protein isolate is a purified form of soy protein with the exclusion of nonprotein components. They are used to increase protein content and enhance moisture retention , Mokwa, Niger State, located at the Guinea savannah region of Nigeria. The cassava roots were processed immediately after harvesting into cassava starch powders.Soy protein isolate was obtained from GNC Foods, Ikeja, Lagos, while vanilla, tartrazine, corn starch custard, and sugar were purchased at Kuto Market, Abeokuta, Ogun State, Nigeria.2.2The method described by Oyewole and Obieze  was used2.3Using simplex lattice design for product formulation which provided eight experimental runs, cassava starch powder and soy protein isolate powder were mixed together and analyzed in the following ratios 100:0, 95:5, 90:10, 85:15, and 80:20 .2.42.4.1The method of Coffman and Garcia  was used2.4.2The packed bulk density was determined using the method of Waring and Kinsella .2.4.3Dispersibility was determined using the method of Kulkarni, Kulkarni, and Ingle .2.4.4The method described by Lin, Lin, Lin, and Chou  was used2.4.5The method described by Hirsch and Kokini  was used2.5Pasting properties were determined with the Rapid Visco Analyser (RVA)  using the method described by Maziya‐dixon, Adebowale, Onabanjo, and Dixon .2.6The method of Abdalla, Umsalam, Abdelhalim, and Khalid  with som2.7dislike extremely and 9 = like extremely.Untrained 30 members panelists  from the Federal University of Agriculture, Abeokuta that were regular consumers of corn starch custard participated in the sensory acceptability test. The participants used were those willing to participate in the acceptability test. The samples of gruel produced were cooled before serving the panelists for determination of the following attributes: appearance, flavor, consistency, taste, and general acceptability on a hedonic scale of 1–9, where 1 = 2.8All experimental data obtained were subjected to analysis of variance (ANOVA) procedure of SPSS version 15.0  in the functional properties of the blends. Functional properties are those parameters that determine the application and end use of food materials for various food products.The functional properties of TMS 30572 and SPI blends are presented in Table The least gelation concentration (LGC) of the blends ranged from 2.00% to 8.00%. LGC is the least quantity of starch blends required to form a gel. There was increase in LGC as SPI inclusion increased with 100% CS having the least mean value, while 80% CS:20% SPI had the highest mean value. The reported LGC values showed that the inclusion of SPI affected the gelling ability of the blends. This may be due to the lower gelling ability of SPI in comparison with CS powder, and this implies that more blends will be required to form a gel. This supports the findings of Ogunwolu, Henshaw, Mock, Santros, and Awonorin  who repoPacked bulk density ranged from 0.37 to 0.77 g/ml. Packed bulk density is a parameter that determines the ease of transportation and packaging of powdery products. Increase in SPI inclusion caused a decrease in the bulk density of the blends with 100% CS having the highest mean value, while 80% CS:20% SPI had the least mean. This probably suggests that 80% CS:20% SPI blend will occupy less space during packaging and more products can be transported.Dispersibility ranged from 56.00% to 65.50%. Dispersibility is a property that indicates the rate of reconstitution of flour sample in water. Increase in SPI caused decrease in dispersibility of the blends with 100% CS having the highest mean value, while 80% CS:20% SPI had the least mean value. It was observed that the lower the dispersibility value, the higher the formation of lumps in the gruels, this supports the findings of Akinwale et al. who repoWater absorption capacity measures the amount of water held by the protein matrix at room temperature. Water absorption capacity ranged from 0.96 to 1.34 g/g, 100% CS had the lowest and 80% CS:20% SPI had the highest mean value. The observed increase in WAC is probably due to the increase in SPI inclusion because of its hydrophilic sites which has good affinity for absorbing water. This supports the findings of Otegbayo, Samuel, and Alalade  who repoThe swelling power and solubility index of TMS 30572 CS and SPI blends at different temperatures are presented in Figures The swelling power increased with increase in temperature and it slightly decreased with increase in SPI inclusion. The increased swelling power of the blends with increase in temperature is in agreement with the findings of Agunbiade and Longe  and AdebSolubility index increased with increase in temperature, but decreased with increase in SPI inclusion. Solubility is the percentage of sample dissolved in a solvent. The increase in solubility index with increase in temperature observed in fortified CS‐based custard support the findings of Bolontrade, Scilingo, and Añón  who workAlso, increase in solubility index of the fortified custard paste as SPI inclusion increased observed in this study may be due to the SPI's pH which is above the isoelectric point of protein. Previous work has expressed increase in solubility of material when solution is maintained above the isoelectric point . Increase in SPI inclusion caused reduction in the peak viscosity, trough viscosity, breakdown viscosity, final viscosity, and setback, the values ranged from 155.38 to 279.42 RVU, 75.21 to 117.63 RVU, 78.25 to 161.79 RVU, 98.67 to 153.88 RVU, and 24.46 to 36.25 RVU, respectively.Pasting properties of blends of TMS 30572 CS and SPI is presented in Table Peak viscosity is the highest viscosity attained during starch gelatinization. It is an indication of the extent of starch swelling before its physical breakdown. This probably suggests that as SPI inclusion of the fortified CS‐based custard increased, it interfered with the swelling ability of the fortified CS‐based custard probably because SPI has good water affinity and will compete with the water the starch needs for swelling to occur, thus hindering the extent of swelling.Final viscosity is an indication of the paste stability after cooking and cooling. It indicates the viscous nature of the paste after cooling. The decrease observed in the final viscosity of the fortified CS‐based custard gruel as SPI inclusion increased, probably suggests that 80% CS:20% SPI inclusion may be desirable for infant consumption because of the less dense paste viscosity.Breakdown viscosity is used to determine the stability of starch paste and high breakdown value indicates reduced starch paste stability. Increase in SPI inclusion led to decrease in the breakdown viscosity of the fortified CS‐based custard. This probably suggests that increase in SPI inclusion will increase the stability of the custard gruel.Setback is also a condition that affects the stability of starch paste. Increase in SPI inclusion led to decrease in setback viscosity of fortified CS‐based custard. This probably suggests that fortified custard will have higher resistance to retrogradation and syneresis effect may be reduced, as reported that high setback viscosity indicates lower resistance to retrogradation , except flavor and consistency. Sensory evaluation measures, analyses, and interprets human reactions to characteristic of foods and materials perceived by the senses of sight, smell, taste, touch, and hearing  was observed in the appearance of the gruels. The samples score was rated between 7.50 and 5.73 on 9‐point hedonic scale with corn starch custard having the highest rating, while 100% CS had the least appearance. The higher rating for corn starch custard may be as a result of the yellowish nature of the corn starch.Appearance is an important attribute because of its influence in gruel acceptability. The mean score of the gruel's appearance is presented in Table p > .05) in the flavor of the gruels. All the samples had close similarities in their flavor acceptance.Flavor of a food product is also an important determinant in its acceptance. There was no significant difference (p > .05) was not observed in the consistency of the gruels. The mean score for the custard's consistency ranged between 6.25 and 5.26. Corn starch custard had the highest rating, while 100% CS had the least rating.Consistency is also an important determinant of gruel acceptability. Significant difference (p < .05) was observed in the taste of the gruels. The mean score for the taste perception by the panelists ranged between 7.33 and 5.46 with corn starch custard having the highest mean score, while 100% CS had the least mean score.Taste is another attribute that determines the acceptance of a food product. Significant difference ; this could make cassava starch custard fortified with 20% SPI suitable as weaning food for children.Although, corn starch custard had higher sensory rating than cassava–soy protein isolate custards, the fortified custard samples were acceptable with up to 20% SPI inclusion.None declared.