We report the impact of white bread made from sbeII wheat flour on in vitro starch digestibility and product quality, and on postprandial glycaemia in vivo, compared to an isoglucidic wild-type (WT) control white bread. Starch in sbeII bread was ∼20% less susceptible to in vitro amylolysis leading to ∼15% lower glycaemic response measured in vivo, compared to the WT control bread, without major effects on bread appearance or texture, measured instrumentally. Despite the early termination of the in vivo intervention study due to the COVID-19 outbreak (n = 8 out of 19), results from this study indicate that sbeII wheat produces bread with lower starch digestibility than conventional white bread.High-amylose  sbeII wheat flour on postprandial glycaemic response.Impact of white bread made from  For instance, the postprandial rise in blood glucose and insulin concentrations varies considerably between starch-rich foods and is strongly influenced by the rate and extent of starch digestion by α-amylase in the small intestine.5 Foods eliciting low glycaemic responses (as measured by glycaemic index) are needed for dietary prevention and management of obesity, type 2 diabetes, and cardiovascular disease.6,7 The manipulation of starch structure through non-transgenic genetic approaches provides new opportunities to develop slowly digested carbohydrate staple foods with lower glycaemic potency.Large dietary intakes of high glycaemic starchy foods can exacerbate the metabolic abnormalities of insulin resistance and over time, lead to abnormal glucose tolerance, insulin sensitivity and hyperglycaemia.in planta using Targeting Induced Local Lesions in Genomes (TILLING) technology8 to induce mutations in genes involved in starch biosynthesis.9 Wheat carrying mutations in starch branching enzyme II (sbeII) genes have been generated using TILLING, yielding starch with a higher proportion of amylose and increased resistance to amylase digestion, compared to conventional wheat starch.10,11 This allows the production of high-amylose starch-based food products without the need of post-extraction processing or supplementation with starches from other botanical sources, which was shown to be a suitable approach to lower glycaemic response to high glycaemic foods, such as bread.12,13Starch structure can be manipulated sbeII mutant wheat was used to produce a semolina pudding with lower starch digestibility than the control pudding made from WT wheat. When tested in humans, however, the Glycaemic Index of the sbeII pudding was not significantly different from the WT control.14 Recently, two studies have investigated glycaemic responses to pasta and bread made from sbeII mutant wheat. Sissons et al. (2020),15 reported that a serving of pasta (spaghetti) made from a sbeIIa durum wheat mutant with starch made of ∼58% amylose, corresponding to ∼7% RS, led to a lower starch susceptibility to amylase digestion in vitro and lower glycaemic index measured in vivo, compared to the WT control. Belobrajdic et al. (2019),16 reported that bread made from high-amylose wheat flour lowered the postprandial glycaemic response by 39% and the insulinemic response by 24%, however the bread provided ∼24 g less starch per 100 g and ∼7.5% less energy (kJ per 100 g) than the control bread. Therefore, it is not clear if the attenuated postprandial glycaemic response following high-amylose bread in the Belobrajdic et al. study was due to the lower amount of starch per serving (glycaemic load) or due to a change in the intrinsic starch digestibility.Previously, a sbeII wheat can be used to attenuate the glycaemic potency of different food products. The aim of the present study was to determine the effect of sbeII wheat in bread on postprandial glycaemic response and satiety, compared to WT control bread. Unlike a Glycaemic Index study, this postprandial study was focussed on understanding the effects of sbeII starch in bread on glycaemic response curve size and shape, compared to a conventional white bread, to inform on the potential health effect of replacing conventional white bread with sbeII bread.Further studies are needed to better understand how and to what extent in vitro). A preliminary sensory assessment  and quality analysis of sbeII bread was also conducted however, due to the small number of participants completing the study, results were not conclusive. The methodology was reported in ESI.†The bread rolls were prepared to deliver the same amount of starch per serving (75 g), such that any observed differences in the glycaemic responses to these bread rolls reflect differences in intrinsic starch amylolysis  to assess glycaemic responses, in parallel with fingerprick (FP) sampling for capillary glucose measurements. CGMs were fitted to the upper arm and used enzymatic-amperometric technology to measure glucose concentrations in interstitial fluid (IF) over several days. Recent studies have used CGMs to investigate the effect of dietary interventions on glucose responses in healthy individuals.sbeII mutant bread wheat and a WT control bread wheat (Triticum aestivum L. ssp. aestivum) were sown in spring 2018 in a field trial using a randomised block design at the John Innes Centre Church Farm field station . Wheat was harvested in summer 2018, grains were transported to Campden BRI . Following tempering to 16.4% humidity, grains were debranned and milled on a Bühler mill with feeding rate of 100 g min−1, then sieved through 140 μm mesh size and passed through a bran finisher fitting a 125 μm mesh to produce white flour.A sbeII and WT control wheat bread rolls were produced using a straight-dough method (AACC Method 10-10.03) at the Quadram Institute . The formulation of each bread was adjusted based on the flour's starch content so that both provided the same total starch (∼75 g). Briefly, flour , yeast (35.6 g and 33.9 g for sbeII and WT control), sugar (44.5 g and 42.4 g for sbeII and WT control), salt (22.3 g and 21.2 g for sbeII and WT control), shortening (40.7 g and 38.8 for sbeII and WT control), and water (867.2 g and 781.6 g for sbeII and WT control) were mixed at low speed for 3 min and kneaded for 5 min at increasing speed, in a heavy-duty planetary mixer with hook attachment . Dough was fermented 2 h at 21 °C then portioned into rolls and proofed for 15 min at 38 °C with 100% relative humidity, then baked using a combination oven  for 15 min at 185 °C (40% humidity for 10 min and 10% humidity for the last 5 min). Baked rolls reached ∼95 °C core temperature. After 2 h cooling at 21 °C, breads were packed in resealable, opaque polyethylene bags (3 mm thickness) and stored at −20 °C. Breads were produced in four batches of 12 rolls (48 rolls per type of flour), under identical conditions and paired by their baking position in the oven. Rolls were thawed for 16 h (overnight) and either served to human study participants as part of their breakfast meal or used for in vitro analyses. Breads were consumed within three months of manufacture.The sbeII and WT control) were randomly selected for proximate nutritional analysis and microbiological safety testing before the start of the study. These were performed by UKAS accredited testing at ALS Laboratories Ltd, Chatteris, Cambridgeshire, UK, as described by Bajka et al. (2021).20 Bread rolls were deemed safe for human consumption; details can be found in ESI Table 1.†Two pairs of bread rolls (n = 8. Starch isolation and apparent amylose determination (n = 3) were carried out as described previously, Corrado et al. 2020.14The proportion of digestible and resistant starch in flour were determined using a ‘Total starch kit’ , in vitro analysis of starch characteristics. From each roll, a minimum of three technical replicates were sampled for analysis, as specified below. Rolls were left to thaw in their packaging at room temperature (∼21 °C) for 16 h, then were blended in a food processor (Kenwood CH 180 Mini chopper) for 50 seconds and sieved to obtain a 1 mm fraction, used for moisture determination and in vitro amylolysis assay, and a 500 μm fraction, used to measure digestible and resistant starch. These fractions were weighed into dry-tared aluminium pans in triplicates  for moisture determination, into 2 mL safe lock tubes (STARLAB (UK), Ltd) in quadruplicates  for starch measurements, and into 15 mL Corning centrifuge tubes  in triplicates  for amylolysis analysis. Digestible and resistant proportions of starch were measured using the reagents from ‘Total starch kit’ and ‘Resistant starch kit’ K-RSTAR , using a small-scale version of the method, as described by Edwards et al., 2015.21 Moisture was determined using the AACC (44-15A) air oven method, one stage procedure.Five pairs of each bread type were randomly selected for 22 Briefly, products of amylolysis were collected after 0, 3, 6, 9, 12, 15, 18, 21, 25, 30, 45, 60, 75, 90 min of incubation with porcine pancreatic α-amylase, as in a previous study on sbeII semolina pudding,14 and reducing sugars  were then quantified by the ‘PAHBAH’ (p-hydroxybenzoic acid hydrazide) assay. The resulting amylolysis curves were then baseline corrected by subtracting the value at t = 0 min (Y0) from each replicate. Starch digestibility was expressed as percentage of starch digested Ct, and C90 representing extent of digestion after 90 min of incubation with α-amylase. Values for the first-order rate constant, k and endpoint, C∞,23 were obtained using a non-linear regression model to fit a first order equation Ct = C∞ (1 − ekt−) to the experimental data, using the stats package in Rstudio.24,25 Parameters were compared using independent groups t-tests, values reported throughout are means ± SEMs (n = 5) unless otherwise specified.Starch susceptibility to amylase digestion (amylolysis) was determined after five months of storage using an established amylolysis assay method based on enzyme-kinetic principles, on five bread rolls per type, three technical replicates. Full details of this method, which involves incubation of starch-rich food material with amylase, quantification of amylolysis products by PAHBAH, and calculation of starch digestibility indices, were recently published.sbeII and WT control) were randomly selected for quality assessment which included specific volume, crumb and crust colour, and texture. The rolls were left to thaw in their packaging at room temperature (∼21 °C) for 16 h. Bulk density (g cm−3) was determined using the rapeseed displacement method (AACC method 10-05.01); a metal block (14 × 6.3 × 5 cm) was used as the standard. Mass was determined immediately prior to rapeseed displacement measures on an OHaus  scale to 0.05 g precision. The specific bread volume (cm3 g−1) was determined as the volume/weight ratio of baked breads.Four pairs of bread rolls (n = 4) on a TA-XT2 Texture Analyser ,20 equipped with a five kg load cell using a modified AACC method 74–09. The Texture Analyser was equipped with a 50 mm diameter compression plate (P50); a uniaxial compression with crosshead speed of 100 mm min−1 was applied to 25 × 25 × 25 mm samples to mimic mastication, with crumb hardness corresponding to the force (N) required for 40% compression. Exponent  software for texture profile analysis was used to assess the following texture parameters: hardness, springiness, cohesiveness, gumminess, chewiness and resilience. Four independent measurements of colour were taken for each bread roll type, using a ‘Gretag Macbeth’ twenty-four patch Colour Checker as reference.26Hue angle and Chroma were calculated using the L*, a*, b* parameters.27 Parameters were compared using independent groups t-tests, values reported throughout are means ± SEMs (n = 4) unless otherwise specified.Crumb texture and bread colour were measured on crust and crumb. Texture was measured instrumentally using a ‘two-bite test’  and was registered with