The SPIDER technique for hybrid thoracoabdominal aortic aneurysm repair can avoid thoracotomy and extracorporeal circulation. To improve technical feasibility and safety, the new Thoracoflo graft, consisting of a proximal stent graft connected to a 7-branched abdominal prosthesis, was evaluated in a pig model for technical feasibility testing, before implantation in humans.Retroperitoneal exposure of the infradiaphragmatic aorta, including visceral and renal arteries, was performed in 7 pigs (75-85 kg). One iliac branch was temporarily attached to the distal aorta to maintain retrograde visceral and antegrade iliac perfusion after deployment of the thoracic stent graft segment (SPIDER technique). The proximal stent-grafted segment was deployed in the thoracic aorta via direct aortic puncture over the wire without fluoroscopy. The graft was deaired before flow via the iliac side branch to the visceral and iliac arteries was established. Visceral, renal, and lumbar arteries were subsequently sutured to the corresponding side branches of the graft. Technical feasibility, operating and clamping time, blood flow, and tissue perfusion in the related organs were evaluated before implantation and after 3 and 6 hours using transit-time flow measurement and fluorescent microspheres. Final angiography or postprocedural computed tomography angiography were performed.P = n.s.). In 1 pig, the proximal stent graft section migrated as the guidewire was not removed, as recommended, before release of the proximal fixation wire. Angiography and computed tomography scan confirmed successful graft implantation and transit-time flow measurement confirmed good visceral and iliac blood flow. Fluorescent microspheres confirmed good spinal cord perfusion.Over-the-wire graft deployment was successful in 6 animals without hemodynamic alteration (Over-the-wire implantation of the Thoracoflo graft using the SPIDER technique is feasible in a pig model. No fluoroscopy was required. For safe implantation, it is mandatory to follow the single steps of implantation. The Thoracoflo graft for TAAA hybrid repair shows promising results in animal testing. However, its potential to reduce perioperative mortality and morbidity must be confirmed in a clinical setting.The Thoracoflo graft is designed for hybrid TAAA repair, avoiding thoracotomy and ECC by using the previously established SPIDER technique. The graft design was modified and reevaluated to improve graft deployment, fixation and stability. Feasibility testing, including quantitative assessment of organ perfusion, was performed in an in vivo experimental setting before first implantation in human.,Based on the Thoraflex hybrid graft (Vascutek Ltd), the Thoracoflo graft was developed as a new hybrid device for thoracoabdominal aortic aneurysm (TAAA) repair. The aim was to offer preventive strategies concerning perioperative mortality and morbidity by combined endovascular thoracic and open abdominal aortic approach for TAAA repair in a single-stage operation. The SPIDER technique for graft implantation allows temporary retrograde distal perfusion of visceral and renal arteries via the iliac access side branch following the deployment of the thoracic stent graft of the device. Hereby, thoracotomy and extracorporeal circulation (ECC) can be avoided while enabling reimplantation of visceral, renal, and lumbar arteries.This report describes evaluation of the modified graft design and implantation technique of the new Thoracoflo graft for TAAA repair in an experimental setting in pigs, including quantitative assessment of visceral and spinal cord perfusion.Circulation, April 1985) and approved by the government animal care committee (AZ101/15) and the institutional review board for the care of animal subjects.Seven domestic pigs with a mean body weight of 80 ± 5 kg were operated on at the animal laboratories, University Heart Center Hamburg. The study was performed in accordance with the “Position of the American Heart Association on Research Animal Use”  were previously described and tested in an experimental setting, confirming further need of modification to improve intraoperative handling, graft stability, to enable reattachment of lumbar arteries, and over-the-wire implantation.A and B) in the experimental setting for graft evaluation was adjusted to the aortic diameters of an 80-kg pig observed from postmortem computed tomography scan. The gelatin-sealed polyester prosthesis consists of a long, Y-shaped arm (8 mm each side) for biliary reattachment and 4 prosthetic arms  attached for reimplantation of celiac trunk (CT), superior mesenteric artery (SMA), and both renal arteries . A 10-mm access branch with a length of 20 mm carries the handle with the delivery system and is positioned on the dorsal side of the main body for reattachment of lumbar arteries after graft deployment. Between the stent-grafted thoracic component and the multibranched, gelatin-sealed polyester prosthesis, a Siena collar is included, enabling suture fixation to the native aortic vessel to prevent stent graft migration after deployment.The Thoracoflo graft prototype , A and BThe stent graft is 150 mm in length and 24 mm in diameter. Longitudinal stiffness and stability of the stent graft are achieved by special orientation and formation of the ring stents. The stent graft section is loaded into a 30-F peel-away sheath. The tip at the top of the delivery system is equipped with a side hole, enabling over-the-wire implantation of the graft via direct puncture of the aorta. The handle with the delivery system is externalized via the 10-mm access branch on the dorsal side of the main body of the multibranched graft and fixed with a splitter. The elongated design of the splitter prevents the amount of blood loss during extraction of the delivery system through the access branch. The release wire for fixation of the proximal stent graft component on the introducer tip is inserted through the delivery system and fixed on a release clip at the end of the handle.The single steps of Thoracoflo graft implantation are illustrated in both The stent-grafted section of the Thoracoflo graft was introduced over the guidewire after direct puncture of the aortic wall. Aortic puncture was performed at the level of the CT ostium and the guidewire was established into the descending thoracic aorta. The needle was retracted and the puncture hole was stepwise dilated using a 10-F and a 14-F dilator sheath, while cardiac output (CO) was reduced by in-flow reduction by pulling on a loop surrounding the inferior vena. The guidewire was externalized through the side hole in the nose cone of the introducer-tip. Then, the stent graft component of the hybrid graft was introduced until the collar was just outside the vessel wall. The peel-away sheath was retracted and the guide wire extracted before release of the proximal fixation wire and retraction of the handle.The proximal part of the extended splitter was opened, allowing retraction of the delivery system with minimized blood loss, and the handle was carefully retracted until the nose cone extended into the side access branch. The splitter was completely removed, the handle was extracted, and the access branch was clamped. In-flow reduction was released, and the graft was deaired and flushed with heparinized saline solution via the access branch. Then, retrograde distal perfusion via the previously attached iliac side branch was established, hereby, avoiding ECC and thoracotomy. Fluoroscopy is not mandatory for thoracic graft deployment, as transesophageal echocardiography can be used during stent-graft introduction to ensure correct positioning of the graft. Thereafter, stepwise visceral and renal arteries were reattached in end-to-end fashion to the corresponding branches of the Thoracoflo graft starting with the CT, followed by SMA. The aorta is then crossclamped below the renal arteries and the Siena collar is sutured to the