The capacity of the model to calculate individual predicted probabilities of graft failure over time was evaluated in 80 independent patients. The model classified the patients in three latent classes with significantly different Scr time profiles and different graft survivals. Donor age contributed to explaining latent class membership. In addition to the SCr classes, the other variables retained in the survival model were proteinuria measured one-year after transplantation , pretransplant non-donor-specific antibodies , and dnDSA in patient who experienced acute rejection . In the validation dataset, individual predictions of graft failure risk provided good predictive performances  for the 60 patients who had not developed dnDSA. For patients with dnDSA individual risk of graft failure was not predicted with a so good performance.Identification of patients at risk of kidney graft loss relies on early individual prediction of graft failure. Data from 616 kidney transplant recipients with a follow-up of at least one year were retrospectively studied. A joint latent class model investigating the impact of serum creatinine (Scr) time-trajectories and onset of In kidney transplantation, a new challenge in modelling is individualized prediction of graft failure risk over time. Up to now, no study has reported such a model appropriate for any kidney transplant patients to assess the individual risk and its evolution with time. Numerous risk factors of kidney graft failure are known: factors linked to donor  –6, to trAssociation between graft failure and serum creatinine (SCr) was studied in taking into account SCr levels measured at specific time-points and/or SCr linear evolution with time after transplantation  , 14. Con et al. (2016) showed that the risk of graft failure up to 13 years after transplantation was associated with both current value and current slope of SCr [ de novo DSA (dnDSA) was not considered by the authors and no prediction of the individual graft failure risk was obtained. No joint latent class model has been developed previously to predict graft failure.Latent class models could permit studying the heterogeneity in the individual time-trajectories of SCr . The joie of SCr . Onset o dnDSA on graft survival and (ii) to study the possibility of individualized risk prediction of kidney graft failure within ten years after transplantation.As several papers reported models predictive of graft failure using data collected up to one year after transplantation, it seemed relevant (i) to jointly model the change of serum creatinine over at least the first year after transplantation and (ii) to investigate in such a model the impact of individual potential risk factors on both change of SCr and graft failure risk. Therefore, the objectives of the present study were (i) to develop a joint latent class model investigating the impact of serum creatinine time-trajectories and onset ofData was extracted from the retrospective cohort of kidney transplant recipients grafted at the University Hospital of Limoges (France) between 1984 and the end of 2011 (n=819). Among these patients, 616 who had sufficient data with a clinical and immunological follow-up of at least one year were included in the study. A flowchart showing patient selection is presented in The study database was approved by the French Informatics and Liberty National Commission . All the grafts came from heart-beating deceased donor. More details about the patients included can be found in a previous work of our group .Graft failure, defined as return to dialysis or preemptive retransplantation, was used as the outcome variable. Death was considered as a censored event when the recipient died with a functioning graft. de novo donor specific anti-HLA antibodies (dnDSA) were collected.Donor-specific variables were age and cause of death . Transplantation-related variables included cold ischemia time, retransplantation, and transplantation period . Recipient variables included the following: age at transplantation, gender, nondonor-specific anti-human leucocyte antigen antibodies (NDSA) before transplantation, initial immunosuppressive regimen, and proteinuria levels at month 12 (M12) after transplantation . Additionally, repeated measures of SCr within the first 18 months after transplantation , diagnosis of the first acute rejection episode (AR), and onset ofAnti-HLA antibodies were screened and identified using Luminex® solid-phase assay (One Lambda LABScreen assays) in samples collected before transplantation, at three, six, and twelve months after transplantation and annually thereafter or whenever clinically indicated. Results were expressed as median fluorescence intensity (MFI). MFI>1000 was considered positive. All sera collected and tested using the Complement Dependent Cytotoxicity method prior to availability of Luminex® technology in our center (2007) were reanalyzed using Luminex® as previously described.[Donor, recipient, and transplant characteristics are presented in https://cran.r-project.org/web/packages/lcmm/lcmm.pdf). This model considers the population of subjects as heterogeneous and assumes that the population consists of a finite number of homogeneous subgroups [A joint latent class model for a longitudinal outcome and a right-censored (left-truncated) time-to-event outcome was developed in the “lcmm” R-Package, version 17.8 (available at  classes), 25. Eac classes), 25.The model was constructed in a step-by-step procedure. The first step of model building aimed at defining (i) a mixed-effects model for the SCr trajectories, (ii) the baseline risk function, and (iii) the number of latent classes. Different link functions were compared to transform the observed SCr values into a Gaussian latent variable : (i) a linear transformation, (ii) a rescaled cumulative distribution function of a beta distribution, (iii) quadratic I-splines with equidistant nodes, and (iv) quadratic I-splines with nodes located at the quantiles of SCr distribution. The most appropriate link function was selected on the basis of goodness-of-fit as measured by the discretized Akaike criterion (dAIC) . The ris dnDSA was retained as covariate, its impact would be studied by taking into account several post-dnDSA follow-up periods because associated adverse effects are known to be delayed from their onset. The criteria for final model selection were the BIC and the highest mean posterior class membership probabilities which assess the ability of the model to discriminate between the different latent classes. Finally, the predicted class-specific survivals were compared with the observed survivals within each class using Kaplan-Meier analysis.In the second step, the impact of the available covariates see  as well Because certain research teams studied the factors predictive of short-term graft survival , we also dnDSA randomly selected) grafted since 2002 and followed up in another French transplant center  was used to evaluate the capacity of the model to calculate individual predicted probabilities of graft failure over time [An independent database of 80 patients  and 12 (20%) of them lost their graft. In these 60 patients, the median follow-up time up to graft failure was 6.13 years (range: 1-10). In the 556 patients who did not develop dnDSA, graft failure was observed in 56 patients (11.2%) over the 10 years of follow-up. The median follow-up time to graft failure in these 556 patients was 4.39 years (range: 0.94-10). One hundred and thirty-five patients were treated for the first acute rejection episode over the whole study period, 121 (90%) of which were biopsy proven. T-cell mediated rejection (TCMR) was evidenced in 104 patients, ABMR in 14 patients, and mixed rejection (TCMR+ABMR) in 3 patients. Ninety-four first rejections occurred within the first year after transplantation.Among the 616 patients studied, graft failure was observed in 68 (11%) patients over the 10 years of follow-up . The median follow-up time in patients up to graft failure was 4.97 years (range: 1-10). Among 548 event-free patients, median follow-up time was 7.13 years (range: 1-10). There were 56 deaths with a functional graft. Sixty patients developed dnDSA (yes/no). The estimations related to the proportional hazard submodel of the final joint model are reported in The SCr time-trajectories were fitted after transformation with a I-spline link function with 5 equidistant nodes since it provided the lowest dAIC. The time-trajectories of SCr after transformation were best described using quadratic function of time to allow nonlinear mean trajectories over time. The baseline risk function was modelled parametrically using a two-parameter Weibull baseline risk function. The joint latent class model including three latent classes was retained. The class-specific risks of graft failure were described using presence of NDSA before transplantation, proteinuria at M12 greater than 0.275 g/L (yes/no), and interaction between onset of acute rejection and development of dnDSA data collected over the follow-up outperformed a joint model taking into account data collected up to the end of the first year after transplantation only (p=0.001). This comparison showed the added value of the dnDSA data collected after one year after transplantation.The donor age (categorized as greater or not greater than 60 years) contributed to explaining latent class membership with the recipients of kidneys from donors younger than 60 years having a significantly higher probability to be allocated to class 1 . The mean posterior probability of belonging to each class ranged from 82.6% in patients allocated to class 1 to 89.2% in class 3, indicating a clear discrimination between the latent classes. Of note, this model including acute rejection andμmol/L, a slow decrease in SCr within the first 18 months after transplantation, and a mean risk of graft failure at 10 years after transplantation close to 5%. Class 2 corresponding to the majority of the patients  was characterized by a higher mean SCr baseline value close to 150 μmol/L and a stable mean trajectory over the first 18 months after transplantation while the mean risk of graft failure at 10 years after transplantation achieved 10%. In comparison with class 1, it was associated with a significant increase in the observed incidence of graft failure at 10 years after transplantation . Finally, class 3 with 35 patients (5.7%) was characterized by a mean SCr value close to that of class 2 at baseline followed by a rapid rise of SCr within the first 18 months after transplantation. In comparison with class 1 and class 2, it was associated with a significant increase in the observed incidence of graft failure (p<0.0001). The mean risk of graft failure at 10 years after transplantation in this class was 100%, and no subject in this class had a graft survival greater than seven years. dnDSA and acute rejection was not significant any more.The short-term risk (5 years) of graft failure was also studied using the developed joint three latent classes' model. This 5-year risk was significantly associated with serum creatinine latent classes (p<0.0001), proteinuria at M12 (p=0.003), and pretransplant NDSA (p=0.034). Contrary to the 10-year model, the effect of interaction betweenIndividual predictions of graft failure up to the end of follow-up were computed for 60 patients from the validation dataset who had not developed DSA, according to their observed history of SCr and the covariates retained in the final 10-year joint model. In the 36 tested patients with graft failure, failure risk was adequately predicted in 28 patients as the 95% confidence interval of the predicted probability of graft failure included values greater than 50%. In the 24 patients who did not experience graft failure, the predicted probability of graft failure remained lower than 30%  until the end of the follow-up except for one patient. Thus, using data collected up 12 months after transplantation in this patient subpopulation, sensitivity, specificity, and overall accuracy of the graft failure prediction at ten years were 77.7%, 95.8%, and 85%, respectively.  dnDSA, the model predicted an increased risk of graft failure, but the individual risk of graft failure was not adequately predicted for most of these patients. The best and worth predicted curves of graft failure obtained in this patient subgroup are shown in In the 20 tested patients who had developed dnDSA. In patients with dnDSA, individual prediction of graft failure risk was not obtained with a so good accuracy. The variables retained in the model are patient variables routinely collected and are classically reported to be associated with graft failure  [ dnDSA and acute rejection showing, as previously reported, that dnDSA are more deleterious for graft survival when the patient has also experienced acute rejection [This study presents a new tool which adequately predicts the individual risk of graft failure in patients who did not developnor age) , 14, 27.nor age) , 28. In ejection . This woejection , 30. dnDSA [ de novo DSA [ de novo DSA and who experienced ABMR [ dnDSA on the prediction of graft failure risk in a predictive tool could lead not only to underestimating this risk in patients with dnDSA but also to overestimating the risk in patients without dnDSA, especially in the long term. Herein, taking into account dnDSA it improved on average the long-term survival prediction but not the short-term one . Consistently, Gonzales et al. found that adding presence of dnDSA at 1 year after transplantation to an existing risk model  [ dnDSA occur all over the follow-up and are mostly absent in the first year after transplantation and (ii) graft loss attributable to dnDSA can occur several years after their onset [Although numerous works highlighted the potential impact of certain DSA classes on graft failure , 10, 31, dnDSA , 8, 14. novo DSA . Recentljection) . This reir onset . Recentlir onset , 32, 33. dnDSA.Preliminary tests were performed from our model for making individualized risk predictions in distinguishing patients with and without dnDSA. However, most of the kidney graft failures are observed in this subpopulation. The frequency of graft failure observed herein (in database used for model development) was similar to the frequency reported by Terasaki's team  with a similar follow-up (median of 94 months) [ dnDSA for whom the graft failure was predicted by our model . As a comparison, a sensitivity of 0.72 and a specificity of 0.71 were reported for the Kidney Transplant Failure Score [ dnDSA). It is noteworthy that this prediction can be performed for one year after transplantation using data routinely collected in clinical setting. Interestingly, this prediction tool does not require histologic data, which is in accordance with the current practice to decrease the use of biopsies.The graft failure risk has been less studied in patients who had not developed months) . In thisre Score . AlthougGreat differences between the present model and the previously published tools for graft failure prediction are in (i) predicting the individual risk of graft failure over time contrary to scoring systems which classified the patient in a risk class  , 14, 31 We used for the first time the recently proposed statistical approach of joint latent class models to predict graft outcome. Interestingly, the strengths of this approach have been demonstrated in oncology  and demeWhile we are in an era with very few new therapeutic strategies and new immunosuppressive drugs, individual prognostic tools are necessary for the optimal selection of patients in clinical trials. To demonstrate significant effects of candidate molecules, future trials should focus on patients with poor renal prognoses, and we believe that our model may be a valuable tool for identification of these patients.Last, our findings should be interpreted by taking into account the limitations of current study. We were unable to directly test the impact of immunosuppressive regimens and their blood levels because of dose adjustments and switches from one regimen to another which occurred frequently in patients over such a long study period (from 1984 until 2011). However, we would expect that the different immunosuppressive regimens are at least in part related to different transplantation period, and the period of transplantation was tested but not among the covariates significant in the multivariate model. Similarly, two out of four criteria for expended donation  were missing in the present study but by combining the two remaining criteria in a single dichotomous variable  we did not observe a better performance of our model than using donor age alone.Although allograft histology thanks to repeated biopsies also was found to be associated with transplant outcome , it was Joint models were used to characterize the kinetics of Scr and their link with time-to-event (time-to-graft failure) and to identify relevant covariates linked to graft survival. The individual predictions of graft failure probability obtained in patients without DSA show that this approach could be useful to improve patient's follow-up and the early detection of numerous at-risk patients as approximately half of graft failures are observed in patients without DSA. The graft failure risk would be reevaluated throughout the time after transplantation in case of dnDSA occurrence or acute rejection. In the future, we have the project to include our predictive model in an expert system available for transplant physicians.