ClinicalTrials.gov (NCT04197726). The study was also approved by the Human Research Governance Committee of the Quadram Institute and by the Department of Research and Development (R&D) of the Norfolk and Norwich University Hospitals NHS Foundation Trust . All participants provided written informed consent to take part in this study. All participants’ data were stored in accordance with the General Data Protection Regulation 2018 and biological samples were handled, stored, transported, and disposed of in accordance with the Human Tissue Act (2004).sbeII bread and WT control bread on glycaemic response, measured in capillary blood by FP test and in IF by CGM.We conducted a two arm two period double-blind randomised cross-over study comparing the effect of sbeII or WT control bread on consecutive occasions, in randomised order, with a minimum of four days washout period between visits with no dietary restriction. During intervention visits, capillary blood was obtained by fingerprick test to determine glycaemic response and in parallel, by CGM to measure glycaemic response in IF. The effect of sbeII bread consumption on satiety was determined using a visual analogue scale questionnaire (VAS)28 before and after bread consumption as well as changes in energy intake during the subsequent meal .Enrolled participants were randomised in blocks of four stratified by sex to complete 3 visits: Visit 1, to apply the CGM sensors, followed by Visit 2 and Visit 3, where participants consumed either Between interventions, participants completed a non-consecutive three-day weighted food diary, on two weekdays and one weekend day, to capture habitual diet. The study lasted approximately one month from screening to follow up.24 It was hypothesised that sbeII bread, with lower susceptibility to hydrolysis in vitro than the WT control, would induce a lower glucose response compared to the WT control in vivo (iAUC 0-120). A sample size of 19 was required to detect a 20% relative difference in iAUC difference with 80% power, assuming a 28.7 mmoL L−1 min−1 SD between pairs as observed in a previous study by Rosén et al. 2009.29 Planned sample size was increased to 25 to account for potential dropouts. Ultimately, owing to COVID restrictions, only eight participants could complete the study.The power calculation was carried out using an R core function, power.t.test.−2, fasting glucose < 6.1 mmoL L−1, HbA1c < 42 mmoL L−1, blood pressure < 160/100 mmHg. Participants were excluded if they were smokers, allergic or intolerant to the study foods or to adhesives, were alcohol or substance abusers or had insulin-dependent or non-insulin dependent diabetes, gastrointestinal disorder, anaemia, cardiovascular disease, certain cancers, or unstable body weight during the past 3 months. Women who were pregnant, lactating, or had given birth during the last 12 months were also excluded.Participants were recruited within 40 miles of the Norwich Research Park, Norfolk, UK. Participants meeting the following criteria were deemed eligible to take part: age 18–65 years, BMI 18–25 kg mThe study consisted of three visits.Freestyle Libre sensors  to record IF glucose continuously for 14 days. They then observed a two-day CGM calibration period during which no interventions or restrictions were applied. Then, they completed Visit 2 and Visit 3 as follows: after a standardised evening meal and ∼12 h fast, baseline measurements of capillary blood glucose and satiety were taken while CGM sensors recorded IF glucose concentrations. They were then given the intervention meal as their first meal of the day (breakfast), either sbeII or WT control bread roll, after which glycaemic, sensory  and satiety responses (VAS) were measured over 3.5 h. Four hours after breakfast, a lunch meal was provided ad libitum as satiety challenge. At the end of the satiety challenge, the visit was considered completed and participants left the Clinical Research Facility of the Quadram Institute. A follow up questionnaire was completed the day after Visit 3 to capture participants’ experience wearing and using CGM devices.During Visit 1, participants applied two CGM The standard evening meal consumed the night before Visit 2 and 3 consisted of a ready meal chosen from a pre-selected list of ready meals with similar nutrient composition. The ready meals contained ∼365 kcal, ∼39 g available carbohydrate, ∼8 g sugars, ∼8 g dietary fibre, ∼17 g protein, based on the nutrient declaration on food packaging. Only water was allowed between the evening meal and the intervention breakfast.sbeII or WT control, 10 g of low-fat spread (Flora Dairy Free) and approximately 250 mL of water to drink. The liquid component of the breakfast meal was standardised by adjusting the water drink to the water content of each bread. The total water content each meal was approximately 317 g, the average serving size of sbeII breads was 153.19 g ± 0.49 g (fresh weight) and the average size of WT control breads was 147.28 g ± 0.73 g (fresh weight), .The intervention breakfast consisted of one bread roll providing ∼75 g of total starch, either ad libitum was made of portions of 80 g white rice, served with either ‘Option A’ 85 g of tomato and basil pasta sauce and 7 g of parmesan cheese or ‘Option B’ 90 g of beef Bolognese pasta sauce, and was served with 250 mL of water. Both sauce options had a similar nutrient composition and provided approximately 177.2 kcal ± 0.7 kcal, 27.3 g ± 0.4 g carbohydrate of which, 4.6 g ± 0.9 g sugars, 1.2 g ± 0.2 g fibre, 6.9 g ± 0.8 g protein per serving . Participants were asked in advance to choose from sauce option A and B and consumed the same lunch meal during both visits.The lunch meal  served ad libitum lunch to determine the effect of the intervention on subsequent energy intake . New bowls of rice with the sauce of choice (one portion) were provided at specific times depending on the speed of consumption of each participant (approximately every 3–10 min), to ensure that warm food was served and that finishing a bowl did not act as a cue to stop eating. Participants were instructed to eat until they were “comfortably satisfied”. Satiety changes were estimated based on VAS responses following bread consumption and the total energy intake, measured as the ‘kcal’, during the subsequent lunch ad libitum.Four hours after consuming the intervention meal, participants consumed an sbeII and WT wheat bread rolls.Glucose concentrations measured in capillary blood by FP and in IF by CGM at pre-selected time points were used to compare the postprandial glycaemic responses to Baseline fasting glucose was obtained 15, 10 and 5 min before the breakfast intervention meal by FP and at 30, 15 and 0 min before the breakfast intervention meal by CGM. The average of these three measurements was used as T0.Intervention meals were consumed within 20 min, followed by ten pricks to measure capillary blood glucose concentrations, every 15 min for 3.5 h while glucose concentration in IF was recorded continuously every 15 min by the two sensors worn by each participant. Data up to 3.5 h was collected in order to capture the end of the postprandial response (return to baseline) to these study foods.et al.30 Average glucose peak and glucose dip were calculated as the maximum (peak) and minimum (dip) glucose concentrations achieved after each meal; for capillary glucose this was calculated between 15 and 210 min, for IF glucose, between 15 and 240 min. Time to peak was calculated as the time to reach the maximum glucose concentration following the meal.The glycaemic response to breads (iAUC) was calculated geometrically between 0 and 210 min (primary outcome), by applying the trapezoid rule to the area over the fasting glucose baseline and under the curve, as recommended by Brouns 31 in Rstudio, with bread type as fixed effect and a random intercept per participant. The model used to obtain the IF effect estimates also included the visit nested within participant, as an additional random intercept to account for having data from two CGM sensors. Estimates of effect were obtained with Satterthwaite approximations for degrees of freedom, using the emmeans package v. 1.4.532 in Rstudio and Pearson pairwise correlation  was determined using the stats package previously mentioned.Capillary and IF glucose iAUCs were compared across groups using a mixed-effects model using the lmer package v. 3.1.233–35 in Rstudio.Datasets were curated using package reshape v. 1.4.3, graphs and plots were made using ggplot2 v. 3.3.1 and ggpmisc v. 0.3.5hunger, fullness, desire to eat, and prospective consumption. The scales consisted of horizontal lines 100 mm long, anchored by “not at all” and “extremely” at opposite ends. Satiety VAS questionnaires were scanned using ImageJ ,36 and an appetite score was calculated as follows: appetite score = [desire to eat + hunger + (100 − fullness) + prospective consumption]/4 as described by Anderson et al. (2010).37VAS questionnaires were completed before the meal (T0), and at 30, 90, 180 and 270 min post-prandially to determine changes in sbell and WT control flours had similar total starch . Apparent amylose  was higher in sbell starch  than in WT control starch , hence the proportion of resistant starch  was much higher in sbell flour  compared to WT control . Indicators of performance in the field and extraction rate can be found in ESI Table 2.†The in vitro amylolysis and starch characteristics of bread rolls are reported in sbeII bread was less susceptible to amylolysis compared to the WT control . First order equations fit starch digestion curves well, sbeII bread was characterized by a lower starch breakdown after 90 min of hydrolysis and a lower digestion rate (k), Breads were formulated to deliver approximately 75 g of total starch per serving. Nutrient composition, indicators of Hardness, gumminess, chewiness and cohesiveness were significantly higher in sbeII bread rolls compared to WT control breads while there were no differences in springiness and in resilience between bread types , predominantly derived from starch †Only 8 out of 19 enrolled participants completed the study due to the COVID-19 outbreak and consequent lockdown . Their csbeII and WT control breads on postprandial glucose responses are reported in Participants required approximately 8–10 min to consume the intervention meals. The estimates of effects of sbell bread was 22.9 mmoL L−1 min−1, 95% CI , lower than control bread (corresponding to an average 15.7% reduction following sbeII bread compared to WT control bread). Similarly, the effect size measured in IF by CGM was 19.2 mmoL L−1 min−1 (14.8% difference between sbeII and WT control breads). While these estimates from only 8 participants are not statistically significant, they are consistent with the in vitro findings and our anticipated effect size of 20% difference between groups. Participants’ fasting glucose concentrations measured in capillary blood and IF were within normal range and their fasting glucose values did not vary significantly between the two visits . The overall pattern of postprandial capillary blood glucose was similar after the consumption of sbeII bread and WT control (0–120). Other postprandial capillary glucose indicators are reported in Glycaemic response (iAUC) to  control . Fig. 3CsbeII and WT control bread, as measured by the satiety VAS questionnaires, sbeII bread . Plots showing individual satiety responses can be found in ESI Fig. 1.ad libitum  did not differ following the two intervention meals , Participants indicated that their ‘desire to eat’ and ‘hunger’ were similar after consuming Each participant wore two identical CGM sensors for all intervention visits on the same arm. Two participants lost one sensor after intervention 1 and completed intervention 2 with one sensor only. Two other participants lost one sensor before intervention 1 and had it replaced. Based on the follow up questionnaire completed by the study participants, the experience of wearing two CGM sensors was described as “quick” and “easy”, some participants found the application slightly painful “like a jab” however, most described them “comfortable” to wear and “interesting”.r = 0.91); on average, there was a 10.5 mmol L−1 min−1 difference between sensors’ measurements of glycaemic response (iAUC 0–120) within participant. We observed a 5.25 mmol L−1 min−1, 95% CI  difference between sensors after consuming sbeII bread (n = 8) and 17.6 mmol L−1 min−1, 95% CI  after WT bread (n = 6). Glucose response curves are shown in ESI Fig. 3.