Oxidative and vascular extra-renal damage were, in contrast, determined by the recipient. Post- versus pre-Tx evaluation of grafts showed an increase in glomerular and peritubular capillary rarefaction in healthy but not CKD grafts within a CKD environment. Proliferation of glomerular endothelium was similar in all groups, and influx of eGFP+ recipient-derived cells occurred irrespective of graft or recipient status. Glomerular and peritubular capillary rarefaction, severity of inflammation and macrophage subtype data post-Tx were, however, determined by more complicated effects, warranting further study. Our experimental model could help to further distinguish graft from recipient environment effects, leading to new strategies to improve graft survival of suboptimal Tx kidneys.Kidney transplantation (Tx) is considered the only definite treatment for end-stage kidney disease (ESKD) patients. The increasing prevalence of ESKD has necessitated the introduction of transplantation with kidneys from suboptimal donors. There is, however, still a lack of fundamental and longitudinal research on suboptimal kidney transplants. Specifically, there is a demand for accurate pre-Tx predictors of donor kidney function and injury to predict post-Tx outcome. In the present study, we combine rat models of chronic kidney disease (CKD) and renal Tx to dissect the effects of healthy and CKD renal grafts on healthy and CKD recipients. We show that renal function at 6 weeks post-Tx is exclusively determined by donor graft quality. Using cell tracking within enhanced green fluorescent protein-positive (eGFPThis article has an associated First Person interview with the first author of the paper. Summary: Using experimental kidney transplantation, we dissected donor graft from recipient environment effects, focusing on the endothelium and inflammation. These results can direct strategies to improve graft survival after suboptimal transplantation. Kidney transplantation (Tx) is considered the only definite treatment option for patients with end-stage kidney disease (ESKD). Graft function and survival in kidney transplant patients have improved over time, especially by limiting acute and late acute rejection events . HoweverIsogenic experimental Tx allows researchers to dissect the influence of the CKD recipient environment from graft function in an immunocompetent context. The symmetrical bilateral ablation (BA) rat model we introduced  can be uThe primary aims of this study were to establish differences in graft function/structure in general, with a particular focus on glomerular and peritubular capillary rarefaction and the presence of inflammation in three outcomes: function of suboptimal (CKD) versus optimal  donor grafts in CKD versus healthy systemic environments (cross-Tx), structure of post-cross-Tx grafts versus pre-cross-Tx biopsies, and effects of recipient-derived cells in cross-Tx. Based on previous observations, we had three hypotheses: (1) long-term graft function and injury are determined by interaction between graft quality and recipient environment at the time of Tx, (2) suboptimal donors lead to further decrease in graft function and injury post-Tx, and (3) incorporation of recipient-derived cells is not determined by the status of the recipient environment.+) Lewis rats ; CD-HR, CKD (C) Lewis donor (D) and healthy (H) eGFP+ recipient (R); HD-CR, healthy (H) Lewis donor (D) and CKD (C) eGFP+ recipient (R); CD-CR, CKD (C) Lewis donor (D) and CKD (C) eGFP+ recipient (R) (n=6/group) (Tables S1 and S2).Our experimental cross-Tx design was as follows: HD-HR, healthy (H) Lewis donor (D) and healthy (H) eGFPn=24) that underwent bilateral ablation (BA), on average, had higher systolic blood pressure (SBP) , proteinuria  and plasma urea  than all control rats (n=24). Pre-Tx data for all four donor and recipient combinations ∼1 week prior to surgery are shown in Table S3.At week 1 (baseline) before donation/Tx, all rats (P<0.05) in our cross-Tx set-up. The SBP of the recipient was influenced by the transplanted kidney at week 5 : SBP in the CR was lower with a healthy donor (HD) than with a CKD donor (CD). The HR had a higher SBP with a CD than with an HD (both P<0.05). A comparable pattern was observed for urea and proteinuria. HD-CR rats had lower urea and proteinuria compared with CD-CR, but higher proteinuria compared with HD-HR, at weeks 3 and 5 . CD-HR rats had higher urea and proteinuria compared with HD-HR and CD-CR at weeks 3 and 5 . The lower urea in the CD-CR rats could have been caused by less chow intake than the CD-HR rats, but this was not recorded.Increased body weight was observed in the CKD recipients (CR) compared with the healthy recipients (HR), corresponding to the age difference  