aortic wall using continuous 3-0 or 4-0 PROLENE suture  to prevent back-bleeding of intercostal arteries and distal graft migration. Thereafter, RRA and LRA are anastomosed.Finally, lumbar arteries were reimplanted as patch into the previous 10-mm access branch, which can be used as loop graft by reattachment of the distal part of the branch to the left iliac side-branch. Reattachment of the lumbar arteries can be performed either as a loop graft or in end-to end fashion, depending on the individual surgeon's preference. In case no lumbar arteries are reattached, the introducer branch can be ligated.The initial end-to-side anastomosis of the iliac branch was replaced by end-to-end anastomosis to both iliac arteries.2O with a positive end-expiratory pressure of 7 cmH2O at 16 breaths per minute using 30% oxygen. Activated clotting time of at least 300 seconds was achieved by heparin (400 IU/kg). A 6-F sheath was inserted in the right carotid artery for monitoring of mean arterial blood pressure (MBP) and arterial blood-gas analyses. A Swan–Ganz catheter (Pulsion) was inserted via the right jugular vein for monitoring of central venous pressure, pulmonary arterial pressure, and left atrial pressure. The Swan–Ganz catheter was zeroed to ambient air pressure to calibrate before each measurement. A 5-F PiCCO catheter  was inserted in the left common femoral artery to monitor CO, systemic vascular resistance, and distal MBP. The PiCCO catheter was calibrated at the beginning of each experiment using thermodilution with 20 mL of NaCl at 8°C . For quantitative assessment of organ perfusion using fluorescent microspheres (FMs), a 5-F catheter was introduced into the left atrium to enable FM injection, and a 5-F pigtail catheter is inserted into the descending thoracic aorta via a 7-F sheath in the common femoral artery for retraction of reference blood.After intramuscular premedication with azaperone (4 mg/kg), midazolam (0.3 mg/kg), ketamine hydrochloride (5 mg/kg), and atropine sulfate (0.15 mg/kg), intravenous anesthesia was induced by pentobarbital (8 mg/kg) and maintained by continuous infusion of fentanyl (0.01 mg/kg/h), midazolam (0.1 mg/kg/h), and ketamine hydrochloride (0.06 mg/kg/h). Pigs were endotracheally intubated and pressure-control ventilated at 15 cm HAll hemodynamic and respiratory parameters were continuously monitored and recorded according to a standardized protocol at each intervention. Isotonic and colloidal solutions were continuously infused at a basic flow rate of 400 mL/h to achieve a stable global end-diastolic volume.After retroperitoneal exposure of the abdominal aorta, visceral and LRA as well as both iliac arteries were dissected free from surrounding tissue to place a 6-mm flow-measurement probe , to monitor the blood flow during measurements by transit-time flow-measurement (TTFM) technique.As in the previous experimental studies, graft implantation was performed by the same 2 senior vascular surgeons with high experience in open aortic surgery. Hemodynamic parameters were continuously monitored and recorded during each intervention. Arterial and venous blood-gas analyses and TTFM were acquired before implantation (baseline), after stent-graft deployment and establishment of retrograde visceral perfusion using SPIDER technique for proof of concept of retrograde visceral perfusion, after implantation of the prosthesis with anastomosis of all side branches (operative [OP]), and hourly during an observation time of 6 hours. Final angiogram was performed after 6 hours of observation using the 5-F Pigtail catheter in the descending thoracic aorta.Quantitative assessment of liver, kidney, bowel, and spinal cord perfusion by FMs was performed during baseline, after implantation (OP), and at 6 hours. At the end of the experiments, the pigs were humanely killed by intravenous injection of 20 mL of T61 under deep anesthesia. The Thoracoflo graft was explanted for photo documentation. Two randomly selected representative animals underwent postmortem computed tomography angiography using a 12-F sheath in the distal aorta and the inferior vena cava for postoperative quality control. Finally, one lobe of the liver, both kidneys, 30 cm of bowel, and spinal cord were harvested and preserved for at least 10 days in 4% formalin for later FM assessment.,6 FMs were suspended in physiological saline solution to a volume of 10 mL and injected constantly over 60 seconds at each intervention. Reference blood samples were withdrawn from the descending thoracic aorta through the previously established 50-F pigtail catheter with a constant-rate withdrawal pump at 3.14 mL/min over 3 minutes . At the end of the experiments, the pigs were humanely killed, and one representative lobe of liver, both kidneys, 30 cm of bowel, and spinal cord were harvested and fixed in 4% formaldehyde for at least 10 days. Visceral organs were dissected into 6 tissue pieces weighing 4 g each. Spinal cord was dissected in total length into pieces of equal size weighing between 2.5 and 3 g each. Tissue samples and arterial blood reference were processed for determination of blood blow by spectrofluorometry, according to the standard method described by Glenny and colleagues.As previously described, FM , were injected into the left atrium during withdrawal of a reference blood sample via the descending thoracic aorta, for quantitative assessment of organ perfusion.P < .05. All computations were performed using SAS software, version 9.4 (SAS Institute).Descriptive statistics for continuous variables were calculated using univariate moments of the distributions (median and interquartile range) at each of the 3 time points , with the exception of operative and ischemic times, which are total times reported only once per pig. Tests for variation by time point for hemodynamic and flow measurements were computed using mixed models with a fixed effect for time and a random effect for subject to account for the correlation of repeated measures within pig. Because multiple measures per pig per time point were made for microsphere tissue perfusion assessments, these data were analyzed using hierarchical mixed models that included multilevel random effects for sample nested in pig with repeats by time point. The null hypothesis was rejected at a nominal alpha of Thoracoflo graft implantation was successful in 6 of 7 pigs, including 6-hour follow-up without hemodynamic alteration.Retroperitoneal exposure of the abdominal aorta, including visceral, renal, and iliac arteries, was performed without significant blood loss or hemodynamic events in all 7 animals. Over-the-wire introduction of the Thoracoflo graft after direct aortic puncture next to the CT ostium was fast, easy, and comfortable to perform after dilatation of the puncture side under in-flow reduction. Retraction of the peel-away sheath, extraction of the release wire for proximal fixation of the stent graft, and removal of the splitter were technically easy to perform.In one pig, the guidewire was not removed, as recommended before release of the proximal fixation wire and retraction of the handle, resulting in distal migration and collapse of the proximal stent-grafted section of the Thoracoflo graft. This pig was excluded from further investigation to evaluate the Thoracoflo graft.No stent-graft migration occurred in the remaining 6 pigs when the guidewire was released, as recommended before release of the proximal fixation wire. Retraction of the handle with the elongated splitter of the device required no additional surgical maneuvers or manual help. Deairing of the graft was easy to perform via the introducer branch, but modification of the splitter—including a closure mechanism for the introducer branch to avoid the need for manual branch occlusion during retraction of the handle and to avoid accidental blood loss—was favored by both surgeons implanting the graft.Reattachment of visceral, iliac, and lumbar arteries