†Intra-individual variation (CV%) between the two sensors used in this study by each participant ranged 0–17% during postprandial breakfast phase and 0–22.7% during the postprandial lunch phase. There was a strong correlation between the two CGM sensors worn by each participant , sbeII mutant wheat with a higher amylose proportion for production of slowly digested and/or low glycaemic bread products, compared to conventional wheat starch. Bread rolls made from sbeII mutant wheat contained more RS and showed lower susceptibility to amylase digestion that the WT control, which is consistent with the hypothesis that the presence of mutations in sbeII genes lead to wheat foods with increased starch resistance to digestion.14This study investigated the use of a et al. in 200239 presented a very good overview of the different studies that compared glycaemic responses to high and low amylose food products and showed that different methods used to determine the RS and dietary fibre can contribute to the variability in glycaemic measurements. In this study, we report amylose and RS portion of starch in bread rolls, as well as starch resistance to digestion measured using a single-enzyme system that allowed us to determine the rate and extent of starch digestion, previously shown to correlate with glycaemic index measured in vivo.22Previous studies measuring glycaemic potency of high-amylose food products reported conflicting results. Often, this is derived from inconsistent carbohydrate content across test meals; Behall sbeII and WT control breads from in vitro amylolysis experiments is consistent with the observed difference in glucose concentrations after consumption of sbeII and WT control breads, although our estimate of the effect in vivo is imprecise because only around one third of the planned sample could be completed.Here, we showed that the predicted difference in glycaemic response between et al.16 reported a lower glycaemic response to high amylose wheat bread measured in plasma glucose. The low glycaemic effect was likely due to the reduced amount of carbohydrate provided in the high-amylose bread compared to the control bread. In this study, we showed that sbeII bread has potential to elicit lower glycaemic response compared to a conventional white bread without changing the amount of starch provided.Studies using novel wheat genotypes characterized by a high amylose content have consistently reported a lowering effect on postprandial glucose response. Previously, Belobrajdic et al. (2011)43 showed that bread made from a high-amylose wheat (38% of starch) induced 24% lower glycaemic responses than a control white bread  although all bread types in their study were baked at pumpernickel conditions , which may have modulated starch resistance to enzymatic digestion. In the present study, the difference in starch susceptibility to amylase digestion between sbeII and WT control breads was observed using a shorter baking time (15 min) at higher temperature (185 °C) to produce white rolls, resembling the Chorleywood baking process.Hallström 40 and further starch retrogradation effects thereafter would likely be minimal and negligible with regard to influencing postprandial glycaemia.41It is noteworthy that these breads were stored at −20 °C for ∼2 months longer than those consumed by the human study participants. There is still a limited understanding of the effect of freeze–thaw treatment on starch digestibility in bread and glycaemia, however the largest effects of cold storage occur during the initial stages of freezinget al. in 202015 showed that pasta made from sbeIIa mutant semolina (with 58% amylose starch) produced pasta with lower glycaemic index (compared to an isoglucidic WT control pasta) and acceptable quality, although the authors did report slightly reduced firmness, increased cooking loss and inferior colour compared to the control, likely to be linked to the large increase in amylose. The sbeII bread used in the present study presented minor differences in texture compared to the WT control but was deemed overall acceptable by participants.A study by Sisson 42 however, the IF measurements can vary depending on the type of device and the insertion site.18The results presented in this study showed that the effect of bread on glycaemic response (iAUCs) were measured similarly in IF by CGM on the upper arm and in capillary blood by FP, therefore the conclusions based on iAUCs does not differ between the metrics. Measuring glucose responses in IF by CGM was found to be an effective and minimally invasive method to carry out intervention studies: sensor measurements are reliably taken every 15 min (this may vary depending on the type of sensor), allowing studies to collect larger glucose datasets compared to capillary glucose measured by FP, which is also more painful for study participants. Previous studies have shown a good agreement between measurements taken in blood and IFin vitro amylolysis. The study was underpowered due to the COVID-19 outbreak that forced us to stop all study activities ahead of time. Nevertheless, the results from the human study suggested that bread made with sbeII mutant wheat may lead to lower postprandial IF glycaemic responses.Based on the results collected from eight participants, the effect size measured in this study in IF was consistent with the expected effect size based on sbeII mutant wheat raw material can be used to produce sbeII wheat-based foods with lower starch susceptibility to hydrolysis, without substantially compromising (bread) texture or appearance, measured instrumentally. Future foods made from sbeII mutant wheat may provide an alternative to high glycaemic wheat-based staple foods, including white bread. Further acute and chronic intervention studies are needed to fully explore the potential use of sbeII mutant wheat to reduce the glycaemic potency of bread and other wheat-based foods.Overall, this study showed that the small increases in amylose content in the IFInterstitial fluidFPFingerprickCGMContinuous glucose monitoringsbeIIStarch branching enzyme IIWTWild-typeRSResistant starchiAUCIncremental area under the curveMarina Corrado: Conceptualization, investigation, methodology, project administration, data curation and formal analysis, writing – original draft preparation, writing – review & editing, Jennifer Ahn-Jarvis: investigation, methodology, writing – review & editing, Brendan Fahy: investigation, writing – review & editing, George M. Savva: methodology, data curation, formal analysis, Cathrina H. Edwards: conceptualization, methodology, supervision, writing – review & editing, Brittany A. Hazard: conceptualization, funding acquisition, methodology, supervision, writing – review & editing.The authors declare no conflicts of interest.FO-013-D1FO03085J-s001FO-013-D1FO03085J-s002


Dental implants with tapered conical connections are often combined with zirconia abutments for esthetics; however, the effect of the titanium base on the implant components remains unclear. This study evaluated the effects of a titanium base on the fracture resistance of zirconia abutments and damage to the tapered conical connection implants. Zirconia (Z) and titanium base zirconia (ZT) abutments were fastened to Nobel Biocare (NB) implants and Straumann (ST) implants and subjected to static load testing according to ISO 14801:2016. The experiments were performed with 3 mm of the platform exposed (P3) and no platform exposed (P0). The fracture loads were statistically greater in the titanium base abutments than the zirconia abutments for the NB and ST specimens in the P0 condition. In the P3 condition of the ST specimens, the deformation volume of the ZT group was significantly greater than the Z group. The titanium base increased the fracture resistance of the zirconia abutments. Additionally, the titanium base caused more deformation in the P3 condition. The implant joint design may also affect the amount of damage to the implants when under a load. The mechanical properties of the abutment should be considered when selecting a clinical design. In dental implant treatments, especially in the esthetic zone, zirconia abutments are desired because of their superior esthetics and biocompatibility ,2. CurreA further classification was recently proposed to distinguish between two-piece abutments with a titanium base in the connecting part and one-piece abutments comprised entirely of zirconia . SeveralSeveral in vitro studies have reported that titanium base zirconia abutments were significantly more resistant to a bending moment than zirconia abutments ,7,8,9,10However, these mechanical studies often use different implant bodies ,7,8,9,10When the titanium base is interposed in the connecting portion from the implant body, the component thickness may increase, making it difficult to maintain sufficiently thick surrounding tissue. Additionally, titanium bases tend to affect the color in thin gingiva ,13,14,15Abutments for tapered conical connection implant bodies with platform shifting can prevent bone resorption and soft tissue retraction around the implant body ,17,18. HThis study aimed to clarify the effect of a titanium base on the fracture strength of the abutment for the same implant with a conical connection. In addition, by observing the amount of deformation of the implant body, especially in bone resorption, the magnitude of harmful stress applied to the implant body until the abutment fracture was evaluated.The null hypothesis were no differences in the fracture load or implant deformation between the titanium base and full zirconia abutments.Implant bodies from Nobel Biocare (NB) and Straumann (ST), which possess their own CAD/CAM systems and options to select the inclusion of a titanium base for zirconia abutment with the same taper joint body, were selected. Actual  products were used for all relevant components. The implant bodies were Nobel Replace CC implants  and Roxolid BLT implants . For the test sample abutments, two types of abutments  were prepared for both implant bodies . Titanium abutments were used as controls (T-NB and T-ST) . The ZT-Test conditions were modified for this experiment according to ISO 14801:2016 , which iThe prepared test samples were fastened to each implant body at 35 N cm, the manufacturer’s recommended torque value. The implant body was fixed directly to the load tester  using a collet chuck . After the upper part of the test sample was covered with a hemispherical load made of carbon tool steel material , a static load test was performed under the conditions of an inclination angle of 30°, room temperature (23 ± 1 °C), and a head speed of 0.5 mm/min (n = 3).In the P3 condition, which assumed the progress of bone resorption, the test body was placed with 3 mm of the platform exposed from the testing machine, and the static fracture load was evaluated for the abutment–implant body complex. There is the possibility that deformation of the implant body may occur under load because of platform exposure. Before and after the static load in the P3 condition, the implant body was imaged using micro-computed tomography  