Apios fortunei contained about 75% starch, indicating that it is an important starch resource. Starch displayed spherical, polygonal, and ellipsoidal granules with central hila. Granule sizes ranged from 3 to 30 μm with a 9.6 μm volume-weighted mean diameter. The starch had 35% apparent amylose content and exhibited CA-type crystalline structure with 25.9% relative crystallinity. The short-range ordered degree in the granule external region was approximately 0.65, and the lamellar thickness was approximately 9.6 nm. The swelling power and water solubility began to increase from 70 °C and reached 28.7 g/g and 10.8% at 95 °C. Starch had typical bimodal thermal curve in water with gelatinization temperatures from 61.8 to 83.9 °C. The 7% (w/w) starch-water slurry had peak, hot, breakdown, final, and setback viscosities of 1689, 1420, 269, 2103, and 683 mPa s, respectively. Rapidly digestible starch, slowly digestible starch, and resistant starch were 6.04%, 10.96%, and 83.00% in native starch; 83.16%, 15.23%, and 1.61% in gelatinized starch; and 78.13%, 17.88%, and 3.99% in retrograded starch, respectively. The above physicochemical properties of A. fortunei starch were compared with those of maize A-type starch, potato B-type starch, and pea C-type starch. The hierarchical cluster analysis based on starch structural and functional property parameters showed that A. fortunei and pea starches had similar physicochemical properties and were more related to maize starch than potato starch.The dry root tuber of  Arisaema species [Trapa species [Starch is synthesized in plastid and exists as semicrystalline granules in plants. It contains transient  and reserve starches. The reserve starch  is synthesized and stored in plant storage tissues including seed, fruit, tuber, rhizome, corm, bulb, and some metamorphosis roots ,2,3,4. I species , and ker species ,12 in reA-(closer to A-type), CC- , and CB-type (closer to B-type) [Starch mainly consists of amylose and amylopectin. The amylose is a mixture of linear and poorly branched polyglucans, and the amylopectin is highly branched polyglucans. The amylopectin branch-chains form double helices, which are laterally packed to form crystalline lattice. The crystalline lattice has two crystalline structures of A- and B-type allomorphs in plants . However B-type) . Recentl B-type)  summariz B-type) ,17. (2)  B-type) . (3) A-  B-type) ,20. (4)  B-type) .Apios fortunei, and its A- and B-type allomorphs are distributed in different starch granules. The B-type granules have significantly lower gelatinization temperature than A-type granules, but their morphology, size, and amylose content are similar [The different distribution patterns and proportions of A- and B-type allomorph have important effects on the physicochemical properties and applications of C-type starches ,22, thou similar . To our A. fortunei and its morphology, structure, and functional properties were investigated and compared with those of maize A-type starch, potato B-type starch, and pea C-type starch. Our objective was to characterize the physicochemical properties of starch from A. fortunei and provide some information for its utilization in food industry.In this study, starch was isolated from root tubers of A. fortunei had 75.1% starch and 7.6% soluble sugar in dry root tuber. Usually, plant storage tubers and roots have starch contents from 30% to 88% [A. americana contains 68% starch [Apios root tuber indicated that it is an important resource of starch.% to 88% , and cer% to 88% . In addi% starch . TherefoA. fortunei starch had spherical, polygonal, and ellipsoidal shapes with central hila. Most starch granules of maize were polygonal with central hila; potato starch had small spherical granules with central hila and large ellipsoidal granules with eccentric hila; and most of pea starch granules were elliptical and had central hila. Similar morphology in A. fortunei, maize, potato, and pea starches has been reported in previous literature [A. fortunei starch showed unimodal size distribution, and sizes ranged from 3 to 30 μm. However, maize, potato, and pea starches had bimodal size distribution, and the volume percentages of small granules were 8.1%, 5.8%, and 6.2%, respectively. The sizes of small and large granules ranged from 0.4 to 3 μm and from 6 to 40 μm in maize starch, from 0.6 to 6 μm and from 10 to 100 μm in potato starch, and from 0.6 to 6 μm and from 10 to 70 μm in pea starch, respectively. A. fortunei starch had the smallest granule size, and potato starch had the largest granule size among four starches , iodine blue value , and apparent amylose content (AAC) are presented in max can reflect the average chain length and polymerization degree of amylopectin and amylose, the BV is related to the affinity of starch and iodine, and the AAC indicates the absorbance of iodine by amylopectin longer branch-chains and amylose [A. fortunei and potato starches had similar λmax, which was significantly higher than that of maize starch and lower than that of pea starch. Normally, AAC is an important parameter in determining the properties and applications of starch [fortunei starch was significantly higher than in maize starch and lower than in potato and pea starches. The AACs of maize and potato starches determined by iodine adsorption method are 31% and 43%, respectively [A. americana starch has about 32% amylose determined using an iodine potentiometric autotitrator [The absorbance spectrum of starch-iodine complex is shown in  amylose . A. fortf starch ,21. The ectively , and wertitrator , legume titrator , and tubtitrator ,29,30.A. fortunei starch was similar to that of pea starch, except that the shoulder peak at 18° 2θ was more pronounced in A. fortunei starch, exhibiting a CA-type starch in A. fortunei. Usually, normal cereal seeds have A-type starch, tuber crops have B-type starch, and legume seeds and some plant rhizomes have C-type starch [A. americana and fortunei [A-, CC-, and CB-type starch [A. fortunei starch and the lowest in pea starch  spectrometer. The attenuated total reflectance (ATR)-FTIR spectrum is usually used to analyze the short-range ordered structure in external region of starch granule . The Fign starch . The IR f starch .A. fortunei, maize, potato, and pea starches are presented in −1) to account for variations in sample concentration, causing the spectra to be at the same relative scale and therefore directly comparable [A. fortunei and maize starches and the lowest in pea starch. The peak intensity reflects the degree of ordering in semicrystalline regions [−1 arises from the periodic arrangement of alternating crystalline and amorphous region and corresponds to lamellar repeat distance [A. fortunei and potato starches had similar lamellar repeat distances, which were significantly larger than those of maize and pea starches. The lamellar structure of starch is related to plant origin but has no direct relationship with crystalline type. For the starch from the same plant origin, the amylose content is significantly negatively correlated with peak intensity and positively correlated with lamellar distance of SAXS spectrum [The lamellar structure of alternating amorphous and crystalline regions in starch granule can be detected by small-angle X-ray scattering (SAXS) instrument . The SAXmparable . The lam regions . The maidistance . A. fortspectrum ,35. Lamespectrum .A. fortunei, maize, potato, and pea starches are presented in A. fortunei starch had two gelatinization peaks with peak temperatures at 68.0 and 75.6 °C. The two peaks resulted in the wide gelatinization temperature range (22.1 °C). Usually, A-type crystallinity has high gelatinization temperature and B-type crystallinity has low gelatinization temperature [americana and fortunei [A. fortunei C-type starch contains A-type starch granules and B-type starch granules [A. fortunei and pea starches might result in different gelatinization properties.The thermal properties of perature ,30. C-typerature ,30. Whenperature ,30. The fortunei ,29. For fortunei . Howevergranules . The difA. fortunei, maize, and pea starches increased after 70 °C. After 80 °C, potato starch had the highest swelling power, maize and pea starches had the lowest swelling power, and pea starch had the highest water solubility. The swelling power and water solubility reflect the water-holding capacity and dissolution degree of starch during heating, respectively. Granule size, starch component of amylose and amylopectin, non-starch component of protein and lipid, and amylopectin structure influence the swelling power and water solubility of starch [A. fortunei, maize, potato, and pea starches might account for their different swelling powers and water solubilities.The swelling power and water solubility of starch at different temperatures are shown in f starch ,10. The A. fortunei starch had significantly higher pasting viscosities and lower pasting times and temperatures than maize and pea starches, which might be due to its components of A-type starch granules and B-type starch granules.Pasting properties of starches, an important functional property determining the quality and utilization of starch in food industry, were measured with a rapid visco analyzer (RVA). The Aspergillus niger amyloglucosidase (AAG), and the released glucose was converted to the degraded starch. The starch fractions are classified into rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS), which are digested within 20 min, between 20 and 120 min, and after 120 min, respectively [A. fortunei, maize, and pea starches had similar RDS and were degraded faster than potato starch, and the RS of A. fortunei starch was significantly higher than that of maize and pea starches and lower than that of potato starch. The different digestion properties of four native starches might be affected by granule morphology and size, starch components, and crystalline structure [A. fortunei, maize, potato, and pea had similar RDSs in their gelatinized and retrograded starches, but RS of gelatinized and retrograded starches was significantly lower in A. fortunei and pea than in maize and potato  and ectively . The RDStructure . Gelatintructure . A. fortd potato . The diftructure .A. fortunei, maize, potato, and pea, the hierarchical cluster analysis was conducted based on the structural and functional property parameters including volume-weighted mean diameter, AAC, relative crystallinity, ordered and lamellar parameters, gelatinization enthalpy, pasting viscosities, and digestion properties . Potato fresh tubers and pea mature seeds were obtained from the natural food market . Normal maize starch (S4126) was purchased from Sigma-Aldrich .Fresh root tubers of A. fortunei were washed and sliced into pieces. The samples were freeze-dried and ground extensively through 100-mesh sieve to obtain the flour. The soluble sugar in flour was extracted with 80% (v/v) ethanol, and the starch in flour was hydrolyzed into soluble sugar using 4.6 M HClO4. The extracted and hydrolyzed soluble sugar were measured using anthrone-H2SO4 method and converted to the contents of soluble sugar and starch in dry root tuber following the method of Gao et al. [The fresh root tubers of o et al. .A. fortunei root tuber, potato tuber, and pea seeds following the method of Fan et al. [A. fortunei root tubers and potato tubers were washed and chopped into pieces. The samples were homogenized in water using a home blender  and squeezed through 4 layers of cheesecloth. The starch-slurry was filtered using 100- and 200-mesh sieves . After standing about 5 h, the precipitated starch was washed 3 times using 0.2% NaOH to remove the surface protein from starch granules. The starch was washed 3 times with water and 2 times with anhydrous ethanol, dried at 40 °C, and ground through 100-mesh sieve.Starches were isolated from n et al. , with sow/v) starch suspension in 50% glycerol was observed and photographed under a polarized light microscope  under normal and polarized light. The size distribution of starch granules was measured using a laser diffraction instrument  following the method of Cai et al. [The 1% . The relative crystallinity was calculated following the method of Wei et al. .