showed favorable anatomical orientation of the visceral side branches. Both kidney arteries in the pig model were very small compared with the visceral arteries—and very sensitive toward vasospasm during the anastomosis to the 6-mm branches of the graft. Hemodynamic stability was maintained in all 6 animals throughout Thoracoflo graft implantation and during 6 hours of observation. Intraoperative angiography and postprocedure computed tomography angiography confirmed open-side branches and successful graft implantation in all 6 animals but mismatch in the diameter for the attached kidney arteries.Data are shown as median (interquartile range). Graft implantation lasted 75 minutes (65-83 minutes). Implantation time of the stent-grafted section was 4.5 minutes (4.5-4.5 minutes). Ischemic time of the visceral organs was minimized to the time needed for the anastomosis of the respective side branch and lasted 15 minutes (13-16 minutes) for CT and 8 minutes (8-9 minutes) for SMA. Ischemic time of left kidney was added to right kidney ischemic time due to simultaneous crossclamping of both renal arteries during opening the juxtarenal aortic segment. RRA was first anastomosed from inside. Ischemic time for the RRA was 13 minutes (12-14 minutes) and for left kidney was 26 minutes (21-35 minutes).P < .05), systolic blood pressure, CO, central venous pressure, and global end-diastolic volume did not significantly change throughout implantation and 6 hours observation compared with preoperative baseline values (P = n.s.). No catecholamines were used to increase diastolic blood pressure throughout the protocol, as systolic blood pressure was stable. MBP was successfully decreased by 50% from baseline during aortic puncture, dilatation of the puncture side, and stent graft implantation, until retrograde distal perfusion via the previously attached iliac side branch was established. Relevant hemodynamic parameters during baseline, graft implantation, and 6-hour observation are illustrated in P = n.s.).Hemodynamic stability was maintained in all 6 animals throughout the implantation and the 6-hour observation period. While diastolic blood pressure and consecutively MBP decreased during graft implantation and 6-hour follow-up period compared with baseline (P < .038) but significantly increased during 6-hour observation compared with baseline (P < .050). SMA flow increased significantly during implantation (P < .037) and was stable compared with baseline after 6-hour observation (P < .112). The LRA showed significantly lower values compared with baseline after implantation and throughout the observation, reflecting the vasospasm and the mismatch in diameter of the sensitive porcine kidney arteries to the corresponding branches of the graft (P < .05). The TTFM values during baseline, implantation (OP), and postoperative observation (6 hours) are illustrated in TTFM confirmed sufficient retrograde visceral perfusion by passive shunting using SPIDER technique: TTFM flow for CT was 545 (450-700) mL/min, for SMA 620 (535-780) mL/min, and for LRA was 225 (210-260) mL/min. Due to retroperitoneal aortic access, there was no TTFM value for RRA. After implantation and during 6-hour follow-up, TTFM flow of visceral, renal, and iliac arteries confirmed sufficient blood flow to the visceral arteries. TTFM flow for CT was stable during implantation (P < .02) but increased during 6-hour observation to baseline values (P < .264). Bowel perfusion showed no significant differences throughout implantation and observation time (P = n.s.). FM confirmed reduced kidney perfusion compared with baseline. Spinal cord perfusion remained stable compared with baseline after implantation (P < .693) and during 6-hour observation (P < .101). Rate of change of organ perfusion showed no treatment-by-time interactions for change from baseline to 6-hour postimplantation. FM application for quantitative assessment of visceral and spinal cord perfusion was successful in all 6 animals during baseline, after OP, and after 6 hours of observation. Six specimens of equal size (3.9 ± 0.2 g each) of the formalin-preserved segments of liver and both kidneys were dissected and processed for fluorospectrometry. The spinal cord was dissected in toto into 9 to 15 samples, weighing 3.7 ± 0.3 g each, depending on the lengths and thickness of the spinal cord. Liver perfusion decreased significantly during implantation  remains the standard of care in many centers, especially in patients with genetic aortic syndromes. Despite this, OAR has undergone major improvements, such as multimodal access to organ protection, consisting of mild hypothermia, cerebrospinal fluid drainage, left heart bypass, sequential clamping of the aorta, and selective reimplantation of the intercostal as well as lumbar arteries. This complex procedure remains challenging and carries high morbidity and mortality, which globally has led to centralization of these highly demanding procedures.14While the aneurysm extent and rupture presentation are not modifiable risk factors, hypotension, blood loss, intraoperative clamp time, and patent lumbar and intercostal artery sacrifice are factors that can be potentially influenced. Avoiding ECC for selective visceral perfusion can reduce the perioperative risk of bleeding, the need for blood products, the inflammatory response, and the risk of ECC related hypotension. Preventing the risk of ECC, as well as ischemia reperfusion-related hypotension, might also reduce the risk of perioperative spinal cord ischemia.Intraoperative clamping time and ischemic times can be minimized using the SPIDER technique. Ischemic time is reduced to the time needed for each side-branch anastomosis added to the time needed for stent graft deployment. This was also confirmed previously by significantly shorter ischemic times compared with conventional OAR.The Thoracoflo graft enables reattachment of lumbar arteries, which might reduce the risk of spinal cord ischemia compared with endovascular techniques that lack this option. Quantitative assessment of spinal cord perfusion by FM showed stable spinal cord perfusion throughout the entire 6-hour observation period. This was also confirmed in a previous study of our group.By hybrid TAAA repair using the Thoracoflo graft thoracotomy and ECC might be avoided. This could significantly reduce pulmonary complications in the clinical setting, which are a major cause of morbidity and mortality.The Thoracoflo graft for TAAA hybrid repair shows promising results in experimental testing. However, the potential to reduce perioperative mortality and morbidity during open TAAA by avoiding the ECC and thoracotomy while enabling reattachment of lumbar arteries must be confirmed in the clinical setting.The Thoracoflo graft is designed for hybrid TAAA repair, avoiding thoracotomy and ECC by using the previously established SPIDER technique. The graft design was modified and re-evaluated to improve graft deployment, fixation and stability, and deairing as well as to enable reattachment of lumbar arteries and establish over-the-wire implantation. Feasibility testing, including quantitative assessment of organ perfusion, was performed in an in vivo experimental setting before first implantation in human.Terumo Aortic. All other authors reported no conflicts of interest.Drs Estrera and Sandhu are consultants for W. L. Gore & Associates, Inc. Dr Estrera is also a consultant for CryoLife, Edwards Lifesciences, and Terumo Aortic. Drs Kölbel and Debus report intellectual property rights and travel grants with Journal policy requires editors and reviewers to disclose conflicts of interest and to decline handling or reviewing manuscripts for which they may have a conflict of interest. The editors and reviewers of this article have no conflicts of interest.The