under a tube voltage of 90 kV, tube current of 160 µA, and voxel size of 40 µm. A 3D model was prepared for each implant. Additionally, the controls were only the implant body attached to the load tester . Before and after the experiment, the models were aligned with the analysis software  . The volIn the P0 condition, the static breaking load of the zirconia abutment alone was evaluated without exposure of the platform from the tester. Additionally, each component after the P0 condition was observed with a digital microscope . The loading geometry is shown in In the P3 and P0 conditions, all specimens in the same group showed similar failure modes. In the Z-NB group, fracture of the zirconia was observed near the platform of the implant connection . In the The relationship between the compressive load and vertical displacement of the loaded part in the Z-NB, ZT-NB, and T-NB specimens is shown in p < 0.001). No significant difference was observed in the NB groups (p = 0.082).In the P3 condition, in which the static fracture load of the abutment–implant body complex was evaluated, the fracture load of the titanium base type was significantly greater than the full zirconia base type in the ST groups  (The deformation volumes of the Z-NB and ZT-NB groups were 3.536 ± 0.327 mm< 0.001) .p = 0.032, ST: p < 0.001).In the P0 condition, in which the static fracture strength of the zirconia abutment alone was evaluated, the fracture load of the titanium base type was significantly greater than that of the full zirconia type in both NB and ST groups  has greater strength and mechanical properties than other zirconia-based ceramic materials ,21,22,23The tapered conical connection implant, which has been frequently used in recent years, is advantageous in securing the thickness of the soft tissue around the implant body and preventing bone resorption and soft tissue retraction ,17,18. TZirconia abutments are classified into two types: zirconia abutments and titanium base abutments . Many clIt is often reported that titanium-based zirconia abutments have significantly greater resistance to a bending moment than full zirconia abutment ,7,8,9,10The room temperature was set to 23 ± 1 °C, which is often adopted as the standard condition in laboratories. When conducting experiments that simulate intraoral conditions, such as cyclic loading tests, it is necessary to use temperatures and humidity that are closer to that of actual patients.In all samples, fracture occurred at the interface between the implant body and zirconia or between the zirconia and titanium base . After tP0 experiments rejected the null hypothesis that the two types of abutments would have similar fracture loads. The static fracture load of the titanium base abutment was significantly greater than the full zirconia abutment, possibly because of the increased resistance to compressive and tensile stresses near the platform caused by the titanium base .In both P3 and P0 conditions, the load–displacement curve of the ZT-ST group in the low load region showed similar behavior with a T-ST group. The cemented zirconia abutment was an example of an integrated titanium base and zirconia with mechanical properties similar to those of a titanium abutment.Additionally, the breaking load of the ZT-ST specimens greatly exceeded that of the ZT-NB specimens. Given that the height of the titanium base can affect the failure load of a zirconia abutment , differeComparison of the P3 and P0 conditions revealed that the static fracture strength decreased when the height of the implant component increased. This decreased fracture strength is related to the fact that immense stress was applied to the weakest part, even at relatively low loads, by the bending moment . ClinicaOne of the limitations of this study was that the number of samples was small (n = 3). A post hoc test to determine the adequacy of the sample size was conducted using a power analysis program (G*Power) with the fracture load of each group. In ST, both the P3 and P0 conditions showed a power of 1.0, indicating a significant number of samples. For NB, the power was 0.42 for the P3 condition and 0.67 for the P0 condition. The sample size for NB was small, and it was highly likely that a Type 2 error occurred. To obtain statistically reliable results, experiments should be repeated with more samples, especially in NB.One of the features of this study was that the presence and amount of deformation of the implant body before and after the P3 experiment could be evaluated in 3D. However, the obtained 3D model can be affected by artifacts. Since each sample was taken independently and under the same conditions, the effect of artifacts and the difference between them was considered to be very small. However, the numerical values of the volume regarded as the amount of deformation in this study were not reliable. Overall, the null hypothesis that there was no difference in the amount of deformation of the implant body after the test for the two types of abutments was rejected at ST. The results of the Z-NB and Z-ST groups suggest that the full zirconia abutment was destroyed only inside the zirconia abutment; other components were hardly affected.Additionally, from the results of the ZT-NB group, the shape of the titanium base unique to the NB limits damage to the implant body even when the zirconia abutment was destroyed. Alternatively, the results of the ZT-ST group indicate that the abutment could cause damage to the implant body instead of breaking, even under a heavy load, because of the titanium-based design. However, in this experiment, only the appearance of the implant body after loading that significantly exceeds the average occlusal force of an adult male  was confCombined with the P3 results, the design of the titanium base can be enhanced to ensure that the strength of the zirconia abutment is similar to that of the ST type. It may also be possible to make a fail-safe design that does not damage the implant body at the time of zirconia abutment destruction, as in the NB type.In this study, the mechanical properties were examined related to the presence of a titanium base in a zirconia abutment and the fracture resistance in a tapered conical connection implant. The following conclusions were obtained under the limitation of the insufficient number of samples and static load test.The presence of a titanium base could increase the fracture resistance of zirconia abutments.The titanium base abutment showed higher fracture resistance than the full zirconia abutment but might damage the implant body.The full zirconia abutment showed lower fracture resistance than the titanium base abutment but is less likely to damage the implant body.Clinicians should select and design zirconia abutments after considering the characteristics mentioned above.


International Journal of ObesityCorrection to: 10.1038/ijo.2016.206The original version of this article unfortunately contained a mistake in Table


Solanum lycopersicum) and pepper (Capsicum annuum). These grafted plants express signs of anatomical junction failure within the first week of grafting. By generating a detailed timeline for junction formation, we were able to pinpoint the cellular basis for this delayed incompatibility. Furthermore, we inferred gene regulatory networks for compatible self-grafts and incompatible heterografts based on these key anatomical events, which predict core regulators for grafting. Finally, we examined the role of vascular development in graft formation and uncovered SlWOX4 as a potential regulator of graft compatibility. Following this predicted regulator up with functional analysis, we show that Slwox4 homografts fail to form xylem bridges across the junction, demonstrating that indeed, SlWOX4 is essential for vascular reconnection during grafting, and may function as an early indicator of graft failure.Grafting has been adopted for a wide range of crops to enhance productivity and resilience; for example, grafting of Solanaceous crops couples disease-resistant rootstocks with scions that produce high-quality fruit. However, incompatibility severely limits the application of grafting and graft incompatibility remains poorly understood. In grafts, immediate incompatibility results in rapid death, but delayed incompatibility can take months or even years to manifest, creating a significant economic burden for perennial crop production. To gain insight into the genetic mechanisms underlying this phenomenon, we developed a model system using heterografting of tomato ( Gene regulatory networks for compatible self-grafted and incompatible heterografted pepper/tomato plants identify a role for SlWOX4 during junction formation. Plants have robust systems for self-regeneration following wounding . GraftinSolanum tuberosum), tobacco (Nicotiana tabacum), and eggplant (Solanum melongena) are routinely grafted with tomato (Solanum lycopersicum) for horticultural purposes (Capsicum annuum) graft combinations have been described as “severely” incompatible  and vascular connections must be made between the scion and stock . Within purposes . Unlike mpatible . The capmpatible . Stuntedmpatible . Graft cmpatible .Despite the long history and wide-spread use of grafting, only eight genes have been directly implicated in junction formation. These genes are involved in cell proliferation and vascular specification .Arabidopsis thaliana roots is regulated by a dynamic transcription factor (TF) network coordinated with hormonal inputs. CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) peptides 41 and 44 encode identical peptides that act as TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTORs (TDIFs), which are produced in the phloem and bind to the PHLOEM INTERCALATED WITH XYLEM (PXY) cambial receptor  and Capsicum annuum var. Big Dipper (pepper). In agreement with previous work on tomato and pepper heterografting  for graft formation in compatible self-grafts and incompatible heterografts. Using our anatomical timeline, we selected informative sample points that are associated with crucial steps during graft formation: graft adhesion (1 DAG), callus formation (3 DAG), and protoxylem differentiation 5 DAG; .2[fold change] >2 or < −2, FDR <0.05) 1, 3, and 5 DAGs in the tomato self-grafts, respectively  terms (372) critically selected based on our observations from the anatomical timeline and published studies on grafting . We inclA. thaliana genes that are functionally implicated in grafting: RELATED TO AP2 6L (RAP2.6L) (Solyc12g042210) and HIGH CAMBIAL ACTIVITY 2  and TOMATO HOMEOBOX GENE 1 (THOM1) (Solyc01g090460). Notably, LBD18 functions in callus specification and maintenance, and THOM1 marks meristematic cells, both of which are crucial developmental processes during junction formation (XTH) gene (Solyc07g052980); XTHs have been shown to function in the proliferation of the pith during tissue regeneration in A. thaliana  converged to regulate two XTH genes: XTH22 (CA07g00520) and XTH38 , LBD18 (CA01g11210), NGATHA-LIKE 1 , and MYB DOMAIN PROTEIN 86  as major regulators of junction formation 3 DAGs . Notably4 HAT14; . We predNext, we compared the regulations of the pepper and tomato self-grafts to (1) align the networks with our anatomical timeline, (2) identify the specific transcriptional regulations involved in the differential progression of junction formation, and (3) contrast gene networks for self-grafted pepper and tomato. To this end, we used a Sankey diagram, which allows for the comparison and visualization of the number of target genes across different samples, time points, hub TFs, and TF families . To connIn the diagram, the species, TF family, TFs with at least one downstream target, and the 10 GO clusters related to grafting, are connected based on the number of their downstream target genes. As