−1 before deconvolution. For deconvolution, the assumed line shape was Lorentzian with a half-width of 19 cm−1 and a resolution enhancement factor of 1.9.The starch-water slurry was analyzed using a FTIR spectrometer  with a DTGS detector equipped with an ATR cell following the method of Wei et al. . OriginaThe starch-water slurry was kept in sealed cell and analyzed using a SAXS instrument  equipped with Vantec 2000 detector and pin-hole collimation for point focus geometry. The test condition setting and lamellar parameter analysis were described previously by Cai et al. .w/v) starch-water slurry was heated in a ThermoMixer C  for 30 min, cooled to room temperature, and centrifuged . The supernatant was removed for measuring the soluble carbohydrate using anthrone-H2SO4 method to calculate the water solubility, and the precipitate was weighed to calculate the swelling power.The swelling power and water solubility of starch were measured from 50 to 95 °C at an interval of 5 °C following the method of Lin et al. . BrieflyThe five milligram of starch and 15 μL of water were mixed and sealed in an aluminium pan , held at 4 °C overnight, and equilibrated for 2 h at room temperature. The sample was heated from 25 to 130 °C at a rate of 10 °C/min using a differential scanning calorimeter .w/w) starch-water slurry was heated and cooled using a rapid visco analyzer . The temperature program included holding for 1 min at 50 °C, heating at 12 °C/min to 95 °C, holding for 2.5 min at 95 °C, cooling at 12 °C/min to 50 °C, and holding for 1.4 min at 50 °C.The 7%  at 37 °C using a ThermoMixer C  with shaking at 1000 rpm. The hydrolysis was terminated by adding 240 mL of 0.1 M HCl and 2 mL of 50% ethanol, and then centrifuged . The glucose in the supernatant was measured using a glucose assay kit .The native, gelatinized, and retrograded starches were prepared and degraded by both PPA  and AAG  following the method of Lin et al. . Brieflyp-values were less 0.05. Hierarchical cluster analysis was employed using between-groups linkage as the cluster method and Pearson correlation as the interval measure.The data reported in all the tables were means ± standard deviation. The one-way analysis of variance by Tukey’s test was evaluated using the SPSS 19.0 Statistical Software Program . Differences were considered statistically significant if A. fortunei is an important starch resource. Starch exhibited spherical, polygonal, and ellipsoidal granules with central hila and had a unimodal size distribution that ranged from 3 to 30 μm. Starch had 35% AAC and exhibited CA-type crystallinity. Starch had two gelatinization peaks in water. The swelling power of A. fortunei starch was significantly lower than that of potato starch and higher than that of maize and pea starches at 95 °C, but its water solubility was the lowest among the four starches. The peak, hot, breakdown, and final viscosities of A. fortunei starch were significantly lower than those of potato starch and higher than those of maize and potato starches, but its setback viscosity was the highest among the four starches. The RDS of native starches from A. fortunei, maize, and pea was similar but significantly higher than that of potato starch. The RS of gelatinized and retrograded starches from A. fortunei and pea was similar but significantly lower than that of maize and potato starches. This study could provide important information for the utilization of root tuber starch of A. fortunei.The root tuber of


Different technique for ceramic veneering and thermal tempering process are expected to be a reason for alteration in strength of ceramic veneered zirconia. This study evaluates the effect of different veneering technique and varied thermal tempering process on flexural strength of ceramic veneered zirconia.Ceramic veneered zirconia bars  were prepared from zirconia block (e.max® ZirCAD), sintered at 1500°C for 4 hours, and veneered with ceramics with different techniques including CAD-fused using e.max CAD® (C), Pressed-on using e.max® Zirpress (P), and layering using e.max® ceram (L), with different tempering process through fast (F), medium (M), and slow (L) cooling (n=15). The specimens were determined for flexural strength on a universal testing machine. ANOVA and Bonferroni’s multiple comparisons were used to determine for significant difference (α=0.05). Weibull analysis was applied for survival probability, Weibull modulus (m), and characteristics strength (σc). The interfaces were microscopically examined. The phase transformation of zirconia was determined using X ray diffraction.p<0.05). No significant effect on flexural strength upon tempering process was evidenced (P >0.05). Phase transformation from t→m related with veneering and tempering procedure.The mean±sd (MPa), m, σc of flexural strength were 922.06±83.45, 12.78, 958.32 for CF, 924.26±74.64, 14.28, 959.62 for CM, 930.25±92.42, 11.83, 970.83 for CS, 518.29±59.97, 10.11, 542.97 for PF, 516.50±67.51, 8.75, 539.17 for PM, and 520.51±42.38, 14.59, 544.51 for PS, 604.36±64.09, 11.28, 630.67 for LF, 583.81±56.95, 11.67, 609.81 for LM, 547.33±52.23, 12.19, 569.36 for LS. The flexural strength was significantly affected by veneering technique (Strength of ceramic veneered zirconia associated with different veneering techniques, but not directly related with tempering process. CAD-on ceramic veneering zirconia is benefit for enhancing the strength of ceramic bilayer and was recommended as a method for ceramic veneering zirconia. Key words:CAD-CAM, cooling process, flexural strength, thermal tempering, zirconia. The increasing demand for highly esthetic and predictably long-lasting restorations has led to the development of several ceramics for restorative dentistry, based on computer-aided design and computer-aided manufacturing (CAD-CAM) technology . The CADCeramic-veneered zirconia is generally constructed through the traditional layering technique, using feldspathic porcelain or other glass ceramics, for instance nano-fluoroapatite-, leucite reinforced-, and lithium disilicate-glass ceramic . These cThe strength of ceramic-veneered zirconia is affected by the thermally related sintering procedure. The sintering process is a thermally generated crystallization of veneering ceramic to be adhered to the zirconia substructure. The promptly applied heat needs to be radiated from the muffle of the sintering furnace to reach the external surface of restoration and properly conducted through the inner surface to derive for suitable maturation of ceramic. Similarly, the veneering ceramic and zirconia substructure need to release the heat from the matured stage to room temperature (RT) during the cooling phase as a tempering procedure. This stage generates a thermal effect and crucially developed residual stress, affecting the strength of the restoration. The tempering process directly relates to the rate and method of cooling from the sintering temperature to RT, which induces appropriate residual stress in the ceramic-veneered zirconia that directly affects the reliability of strength . PreviouThe residual stresses are generated as a result of the thermal gradient from the CTE difference between zirconia and veneering ceramic during the cooling period until the glass transition temperature (Tg) is reached . Such coThe specimens for evaluation of flexural strength of ceramic veneering Y-TZP  were pre-Preparation of Zirconia SpecimenA hundred and thirty-five Y-TZP  bars [width (W), length (L), thickness (T) = 5, 31.25, 0.9 mm) were prepared using sectioning apparatus , and fired in the furnace  at the recommended temperature of 1500ºC for four hours, using a heat rate of 5 ºC/min, to derive for fully sintered bars  due to 20% sintering shrinkage. All bars were randomly segregated into nine groups (15 bars each) to be veneered with ceramics  according to three different veneering and tempering methods.-Conventional Ceramic Layering TechniqueA thin layer of special liner  was applied to the zirconia bars, which were sintered in a furnace  twice to reach a thickness of 0.1 mm. Then, the creamy mixed consistency of dentine ceramic  was applied, condensed with ultrasonic condenser , and fired for no more than three times to derive for the final dimensions.-Press-on Ceramic Veneering TechniqueThe zirconia bars were applied with a thin layer of special liner and fired in a furnace as previously described. The wax pattern was applied on the sintered liner surface of zirconia using blue inlay wax , invested in the investment mold , and then heat-pressed with pressable ceramic  at 910ºC in the pressing furnace . After divestment, the samples were finished and polished to derive for the final dimension.-CAD-on Ceramic Veneering TechniqueThe bar-shaped veneering ceramics  were prepared from pre-crystallized lithium disilicate blocks  using a precision machine. The zirconia bar and ceramic bar were joined with the Crystall/Connect (Ivoclar Vivadent), and fired at 840ºC as per manufacturer’s instruction.-Thermal Tempering ProcessThe specimens were subjected to different tempering processes through F-, M-, and S-cooling. F-cooling was performed by fully opening the furnace’s muffle after sintering to allow for cooling to RT. M-cooling was performed by leaving the specimen to cool down in the closed muffle until Tg of each veneering ceramic was reached, and then fully opening the muffle to cool to RT. S-cooling was performed by leaving the specimen to cool in the closed muffle until a temperature of 200 ºC, and then fully opening the muffle to cool down to RT.-Evaluation for Flexural StrengthControlled cracks were initiated on the veneering ceramic surface with Vickers diamond indentation of the microhardness tester  at 4.9 N load, for 15 seconds dwelling time, to produce transverse and longitudinal crack lines, and optically measured for crack length (×50 magnification) at 24 hours after initiation  with vacuum 130 m-torr, at 10 mA current, for three minutes. The fracture surfaces and zirconia-ceramic interface of specimens for each group were determined for scanning electron microscopy  and energy dispersive analysis of x-ray  for characterizing the failure surface and interfacial zone.