that died, possibly of heart failure, during the terminal experiment. This rat had no cardiac output immediately after being anesthetized and could not be resuscitated. Postmortem, we observed a markedly dilated heart with multiple fibrotic scars in the myocardium.Fig. S2). A comparable pattern was observed for renal hemodynamics  and fractional excretions of sodium and potassium .Anemia (decreased hematocrit) and decreased renal function (increased plasma urea and decreased GFR measured by inulin clearance) at termination confirmed marked impairment after ablation. This was exclusively determined by donor status: recipient environment did not influence outcome  ; Fig. S2P<0.05 for CR versus HR; P<0.05 for CR versus HR; Thiobarbituric acid reactive substance (TBARS) excretion was exclusively determined by recipient environment and was higher in the CR versus HR, irrespective of the transplanted kidney .Representative histology [periodic acid Schiff (PAS) staining] and immunohistochemistry [aminopeptidase P (JG12) and ED1 ] pre- and post-Tx of a HD-CR combination  is shown in +) post- versus pre-Tx, showed marked decreases in HD-CR (P<0.05), and in CKD versus healthy donor grafts in a healthy recipient environment (P<0.05). Donor and recipient environment effects on endothelium were more pronounced in glomeruli compared with peritubular capillaries. The strongest donor-recipient interaction effect was observed for glomerular endothelial area relative to glomerular area . Note that the glomerular area size distribution within CD kidneys was much broader than in HD kidneys .Comparisons of renal peritubular capillary and glomerular rarefaction (JG12in HD-CR A-F endotin HD-CR K. Post-T+) were significantly more evident in post- versus pre-Tx in CD-HR tubular fields (+ cells per tubular field and glomerulus in CD-HR compared with CD-CR (P<0.05) and in HD-CR compared with CD-CR (P<0.05). ED1 inflammation was primarily determined by donor status in tubular fields and by donor and donor-recipient interaction in glomeruli.Signs of inflammation . HD-CR also had significantly increased tubular expression of CD163 compared with HD-HR (P<0.001). For iNOS, and M1 marker, both CD versus HD and CR versus HR increased glomerular expression. Here, either donor or recipient environment status, but not donor-recipient interaction, determined the outcome .Next, we used inducible nitric oxide synthase (iNOS) and CD163  immunohistochemistry to determine relative contributions of M1 and M2 macrophages (MQs) post-Tx. For CD163, an M2 marker, CD versus HD in a HR increased the number of positive cells per glomerulus and tubular field  immunofluorescent staining. No significant differences were found in kidneys of the recipients for eGFP+ cells/glomerulus or eGFP+ area/tubular field (. Peritubular expression of JG12/Ki67 (P<0.05) and was higher in the CKD donors than in healthy donors in the CR. However, glomerular JG12+/Ki67+ . The presence of eGFP−/ED1+ cells per tubular field was exclusively determined by donor status (P=0.019), being higher in CD independent of recipient status. The number of eGFP−/ED1+ cells per glomerulus was influenced by donor-recipient interaction  and glomerular ] or vice-versa [tubulo-interstitial  and glomerular ] .Using eGFP/ED1 double staining, the relative contribution of recipient environment and graft to eGFPironment A-E. In geraction L, althou+ recipients in combination with cross-transplantation, allowing identification of impact of recipient environment. Using this model, we could investigate three different conditions: (1) comparing optimal  versus suboptimal (CKD) donor grafts in healthy and CKD systemic environments (cross-Tx), (2) comparing post-cross-Tx grafts versus pre-cross-Tx biopsies, and (3) exploring the effects of recipient-derived cells in cross-Tx. Our study was descriptive in the sense that there was no donor and/or recipient treatment prior to surgery.The primary aim of this study was to establish differences in graft function and structure, with particular attention to peritubular and glomerular capillary rarefaction and inflammation. This could only be studied in the context of our CKD/Tx model that combines two unique features : symmetr+ inflammation in the tubulo-interstitium.For the first time, our symmetrical study design shows that post-Tx graft function is solely determined by donor status. Despite significant improvement of SBP, proteinuria and urea when comparing healthy with CKD donors in CKD recipients, recipient environment or interaction effects between recipient and donor did not affect renal function. Suboptimal donors in both healthy and CKD recipient environments had significantly lower kidney clearance function – as measured by GFR – than