expected, AP2/ERF TFs, which were prominent hubs in both pepper and tomato GRNs, are uncovered as key regulators for self-grafting in both species in the Sankey diagram . InteresThe Sankey diagram indicates that the difference in the developmental timing of junction formation between self-grafted pepper and tomato originates from the delayed induction of key TF families, such as the LBD family, and/or the absence of other key families at specific time points, for example, the AP2/ERF and NAC families. Additionally, the regulation of DEGs associated with callus formation and wounding response is delayed in the pepper self-grafts. Thus, our network analysis informs us on the molecular underpinnings for the developmental delay in pepper graft formation.As shown in the network analysis, tomato and pepper self-grafts utilize distinct regulatory pathways to heal following grafting. Because of this, we hypothesized that the inability of tomato and pepper heterografts to form vascular connections could be due to misaligned genetic processes required for vascular differentiation across the junction. This hypothesis is further supported by the disruption of expression dynamics from the self-graft DEGs, as well as heterograft-specific DEGs in the heterografts compared to the self-grafts . To fullFirst, to identify shared genetic components, we compared the graft-related DEGs from self- and heterografted plants, which uncovered 185 shared tomato genes and 401 shared pepper genes . To idenA. thaliana were identified, but rarely in both reciprocal graft combinations, highlighting the fact that the incompatible grafts are disrupted in genetically distinct ways  were plotted from the self-grafted and heterografted data sets. Notably, these plots show highly perturbed expression in the heterografts, compared to the self-grafts  and CaWOX4 (CA04g18420) in more detail. While these WOX4 orthologs exhibit patterns of gradually elevated expression in self-grafted plants, their expression becomes disrupted in the heterografts , the grafts formed mature xylem connections that spanned the junction and thus did not exhibit graft incompatibility , programmed cell death (NAC104—tomato), and ethylene signaling (ERFs—pepper). At later developmental stages, 3 and 5 DAG, we identified hubs involved in callus production (LBD18—tomato and pepper), meristematic activity , and hormonal signaling . Despite genetic diversity in these regulators between tomato and pepper, we showed that our hub genes converge on the regulation of functionally related targets that are essential for grafting in A. thaliana  and Solanum lycopersicum (tomato) seeds were treated with 50% bleach for 30 s and then rinsed five times with sterile distilled water. Tomato seeds were germinated on wet paper towels in Phytotrays (Sigma-Aldrich) that were placed in the dark for 72 h, transferred to the light for 72 h, and then transplanted into Lambert LM-111 soil. Pepper seeds were immediately planted 1 cm deep into LM-111 soil. Tomato and pepper seedlings were grown in climate controlled chambers set to 23°C with 16:8 day/night light cycles under F54T5/841/HO fluorescent bulbs (500–800 µmol·m-2·s-1).To trigger germination, Capsicum annuum (var. Big Dipper) seeds were grown as described above. Seven days later Solanum lycopersicum (Var. M82) seeds were grown as described above. Twenty-one-day-old pepper seedlings and 14-day-old tomato seedlings, which have the same stem diameter, were joined with a slant or wedge graft on the internode between the cotyledons and first leaf (−2·s−1).rst leaf . Grafts Capsicum annuum (var. Big Dipper) and 50 Solanum lycopersicum (Var. M82) seeds were grown as described above and slant grafted (Fifty  grafted . PlasticSolanum lycopersicum (var. M82) and 180 Capsicum annuum (var. Big Dipper) were grown and grafted as described above. Nine independent plants grown and grafted at the same time were collected for each graft combination, 3–6 days after grafting. Stems were fixed in formalin–alcohol–acetic acid (FAA), stained with PI, and cleared in methyl salicylate (as described below).One hundred eighty Agrobacterium tumefaciens-mediated transformation into an indeterminate (Sp+) tomato cultivar M82 at the Boyce Thompson Institute Center for Plant Biotechnology Research  M82 was used for our WT control. Fifty wox4 mutants were imaged 2 weeks after germination (3 weeks after imbibition), 5 weeks after germination, 8 weeks after germination, during reproductive flowering, and fruit maturation. Stem cross sections were stained with Ruthenium Red and Toluene Blue O as described as below.Sp+ WT and Newly formed protoxylem was identified in junctions at 5 DAG. Total area was calculated by measuring 500 μm above the graft junction and 500 μm below the graft junction, representing the approximate harvest area used for RNA Seq. All stem tissue within this area was calculated in Fiji . Newly f), and immediately transferred into ice-cold FAA  fixative, and infiltrated under a vacuum for 2–4 h. The tissue was moved to fresh FAA and stored at 4°C overnight. The following day, tissue was moved through an ethanol dehydration series, followed by a rehydration series.Graft junctions were harvested by cutting approximately 1 cm above and below the cut site. Tissue was placed into tissue cassettes  for 1 h and rinsed with phosphate buffered saline. Tissue was then dehydrated again in the dark, and gradually transferred into methyl salicylate clearing agent. Finally, the tissue was cleared in 100% methyl salicylate at 4°C for 2 weeks. Fully cleared graft junctions were imaged on a Zeiss LSM880 Confocal Microscope using an Argon Laser 514 nm beam.After fixing in FAA, and dehydrating and rehydrating tissue, the samples were stained with 20 µg·mLAfter fixing in FAA, and dehydrating and rehydrating, tissue was stained with 0.01% Auramine O  in 0.05-M Tris–HCl pH 7.2 for 15 min. The tissue was rinsed with water and immediately imaged on a Leica M205 fluorescent dissecting microscope using an EL6000 Mercury Metal Halide light source.n = 3), pepper (n = 3), Sp+ WT (n = 3), and Slwox4 (n = 3), respectively.After fixing in FAA, and dehydrating, tissue was embedded in Steedman’s Wax  as previously described . Wax andn = 16) and pepper:pepper (n = 16). Twelve independent plants grown and grafted at the same time were tested for tomato:pepper (n = 12) and pepper:tomato (n = 12). Eight and five independent plants grown and tested for WT:WT and Slwox4:Slwox4, respectively.Graft junction integrity was tested using manual bending. Each stem portion was held 1–2 cm away from the graft site. Even pressure was applied to bend the stem at a 45° angle. Stems that broke at the graft junction were marked as broken, stems that did not break or broke at a different point of the stem were considered not broken. Sixteen independent plants grown and grafted at the same time were tested for tomato:tomato  and 50 Capsicum annuum (var. Big Dipper) seedlings were grown as described above. Seedlings were wedge grafted. Graft junctions, consisting of 1 cm from the scion and 1 cm from the stock, were harvested between 8 and 10 PM at 1 day, 3 days, and 5 days post-grafting. Junctions were immediately flash frozen in liquid nitrogen, with one junction harvested per replicate and four independent plants per time point. RNA was extracted using TRIzol reagent . The purified RNA was treated with DNAseI , and quantified and quality checked on a DeNovix DS-11  spectrophotometer. RNA-seq libraries were constructed using 2.5 µg of total RNA per sample. Briefly, mRNA sequencing libraries were constructed by isolating mRNA with the NEBNext Poly(A) mRNA Magnetic Isolation Module , followed directly by the NEBNext Ultra Directional RNA Library Prep Kit for Illumina using NEBNext Multiplex Oligos for Illumina. Six libraries were pooled per lane and run as a single-end sequencing run with 101 cycles on an Illumina HiSeq 2500 at the University of Delaware Sequencing and Genotyping Center. All sequencing data are available on GEO at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE167482 .Library construction: 50 Solanum lycopersicum cv Heinz  that clustered the samples based on the similarity. The PCA was performed in R (version 4.0.2) using the  the PCA . MoreoveTo visualize gene expression, heatmaps were generated using the scaled expression. Specifically, the expression was scaled for each gene using a min–max scaler formula, where the minimum  for DEG selection using FC and FDR values. Specifically, an FDR threshold of 0.05 and logted DEGs . To infeted DEGs  as well ted DEGs . A totalted DEGs . To idented DEGs . For infted DEGs . Only puted DEGs . The regted DEGs . The netted DEGs . Specifihttps://github.com/rspurney/TuxNet and video tutorials regarding installation, analysis, and network inference are freely available at https://rspurney.github.io/TuxNet/. All networks were visualized in Cytoscape 3.8.0  within the TuxNet interface was used for network inference . Similarpe 3.8.0 . Sankey etworkD3 . For theCapsicum annuum, A. thaliana, and Solanum lycopersicum using the default settings for OrthoFinder , PTI5 (Solyc02g077370), RAP2.6L (Solyc12g042210), HCA2 (Solyc06g071480), LBD18 (Solyc01g091420), THOM1 (Solyc01g090460), XTH (Solyc01g090460), XTH (Solyc11g065600), ERF4 (Solyc01g090560), JRE4 (Solyc01g090340), ERF (CA01g01830), ERF (CA01g01880), MYC2 (CA01g00280), LBD18 (CA01g11210), NGAL1-like (CA01g00060), MYB86 (CA01g20220), HD-ZIP14 (CA10g19210), LBD4 (CA02g00820), LBD25 (CA02g30000), XTH22 (CA07g00520), XTH38 (CA11g08350), WOX4 (Solyc04g078650), WOX4 (CA04g18420), NST2 (CA01g34750), LBD4 (CA02g00820), BIN2/BIL1 (CA02g13610), WOX14 (CA02g19960), BIN2/BIL1 (CA02g29760), VND6 (CA03g15410), PXY (CA03g15770), PXY (CA03g15770), ER-like (CA03g17140), BIN2/BIL1 (CA04g09860), SERK1/SERK2 (CA04g14890), KNAT1 (CA04g16610), STZ (CA04g17920), WOX4 (CA04g18420), BES1 (CA04g20150), MP (CA04g23050), ATHB8 (CA04g23050), PXY (CA05g14410), PXY (CA05g14410), ERF-1/ERF2/105 (CA05g14470), VND6 (CA06g06410), MYB15 (CA06g10340), WRKY33 (CA06g13580), VND7 (CA06g14050), TMO6 (CA06g23590), MOL1 (CA07g02560), BIN2/BIL1 (CA07g14640), ER (CA08g03060), WRKY46 (CA08g08240), WRKY33 (CA09g11940), WRKY33 (CA09g11950), NST1 (CA11g08290), LHW (CA11g16160), BES1 (CA12g17430), LBD4 (CA12g22480), WRKY46 (Solyc01g095630), BIN2/BIL1 (Solyc01g101000), LBD4 (Solyc02g069440), BIN2/BIL1 (Solyc02g072300), WOX14 (Solyc02g082670), BIN2/BIL1 (Solyc02g086670), MYB15 (Solyc03g005570), BIN2/BIL1 (Solyc03g006070), ER-like (Solyc03g007050), VND6 (Solyc03g083880), PXY (Solyc03g093330), ERF-1/ERF2/105 (Solyc03g093610), SERK1/SERK2 (Solyc04g072570), KNAT1 (Solyc04g077210), STZ (Solyc04g077980), WOX4 (Solyc04g078650), BES1 (Solyc04g079980), MP (Solyc04g081235), ATHB8 (Solyc04g081235), PXY (Solyc05g051640), ERF-1/ERF2/105 (Solyc05g052050), TMO6 (Solyc06g005130), VND6 (Solyc06g034340), MYB15 (Solyc06g053610), VND7 (Solyc06g065410), WRKY33 (Solyc06g066370), LHW (Solyc06g074110), TMO6 (Solyc06g075370), MOL1 (Solyc07g005010), BIN2/BIL1 (Solyc07g055200), NST2 (Solyc08g008660), ER (Solyc08g061560), WRKY18 (Solyc08g067340), WRKY18 (Solyc08g067360), WRKY33 (Solyc09g014990), TDIFs (Solyc09g061410), MYB15 (Solyc09g090130), NST1 (Solyc10g005010), VND7 (Solyc11g018660), LBD4 (Solyc11g045530), LHW (Solyc11g068960), TMO6 (Solyc11g072500), BIN2/BIL1 (Solyc12g062870), STZ (Solyc12g088390), BES1 (Solyc12g089040), LBD4 (Solyc12g100150).The following materials are available in the online version of this article.A. thaliana are disrupted during tomato and pepper heterografting  (supports supports .Slwox4 fail to form xylem bridges and thus exhibit graft incompatibility (supports supports .Slwox4 vegetative and reproductive organs.Slwox4 (yellow) and break tests from self-grafted WT and self-grafted Slwox4 (gray).koab246_Supplementary_DataClick here for additional data file.