GBSSI and their isoforms) in the high resistant starch mutant lines, in comparison to the parent variety (J411). The expression of SBEs genes was higher in the low resistant starch mutants. The other three candidate genes showed overexpression  and four had reduced  expression in high resistant starch mutants. The overexpression of AMY and ISA1 in the high resistant starch mutant line JE0146 may be due to missense mutations in these genes. Similarly, there was a stop_gained mutation for PHO2; it also showed overexpression. In addition, the gene expression analysis of 21 starch metabolizing genes in four different mutants (low and high resistant starch mutants) shows that in addition to the important genes, several other genes  may be involved and contribute to the biosynthesis of resistant starch. There is a need to do further study about these new genes, which are responsible for the fluctuation of resistant starch in the mutants.Several health benefits are obtained from resistant starch, also known as healthy starch. Enhancing resistant starch with genetic modification has huge commercial importance. The variation of resistant starch content is narrow in wheat, in relation to which limited improvement has been attained. Hence, there is a need to produce a wheat population that has a wide range of variations in resistant starch content. In the present study, stable mutants were screened that showed significant variation in the resistant starch content. A megazyme kit was used for measuring the resistant starch content, digestible starch, and total starch. The analysis of variance showed a significant difference in the mutant population for resistant starch. Furthermore, four diverse mutant lines for resistant starch content were used to study the quantitative expression patterns of 21 starch metabolic pathway genes; and to evaluate the candidate genes for resistant starch biosynthesis. The expression pattern of 21 starch metabolic pathway genes in two diverse mutant lines showed a higher expression of key genes regulating resistant starch biosynthesis ( Starch is the main part of the wheat grains (70% to 80% of the dry weight of seed) and any change in their nutritional composition can help to provide benefits to a lot of people. The composition of the nutrients in starch depends upon the ratio of amylopectin and amylose . AmylopeSSIIa gene [SBEIII knocking out or silencing the gene helps to increase resistant starch [GBBSS decreases the percentage of resistant starch in wheat grains [The portion of the starch that passes through the small intestine in undigested form from the healthy person is called resistant starch . By usinIIa gene . Similart starch ,11,12,13t grains . It is at grains ,16. In at grains . Moreovet grains . Two addt grains .GBSSI loci in wheat, many partial waxy and full waxy phenotypes have been produced [SBEIIa, SBEIIb, and SSIIa, have been studied in wheat for the development of high resistant starch and low resistant mutant lines [Variations have been produced in many crops through different chemical agents. Ethyl methane sulphonate (EMS) is the main agent for producing variations in different crops . It helpproduced . Similarnt lines ,23.ZIP TF family is preferentially expressed to regulate starch synthesis [TaMYB13-1 and bHLH helps to increase starch in wheat [In wheat, seed formation is divided into three stages: (1) the division and expansion stage (0–14 days after anthesis); (2) the grain-filling stage (14–24 days after anthesis); and (3) the maturation stage (28 days after anthesis) . The genynthesis . In tranin wheat ,27. DiffIn the present study, the EMS stable population was screened, whose parent variety was J411; it showed variation for resistant starch content. This stable population consisted of one hundred and fifty mutant lines. Further, two high resistant mutant lines and two low resistant starch mutant lines were used to study quantitative gene expression patterns of 21 starch metabolic pathway genes during seed development. The overall workflow is given in The EMS mutant library was screened, which was the uniform population. This mutant population was consisting of 150 mutant lines. These mutant lines were sown in the experimental area of the Institute of Crop Sciences, Beijing with three replications. From these 150 mutant lines, samples of each replication were collected at the maturity stage of the crop. Resistant starch content, digestible starch, and total starch content were measured from these samples by using the Megazyme kit. One-way ANOVA analysis showed significant variation in the resistant starch content of the lines in the three biological replications. Some lines showed resistant starch of more than 17% and low resistant starch in lines of 0.7%; while the resistant starch of J411 was 1.3%. Similarly, the maximum digestible starch in some mutant lines was 76.2%; while the lowest digestible starch in some mutant lines was 27.4%. The overall results of all the mutant lines are given in Firstly, two high resistant starch lines (JE0146 and JE0296) and two low resistant starch (JE0244 and JE0213) lines were selected after screening the mutant population. These lines showed a significant difference from WT (J411) whose value was 1.2% in 2019–2020 and 2020–2021 . In bothThe images of high starch mutant lines and low starch mutant lines were taken to observe the length and width of the mutant lines . The graPure starch was extracted from wheat flour; a TM4000 scanning electron microscope observed the difference in the morphology of starch granules between the wild-type (J411) and resistant starch mutants . The resAGPaseL and AGPaseS): two were granule-bound starch synthase (GBSSI and GBSSII); four were soluble starch synthase isoforms ; three were isoforms of branching enzymes ; and the other four were debranching enzymes with pullulanase . Further, four starch-degrading genes and two transcription factors  were selected for expression analysis.Expression analysis was studied in 21 starch metabolic genes, including the genes responsible for high resistant biosynthesis. Expression analysis was conducted at four seed development stages  between four selected mutants and J411 (WT). These four mutant lines contain about 0.8% and 16.5% resistant starch content. There were fifteen starch biosynthesis genes, in which of the large and small subunits of ADP-glucose pyrophosphorylases  and six genes in JE0296  showed overexpression; moreover, two genes of JE0146 (SBEI and SBEIII) and three genes of JE0296  showed reduced expression during all the seed development stages. The expression of the remaining starch metabolic genes was inconsistent; i.e., either high or low expression during the seed development stages  and seven  genes showed overexpression, respectively. In addition, eight genes  of JE0244 and six genes  of JE0213 showed reduced expression during the seed development stages in the low resistant starch mutant lines  and low resistant starch mutant lines (JE0244 and JE0213). It was depicted that in the mutant lines, the number of SNPs were more in all four mutant lines than in the Indels. The number of SNPs in JE0296 was higher than the other three mutants. However, the Indels were almost in an equal amount in all the mutant lines. The different types of SNPs between the mutants suggested that there were more transitions than transversions in the four mutants . There wFrom the four mutant lines, seventeen variants were identified in twenty-one starch biosynthesis genes. Eight variants were discovered in JE0146: five were missense mutations; two were synonymous mutations; and the remaining one was a stop_gained variant. JE0296 had five variants that consist of three synonymous and two missense mutations. Similarly, JE0244 and JE0213 both had two variants .AMY and ISA1 in the high resistant starch mutant line JE0146 may be due to missense mutations in these genes. Similarly, there was a stop_gained mutation for PHO2 and it also showed overexpression. SBEIII had a missense mutation with reduced expression. These mutations could be responsible