healthy donors. Donor status also exclusively influenced progression of GS, and increased peritubular capillary rarefaction and injury of general ED1Clustering of vascular calcification, oxidative stress and endothelial dysfunction is characteristic of CKD and might contribute to enhanced mortality . Strikin+  infiltrating MQs post-Tx did, however, correlate with the severity of acute rejection, renal function (decline in eGFR) and long-term allograft function  compared with normal histology (no rejection or IFTA) after Tx . The numfunction . Within + subpopulation of ED1+ cells was highest in the CD-HR. This trend was associated with lack of TI and GS progression, as well as preservation of glomerular and peritubular endothelial area compared with reference kidneys. At this time point, presence of CD163+ MQs could promote anti-inflammation and healing, preventing further tissue damage. The presence of interstitial M2 MQs (CD163+) in kidney allograft protocol biopsies was previously correlated with both interstitial fibrosis and kidney function, thereby introducing an important pro-fibrotic role for these cells in persistent lesions . Endothelial damage can originate from systemic and/or local factors. The association between oxidative stress, inflammation and CKD is well established  capillary rarefaction and general inflammatory status in post-Tx grafts versus pre-Tx (reference kidney). In HD-CR kidneys, GS and TI generally became more severe, glomerular endothelium and peritubular capillaries were lost, there were more glomerular ED1ablished . Althougablished , renal tablished . Immunosablished . As arteablished .We also observed that the CKD kidneys transplanted into CKD recipients do not appear to be additionally damaged as shown by no post- versus pre-Tx difference in the CD-CR group. Vercauteren et al. found that the remnant kidney is less susceptible to ischemia-reperfusion injury (IRI) in comparison to a healthy kidney . In a suProteinuria is an important marker for kidney dysfunction. Patients with moderately reduced GFR without proteinuria have better clinical outcomes compared with patients with heavy proteinuria without abnormal GFR . Loss of+ recipients, we found no recipient effects on graft function or renal damage (see Condition 1). Preservation of glomerular endothelium was not associated with a difference in eGFP+ cell number, increases in cell proliferation, or differences in glomerular endothelial proliferation (JG12/Ki67). This suggests that the incorporation and proliferation of (circulating recipient-derived) cells is not a major factor in preservation and regeneration of glomerular endothelium in CKD. This is consistent with previous observations that renal artery injection of bone marrow cells protected against glomerular damage in experimental CKD, but was not related to incorporation or transdifferentiation of these cells into glomerular endothelium  (T-cell), CD20  (B-cell) or CD209  (dendritic cell) (Regarding recipient-derived eGFPo injury . In bothic cell) .Our study has some limitations. First, we did not perform time- and transplant-matched control testing for effects of the recipient environment. This is due to the chosen model of orthotopic left kidney transplantation: the same procedure for the right kidney is technically impossible. Transplanting two kidneys from one donor into two recipients would have required two microsurgeons to avoid systematically introducing differences in ischemia time between the first and the second kidney. However, to standardize ischemia times, all transplantations were performed by the same microsurgeon (D.A.P.). Second, the difference in median age between donors and recipients and the large variation in the ranges within the groups could be confounders in interpreting the results. These differences arose for logistic reasons because of the duration of the study. It should, however, be noted that there was practically full overlap in these ranges.in vivo or ex vivo treatment prior to transplantation surgery, and recipient cardiovascular status prior to and after transplantation surgery. In the long run, this will help with donor/recipient selection decisions as well as open avenues for novel therapeutic developments.Our study provides a first glimpse into the complex regulation contributing to Tx outcome. Donor, recipient and donor-recipient interaction effects all play important roles in determining the status of renal peritubular and glomerular endothelium and inflammation. Implications of several of our findings regarding dynamics of inflammation and glomerular capillary rarefaction warrant further study in suboptimal versus optimal kidney grafts. A second focus could be on strategies to improve graft quality