In the Eurotransplant, 12.6% of kidney transplantations are a repeat procedure. Third transplants are significantly more complex than first and second ones. We compared the results of first (PRT) versus third (TRT) transplantations.Between 2011 and 2016, we performed 779 deceased donor adult kidney transplantations, 14.2% out of them were second, 2.6% (20) third, and 0.3% fourth. We compared the pre-, intra-, and postoperative data, kidney function, and survival rate.p = 0.02). HCV infection rate  is ten times higher. The operation time is longer , and delayed graft function is much more frequent . Induction therapy was given to every TRT (7.9% vs.100%), but as a result, the rejection rate was the same (~ 15%). Hospital stay is a week longer. Patient’s survival at 1, 3, and 5 years for PRT is 96.4%, 93.9%, and 91.2% and for TRT is 90%, 85%, and 78.4%, respectively (p = 0.023). TRT’s odds ratio of fatal outcome is 4.35 (1.5–12.5). Graft survival at 1, 3, and 5 years for PRT is 93.1%, 91.4%, and 90.3% and for TRT is 75%, 75%, and 75%, respectively (p = 0.020). TRT’s odds ratio of graft loss is 3.14 (1.1–8.9). Of PRT 85.76%, out of PRT 85.76%, while out of TRT 60% live with a functioning graft, p=0.00149.Recipients of TRT were younger (53.4 vs. 47.3 In a third transplant, both graft and patient survival are significantly inferior to primer ones. Careful selection is required to minimize the patient risk and graft loss. Despite significant improvements in the last five decades, the real half-life of kidney grafts is still around 8 years, substantially shorter than projected half-lives . Chronichttp://statistics.eurotransplant.org).In the Eurotransplant, 17.9% of those on the waiting list are listed for repeat kidney transplantation, and 13.7% of the procedures performed was a repeat one in 2019  were first, 14.2% (111) second, 2.6% (20) third, and 0.3% (2) fourth transplantations.Nine TRT recipients shared the donor with a first or second recipient. We compared the outcomes of these nine pairs as “selected third” (sTRT) vs. “non-third” (nTRT), as a mixed group of 7 first and 2 second recipients. We have no data about the kidney pair of the other eleven third recipients.Sex, age, BMI, ECD, virologyPRA, HLA mismatch, immunosuppressive therapy, rejectionSurgical details and complications, hospital stayDGF, kidney function, graft, and patient survival. Graft survival was recorded from transplantation to graft failure: graftectomy or return to dialysis. Death censored graft survival was counted.We prospectively registered the pre-, intra-, and postoperative data:The end of the observation period is March 2019.Immunosuppression was given as per protocol. Maintenance therapy generally consists of tacrolimus, mycophenolic acid, and prednisolone triple therapy.p = 0.000.Induction therapy was given to all third and 7.9% of primer recipients, considering previous immunization, According to our centre policy, we require preoperative pelvic angio-CT to visualize the vascular anatomy of the potential TRT recipient. At least one of the previous grafts has to be removed prior to waitlisting. Removal of the specific kidney graft depends on clinical circumstances. Our centre’s preferred site is the right iliac fossa for primer, and if possible, for third transplant as well. Third transplantations were performed by experienced senior transplant surgeons.U or Kruskal-Wallis test. Survival was analysed by the Kaplan-Meier method and compared with log-rank test. A p value of less than 0.05 was considered to be significant. Propensity score matching analysis was performed using logistic regression analysis, and then thirds were matched to primers with a 1:1 matching in propensity scores without replacement. The match tolerance was set to 0.1. Statistics were calculated by TIBCO Software Inc. (2018), Statistica , version 13, and by IBM SPSS version 25.For descriptive statistical analysis, mean and median values, standard deviations, and absolute and relative frequencies were calculated. Qualitative data were compared by the Pearson Chi-square test. Quantitative variables were compared using Mann-Whitney The demographic data of donors are presented in Table None of our third recipients received an ECD graft, while this rate is about 34% for primer grafts.Recipients’ data are presented in Table Details and exact numbers are presented in Table We did not observe differences in vascular or ureter-related complications, and there were no more lymphoceles. In the TRT group, one arterial thrombosis caused graft loss; in the PRT group, we could save three kidneys out of 19 having vascular complications. The follow-up US revealed several perirenal hematomas, but they did not require more reoperation.There were no differences in acute rejection rate (PRT: 15.9% vs. TRT: 15%).The DGF rate proved to be much more frequent: 22.4% in the PRT and 60% in the TRT group, which is highly significant. In case of TRT, the OR of DGF is 5.2 (2.1–12.9).Hospital stay proved to be roughly a week longer (PRT 14.8 vs. TRT 21.4 days); propensity analysis verified the significance.Out of PRT, 78.2% got end-to-side uretero-ureteral anastomosis, while in the TRT group, 70% of recipients received neocystostomy; this difference is highly significant (see Table p = 0.023) . Until March 2019, we lost 56 more patients in the PRT group  and 4 more in the TRT group , resulting in a significant difference (p = 0.01).In the first 30 days, there were 3 (0.46%) deaths in the PRT and 1 (5%) in the TRT group  compared to PRT.p = 0.065).Patient survival at 1, 3, and 5 years for nTRT is 100% and for sTRT is 77.7%, 66.7%, and 66.7% (p = 0.020) . One graft had arterial thrombosis caused by acute accelerated rejection; one had acute irreversible rejection; in the others, ischaemic lesions were observed even though ultrasound showed proper circulation.In the first 30 days, there were 26 (4.02%) graft losses in the PRT and 4 (20%) in the TRT group .From 2011 to March 2019, graft loss in surviving patients was 9.6% in the PRT and 25% in the TRT group (The odds ratio of graft loss for TRT recipients is 3.14 (1.1–8.9) compared to PRT.p = 0.028).Graft survival at 1, 3, and 5 years for nTRT is 100% and for sTRT is 55.6%  would be refused, as obesity poses a significant risk of surgical complications –23.Many centres have observed that TRT patients are younger , 16, 20.The rate of HCV positivity in the PRT group is slightly higher than the population-based frequency in Hungary (0.5–0.7%) , 28. TheThe PRA level, a marker of prior sensitization, proved to be much higher in the TRT population. This is expected and consistent with all the known literature. Several papers report a trend that a PRA of more than 80% is associated with poor long-term graft function , 20, 29.The mean cold ischaemic time is the same for PRT and TRT. Comparing the nTRT and sTRT groups, there is an almost 2-h difference in favour of third transplantations.TRT needs significantly longer operative time , 26, 30.Surgical opinions vary in the question of previous transplant nephrectomy. Kienzl-Wagner et al. claim it is not necessary at all . AnotherOur policy is that at least one of the previous grafts has to be removed prior to waitlisting. We strongly believe that this is rational as both presence and removal might cause unexpected complications. TRT itself is a demanding long operation performed on a comorbid patient, and there is no need for any extension of the procedure.Another potential surgical challenge is creating ureter anastomosis. The majority of centres perform neocystostomy for TRT patients , 25, 32,Induction therapy was given after immunologic consideration in a few cases in PRT and to everyone in the TRT group with the likely result that there was no difference in acute rejection rates. This is important as the negative impact of even a single episode of acute rejection is well documented , 36–39. The DGF rate proved to be significantly higher in TRT. Most authors assume that the increased rate of DGF is driven by recipient factors, namely, sensitization, rather than by donor factors , 40, 41.Remarkably more indicative biopsies were obtained in TRT. We do not perform protocol biopsies.Induction therapy, prolonged DGF, more frequent biopsy, and other factors resulted to a 1-week longer hospital stay, reaching a high significance.All these factors together count for much higher expenses in case of a third transplantation , 43.Death in the first 30 days occurred in very few cases, due to cardiac failure or pneumonia with sepsis and ARDS. On the long run, cerebro- and cardiovascular events caused the death of our TRT patients, who are at a higher risk of fatal outcome. Our finding corresponds to the multicentre ET study . Many otOn the other hand, even third transplantation is associated with a significant survival advantage relative to remaining on dialysis, provided that an SCD donor organ was used , 16, 19.Graft losses occurred in the first 6 months, and in our paired subpopulation (sTRT vs. nTRT), the same was observed. This early graft loss is likely the cause of inferior graft survival , 30, 31.We had no TRT graft loss due to surgical reasons. Graft loss occurred as the result of either early rejection or chronic allograft dysfunction, which is in agreement with others , 25, 30.We introduced a not too scientific, but practical parameter to assess the efficiency of our labour investment into third transplantations. The rate of working kidneys in living recipients per performed transplantations is much higher following PRT.TRT and sTRT recipients show a lower GFR until the end of the first post-transplant year. This might have a clinical importance, although mathematically it is not significant.In a third transplant, younger recipients receive a younger, good quality kidney, and still both graft and patient survival are significantly inferior to primer ones. Third kidney transplantations may be performed safely by experienced, senior surgeons, but they represent an intense surgical challenge. The main cause of graft loss is rather immune mediated than surgical. This sensitized patient population requires profound immunosuppression with all its risks and consequences.Patients are at a high mortality risk receiving a third transplant, but this is probably less than remaining on dialysis. A meticulous patient selection is mandatory with a view to reducing post-transplant mortality. Immunotherapy, postoperative dialysis, and prolonged hospital stay cause remarkable expenses. Further prospective studies should be performed to compare the third transplantation with the continuation of dialysis.