-Evaluation of Crystal StructureThe crystal phases of ceramic-veneered Y-TZP were determined for the quantity of tetragonal (t) and monoclinic (m) crystalline structures of Y-TZP with the X-ray diffractometer . The specimens were scanned with copper k-alpha (Cu Kα) radiation from 2-theta  of 20–40o with 0.02o stepwise for two-second intervals. The crystalline phase was determined and compared to the standard database on powder diffraction, and calculated for respective d-values with Bragg equation 7 .In which: λ: wavelength (0.154058 nm for CuKa), d: distance of planes in the hkl-Miller indices, θ: Bragg angleThe proportion of m- to t- crystal was quantified from the spikes’ magnitudes using X-Pert Plus software . The amount of m-phase to overall zirconia phase content was computed from Garvie-Nicholson equation and further adjusted for nonlinearity with the Toraya equation, as shown in equations 8, 9, and 10 .In which: It and Im: essential intensities of t- and m- phase, C: compositional corrected factor (C = 1.32), Xt and Xm: the Toraya-corrected portion of t- and m- phase of Y-TZP-Statistical DeterminationThe data was statistically evaluated with software SPSS/PC+ Version-20 . An analysis of variance (ANOVA) was determined for significant differences of fracture strength relative to the ceramic veneering method and tempering procedure. Bonferroni post-hoc multiple tests were evaluated for the difference between variables at α=0.05. An analysis of fracture strength’s reliability was determined by Weibull++®statistics , and appraised for Weibull modulus (m) using equation 11 in conjunction with the slope of graph sketched between ln{ln(1/Ps(Vo))} and m ln(σ/σo).Where: Ps (Vo): survival probability of identical sample, Vo: volume of sample, σf: flexural strength; σo: characteristic flexural strength, m: Weibull modulus.p<0.05). No statistically significant differences were indicated on mean flexural strength due to the effect of the tempering process and the interaction between these two factors (p>0.05) as presented in p<0.05). The C-veneering technique provided significantly higher flexural strength than L- and P-veneering (p<0.05). However, the tempering process did not initiate a significant impact on flexural strength (p<0.05). No significant differences in flexure strength were indicted among ceramics fabricated from either P- or L- veneering technique upon different tempering processes (p>0.05), except for the LF group, which was significantly different from PF, PM, and PS (p<0.05) , 95% confidence interval (CI), Weibull modulus (m), characteristic flexural strength, and relative phase transformation for every group is demonstrated in strength . The CAD(p<0.05) . The chaThe SEM-micrographs revealed a harmonized inter-digitation between zirconia and veneering ceramic for every group, which indicated favorable bonding of ceramic-veneered zirconia upon different veneering techniques and tempering processes Fig. A. HoweveThe XRD patterns mostly indicated t-crystalline phase and a minimal amount of m-crystalline phase for each group as indicated in Figure In this investigation, the flexure strength of ceramic-veneered zirconia was influenced by the veneering technique but not disturbed by the tempering process. Thus, a null hypothesis was rejected for the effect of the ceramic veneering technique, and accepted for the effect of the tempering process. The CAD-on veneering technique significantly engendered higher flexural strength for ceramic-veneered Y-TZP than other veneering techniques as supported by other studies ,27. The The thermal tempering processes indicated no significant effect on flexural strength. However, it seems to demonstrate that prolong tempering through S-cooling exhibited a higher impact on enhancing flexural strength than M- and F-cooling for both CAD-on and press-on techniques. Conversely, a short tempering process through F-cooling exhibited higher flexural strength than M- and S-cooling for the L-veneering technique. This probably indicated that S-cooling for the CAD-on technique was capable of inducing appropriated residual stress to enhance flexural strength as supported by another study . This waThe microscopic investigation indicated that the fracture initiated at the veneering ceramic propagated to the interface and penetrated through the zirconia substructure until failure. The fracture patterns of CAD-on groups were rather perpendicular to the surface of zirconia, whereas the others were slightly oblique. This possibly related with the lower fracture toughness and amount of porosities in different ceramic veneering techniques, which resulted in the different paths of crack propagation. A number of porosities found in layering- and press-on groups could act as stress concentration, leading to crack propagation. The crack usually propagates along the veneer layer, where the fracture toughness is low, as well as along the core-veneer interface due to possessing a lower strain energy release rate compared to zirconia core . The betThe stress generated from different veneering techniques and cooling methods can induce phase transformation. The CAD-on technique was capable of inducing higher t→m phase transformation than layering and press-on techniques. This implied that the capability of generating residual compressive stress for CAD-on was higher than other techniques, which resulted in better enhancing of flexural strength. To derive for a suitable amount of residual stress for prompt flexural strength, the appropriate ceramic veneering technique needs to be considered primarily . This stThis investigation described the role of ceramic veneering techniques and thermal tempering processes for the flexure strength of ceramic-veneered zirconia. The experiment indicated that flexure strength of ceramic-veneered zirconia is primarily influenced by veneering technique. The CAD-on technique rendered favorable flexural strength, as it is capable of inducing residual stress to enhance fracture resistance for ceramic-veneered Y-TZP. The tempering process did not impair flexural strength. However, S-cooling tends to favor providing suitable residual stress for CAD-on and press-on techniques, whereas F-cooling seems favorable for layering techniques. Proper selection of ceramic-veneering Y-TZP is extremely crucial to assure durable fracture resistance. CAD-on was suggested as a suitable veneering technique that conferred favorable fracture resistance for ceramic-veneered Y-TZP.


Their structural and functional properties were investigated and compared among different extraction media. The results showed that the granule size, apparent amylose content, lamellar peak intensity, thermal properties, and pasting properties were different among different-colored sweet potatoes due to their different genotype backgrounds. The four extraction media had no significant effects on starch structural properties, including apparent amylose content, crystalline structure, ordered degree, and lamellar peak intensity, except that the NaOH and Na2S2O5 treatment were able to increase the whiteness of purple and yellow sweet potato starches. The different extraction media had some effects on starch functional properties, including thermal properties, swelling power, water solubility, and pasting properties. The above results indicated that the H2O was the most suitable extraction medium to simply and fast isolate starch from root tubers of different-colored sweet potatoes.Different-colored sweet potatoes have different contents of pigments and phenolic compounds in their root tubers, which influence the isolation of starch. It is important to justify the identification of the most suitable isolation medium of starch from different colored root tubers. In this study, starches were isolated from root tubers of purple, yellow and white sweet potatoes using four different extraction media, including H Ipomoea batatas (L.) Lam.] is one of the world’s important starch-producing crops. Its dry root tuber contains 50 to 80% starch, and is also rich in dietary fiber, vitamin C, provitamin A, iron, and minerals [Sweet potato [minerals ,2. Threeminerals ,4,5. Theminerals ,5.2O extraction [w/v) NaOH extraction [w/v) Na2S2O5 extraction [w/v) NaHSO3 extraction [w/v) Na2S2O5 extraction is also used to isolate starch from maize endosperm [2S2O5 and NaHSO3 treatment can effectively minimize browning of the sample during starch isolation [The root tuber of sweet potato is a good resource for starch . The strtraction ,7,8, 0.2traction ,10, 0.2%traction ,12, and traction . The appndosperm . The NaOsolation ,12,15.Many studies have shown that extraction media affect the structural and functional properties of starch ,17,18. B2O, 0.5% Na2S2O5, 0.2% NaOH, and both 0.5% Na2S2O5 and 0.2% NaOH extraction media. Their structural and functional properties were investigated and compared. Our objectives were to evaluate the effects of extraction media on starch properties, and find a suitable medium for isolating starch from different-colored sweet potatoes. This study could provide an important reference for the isolation method of starch from different-colored sweet potatoes, and would be helpful in the applications of sweet potato starches in food and nonfood industries.In this study, starches were isolated from purple, yellow and white sweet potatoes using H2O, the color was significantly different among the three varieties. L*-value was the highest for white-fleshed sweet potato and the lowest for purple-fleshed sweet potato , OD620/550 and apparent amylose content are presented in 2S2O5 and NaOH treatment had no significant effect on the absorption spectrum of starch-iodine complex, including OD620/550 and apparent amylose content. No significant differences were also reported in the apparent amylose contents of starches isolated with different extraction media in acorn starches [The absorption spectrum of the starch-iodine complex is shown in starches  and pea starches . Howeverstarches , and froA- (closer to A-type), CC-  and CB-type (closer to B-type) starches according to the proportion of A- and B-type crystallinity from high to low. The CC-type starch has strong diffraction peaks at about 17° and 23° 2θ, and small peaks at about 5.6° and 15° 2θ. The peak at 5.6° 2θ is the characteristic of B-type crystallinity. Compared with CC-type starch, the CA-type starch has a shoulder peak at about 18° 2θ, which is the characteristic peak of A-type crystallinity [2O, the Ningzi 2, Su 16 and Su 29 starches all showed CA-type XRD patterns due to the existence of shoulder peak at 18° 2θ. It is noteworthy that the intensity of the shoulder peak at 18° 2θ was the weakest and that of the peak at 5.6° 2θ was the strongest in Su 16 starch, indicating that the proportion of B-type crystallinity was higher in Su 16 starch than in Ningzi 2 and Su 29 starches. The A-, CA-, CB- and C-type starches have been reported in different sweet potato varieties in previous papers [2O, Na2S2O5, NaOH, and both Na2S2O5 and NaOH treatment were 22.2%, 23.2%, 22.1%, and 23.3% for Ningzi 2, 20.8%, 21.5%, 21.2%, and 21.4% for Su 16, and 22.8%, 22.4%, 21.9%, and 22.2% for Su 29. These results showed that the Na2S2O5 and NaOH treatment had no effect on the starch crystalline structure.The X-ray powder diffraction (XRD) patterns of starches are shown in allinity . Accordis papers ,22,26. T−1 including at approximately 1150, 1125 and 1105 cm−1 , and 1100–900 cm−1 including at approximately 1078, 1045, 1022, 995 and 925 cm−1 (C–O–H bending). The bands in the region 1100–900 cm−1 are sensitive to changes in starch structure; the bands at 1047 and 1022 cm−1, especially, are widely used to reflect the ordered structure [−1 are associated with ordered/crystalline and amorphous regions in starch, respectively. The ratio of absorbance 1045/1022 cm−1 can be used to quantify the ordered degree [−1 of H2O, Na2S2O5, NaOH, and both Na2S2O5 and NaOH treatment were 0.70, 0.70, 0.69, and 0.71 for Ningzi 2, 0.71, 0.70, 0.71, and 0.71 for Su 16, and 0.71, 0.70, 0.70, and 0.70 for Su 29. These results indicated that the Na2S2O5 and NaOH treatment had no effect on the double-helical order of starch in granule external region. Similar results are also reported in parota starches isolated using H2O, sodium bisulfite and acid steeping [The short-range ordered structure of starch, defined as the double-helical order, can be detected using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrometer. The ATR-FTIR spectrum can reflect the short-ranged ordered structure in the starch external region . The dectructure . The band degree . The stasteeping .