for the high resistant starch in JE0146. JE0296 had two missense mutations in the AMY and SSII genes; however, their expression was inconsistent. Thus, we can say that there may be some other starch-related genes that may be responsible for high resistant starch content in this line, and that change the expression of other genes. In the low resistant starch mutant line JE0244, there was one missense mutation in SBE1, with inconsistent expression; wherein, in the early stages, there was reduced expression and later, overexpression. The overexpression of SBE genes helps to reduce the resistant starch. There was also one conservative_inframe mutation in SSII and gene expression increased in the mutant line JE0244. Similarly, there were two mutations in the known 21 genes of JE0213; in which one was a 5_prime_UTR mutation in SBEI and due to this, mutation expression was inconsistent. The second mutation was a disruptive_inframe mutation in the SSII gene and the expression was reduced.The overexpression of Wheat is the major staple crop and it is the main source of cereal-based products. The modification of starch composition in wheat by increasing the resistant starch content helps to improve the quality and yield of crops . There hDifferent pieces of published literature have shown that the resistant starch contents were in the range from 1% to 18% in different wheat mutants . In thisGBSSI in the present study supports the previous results that may be the accumulation of high amylose; this is because GBSSI plays an important role by elongating the a-amylase chain, which ultimately increases the resistant starch content [GBSSI in wheat and rice; while the silence of this gene or null mutants produced waxy and partial waxy wheat with a low amount of resistant starch [SSIII helps to increase the resistant starch content [ISA significantly increased the amylopectin content in rice [SBEII silencing helped to increase the amylose content in wheat, which ultimately increased the resistant starch content [The expression analysis of 21 starch metabolic genes was conducted by including genes responsible for changing the resistant starch of four altered resistant starch mutants and a parent variety (J411) at four different stages of grain formation . The ove content . Amyloset starch ,36,37. D content . Similar in rice . SBEII s content . TherefoAMY and BMY) with starch phosphorylases  plays an important role in starch metabolism with degradation and hydrolysis [AMY and BMY overexpression also altered the starch structure and starch granules. Morphology, with changing the structure of starch granules, affects the baking quality of wheat [SSI also plays important role in the increment of resistant starch. In wheat and rice, losing the function of SSI helped to increase the resistant starch as well as amylose content [SBEII decreased the amylose content in potato and increased the amylopectin content [RSR1) was identified [Amylase  and digestible starch (DS) of 150 mutant lines were measured by using a commercial Megazyme kit  following the manufacturer’s protocol; this being the detailed protocol followed by McClary et al. . Three bΔA = absorbance of sample solution read against reagent blank.F = factor to convert absorbance values to mg glucose.EV = sample extraction volume (10.3 mL or 100 mL).0.1 = volume of sample analysed.1000 = conversion from mg to mg.100/W = conversion to 100 mg sample.W = sample weight in mg.162/180 = factor to convert from free glucose, as determined, to anhydroglucose, as occurs in starch.The total starch content was determined using the Megazyme International Starch Assay Kit (K-STAR) as per the manufacturer’s protocol . Three bΔA = absorbance of sample solution read against reagent blank.F = factor to convert absorbance values to μg glucose .EV = sample extraction volume (10.2 mL for the procedure).0.1 = volume of sample analysed.D = further dilution of sample solution.1/1000 = conversion from μg to mg.100/W = conversion to 100 mg sample; W: sample weight in mg.A grain analyzer was used to measure the protein, gluten content, and hardness of the seed. A FOSS infratecTM 141  grain analyzer was used to measure these traits by using WinISI II v1.50  . All theSamples of seed from each of the mutant lines and WT were collected with three replications for measuring the grain width, grain length, grain area, and thousand-grain weight (TGW). SmartGrain was used for evaluating these traits by following the methodology described in previous studies . TGW wasSeed starch purification was conducted by following the method described by Hawkin et al. . To studSelected mutants were used to capture the genetic diversity. The four mutant pools were subjected to exon capture and sequencing to compare the polymorphisms between different extreme pools. The process of exon sequencing was followed as described by King et al. . First, Actin (GenBank accession no: AAW78915), a housekeeping gene, was used as an internal control. We followed the method of Singh et al. [Twenty-one starch metabolic genes were used to perform quantitative expression analysis at four seed development stages  in two low resistant mutant lines (JE0244 and JE0213) and two high resistant mutant lines (JE0146 and JE0296), and the parent wheat variety ‘J411’. The samples were collected from the selected line at 6, 12, 18, and 24 days after anthesis and stored quickly in liquid nitrogen; they were then stored at -80 degrees centigrade for the extraction of RNA. The detailed protocol for RNA extraction and cDNA formation used in this study followed that of Singh et al. . Primer h et al. . The ΔΔCMicrosoft Excel formulas were used to calculate the mean value and standard deviation. In order to study the variations in resistant starch and other traits, a one-way analysis of variance (ANOVA) was used. To find the significant difference between the mutant lines and parent variety for all the traits, the Dunnet test was used. For the purpose of these analyses, three biological replications were used for studying all of these traits.In this study, 150 mutant lines were used that showed variations in resistant starch content; this useful germplasm can be used for a genome-wide study and help to improve the starch-based nutritional quality in wheat. This study indicates that low resistant starch accumulation in mutant lines may have resulted from the reduced expression of key genes for resistant starch and starch biosynthesis. The differential gene expression analysis in the low and high resistant starch mutant lines in comparison to the parent variety supports the involvement of other starch metabolic pathway genes; these include phosphorylases, isoamylases, etc. in resistant starch biosynthesis. There is a need to find out about starch metabolic genes through the backcrossing of these mutant lines and a developed NILs population for mapping.