The outcome of the reactions varied markedly in terms of reaction efficiency (RE), molecular weight distribution, and average hydrodynamic diameter, for the products derived from the different maleated reagents used, as well as for the different reaction protocols.Modified starches offer a biodegradable, readily available, and cost-effective alternative to petroleum-based products. The reaction of alkenylsuccinic anhydrides (ASAs), in particular, is an efficient method to produce amphiphilic starches with numerous applications in different areas. While ASAs are typically derived from petroleum sources, maleated soybean oil can also be used in an effort to produce materials from renewable sources. The reaction of gelatinized waxy maize starch with octenylsuccinic anhydride (OSA), dodecenylsuccinic anhydride (DDSA), a maleated fatty acid (TENAX 2010), phthalic anhydride (PA), 1,2,4-benzenetricarboxylic acid anhydride , and three maleated soybean oil samples, was investigated under different conditions. To minimize the reaction time and the amount of water required, the outcome of the esterification reaction was compared for starch dispersions in benchtop dispersed reactions, for starch melts in a heated torque rheometer, and for reactive extrusion in a pilot plant scale twin-screw extruder. The extent of reaction was quantified by  Starch is a biopolymer that is renewable, readily available, biodegradable and cost-effective . These aAmylose and amylopectin chains in starch granules are deposited as alternating semi-crystalline and amorphous layers ,12. The TM, Purity GumTM, CAPSULTM, and Hi-CAPTM [Native starch has several drawbacks for industrial applications; for example, it is brittle unless it is suitably plasticized , but canHi-CAPTM . For fooHi-CAPTM , which iHi-CAPTM . The proHi-CAPTM . The amoHi-CAPTM . MechaniHi-CAPTM . Starch Hi-CAPTM . GelatinHi-CAPTM . GelatinHi-CAPTM .OSA and other ASAs are produced by the reaction of maleic anhydride (MA) and unsaturated hydrocarbons, typically containing a terminal alkene . They re1H NMR spectroscopy and gel permeation chromatography (GPC). The hydrophobically modified starch products synthesized have numerous potential uses as food thickeners, binders in paper making, or as adhesives [This study concerns the reaction of starch with different cyclic anhydrides, namely two commercially available ASAs , TENAX 2010 , phthalic anhydride (PA), 1,2,4-benzenetricarboxylic acid anhydride  and three maleated soybean oil products developed in our laboratory containing 1.1, 2.0 and 2.3 anhydride rings per TG. These reactions are often completed on granular starch in batch stirred reactions for extended times. With granular starch, the reaction is heterogeneous, and requires a lot of water with respect to the starch and a base. In the current study, gelatinized waxy maize starch was used first in “classical” benchtop batch reactions, following methodologies previously reported for modification with ASAs. To implement the procedures on an industrial scale, the reactions were then transferred to a melt mixer, also referred to as a heated torque rheometer or internal roller mixer, to modify the TPS at high solids (80 wt% starch), with and without NaOH. Finally, starch was modified by a continuous pilot plant scale twin-screw extrusion process using DDSA, TENAX and maleated soybean oil containing, on average, 1.1 anhydride rings per TG. While the removal of solvents and contaminants such as catalysts in starch modification often impairs the economic viability of processes , the prodhesives . In compWaxy maize starch (waxy pearl 1108) was purchased from Cargill Inc. . OSA and DDSA were purchased from Dixie Chemicals , and TENAX 2010 was acquired from MeadWestvaco Corporation . The remaining chemicals were purchased from Sigma Aldrich . All chemicals were used as received. Native waxy maize starch was gelatinized by extrusion in a pilot plant scale twin-screw extruder.w/v NaOH. The mixture was stirred with an overhead mechanical stirrer until the starch solution was homogenous. OSA  was dissolved in acetone (approximately 50 wt%) before slow addition (over 10 min) to the stirred reaction. The pH was monitored with a pH meter  and maintained between 9 and 10 over 60 min by addition of 20% NaOH solution. The reaction was stopped by addition of HCl (1.5 M) to adjust the pH to 6.5–7.0. The crude product was dried by heating under an air stream, while a portion was also purified by precipitation in acetone followed by Soxhlet extraction with acetone for 48 h. The solid products were then dried in an oven at 80 °C at reduced pressure overnight. The crude and purified products were analyzed by 1H NMR spectroscopy, and the purified product by GPC. The procedure was repeated for anhydride loadings of 5, 7.5, and 10 wt%. The procedure was also repeated with the other cyclic anhydrides .Gelatinized waxy maize starch  was dispersed in 120 mL of distilled water (33 wt%) in a glass beaker and the pH was adjusted to 10 using 20% 1H NMR spectroscopy , and by GPC for the purified product. The procedure was repeated for anhydride loadings of 5, 7.5, and 10 wt%. The procedure was also repeated with the other cyclic anhydrides . All the reactions were completed either without added base or with 1.1 equiv of NaOH with respect to the anhydride. When NaOH solution was added, the amount of distilled water used in the procedure was decreased so as to maintain the overall water concentration constant.Uncooked waxy starch  and distilled water  were loaded into a melt mixer  fitted to an ATR Plasti-Corder Torque Rheometer (C. W. Brabender) preheated to 90 °C by circulating oil. To measure the internal temperature over the duration of the whole reaction (up to 15 min at 40 rpm), the chamber was fitted with a thermocouple at the bottom. After 4 min, OSA  was added slowly to the mixing chamber. After the reaction, the product was removed from the mixing chamber and ground to a fine powder in a coffee grinder. A portion of the crude product was purified by Soxhlet extraction with acetone for 48 h. The solid products were then dried in an oven at 80 °C at reduced pressure overnight. The crude and purified products were analyzed by w/w (1.1 equiv wrt anhydride) to the starch melt during extrusion. The overall amount of water added was adjusted to remain at a consistent level for all the products. Extrusion-modified starch samples were ground into a fine powder with a coffee grinder, a portion was purified by Soxhlet extraction with acetone for 48 h, and the samples were dried at reduced pressure in an 80 °C oven overnight. The crude and purified products were analyzed by 1H NMR spectroscopy , and the purified product by GPC.The modification of waxy maize starch with DDSA, TENAX 2010 or maleated soybean oil containing, on average, 1.1 MA units per TG (1.1 MA/TG) was accomplished by reactive extrusion, similarly to a reported procedure ,29. Reac1H nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker 300 MHz instrument . The concentration of all the samples was 15–30 mg/mL in DMSO-6d with 7 drops of trifluoroacetic acid (TFA), or deuterium oxide for PA- and TMA-modified samples, and 64 scans were averaged. The reported chemical shifts are relative to the residual solvent protons at 2.50 ppm for DMSO-6d and 4.79 ppm for deuterium oxide.p = 334,000 Da and Mw/Mn = 1.10  was used to calibrate the instrument. The Analytical gel permeation chromatography (GPC) measurements for modified starch samples were performed on a Malvern GPCmax instrument with a TDA 305 triple detector array  and one 300 mm × 8.0 mm I.D. PolyAnalytik SuperesTM column  with a theoretical linear PS molar mass range of up to 200 MDa. A flow rate of 0.6 mL/min was used with 0.05 M LiBr in DMSO as the mobile phase at 50 °C. The raw data were collected and analyzed with the OmniSec software package version 5.00. Samples were prepared at a concentration of 2 mg/mL in 0.05 M LiBr in DMSO and filtered through 0.2 μm nylon filters. To obtain absolute molecular weight (MW) values, a pullulan standard with a peak molecular weight M1H NMR analysis, while GPC measurements were used to determine whether the starch products suffered chain scission during the modification procedure.The reaction of starch with cyclic anhydrides in the presence of water has been widely investigated, starting with Caldwell and Wurzburg  in 1953 1H NMR analysis was completed on the unpurified and purified products for each reaction. The spectra obtained for OSA-modified starch  at 40 rpm. The time and torque recording was started as soon as a torque of 1.0 Nm was obtained . Upon loIncreasing the OSA loading led to decreased RE within the 2.5–10 wt% range tested. While the RE at 2.5 wt% OSA loading was 92.6%, close to the 95% RE obtained for the dispersed phase reactions, it did not plateau at higher loadings, as seen in other studies ; rather,When the procedures described above were repeated with DDSA, different trends were observed B. For thThe RE for DDSA-modified starch in the melt phase without base followed the same decreasing trend as the melt reactions with OSA without base as a function of anhydride loading, except for 5 wt% loading, which was highest. When NaOH was used, 100% RE was achieved for both 2.5 and 5 wt%, but the efficiency dropped to 70% at higher loadings B.n = 2.2 6 g/mol and an absolute weight-average molecular weight Mw = 4.5 6 g/mol, corresponding to a polydispersity index Ð ≡ Mw/Mn = 2.0. The weight-average hydrodynamic diameter (Dh) of the molecules was 50 nm. The fact that the elution curves and the molecular weight averages  and Dh = 80 nm, all higher than the materials used in the dispersed phase reactions. The same starch grade was used for the dispersed phase reactions and the melt mixer reactions; however, the starch originated from different lots. The observed differences in absolute molecular weight among the lots are attributed to a combination of variance in year-to-year growth conditions, which have previously been shown to result in more than one order of magnitude difference in Mw for maize starch, and the fact that the gelatinized starch was prepared under higher shear conditions in a twin-screw extruder, rather than in a melt mixer [To generate reference samples for reactions completed in the melt mixer, granular waxy maize starch employed in the melt mixer experiments was processed with water for 15 min under the same conditions as the anhydride reactions. The processed starch prepared at 40 rpm had an absolute Mlt mixer .6 g/mol), the intermolecular formation of ester or anhydride bonds could certainly explain the appearance of the shoulders in the GPC traces of Absolute molecular weight analysis was attempted using the light scattering detector on the GPC system for the starch modified on the melt mixer. Unfortunately, the low-angle light scattering (LALS) detector signal was saturated by a large high-molecular weight shoulder , prevent1H NMR analysis was completed in D2O for both PA- and TMA-modified products, because the peak from TFA in DMSO-6d overlapped with the aromatic signal used for quantification at 7.37 and 7.43 ppm. The RE for the PA derivatives (1H NMR spectrum) at 2.6 wt% loading, decreasing to 39.9% at 10.1 wt% loading. The corresponding PA reactions in the melt mixer with base also had an RE > 100% for 2.5 wt%, remaining above 85% at the higher loadings. Only one report has been published on the reaction of PA with starch in the presence of water [While the reaction of starch with OSA or DDSA introduces a hydrophobic alkyl tail, PA and TMA introduce an aromatic ring onto starch. Dispersed phase reactions with PA and TMA were completed similarly to OSA and DDSA, except that PA was not completely soluble in acetone at 50 wt%. For that reason, PA was dissolved in THF (50 wt%) rather than acetone. ivatives A followeof water , and it The RE for TMA in the dispersed phase B was aboh for the PA- and TMA-modified products prepared in the dispersed phase, as with OSA and DDSA, were similar to the unmodified starch. The corresponding RI elution profiles were likewise identical to unmodified starch, indicating that no degradation or cross-linking occurred during the reaction.The absolute molecular weight averages  and Dh fh of PA-modified starch prepared in the melt mixer without base decreased considerably with respect to unmodified starch  and chain cleavage occurring during the reaction.The molecular weight