−1) to account for variations in sample concentration, leading to the spectra being at the same relative scale, and therefore directly comparable [max) and intensity, can be measured by a simple graphical method, and the lamellar repeat distance (D) can be calculated from the Smax according to D = 2π/Smax [−1, corresponding to a lamellar repeat distance of approximate 10.2 nm. The different extraction media had no effect on lamellar repeat distance. However, the peak intensity was significantly different among the three sweet potato varieties, at about 320, 220, and 260 for Ningzi 2, Su 16, and Su 29, respectively. The peak intensity depends mainly on the degree of ordering in semicrystalline regions [2S2O5 and NaOH treatment had no effect on lamellar peak intensity.The lamellar structure of alternating amorphous and crystalline regions in starch granules can be detected by a small-angle X-ray scattering (SAXS) instrument . The SAXmparable . The par 2π/Smax . The amo2O extraction, significant differences were detected in the three sweet potato starches. Ningzi 2 starch had a single gelatinization peak with high gelatinization temperatures and enthalpy and low gelatinization temperature range, and Su 16 starch had a wide gelatinization peak with low onset gelatinization temperature and wide gelatinization temperature range. Similar results have also been reported in some sweet potato starches [The thermal properties of sweet potato starches isolated using four extraction media are presented in starches ,22. In tstarches . For thestarches .2S2O5 extraction had an effect on the swelling power and water solubility of starch. Similar results have also been reported in acorn starches isolated with alkaline and enzymatic extraction media [The swelling powers and water solubilities of starches at 95 °C are shown in on media .The pasting properties of starches, an important functional property determining the quality and utilization of starch, were analyzed using a rapid visco analyzer. The changes in pasting profile are influenced by structural, thermal, and morphological changes taking place as a result of temperature . The pasPurple sweet potato variety Ningzi 2, yellow sweet potato variety Su 16, and white sweet potato variety Su 29 were used in this study. They were grown under normal agronomic practices in the experimental field of Jiangsu Academy of Agricultural Sciences, Nanjing, China in 2017. The root tubers after harvest were stored in dark with about 85% humidity at 12 °C for about one month before use.Starch was isolated from the root tubers using the following four extraction media according to the methods of Lai et al. , OsundahThe color of starch was measured using a portable colorimeter  in which L*, a*, and b* values were measured. The L*, a*, and b* values state the position on the white/black, red/green, and yellow/blue axis, respectively.The nitrogen content in dry starch was measured using an elemental analyzer , and then converted to protein content using a 6.25 conversion factor.Starch powder was directly mounted on an aluminum stub using double-sided adhesive tape. The sample was coated with gold using a sputter coater and observed using an environmental scanning electron microscope . Starch granule size was analyzed using a laser diffraction particle size analyzer  following the methods of Zhang et al. . BrieflyThe starch-iodine absorption spectrum and apparent amylose content were measured as previously described by Zhang et al. . BrieflyThe crystalline structure of starch was analyzed using an X-ray powder diffractometer . The signal-to-noise ratio of XRD spectrum is influenced by water content of starch . The sam−1 before deconvolution. For deconvolution, the assumed line shape was Lorentzian, with a half-width of 19 cm−1 and a resolution enhancement factor of 1.9.The short-range ordered structure of starch was analyzed using a FTIR spectrometer  with a DTGS detector equipped with an ATR cell. The sample preparation and test condition setting were described previously by Wei et al. . The oriThe lamellar structure of starch was analyzed using a SAXS instrument  equipped with Vantec 2000 detector and pin-hole collimation for point focus geometry. The sample preparation, test condition setting, and lamellar parameter analysis were described previously by Cai et al. . BrieflyThe thermal properties of starch were measured using a differential scanning calorimetry  following the method of Zhang et al. . Brieflyw/v) starch-water slurry was heated in a water bath for 30 min, cooled to room temperature, and centrifuged . The supernatant was removed to measure the soluble carbohydrate using anthrone-H2SO4 method, and the precipitate was weighed to calculate the swelling power. The swelling power was the ratio of precipitate weight and starch weight after subtraction of the soluble carbohydrate weight, the water solubility was the amount of soluble carbohydrate by the original starch.The swelling power and water solubility of starch were measured at 95 °C as previously described by Lin et al. . BrieflyThe pasting properties of starch were measured using a rapid visco analyzer  following the method of Zhang et al. . BrieflyThe data reported in all the tables are means ± standard deviation. One-way analysis of variance by Tukey’s test was evaluated using the SPSS 16.0 Statistical Software Program.2O, 0.5% Na2S2O5, 0.2% NaOH, and both 0.5% Na2S2O5 and 0.2% NaOH extraction media. Starches from different colored root tubers had differences in granule size, amylose content, thermal properties, swelling power, water solubility, and pasting properties due to their different genotypes. Na2S2O5 and NaOH extraction were able to increase the whiteness of starch from purple and yellow sweet potato, but had no significant effect on apparent amylose content, crystalline structure, ordered degree, and lamellar structure of starches. The different extraction media had a slight effect on the thermal properties, swelling power, water solubility, and pasting properties of starches. This study indicates that H2O is the most suitable extraction medium for simply and quickly isolating starch from root tubers of different-colored sweet potatoes.In conclusion, starches were isolated from root tubers of purple, yellow and white sweet potatoes using H


Triticum aestivum) is a 3:1 mixture of the two polysaccharides amylopectin and amylose. Altering the activity of certain key starch synthesis enzymes  has succeeded in generating starches containing a different polysaccharide ratio. Here, mutagenesis, followed by a conventional marker‐assisted breeding exercise, has been used to generate three mutant lines that produce starch with an amylose contents of 0%, 46% and 79%. The direct and pleiotropic effects of the multiple mutation lines were identified at both the biochemical and molecular levels. Both the structure and composition of the starch were materially altered, changes which affected the functionality of the starch. An analysis of sugar and nonstarch polysaccharide content in the endosperm suggested an impact of the mutations on the carbon allocation process, suggesting the existence of cross‐talk between the starch and carbohydrate synthesis pathways.Modifications to the composition of starch, the major component of wheat flour, can have a profound effect on the nutritional and technological characteristics of the flour's end products. The starch synthesized in the grain of conventional wheats ( Amylose‐free wheat starch  is more crystalline than wild‐type starch, is more readily degraded by amylases, and absorbs and retains more water. These characteristics increase the utility of waxy wheats both in the formulation of animal feed and in the production of biofuel. In chilled and frozen dough‐based food products, the inclusion of waxy wheat flour offsets some of the decline in quality experienced during storage , an enzyme responsible for the elongation of amylose chains. Because bread wheat is hexaploid, independent mutations at each of the three homeoloci et al., et al., Here, a description is given of the identification and characterization of three wheat lines in which the grain amylose content varies from 0% to 79%. The lines were bred by combining mutations at all three homoeologs encoding either GBSSI, SSIIa, or SBEIIa, based on independent induced mutants for each locus, as described elsewhere , SSIIa null (Cad‐SSIIa*) and SBEIIa null (Cad‐SBEIIa*)  Figure . As expeAs expected, the mutant lines generated in this work contain widely different levels of amylose in their grains. In respect to the amylose content of the cv. Cadenza , GBSSI mutant had no detectable amylose, while SSIIa and SBEIIa mutants were amylose rich .Grains of the cv. Cadenza have an average weight of 47 mg and 59% of this weight is constituted by starch. One‐third of total starch is amylose  for the starch isolated from Cad‐GBSSI* compared with the one isolated from the cv. Cadenza , were smaller in weight (−30%) and contained less total starch (−33%) than Cadenza grains Table . The incFor Cad‐SSIIa*, a further limit to the increase in viscosity could be represented by the decreased percentage of total starch content.et al., et al., Grains of Cad‐SBEIIa* were the most enriched in amylose , water‐soluble alpha‐glucans (2.05%) and sucrose (1.7%). The remaining soluble carbohydrates constituted together <1% of the grains’ dry matter  and small (S) subunits. An ADP‐glucose transporter (ADPG‐t) is required for the import of cytosolic ADP‐glucose into the amyloplast for starch biosynthesis.The expression of a set of genes involved in starch and nonstarch carbohydrates metabolism in wheat grains was evaluated by qRT‐PCR in wild type, Cad‐GBSSI*, Cad‐SSIIa* and Cad‐SBEIIa*. Starch biosynthesis initially involves sucrose synthase (Susy), UDP‐glucose pyrophosphorylase (UGPase) and ADP‐glucose pyrophosphorylase (AGPase) for the conversion of sucrose into ADP‐glucose. AGPase exists in two isoforms in wheat endosperm, the cytosolic isoform is coded by genes Susy and three AGPase genes  were up‐regulated more than 2‐fold in Cad‐GBSSI*. Two AGPase genes (AGPaseS1 and L2) were significantly up‐regulated also in Cad‐SSIIa*, together with ADPG‐t coding for the ADP‐glucose transporter. In Cad‐SBEIIa*, all genes were up‐regulated more then 2‐fold except for the gene coding the small subunit of plastidic AGPase whose expression was genotype‐independent .Inside the amyloplast, ADP‐glucose is polymerized by GBSSI to amylose. Amylopectin formation and modelling require a large set of enzymes belonging to starch synthase , starch branching enzyme  and starch debranching enzyme families . The expression of some of these genes was not significantly affected by the genotype (GBSSI was up‐regulated in the amylose‐rich Cad‐SSIIa* mutant. GBSSI up‐regulation was even more pronounced (4.5‐fold) in Cad‐SBEIIa* containing the highest levels of amylose, and genes involved in the metabolism of amylopectin such as SSI, SBEIIb (possibly compensating the lack of SBEIIa), ISAI and ISAII were all strongly up‐regulated  up‐regulated in mutants. The transcripts for one starch branching enzyme  and two debranching enzymes  were found increased in the amylose‐free Cad‐GBSSI* mutant. On the contrary, onounced .5‐fold iet al., Besides starch, sucrose is also the precursor of fructan biosynthesis. The transcription intensity of the two fructan synthesis genes (1‐SST and FFT 6SST) was too low in the developing grain to reveal any genotypic variation. Gene FEH was selected as a representative of the genes encoding fructan exohydrolases. This gene is known to be transcribed at appreciable levels in the maturing grain  