CCT decreased from a pre-operative 742 ± 118, to 546 ± 87 and 512 ± 67 at three and six months, respectively (p < 0.01). ECD decreased from 2878 ± 419 cells/mm2 to 1153 ± 466 cells/mm2 at three and six months, respectively (p < 0.01). The “Wave maneuver” may be very beneficial in DMEK cases where the AC is either very deep or the bottom of the AC is compromised. The “Wave maneuver” learning curve was brief.A novel technique for Descemet membrane endothelial keratoplasty (DMEK) graft handling and centration without the endothelium touching the posterior part of the anterior chamber (AC), is presented here. It is particularly suitable for vitrectomized eyes, deep AC, and AC intraocular lenses (ACIOLs), potentially reducing surgery time and endothelial cell loss during surgery. This retrospective interventional case series includes 27 eyes with complex ocular pathology. All utilized a “Wave maneuver” to center an early elevated graft without completing graft centration on the bottom of the AC. Successful graft attachment and centration were evaluated intra and post-operatively. Best-corrected visual acuity (BCVA), central corneal thickness (CCT), and donor endothelial cell density (ECD) were measured pre-operatively, and three and six months post-operatively. DMEK grafts were successfully attached and centered in all cases. No maneuver-related complications were observed intraoperatively. BCVA improved from a pre-operative 0.2 ± 0.63, to 0.43 ± 0.49 and 0.76 ± 0.51 at the three- and six-month follow-ups, respectively ( Descemet membrane endothelial keratoplasty (DMEK) is currently the first-line procedure for many corneal surgeons to treat corneal endothelial dysfunction . The proHowever, technical challenges such as careful graft preparation and meticulous graft orientation techniques demand a steep learning curve, preventing larger, more widespread DMEK adoption. Various modifications have been introduced, gradually improving surgical technique or donor preparation to succeed in challenging situations ,7,8.DMEK is a preferred and predictable procedure in most cases for eyes with Fuchs endothelial dystrophy (FECD) ,12,13,14To overcome these difficulties, this maneuver is presented to reduce graft contact and manipulation over the ‘bottom’ of the AC.This study followed the tenets of the Helsinki Declaration. The Hadassah Medical Center Institutional Review Board (IRB) number 0418-22HMO approval was obtained for this study, and all procedures were carried out per their guidelines.A retrospective review of the medical records of 27 patients who underwent a challenging DMEK surgery with the help of the “Wave maneuver” due to corneal endothelial dysfunction between 2018–2021. The surgeries took place at the Department of Ophthalmology, Hadassah Medical Center, Jerusalem, Israel, by two corneal surgeons (I.L. and E.N.N.). Surgical notes and recordings were reviewed for intraoperative complications, and the medical files were reviewed for post-operative follow-up. Post-operative follow-ups were performed at 1–3 days, one week, one month, three months, and six months after the procedure. Corneas were assessed for clearance using the slit lamp. Corneal graft attachment and central corneal thickness (CCT) were measured with Casia II anterior segment optical coherence tomography .Endothelial cell count (ECC) was measured with Konan CellChek specular microscopy . Best-corrected visual acuity (BCVA) was measured with a Snellen chart.Graft preparation was performed using a Sinskey hook previously described  ,30, dyedA small air bubble is injected underneath the graft to elevate it. The graft is faced against the posterior corneal surface in the correct orientation, placed in the mid periphery towards the angle to ‘lock’ it so it will not move. At that point, an air bubble is placed under the graft, and several peripheral taps are made using the bubble bumping technique to open the graft as much as possible. As soon as the graft is partly open, a small air bubble is left under the graft, the anterior chamber is deepened with Balanced Salt Solution, and swiping fast taps on the cornea towards the area of the desired movement, the graft is moved on the posterior corneal surface with mostly posterior stroma Descemet membrane touch.Patients were treated with dexamethasone 0.1%, ofloxacin, and diclofenac eye drops in the post-operative period. Ofloxacin eye drops were discontinued after five days, diclofenac eye drops were discontinued after 1–3 months (according to macular OCT), and dexamethasone eyedrops tapered down during the six months of follow-up.t-tests and with one-way analysis of variance (ANOVA) tests.The statistical analysis between pre-operative and post-operative patients after three and six months with the DMEK “Wave maneuver” was performed using the Statistical Package for Social Sciences software 25.0  with two-tailed During the study period, 27 eyes of 27 patients aged 22 to 90 were operated on using the DMEK “Wave maneuver” . The ocup < 0.01).Concerning visual acuity, 84% of eyes reached a BCVA of 0.50 (20/40) or better, six months after the “Wave maneuver” DMEK surgery. Increased BCVA was measured in 36%, achieving 1.0 (20/20) or better . The patp < 0.01)  .p < 0.01), respectively  decreased from a pre-operative 742 ± 118 to 546 ± 87 and 512 ± 67 at three and six months  decreased (from a pre-operative DMEK “Wave maneuver” operation) in measurement count of 2878 ± 419 cells/mmThis series presents a novel method of performing the manipulation and placement of grafts during DMEK surgery in challenging cases. It has been called the “Wave maneuver” due to the wave-like appearance of the graft unfolding as the surgeon gently utilizes an air bubble to maneuver the graft. This maneuver aided the successful outcome of these patients, providing a statistically significant improvement in visual acuity, a statistically significant decrease in corneal thickness, and acceptable endothelial cell density at six months post-operation.The primary complications of DMEK are partial or total graft detachment , which sAs with other endothelial keratoplasty procedures, using air or gas in the AC is fundamental to sutureless adherence of the donor graft to the host cornea ,10. StudDMEK graft unfolding often relies on a shallow and stable AC ,36. Due Despite the many advantages of DMEK, technical difficulties in graft insertion and unfolding led to other surgical techniques. The “Wave maneuver” was developed to circumvent the “floor” problem of the anterior segment in particularly challenging eyes. While it is preferred to open the graft on the iris before elevating it onto the posterior cornea with an air bubble, the patients described here all lacked the support required due to vitrectomy (the graft could slip through the pupil posteriorly) or an AC too deep, or the risk of an AC IOL touching the endothelial cells and damaging the graft. There is a risk when moving or opening the graft in patients with synechiae. While many surgeons prefer to divert to DSAEK in these cases, the graft size is limited, and the outcome preference still lies with DMEK with the advantage of the ability to exchange almost the entire corneal endothelial layer. In this maneuver, the graft is raised at the beginning of the procedure and dragged from the Descemet’s side, not the endothelial side, avoiding cell loss.Similarly, other surgeons have developed techniques to open or position the graft. A study described a modified double-bubble technique in DMEK for vitrectomized eyes . It was A slightly more complicated yet effective technique was devised whereby a corneal graft with a tail was created. A 3 mm pedicle was used to orient and drag the graft into the anterior chamber, after which the tail was extravasated. The graft was unrolled and centered using the pedicle, and gas was injected to planate the graft .Another technique is called EndoGlide-DMEK ,7. Once Most techniques described in the literature either aim to facilitate centration and movement of the graft, or help unscroll it. This maneuver achieves both objectives, and is implemented from the insertion of the graft, not after centration.The average six-month endothelial count did decrease compared with pre-surgery, and a repeat DMEK procedure may be required at some point. Various risk factors for endothelial decompensation include, among other things, donor status, graft size and recipient factors such as glaucoma, or glaucoma surgery . That beThere are limitations to this study. A longer follow-up period would further establish the efficacy of the “Wave maneuver”. The ages and ocular histories of the patients are very heterogeneous. The stability of refractive error and possible fluctuations in astigmatism were not monitored, nor was the corneal tomography.To conclude, presented here is a valuable technique to assist during DMEK surgeries in challenging cases to minimize graft-AC bottom contact, which aids in maintaining control over the graft and helps preserve endothelial cells.