and Dd starch : the Mn n and Mw of the products without base decreasing by more than one order of magnitude, and Dh decreasing more than 2-fold. The Mn, Mw, and Dh of the TMA-modified products prepared without base were, nevertheless, larger than the corresponding PA derivatives. As for the PA reactions, the decrease in molecular weight and Dh of the products was minimized with base addition, the reactions with 2.2 equiv of base having Mn, Mw, and Dh values most comparable to the starch substrate.The reactions with TMA followed the same trends observed for PA, with the Mh, the TMA derivatives prepared without base should have a lower molecular weight since unreacted TMA contains a free carboxylic acid group. As that was not the case, hydrolysis cannot be the only factor coming into play. Similarly, the decrease in molecular weight and size did not scale linearly with the TMA loading, as the 5 and 10 wt% TMA products had nearly identical characteristics. This suggests that acid-catalyzed chain cleavage and cross-linking both played a role in the trends observed.While previous reports on starch modification with a base showed signs of degradation , this wa18 fatty acids, maleated soybean oil is an entire TG containing over 50 carbons. The hydrophobic domains introduced in starch by reaction with one mole of maleated soybean oil would, therefore, be much larger than for one mole of the ASAs reported above. While the reaction with starch involved anhydride rings, further reactions of the modified starch could focus on the carbon-carbon double bonds; for example, cross-linking with atmospheric oxygen for coating applications [All the starch derivatives reported so far were synthesized using anhydrides derived from petroleum products . TENAX 2ications .12 alkyl tail) was not observed for reactions with TENAX. Foaming was likely suppressed due to the higher RE for the TENAX reactions, leading to a lower succinate salt concentration in the solution acting as a surfactant, in addition to the increased viscosity. The higher viscosity also suggests that reactions with TENAX may be more suitable for melt mixer or extruder operations, designed for these conditions. To the authors’ best knowledge, this is the first report on reactions between TENAX 2010 and starch.Reactions between starch and TENAX in the dispersed phase were completed in the same manner as described above, except that the starch was dispersed at 25 wt% instead of 33 wt%. This is because for reactions with 5 wt% TENAX loadings and above, the viscosity of the reaction increased to the extent that the reaction formed a solid mass around the impeller of the mechanical stirrer. This impeded mixing and pH control in the reactions but did not result in a drop in RE, which remained above 90% at all loadings A. FoaminModifications to the reaction conditions were also required for the vegetable oil-based anhydrides in the melt mixer, since, at 40 rpm, the torque immediately dropped to zero upon addition of the oil, indicating that homogeneous mixing was not achieved. This problem was avoided when the reactions with vegetable oil-based anhydrides were completed at 60 rpm. The reactions in the melt phase, with or without base, followed trends similar to DDSA since increasing the loading of anhydride resulted in a decrease in RE. The highest RE achieved for reactions without base varied from 72.9 to 22.3%, and from 98.6 to 43.0% with base. In contrast to reactions completed in the dispersed phase with gelatinized starch, reactions in the melt mixer had uniform mixing throughout the whole procedure. Similarly to the OSA, DDSA and PA reactions, a base led to higher RE values.Dispersed phase reactions between starch and the maleated soybean oils were completed as described for TENAX since at weight loadings of 5 wt% and above, the reaction mixture likewise formed a solid mass around the impeller of the stirrer. For dispersed phase reactions with MSO-1.1, an RE of 77.2% was achieved at 2.8 wt%, increasing to 97.3% at the highest loading of 10.0%. The two remaining maleated oils did not show the same RE dependence on oil loading, as, for MSO-2.0, an RE of 89.7% was achieved at 2.7 wt%, decreasing to 78.0% at higher loadings, while for MSO-2.3, the lowest RE of 81.4% was obtained at 7.6 wt% loading and the highest RE was 95.9% 10.0 wt% loading.The melt phase reactions with maleated soybean oil also proceeded differently: while a small decrease in torque was observed upon addition of the other reagents to the melt mixer, maleated soybean oil yielded an increase in torque . Such ann, Mw and Dh values comparable with unmodified starch, and the elution profiles were essentially identical, without the high molecular weight shoulder or large Dh increase that would be expected in the presence of cross-linking.The average molecular weight  of the Th values  should increase the likelihood of cross-linking. For the lowest MA/TG ratio (MSO-1.1), the starch modified in the melt phase only displayed a slight increase in molecular weight and Dd starch . There wh for MSO-2.0-modified starch at 5 wt% loading prepared without base in the melt phase were slightly lower than for unmodified starch, but these values were much lower at 10 wt% loading without base, with Mn decreasing 4-fold, Mw decreasing 6-fold, and Dh decreasing 2-fold. The corresponding RI peak was shifted to noticeably higher elution volumes, consistently with the observed decrease in Dh. It should also be noted that the decreases observed were apparently mainly due to degradation of the longer chain components, as there was a major loss in the high molecular weight portion of the distribution. When base was added to the reaction for MSO-2.0-modified starch at 5 wt% loading prepared with base, there was no significant change in either molecular weight or Dh, and the corresponding RI peak elution volume was similar to unmodified starch. The RI elution curve for the MSO-2.0-modified starch at 10 wt% loading prepared with base had a noticeable high molecular weight shoulder, which is likely responsible for the increased Mw value.The molecular weight and Dn and Mw both decreasing by over one order of magnitude and Dh decreasing 4-fold. The RI peak elution volume increase was consistent with the Dh reduction also observed. As with the MSO-2.0 starch product at 10 wt% loading, the high molecular weight population was strongly affected. Similarly to the MSO-2.0 reactions, the addition of a base to the MSO-2.3 reactions compensated for the molecular weight and Dh reductions, leading to molecular weight and Dh values comparable to unreacted starch.The MSO-2.3-modified products without base in the melt phase suffered substantial reductions in molecular weight, with Mh correlated with the substitution level. Our findings that reactions carried out at high torque led to lower molecular weight products but no increase in substitution level are consistent with previous reports on cross-linked starch prepared under high shear conditions. Song and coworkers [h. While MSO-1.1 also had a functionality greater than one, it likely did not form enough intermolecular cross-links to produce significant torque increases and fragmentation, regardless of whether a base was used.For the MSO-2.0- and MSO-2.3-modified starch products, the decreases in molecular weight or Doworkers  indeed doworkers  also invBoth single and twin-screw extruders can be used to produce modified starch, in a continuous process, at an industrial scale . They caWhen DDSA was used to modify starch in a twin-screw extruder , a trendFor the reactions of starch with TENAX in the twin-screw extruder, RE values higher than for DDSA were obtained. Without base, an RE of 93.8% was achieved at 1.6 wt% loading, decreasing to 83.1% at 5.2 wt% loading. The addition of a base to the reaction did not improve on the 86% RE at 3.7 wt% loading, but increased it to 94.4% at 5.2 wt% loading.tert-butylperoxy)-2,5-dimethylhexane (Luperox 101) as catalyst and glycerol as plasticizer, although some water was also present in the starch. A higher conversion was achieved herein without catalyst, and using only water as plasticizer.For reactions between starch and MSO-1.1, an apparent RE > 100% was achieved at the lowest weight loading (1.6 wt%), decreasing to 92.2% at a 5 wt% loading. Interestingly, when base was added, a drop in RE was observed, in contrast to the melt phase reactions and the previously discussed extrusion reactions: the RE decreased to 81.3% at 5 wt%, and to 34.9% at a loading of 7.5 wt%. A possible explanation for the drop in RE observed is that the addition of base made the starch melt more hydrophilic, such that the hydrophobic anhydride did not have sufficient time to fully react in the extruder. The conversion of maleated vegetable oil achieved in the twin-screw extruder without base was higher than in the only previous report on that topic. Narayan and coworkers , indeed,h of DDSA-modified starch reactive extrusion products were essentially independent of the DDSA loading  RE but noticeably higher molecular weight and Dh than the other MSO-1.1-modified products. A possible explanation for the higher molecular weight and Dh of the 7.5 wt% MSO-1.1 product is that since more NaOH was added to the system due to the higher anhydride loading, the addition of NaOH decreased the torque. The addition of NaOH to starch in the melt mixer resulted in a larger drop in torque as compared to deionized water, and it has indeed been shown before that the addition of NaOH lowers the starch viscosity more than pure water [In the reactions carried out in the melt mixer, the decrease in molecular weight and Dre water . The droHydrophobically modified starch derivatives were successfully prepared by different methods , by reaction with cyclic anhydrides derived from either petroleum products  or vegetable oils (TENAX and MSO), through environmentally friendly procedures. The reaction efficiency (RE) was found to depend on the state of the reaction, as well as the structure of the maleated reagent used.Reactions completed with starch dispersed in water (33 wt% starch) had RE values above 80%, except for DDSA and maleated soybean oil samples. At moderate to high maleated vegetable oil loadings, the reactions were no longer homogeneous. GPC analysis revealed that the molecular weight and hydrodynamic diameter did not increase; therefore, the viscosity increases observed are attributed to the hydrophobic modification.h of the modified starch products were greater than for the products prepared without base, in contrast to previous literature reports.Reactions completed in a heated melt mixer on starch plasticized with water (80 wt% starch) had decreasing REs for increasing anhydride loadings, except for TMA, which maintained a high RE at all loadings. Reactions completed with a base had higher REs for all the anhydrides tested, indicating that esterification is favored over hydrolysis. Interestingly, the molecular weight and DReactions with maleated soybean oil in the melt mixer led to a significant increase in the measured torque. The base-promoted reactions, in particular, were no longer plasticized, whereas the reactions without base displayed a torque increasing throughout the reaction. GPC analysis revealed that the products of the base-promoted reactions had molecular weight and size characteristics similar to unmodified starch, while the products obtained without base had undergone extensive shear-induced chain scission. The decrease in size observed was attributed to the high shear forces experienced by the starch derivatives, due to the increased torque, rather than to the substitution level.When comparing the reactive extrusion results with DDSA, TENAX, and MSO-1.1, it should be kept in mind that while the hydrophobicity of the anhydrides increased in the order DDSA < TENAX < MSO-1.1, the RE varied in the same order. Noteworthy is the fact that the MSO-1.1-modified product at 1.6 wt% loading reached 100% RE without a base. The addition of a base increased the RE for DDSA and TENAX while decreasing the RE for MSO-1.1. Reactive extrusion proved to be the most advantageous technique to readily produce hydrophobically modified starch in an environmentally friendly and scalable way. The RE is high enough that the reaction products would not need to be purified before use. A major economic obstacle to the industrial implementation of this modification method is, therefore, removed.