whose expression was determined in the grains of the three null mutant lines and in the cv. Cadenza. None of these genes were significantly (<2‐fold) up‐ or down‐regulated in any genotype, although expression of all of them tend to be consistently higher in Cad‐GBSSI* and possibly lower in Cad‐SSIIa*  on the representation of extra‐long chains (DP > 77) in Cad‐GBSSI*, as these were not assayable using the present detection system: other SSs may act to form such extra‐long chains when GBSSI is absent , while the other two null mutant lines’ starch was deficient in A‐type crystallinity (and enriched in V‐type). The pattern of crystallinity seen in the starch of Cad‐SBEIIa* and Cad‐SSIIa* has also been noted in high amylose rice and barley starch  limiting the amount of carbon which can be fixed as starch is located downstream of ADP‐glucose formation  Cadenza, using the TILLinG  procedure  of water and kneaded until it formed a dough. Starch was washed from the dough with 300 mL water, then, following a centrifugation (1500 g), the pellet was rinsed twice in 20 mL water. The thin yellow‐grey layer deposited on the surface of the starch pellet was removed with a spatula, and the starch left to dry for 2 days at 25 °C. For the SSIIa mutant, starch was isolated by crushing individual grains, followed by application of the starch granule extraction method  Hitachi S‐4000 , as reported by Sparla et al. .et al. , as reported by Botticella et al. .The starchs’ amylose contents were determined from a 15 mg aliquot of purified starch using a colorimetric assay based on the iodine–amylose reaction  with its guard column. The detection cell contained a gold working electrode (1.0 mm in diameter) and an Ag/AgCl reference electrode. Elution was carried out at 30 °C with a solute flow rate of 1 mL/min. Sugars were eluted as described by Cimini et al. . The total fructan content was determined as described by Verspreet et al.  , consisting of an isocratic quaternary pump, a pulsed amperometric detector, an injection valve with a 15 mmi et al. . The gal .Wholemeal flour  was subjected to RVA, as described by Sestili et al. . For theet al.  from snap‐frozen grains harvested 18 days postanthesis developing on field‐grown plants. Each genotype was represented by a bulk of four grains. One μg of the extracted RNA was used as template for the synthesis of cDNA, following the protocol of the QuantiTect Reverse Transcription Kit . The subsequent qRT‐PCRs were run using a CFX 96 Real‐Time PCR Detection System device , and the 10 μL reactions were formulated with SsoAdvUniver SYBR GRN SMX (Bio‐Rad). The reactions were initially denatured (94 °C/30 s), then cycled 40 times through the sequence 94 °C/5 s, 60 °C/30 s. A melting curve was generated over the range 65–95 °C, with 0.5 °C increments @ 5 s. Transcript quantification was enabled by CFX manager software. Each estimate of transcript abundance was based on the mean of three technical replicates, and each genotypic value was based the mean of two biological replicates. The list of genes analysed, along with the housekeeping gene, and the sequences used as primers are shown in Table post hoc Tukey HSD (Honestly Significant Difference) parameter to assess the significance of differences between means.One‐way analyses of variance (ANOVAs) were conducted, using the The authors declare no conflict of interests.Figure S1 Scheme of crosses between single mutants to generate complete null genotypes.Table S1 List of primers used for tetra primers PCR.Table S2 List of primers used in qRT‐PCR.Table S3 RVA and hardness parameters.Click here for additional data file.


RNA‐seq analysis of Della stem gene expression during the starch accumulation phase of development. Genes differentially expressed in stems were identified that are involved in starch biosynthesis , degradation  and amyloplast sugar transport (glucose‐6‐P translocator). Transcripts encoding AGPase SS and LS subunits with plastid localization were differentially induced during stem starch accumulation indicating that ADP‐glucose for starch biosynthesis is primarily generated in stem plastids. Cytosolic heteroglucan metabolism may play a role in stem sucrose/starch accumulation because genes encoding cytosolic forms of the disproportionating enzyme and alpha‐glucan phosphorylase were induced in parallel with stem sucrose/starch accumulation. Information on the stem starch pathway obtained in this study will be useful for engineering sorghum stems with elevated starch thereby improving forage quality and the efficiency of biomass conversion to biofuels and bio‐products.Sweet sorghums were identified that accumulate up to ~9% of their total stem dry weight as starch. Starch accumulated preferentially in stem pith parenchyma in close proximity to vascular bundles. Stem starch accumulated slowly between floral initiation and anthesis and more rapidly between anthesis and 43 days post‐anthesis before declining in parallel with tiller outgrowth. Genes involved in stem starch metabolism were identified through phylogenetic approaches and  Stems also serve as temporary depots for nonstructural carbohydrates such as sucrose, monosaccharides, starch, fructans, and isoprenoids  Moench] stems after grain maturity was conducted from plants grown in the experimental field plots in College Station, Texas in 2014. Before planting, ammonium nitrate (32‐0‐0) was applied at a rate of 140 kg N/ha. Samples were harvested when all plants had passed grain maturity. The genotypes in this study were at variable stages of post‐grain maturity development. Three plants per plot were harvested, avoiding the end of the plots to avoid edge effects, and these three plants were used in subsequent analyses. After harvesting, leaves and leaf sheaths were removed from the stems. A stem section spanning three internodes of which the middle internode was located at 50% of the total stem length was removed by excision at the nodes. These internode sections were cut into smaller sections and dried in a forced air oven at 60°C for 3 days; the tissue was coarsely ground in a Wiley Mill  and subsequently finely ground in a Cyclone Sample Mill  until the tissue exhibited the consistency of a powder. Then, this powered tissue was used for starch quantification.The survey of starch in sorghum [2.2Della , a sweet sorghum developed from a cross of Dale and ATx622  using an analytical balance and transferred to a 15‐ml conical tube. Alternatively, to quantify nonstructural carbohydrates in tissue that gave rise to the RNA‐seq data, approximately 0.5 g of frozen, pulverized tissue was placed into a previously weighed 15‐ml conical tube and lyophilized until desiccated. The tube and tissue were reweighed, and tube weight subtracted to obtain the tissue weight. Both tissue types were subjected to the following extraction protocol. Water‐soluble NSCs were extracted in 10 ml of water/Na azide (200 mg/L) solution at 50°C for 48 hr with agitation. This length of incubation was experimentally determined to be optimal for this experimental set‐up. This extraction time allowed the extraction solvent to fully penetrate all of the biomass particles and extract the soluble carbohydrates. Aliquots of 20 μl were diluted 50 ×  into 980 μl of deionized water. All HPLC samples were filtered using 0.45‐μm cellulose acetate sterile filters. Sucrose, glucose, and fructose concentrations were quantified using high performance anion exchange‐pulsed amperometric detection (HPAE‐PAD) with a Carbopac PA1 analytical column  as well as a Borate trap (Dionex) and an Aminopac column (Dionex) to reduce borate and amino acid interference  chromatography with the use of a Carbopac PA1 analytical column . Curve validation samples were randomly interspersed among the analytical samples during chromatography to validate the glucose, fructose, and sucrose standard curves. The percentage of starch in the dry biomass was calculated from the glucose released during digestion.The starch in stem biomass was quantified using the NREL Laboratory Analytical Procedure for extracting starch from solid biomass . Photographs were captured using the Zeiss AxioVision 3.0.6 software.To visualize the location of starch in the stem by stereomicroscopy, field grown plants were harvested at anthesis and post‐grain maturity. A 1‐mm slice of stem tissue was excised with a razor blade from the middle of internode 11. These 1‐mm internode sections were infused with Lugol's solution in a vacuum. After infusion, the internode sections were stored in this solution overnight. Excess staining solution was removed by incubating the tissue slice in water for 24 hr, changing the solvent every 6 hr. The sections were imaged under white light using a Zeiss M2.7https://www.mrcgene.com/rna-isolation/tri-reagent). The high salt precipitation step was included because of the potential high concentration of soluble carbohydrates in the sample. Total RNA was further purified using the RNeasy kit  which isolates RNAs >200 nucleotides in length. An on column DNase1 digestion was performed using the RNase‐free DNase set  to remove any contaminating DNA. The quality of the RNA libraries was assessed using an Agilent Bioanalyzer. The mean RNA integrity number (RIN) from this quality control step was 8.9, and the minimum RIN was 8.3 indicating that the quality of the RNA libraries was high. RNA libraries were prepped for sequencing using the Illumina TruSeq® V2 mRNA Sample Preparation Kit , which selects for polyadenylated RNA and excludes rRNA and nonpolyadenylated RNAs from the sample. The 24 libraries were sequenced on one lane of an Illumina HiSeq 2500 in single‐end mode. The average sequence depth was 5.8 M reads.RNA extraction was performed using the Trizol extraction method   was used to predict the presence of chloroplast targeting sequences in peptides sequences.Peptide sequences were obtained from Biomart, hosted by the Phytozome database. The primary transcript was used for the analysis. The ChloroP algorithm  through 80 days post‐anthesis (~175 DAE). Genes previously identified in other plants that are involved in starch synthesis, turnover, and plastid sugar transport were used to identify sorghum homologs based on sequence similarity and phylogenetic analysis of gene families , turnover , and transport  were upregulated between floral initiation and 43 days post‐anthesis in parallel with stem starch accumulation.To help visualize patterns of gene expression in the context of the entire starch pathway, a working model of stem starch metabolism was developed based on prior knowledge of this pathway obtained in other plants/organs  and one gene encoding AGPase LS  were expressed at lower levels in stems at floral initiation and at gradually increasing levels during development with peak expression between 25 and 43 days post‐anthesis  was differentially expressed 47‐fold higher in stems post‐anthesis and Sobic.002G160400 (APS1) eightfold higher  and four genes encoding AGPase large subunits (LSs) were identified in the sorghum genome , Sobic.002G116000.1, was the most highly expressed gene in the family (max TPM = 737), and the gene was differentially expressed in stems between floral initiation and 43 days post‐anthesis (DE = 11‐fold) , and the third was a STARCH SYNTHASE II   was induced ~96‐fold between floral initiation and grain maturity in parallel with the accumulation of starch in the sorghum stem  with peak expression post‐anthesis  showed twofold higher expression in stems post‐anthesis  were expressed at similar levels, and gene expression was induced ~21‐fold and ~37‐fold, respectively, with peak expression 43 days post‐anthesis  was higher in stems vs. leaves