To understand the effect of gluten on starch digestion characteristics, the structural characteristics of protein, starch, and starch digestion attributes were explored by using flours of four wheat near-isogenic lines. Protein and starch fractions from the four flours were used to form so-called recombinant flours where glutenin and gliadin protein fractions, in different ratios, were combined with starch and heated in a water slurry at 80 °C for 5 min. We found that starch digestibility of the recombinant flours could be reproducibly modified by altering the long- and short-range molecular order of starch through varying the attributes of the gluten protein by virtue of the gluten strength as well as the proportions of glutenin and gliadins. The gluten composition changes of strong-gluten flour did not improve the starch digestion resistibility, however, for the moderate- and weak-gluten flours, the proportional increase of glutenin improved the resistance of starch to digestion through the increased long- and short-range molecular order of starch. The resistance of starch to digestion could also be enhanced with increasing gliadin, and was associated with the modified short-range molecular order of starch. We propose that flour mixtures can be optimized for specified product quality by manipulating the amounts of both gliadin and glutenin. Starch, as the principal storage polysaccharides in most plant-derived foods, is an important component of nutritional products that provides energy for humans and animals ,2. CookeStarch digestion is not only related to its intrinsic structure, such as the size and shape of the starch granules, the proportion of amylose and amylopectin, the chain length distribution and the crystallinity of starch , but alsGlutenin and gliadin, as the main components of the gluten network in wheat-based food products, have been proven to affect starch digestion properties in food matrix through different mechanisms due to their differences in molecular weight and structural characteristics. Glutenin elasticity forms the functional basis of the gluten network, and acts as a physical barrier to retard the access/binding between starch and enzymes . MoreoveIt is well known that a high proportion of glutenin in wheat gluten contributes to a stronger gluten network, while the high percentage of monomeric gliadin leads to a weaker gluten with better extensibility ,9. As meGlu-D1 were created by crossing Chinese winter wheat cultivar Xiaoyan-22  and common French wheat cultivar 738 . Based on the continuous backcross and self-crossing after the initial cross, four BC7F5 NILs designated as 2001 (N/7+9/5+10), 2002 (N/7+9/2+12), 2003 (N/7+8/2+12), and 2006 (N/14+15/5+10) were developed for the separation of protein and starch. The time to forming a dough complex of the 2001, 2002, and 2003 flour samples was measured by Mixolab  as 3.33 ± 0.18 a min, 1.74 ± 0.08 b min, 1.19 ± 0.01 c min, respectively. Therefore, we defined the flour from 2001, 2002, and 2003 as strong-strength flour, moderate-strength flour and weak-strength flour, respectively. Seeds of the four wheat NILs were milled into flour by a Brabender Quadrumat Senior mill using the AACC method 26–20.01 [As described in our previous paper , wheat Nd 2006 N/+15/5+10 Wheat flour and distilled water were mixed to form a dough and kept at room temperature for 30 min. The dough was then rinsed with distilled water until the water was clear to get the gluten. Starch was obtained by centrifugation of the collected starch water .w/v), followed by stirring magnetically for 4 h at room temperature to fully dissolve gliadin. After centrifugation at 4000 rpm for 20 min, the collected supernatant was evaporated using a rotary evaporators  to remove most of the ethanol, and the residue was placed in an oven at 50 °C until completely dry to obtain gliadin-rich fractions. The centrifuged precipitate, after gliadin extraction, was dried to obtain glutenin-rich fractions [In order to isolate gliadin from gluten, 200 g of the gluten fractions were extracted from the 2001, 2002, or 2003 flour samples by resuspending in with 70% aqueous ethanol at a ratio of 1:15 (ractions . The purw/v) and stirred at room temperature for 20 min, immersing in boiling water on a magnetic stirrer for 5 min  before it was transferred to a constant temperature magnetic stirrer at 37 °C for 15 min. Finally, the sample was lyophilized, powdered, and sieved through 200 mesh.Firstly, the gluten separated from 2001, 2002, or 2003 was mixed with starch from 2006 in the ratio of 1:5 to prepare three recombinant flours named 200115, 200215, and 200315. Secondly, the glutenin and gliadin from the gluten of each NIL were blended in the ratios of 3:1 and 1:3 to prepare two reconstituted gluten proteins, and six recoconstituted gluten proteins were obtained using the three NILs. Finally, each of the recomposed gluten protein was mixed with 2006 starch in the ratio of 1:5 to develop six recombinant flours named 200131, 200113, 200231, 200213, 200331, and 200313, respectively. The formulations of the nine recombinant flours are provided in The crystalline properties of the recombinant flours were characterized by an X-ray diffractometer  and the test parameters were set at 40 mA and 40 KV with Cu Kα radiation (λ = 1.5406 Å) . The X-rw/w), dried and ground until the mixture was homogeneous. The spectra were obtained using a scanning band between 400 and 4000 cm−1 at a resolution of 4 cm−1. An empty cell was used as the background [−1) [−1) band.FTIR spectra of the samples were measured using a Vertex 70 FTIR spectrometer  equipped with the deuterated triglycine sulfate detector. All samples were mixed with potassium bromide in a ratio of 1:100 (ckground . Accordiund [−1) . Omnic  equipped with CP-MAS accessories. Dipolar decoupling was systematically used during the acquisition sequence. The recombinant flours sample of 200 mg was scanned at a frequency of 125.7 MHz. The optimal contact time was 13 ms, spectral width 40 kHz, Scan rate 20 Hz, Scan number 4096. PeakFit™  was used to fit characteristic peaks in the spectra the content of double helix was defined as the ratio of fitting peak area of C2,3,5 (70–75 ppm) to the total fitting peak area, the single helix proportion was defined as the ratio of the fitting peak area of V-type characteristic peak (94 ppm) to the total fitting peak area, and the content of the amorphous phase (PPA) was defined as the ratio of the fitting peak area of C4 (80–84 ppm) to the total area [The solid-state −1 with a resolution of approximately 7 cm−1 was obtained. The full width at half maximum (FWHM) of the band at 480 cm−1, which represented the short-range molecular order of starch, was determined by using the WIRE 2.0 software [The short-range molecular orders of recombinant flours were obtained by using a Laser Raman spectrometer with a 785 nm green diode laser source . Raman spectroscopy of each sample in the range of 100–3200 cmsoftware .g for 20 min at room temperature. The protein concentration in supernatants was determined by the Coomassie bright blue method. The proportion of ionic bonds was determined by the content difference between proteins extracted by SB and SA, hydrogen bonds was quantified by the content difference of proteins extracted by SC and SB, hydrophobic interactions was determined by the proteins dissolved in SD and SC, and disulfide bonds was quantified according to the content difference of proteins dissolved in SE and SD.The molecular force between gluten molecules was evaluated by the solubility of the protein in different denaturation solvents using the method described by Gómez-Guillén with some modification . The solDissolve recombinant powders (50 mg) in 15 mL PBS . Sample solutions were placed in a cuvette under temperature control at 25–50 °C. The corresponding steady-state fluorescence spectra of the samples were obtained by a fluorescence spectrometer  upon 295 nm excitation at a spectral resolution of 2.5 nm, and emission spectrograms between 300 and 400 nm were acquired at a rate of 100 nm/min .According to previous research with minor modifications , the app5 U/mL, A107823—10 mL, Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China) were transferred into each reaction flask and incubated in a 37 °C water bath for 180 min with constant shaking (120 rpm). Hydrolyzed solution (1 mL) was withdrawn at specific time points , then boiled for 5 min to stop the enzyme reaction. The solution was centrifuged at 4000 rpm for 10 min and, after proper dilution, the glucose concentration in supernatant was measured by a biosensor  after calibrating with a standard glucose solution of 1 mg/mL. Digestibility and contents of rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) were calculated by the following formulas:TS is the total starch quality, mg; NG is the content of native glucose in starch; G20 and G120 are the glucose content respectively released within 20 min and 120 min of hydrolysis, mg.In vitro digestibility of recombinant flours was tested according to an existed method with a slight modification . SamplesAll tests were performed in triplets unless stated otherwise, and the results were presented as mean ± standard deviation. The one way analysis of variance (ANOVA) was processed by SPSS statistical software  with the Duncan’s test at a