during the phase of development analyzed whereas the related family member Sobic.001G100000.1 (AGL2) was expressed at higher levels in leaves compared to stems  was expressed at higher levels in stems vs. leaves whereas the family member Sobic.007G101500.1 (AGS1) was expressed at higher levels in leaves compared to stems during these same developmental stages , the maltose transporter (MEX1), the plastidial glucose transporter (pGlcT), and/or the adenylate translocator. RNA‐seq analysis showed that a gene encoding a member of the GLUCOSE‐6‐PHOSPHATE/PHOSPHATE TRANSLOCATOR 2 (GPT2) family was induced ~20‐fold in parallel with increased stem starch accumulation during development   encodes a protein that transports glucose released from starch degradation out of the plastid. This gene showed increased expression in stems post‐anthesis; however, the relative expression of pGlcT (max TPM = 150) was much lower than GPT2   encodes a protein that transports maltose out of plastids. This gene was expressed at low levels in stems (max TPM = 8) and was not differentially expressed between floral initiation and 43 days post‐anthesis  Figure . Inducti) Figure . The pla) Figure   Figure . One mem) Figure . The gen4The overall goal of this study was to learn more about the regulation of starch accumulation in sorghum stems because increasing stem starch levels could improve biomass yield and the composition of high‐biomass sorghum that is used for forage and production of biofuels and bio‐products. While prior studies have noted the presence of starch in sorghum stems  and ~30‐fold  from floral initiation to 43 days post‐anthesis. This indicates that generation of ADP‐glucose for starch biosynthesis occurs in plastids similar to other monocot vegetative tissues. This conclusion is consistent with elevated expression of GPT2 that could transport G‐6‐P into plastids thereby providing a source of G‐1‐P for ADP‐glucose biosynthesis. Eight genes encoding starch synthases were expressed in stems, and three of these genes showed increased expression (11–29‐fold) in parallel with stem starch accumulation between floral initiation and 43 days post‐anthesis. Two of the three genes encoding starch‐branching enzymes and a gene encoding isoamylase 1 showed similar dynamics of gene expression. Taken together, this study identified genes in gene families that encode enzymes for known steps in starch biosynthesis that are differentially expressed in parallel with stem starch accumulation.In monocot grain endosperm tissue, most of the ADP‐glucose used for starch biosynthesis is synthesized in the cytoplasm and imported into amyloplasts , which generates G‐1‐P from linear malto‐oligosaccharides released from the starch granule, was induced 37‐fold. Subsequent conversion of G‐1‐P to G‐6‐P would facilitate export of hexoses from the plastid via GPT2. Genes encoding other enzymes involved in starch turnover were induced to lower extents. The expression patterns of the ten gene family members encoding β‐amylase that were expressed in stems was especially complex, with some family members differentially expressed in stems at floral initiation followed by decreased expression, and others showing differential expression post‐anthesis. Further studies on the biological function of this complexity are warranted.As stem starch is a carbohydrate reserve, it was not surprising that genes encoding enzymes involved in starch turnover were induced in parallel with stem starch accumulation. A gene encoding alpha‐glucan‐water dikinase that mediates an early step in starch turnover showed the highest differential expression during the developmental time course (~96‐fold). Expression of starch phosphorylase  and glucan phosphorylase (PHS) were expressed in stems. These genes encoded proteins with predicted plastid localization  as well as proteins with predicted cytosolic localization . Prior studies have shown that the cytosolic forms of these enzymes are involved in the synthesis/turnover of cytosolic heteroglucans  regulate the expression of genes in the stem starch pathway. Additional levels of regulation that occur in chloroplasts and amyloplasts  (Cho et al., 4.3In this study the temporal dynamics and spatial distribution of starch accumulation in stems of the sweet sorghum Della was characterized. Transcriptome and phylogenetic analyses helped identify differentially expressed genes involved in stem starch metabolism and sugar transport through the outer membrane of amyloplasts. Specific gene family members involved in stem starch metabolism were differentially expressed in stems and leaves.B.A.M. and J.E.M. designed the research; B.A.M. and W.L.R. cultivated plant material; B.A.M. and A.L.C. performed experiments; B.A.M. and J.E.M. performed the analysis of data; B.A.M. and J.E.M. wrote the manuscript. Click here for additional data file. Click here for additional data file.


Various techniques have been used for the repair of oroantral fistula (OAF) but majority of them have focused on the soft tissue closure alone, and most of the time, the osseous floor of the sinus was ignored. Existing literature supports that bone grafts supported by Buccal Fat Pad (BFP) heal well without undergoing significant resorption and necrosis. Through this case report, we wish to elaborate on the clinical success of using BFP and autogenous chin graft for simultaneous reconstruction of a large long-standing oroantral fistula with underlying osseous defect. The combination technique can prove beneficial for osseous regeneration of sinus floor and improve chances for future implant prosthetic rehabilitation. An oroantral fistula can develop as a sequel of dental extractions, infection, maxillary cyst/tumor excision, persistent infection/abscess, or radiotherapy , 2. A smManagement of established OAF can be classified into nonsurgical and surgical. Nonsurgical methods employ placing materials into the defect to act as a mechanical barrier without attempting flap closure. Synthetic graft materials, fibrin glue, xenograft, absorbable implants, and acrylic splints have all been used.Surgical principle involves raising the adjacent or distant flap and advancing into the defect, for example, buccal flaps, palatal flaps, tongue flap, and nasolabial flaps . All the2. The mean thickness is about 6 mm [The first clinical application of BFP was described by Egyedi in 1977 where he used it for reconstruction of palatal defect following tumor excision [out 6 mm , 9. CareIgnoring the underlying bony defect can have serious consequences with respect to prosthetic rehabilitation. Simultaneous use of autogenous bone graft for OAF closure is recommended to facilitate good prosthetic treatment. Bone grafts from different sites have been used. In 1969, Proctor reported the use of iliac crest graft for large OAF closure . This ofThe use of chin grafts in OAF closure has been reported with successful outcomes. Haas et al. in their preliminary study showed successful results with chin grafts . It is iSeveral techniques have been utilized to reconstruct the bony defect. Most of the published articles are in the form of a case report or case series. There is a lack of large prospective randomized studies comparing the reliability and outcomes of these existing methods. Kapustecki et al. in their series of 20 cases used autogenous grafts from symphysis mandible 14 patients) and external oblique line (6 patients) covered with PRF (Platelet Rich Fibrin) membrane  patients.Er et al. performed two-layered closure with simultaneous bone grafting using graft from different intraoral sites . They noA 45-year-old male patient, chronic smoker, reported to us with the chief complain of leakage of liquids through his nose while drinking. Otherwise, he was asymptomatic. He underwent extraction of his tooth number 16 about 6 months back elsewhere. On clinical examination, there were no signs and symptoms suggestive of acute maxillary sinusitis. Intraorally, a fistulous opening round in shape with normal surrounding mucosa and an obvious bony defect was seen along the maxillary alveolus molar region . No actiRadiographic examination was done to define the underlying bony defect and also to rule out any foreign body (fractured root tip). The sinuses showed normal appearance. The bony defect of size 2 × 1.8 cm along the sinus floor was confirmed .After correlating the clinical and radiographic findings, surgical closure using buccal advancement flap (BAF)+BFP+autogenous bone graft from the chin was planned. Preoperatively, the patient was started on antral regime  for 5 days.The entire procedure was performed under local anesthesia  using Posterior Superior Alveolar block supplemented by infiltration+greater palatine nerve block. The fistulous tract was excised in a circumscribed manner along the defect. A modified buccal mucoperiosteal flap was elevated in trapezoidal outline starting from the mesial and distal end of the fistulous opening. The periosteum was incised along the posterior aspect of the flap to identify BFP. Gentle blunt dissection was done to harvest the BFP till we obtained the sufficient amount of fat to cover the graft and the defect. Next, the cortical bone graft was harvested from the chin of size matching the defect. The graft was press fit into the defect and did not require screw fixation. Then, the BFP was mobilized to cover the graft, and finally, the mucoperiosteal flap was repositioned and sutured in place Figures  and 4. TThe patient was observed for signs of infection, dehiscence, and necrosis. Postoperative radiograph showed bone graft well in place . No signThe commonest cause for the development of OAF includes dental extractions of posterior teeth  due to close proximity of the root apices to the sinus floor. Frequencies of such occurrences have been reported to be between 0.31% and 4.7% . Based oEver since its first clinical use by Egyedi in 1977, BFP has gained popularity and rightly so because of its advantages: rapid epithelialization, excellent reliable blood supply, anatomically favorable position, ease of harvest, low rate of complications, and minimal to no donor site morbidity.Problems that we can come across mainly at the time of harvesting BFP are perforation or shrinkage. Egyedi advocated covering the fat pad with split skin graft to overcome these issues, but Tideman et al. have shown that BFP was capable of self-epithelialization within 3-4 weeks of its inset , 21. NevOur idea of three-layer closure is supported by the studies of Er et al. and Weinstock et al. , 18. Er There is so much heterogeneity in the methods for OAF closure, and no particular technique is superior or inferior to other. Each case is at the discretion of the treating surgeon based on his experience and preference. The choice of the technique must be guided by 4 important assessment criteria: (a) size and type of defect, (b) presence or absence of sinus disease, (c) minimal donor site morbidity to the patient, and (d) prosthetic considerations. The described technique has the added advantage for graft support and minimizing the chance of graft resorption or wound dehiscence.Repair of OAF is essential to prevent further complications related to the maxillary sinus. The routine flap based techniques will not be sufficient alone due to the continued demand and necessity for implant rehabilitation. Due consideration should be given to the underlying osseous defect as well.Therefore, we used autogenous bone graft harvested from the chin for the closure of bone. BFP and chin graft may be adequate to cover a large defect, but BAF may serve as an additional support and will help in minimizing any complication related to the grafts.


Two dual-modified rice starch samples (oxidized cross-linked rice starch and cross-linked oxidized rice starch) were obtained by the oxidation of cross-linked rice starch and the cross-linking of oxidized rice starch at the same level of reagents. The physicochemical properties of native rice starch, cross-linked rice starch and oxidized rice starch were also studied parallel with those of the two dual-modified rice starch samples using rapid visco analysis (RVA), differential scanning calorimetry (DSC), dynamic rheometry and scanning electron microscopy (SEM). It was found that the levels of cross-linking and oxidation used in this study did not cause any significant changes in the morphology of rice starch granules. Cross-linked oxidized starch showed lower swelling power (SP) and solubility, and higher paste clarity in comparison with native starch. Cross-linked oxidized rice starch also had the lowest tendency of retrogradation and highest ability to resistant to shear compared with native, cross-linked, oxidized and oxidized cross-linked rice starches. These results suggest that the undesirable properties in native, cross-linked and oxidized rice starch samples could be overcome through dual-modification.  Starch is a common ingredient used widely in many food and non-food applications. However, the application of native starches is often limited because of shortcomings in their physiochemical properties such as low shear resistance and thermal stability, thermal decomposition and high retrogradation tendency ,2,3. ConNative rice starches have poor freeze-thaw stability, resistance to shear, and fair stability to retrogradation with moderate clarity and soft texture . The perOxidized starch is widely used in both food and non-food industries where film formation and adhesion properties are desired. The major application of oxidized starch is as a surface sizing agent and a coating binder in paper industry. The food products where oxidized starch is used are neutral tasting and low-viscosity items such as lemon curd, salad cream and mayonnaise . OxidizeModified starch meets the functional properties required in food products, but modified starch still does not overcome some undesirable properties. Cross-linking shows a high retrogradation trend. Oxidation significantly decreases the viscosity of starch paste and the ability to resist shear. Therefore, dual-modification is used further to improve the undesirable characteristics of starch. However, until now, most of the works have been focused on corn, wheat, tapioca, sago and potato starches ,6,7,8,9,Mucuna bean [et al. [et al. [The SP, solubility and paste clarity of native and modified rice starches are presented in una bean  and normuna bean . Leach e [et al.  proposed [et al.  indicate [et al. . The cla [et al. . The stri.e., there were sufficient cross-links to retard the swelling of the starch and cause a decrease in viscosity. As can be observed in The RVA viscosity characteristics of native and modified starches are shown in The transition temperatures and the enthalpy values of gelatinization of the native and modified rice starch samples observed using the DSC are presented in to and tp) as compared to native, cross-linked and dual-modified starch samples, the decreases in transition temperatures might be due to the weakening of the starch granules, which led to the early rupture of the amylopectin double helices [Hgel of dual-modified starch samples (oxidized cross-linked starch and cross-linked oxidized starch) were not significantly different, while the transition temperatures of cross-linked oxidized starch was significantly higher than that of oxidized cross-linked starch, it indicated that at the lower cross-linked level of 0.3% epichlorohydrin, the oxidation reaction occurred in the cross-linked starch, while cross-linked starch was more difficult to oxidize compared with native starch duo to the strengthened structure of starch after cross-linking. The weakened structure of oxidation starch was strengthened through cross-linking. This explained the higher ΔHgel of dual-modified starch as compared with native starch. Heat of gelatinization reflects the energy required for disrupting the starch granule structure and since cross-linking reinforces the starch granules, more heat will be required for gelatinization . Strengt helices . The ΔHgHret) at the end of the storage period dropped significantly compared to transition temperatures and ΔHgel during gelatinization. The retrogradation enthalpy of all modified starch samples, except cross-linked starch, was lower in comparison to native starch. The retrogradation enthalpy of cross-linked oxidized starch was the lowest. The enthalpy values of the retrograded starch reflect the melting of the crystallites formed by the association between adjacent double helices during gel storage [Starch gels are metastable and non-equilibrium systems. Therefore, they undergo structural changes during storage . As show storage . Retrogr storage , Amylose storage . Cross-let al. indicated that high values of shear stress pointed to a high stability of the structure of the starch [The results from the previous studies indicated that rice starch samples were pseudoplastic and shear thinning liquids. As shown by the flow curves in e starch . AccordiScanning electron micrographs of native and chemically modified starch samples at 5000 × magnification are presented in Native unmodified rice starch (made from bad mouth-feeling rice) was purchased from Puer Yongji Biological Technology Co. Ltd. . Sodium hypochlorite containing 5% active chlorine and epichlorohydrin  were purchased from NongHe Co. Ltd. . All chemicals and reagents used in this study were of analytical grade.g, 15 min). After washing with distilled water, the sediment was dried at 45 °C for 48 h in a vacuum oven.Cross-linking reaction of starch was performed according to the method of Reddy and Seib  with a set al. [2SO4. After the addition of NaOCl, the pH of the slurry was maintained at 8.5 with 1 mol/L NaOH for an additional 30 min. The slurry was then adjusted to pH 6.5–7.0 with 1 mol/L H2SO4, the oxidized starch was isolated by centrifugation , washed with water and dried in a vacuum oven at 45 °C for 48 h.The oxidized rice starch was prepared by following the method of Autio et al.  with a s2SO4 and then NaOCl (2.5% active chlorine concentration) was added as previously described. The OCRS was also recovered using the same procedure as for the CRS. Cross-linked oxidized rice starch was prepared by first oxidation with NaOCl (2.5% active chlorine concentration) as described above. After oxidation, the pH of the slurry was adjusting to 10.0 with 1 mol/L NaOH, and then epichlorohydrin  was added as previously described. The cross-linked oxidized rice starch was obtained.Oxidized cross-linked rice starch was prepared by first cross-linking with EPI  as described above. After cross-linking, the pH of the slurry was adjusted to 8.5 with 1 mol/L Hg for 15 min. The supernatant was decanted carefully and retained, and the SP was determined as weight of the sediment according to: The SP of native and modified starches was measured according to the method of Sasaki and Matsuki  with a s2 is weight of tube after supernatant was removed (g); W1 is weight of dry tube (g) and W is weight of db starch (g). The supernatant was dried at 120 °C for 2 h, the solubility was measured according to:where W3 is weight of dried supernatant; W is weight of weight of db starch (g). The paste clarity of native and modified starch was determined based on the method of the Perera and Hoover [where Wd Hoover . For theThe pasting properties of rice starch were measured according to our previous method by using a Rapid Visco Analyzer  . Viscosito) and peak temperature (tp) were determined from the run heating DSC curves. Enthalpy of gelatinization (ΔHgel) was evaluated based on the area of the main endothermic peak. After the DSC run, gelatinized starch samples were stored at 4 °C for 7 days for retrogradation studies, these stored samples were scanned under the same conditions and the transition temperature and retrogradation enthalpy (ΔHret) were determined from the second heating run. The thermal properties of the samples were carried out using 61 DSC-Pyris Diamond . Starch samples were weighed into aluminum DSC pans, and deionized water was added by micropipette to achieve a water-sample ratio of 2:1. The sample pans were sealed and allowed to stabilize at room temperature for 24 h before heating. Samples were heated at a rate of 10 °C/min from 25 to 100 °C, using an empty pan as reference. The onset temperature (−1) was obtained at 20 °C to characterize flow behavior.Starch paste (8%) was put into the testing platform of dynamic rheometer . Viscometry was performed using a controlled strain rheometer using a parallel plate mould (40 mm diameter and 1 mm gap). After trimming off the over-loaded portion of samples around plates, the open side of samples was covered with a thin layer of silicon oil to prevent moisture loss. Shear stress with increasing shear rate  at an accelerating voltage of 10 KV. Starch granules were sprinkled onto double-backed cellophane tape attached to a stub before coating with gold-palladium.p < 0.05). The data reported in the tables were average of triplicate observations. Data obtained were analyzed by single factor analyses of variance (ANOVA) using SPSS for Windows version 13.0. Confidence interval of sample means was reported at the 95% confidence probability. Comparisons of means were made using least significant difference (LSD) and shortest significant ranges (SSR) at 5% significance level (Scanning electron micrographs of native and modified rice starch samples indicated that chemical modification of cross-linking and oxidation had no significant effect on the morphological properties of rice starch granules. At a cross-linking level of 0.3% epichlorohydrin, the obtained cross-linked starch was more difficult to oxidize compared to native starch. The low degree of cross-linking results in an increase in swelling power and solubility, and a decrease in paste clarity, however, a higher degree of cross-linking results in a decrease in swelling power and solubility, and an increase in paste clarity. Oxidation reduced the swelling power, pasting temperature and paste viscosity, and increased the solubility and paste clarity. Cross-linking increased the retrogradation tendency of starch, while oxidation decreased the retrogradation tendency of starch. The tendency of retrogradation of cross-linked oxidized rice starch was the lowest in all native and modified starch samples. Meanwhile cross-linked oxidized starch showed the highest ability to resist shear among all tested starch samples. It can thus be deduced that high quality modified rice starch, cross-linked oxidized starch was obtained by dual-modification, and it can more effectively meet the functional properties demands for food products. Further studies are required to elucidate the detailed structural changes in rice starch during the process of cross-linking and oxidation.