significance level of 0.05. A correlation analysis between starch digestibility and the structural characteristics of protein and starch was performed using an Originpro Learning Edition. The Spearman correlation coefficient was employed to represent the degree of each correlation.Glu-B1 had little if any effect on starch digestion. For the 2001 samples, the proportional increase of glutenin in gluten protein (200131) had no significant effect on the content of RDS, SDS, and RS, however, a significant content decrease in RS and increase in RDS were found in the gluten containing more gliadin (200113). This difference in 2001 samples indicated that the barrier effect of strong-strength gluten protein on starch granules was weakened with the increasing proportion of gliadin, and the inhibiting effect of macromolecular protein network on starch digestibility was greater than that of monomeric protein. For 2002 samples, part of the RDS was converted to RS after the increase of glutenin (200231), while part of SDS was converted to RS after the increase of gliadin (200213). The difference between 200231 and 200213 indicated that for moderate-strength gluten protein, the enhanced coating effect of the stronger gluten network mainly inhibited the rapid digestion of starch, while the additional monomeric proteins were more prone to interacting with amylase or starch and reducing the amount of SDS. Although there was no difference in RS content between 200231 and 200213, more SDS and less RDS were detected in 200231 than those in 200213, which indicated that for the flour with moderate-strength gluten, compared with the proportional increase of monomeric gliadins, the improved strength of gluten containing more glutenins was more conducive to reducing the starch digestibility. For 2003 samples, part of RDS and SDS were converted into RS after the increase of glutenin, and part of RDS was converted to SDS with the increase of gliadin, which indicated that for the flour with weak-strength gluten, the enhancement of gluten strength through quantitative increase of glutenin was more effective to inhibit starch digestion than the proportional increase of monomeric gliadins. In brief, for the flour with moderate or weak-gluten strength, the proportional increase in glutenin and gliadin significantly decreased the starch digestibility. The inhibition effect of an enhanced macromolecular gluten network on starch digestion was stronger than that of gluten protein with more monomeric proteins. However, for the flour with high-strength gluten protein, further enhancement of macromolecular gluten network or incorporation of additional monomeric gliadins did not improve the resistance of starch to digestion.The in vitro digestion curve of all recombinant flours showed faster digestion rates of starch within the first 30 min . CompariThe maximum fluorescence emission of proteins (about 337 nm) varies with the polarity of tryptophan residues in the environment . As showAccording to a previous study , the polFor the three wheat NILs, significant differences in the contents of α--helix and β-sheet under different glutenin-gliadin ratios were found . QuantitFor the recombinant flours containing natural gluten protein and starch, 200115 had the optimal β-sheet/α-helix compared to 200215 and 200315, which contributed to the formation of more compact and stable gluten network that inhibited the enzymatic hydrolysis of starch and produced more RS and less RDS B. For thAs a relatively stable covalent bonds, disulfide bonds play an important role in stabilizing the spatial structure of the peptide chain. As shown in Hydrophobic interactions are the main driving force of protein folding. For the three NILs, the intensity of hydrophobic interaction was increased to varying degrees with the increase of glutenin content, indicating that higher glutenin content could promote the aggregation of hydrophobic residues and stabilize the structure of protein networks. For 2001 samples, the hydrophobic interaction was always weaker than most of the 2002 and 2003 samples, which may be attributed to the stronger protein-starch interaction in 2001 samples that hindered the aggregation of hydrophobic groups in protein. This prediction was also confirmed by the significantly higher hydrogen bond content in 2001 samples . NotablyThe recombinant flours directly prepared by natural gluten and starch showed much higher content of hydrogen bonds compared to the flours with increased proportion of glutenin or gliadin, this difference may be due to the fact that some original hydrogen bonds existing in the gluten extractions were broken in the separation of glutenin and gliadin. Furthermore, when the proportion of glutenin or gliadin was increased, the hydrogen bond content decreased to different degrees and the lower content of hydrogen bond was accompanied by the higher content of gliadin. This phenomenon indicated that the incorporation of additional glutenin or gliadin led to the rearrangement of original gluten structure. As for the gluten with increased content of glutenin, the decreased hydrogen bond was accompanied by the significant increases in both hydrophobic interactions and disulfide bonds, resulting in a more extensive and stable gluten network, however, for the gluten with increased gliadins, significant decreases in disulfide bonds and hydrogen bonds indicated the destruction of gluten structure. The content of ionic bonds in gluten protein was significantly lower compared with the other chemical bonds. Moreover, regardless of the composition of gluten protein, the flours containing 5 + 10 subunits showed relatively high ionic bonds, which was consistent with our previous study .X-ray diffraction  showed t−1 was correlated with the presence of starch-protein complex, the peaks at 1714 cm−1 and 2854 cm−1 indicated the existence of lipid-starch complex, and the simultaneous appearance of the three peaks indicated the presence of starch-lipid-protein complexes in the samples [−1, indicating the formation of starch-protein complexes. No obvious peak was observed at 2854 cm−1 and 1745 cm−1, which possibly indicated that the low content of lipid in wheat flour and the further lipid loss during separating of gluten proteins and starch reduced the possibility for forming starch-lipid or starch-lipid-protein complexes. Compared with other flours, more obvious peaks at 1538 cm−1 were observed in 200115, 200215, 200315, 200113, and 200231, in view of their lower LMP contents  and the degree of double helix (DD) of starch can be characterized by 1047/1022 and 995/1022, respectively [−1 of LCM-Raman spectrum was also used to characterize the short-range molecular order of starch [In the FTIR spectrum, the absorbance at 995 cmectively . Moreovef starch . As show−1 that were sensitive to the anomeric structure around glycosidic bonds [Besides the non-covalent bonding of protein to starch, the covalent interaction between protein and starch was detected according to the shifts of the bands at ~860 cmic bonds . The peaNMR can be used to assess helix content in starch . The wid−1 peak from FTIR, we inferred that the covalent protein-starch complex accounted for a high proportion of the single helix in the recombinant flours containing natural gluten. In contrast, more non-covalent protein/lipid-starch complexes may be involved in the high content of single helix in 200231 and 200331. For the recombinant flours with increased gliadin, there were still more starch fragments linked to the protein . Improvement of the short-range molecular order  of starch, ultimately showed an insignificant influence on starch digestive characteristics. We note that when the proportion of gliadin increased, the short-range order of starch  was improved, although the relative crystallinity of starch decreased significantly due to the reduced strength and stability of gluten network, which ultimately promoted the conversion of RS to RDS. For the flours with moderate-strength gluten, the increase of glutenin content improved the degree of polymerization within the gluten network, which increased the relative crystallinity and content of single/double helix in starch and converted some RDS into RS. However, the proportional increase of gliadin led to the increased content of double helix in starch although the long-and short-range molecular order of starch was decreased. This change promoted the conversion from SDS to RS. For the flours with weak-strength gluten, the increase of glutenin content made up for the lack of gluten network, thus improving the relative crystallinity and single helix content of starch and facilitating the conversion of RDS and SDS to RS. In contrast, the increase of gliadin content decreased the gluten compactness, but led to more glycosidic linkages between monomeric gliadin and starch although both the long- and short-range molecular order of starch were decreased, which ultimately resulted in the partial transformation of RDS into SDS. Generally, the glutenin in gluten mainly affected the long-range order of starch, while the short-range order of starch was affected by both glutenin and gliadin. This study has clarified the mechanism by which the composition and strength of gluten protein can affect starch digestibility and provide a model for developing consumable low-GI foods at a broader level.