by In summary, our study stresses the importance of the recipient environment, oxidative stress, endothelial proliferation, inflammation and vascular injury for graft outcome in renal transplantation, even when hypertension abates because of an initially healthy renal graft. It also raises the question of how to improve the recipient environment nonhemodynamically, to maintain long-term renal graft function and structure.n=21). In the transplantation study, Lewis males were used as donors (n=24) and eGFP+ Lewis males  as recipients. Rats were housed in a climate-controlled facility with a 12:12-h light: dark cycle under standard conditions.The study protocol was approved by the Utrecht University Committee on Animal Experiments (DEC number 2012.II.03.053) and conformed with Dutch Law on Laboratory Animal Experiments. Male inbred Lewis rats  were used in a preparatory study (+ recipient (R); CD-HR, CKD (C) Lewis donor (D) and healthy (H) eGFP+ recipient (R); HD-CR, healthy (H) Lewis donor (D) and CKD (C) eGFP+ recipient (R); CD-CR, CKD (C) Lewis donor (D) and CKD (C) eGFP+ recipient (R) (n=6/group). The cross-Tx design is depicted in Tables S1 and S2.The following groups were used: HD-HR, healthy (H) Lewis donor (D) and healthy (H) eGFPn=21 rats), we investigated whether injury was symmetrical in our bilateral model of CKD. Male Lewis rats (8 weeks of age) underwent two-thirds ablation of each kidney in a one-step procedure (BA); controls underwent bilateral sham surgery. Under isoflurane anesthesia, a median laparotomy was performed; arterial branches of both kidneys were carefully isolated under a Leica operation microscope and branches were sequentially coagulated, leaving only one branch/kidney intact, which supplied approximately one-third of that kidney. The area of ischemia was macroscopically checked on both the ventral and dorsal sides of each kidney to ensure that two-thirds of the renal mass of each kidney was infarcted , respectively . Proteinuria, in comparison with BP and urea, was the best predictor for GFR, RPF, TI and GS . In this pilot study, bilateral ablation led to CKD, renal damage (GS and TI) was symmetrical and the best systemic predictor for function and injury was proteinuria. These findings firmly position this new bilateral ablation model in the field of transplantation.LK-GS and LK-TI correlated strongly with RK-GS and RK-TI -nitro-L-arginine (L-NNA), a NO-synthase inhibitor (200 mg/l) in drinking water . Because proteinuria proved to be the best predictor of kidney damage in our preparatory study, all BA rats were screened and, where appropriate, matched with recipients based on proteinuria before entering the transplantation protocol.We used orthotopic left kidney transplantation as described , with suFor the donor procedure, donor rats were placed on an operating table with a heating pad keeping the body temperature at 37°C. A long abdominal incision was made from the sternum to the symphysis. The bowel was moved slightly to the left side, covered with moist gauze. The renal vessels and ureter were dissected carefully using atraumatic technique. Heparin was administered directly into the spleen, and 5 min after the left kidney was perfused with saline, the renal vessels were cut close to their junction to the aorta and vena cava and the ureter, ∼2 cm distal to the kidney hilum. Finally, the donor kidney was kept on ice with a standardized cold ischemia time of 30 min. All isografts were perfused and placed in organ-preserving solution Viaspan  prior to transplantation. The right kidney of the donor rat was preserved in formaldehyde and then embedded in paraffin to evaluate pre-Tx histology (see above).For the recipient procedure, the recipient rats were prepared in the same way as the donor rats except that heparin was not administered. The renal vessels were clamped using separate microvascular clamps and, including the ureter, were cut close to the kidney hilum. The donor kidney was placed and the following anastomoses were performed: end-to-end arterial anastomosis using eight to ten separate stitches; end-to-end venous anastomosis using continuous suture; end-to-end anastomosis of the ureter with four separate stitches. All anastomoses were performed with 10-0 prolene sutures. After completing the anastomoses at a standardized time of warm ischemia of 30 min, the microvascular clamps were released. The immediate patency of anastomosis was checked 20 min after clamp removal. After 10-14 days, the animals were anaesthetized again, the contralateral kidney was removed, the graft vessels were checked for late patency and long-term complications , and the graft ureter anastomosis was checked for hydronephrosis. Only one case of hydronephrosis was observed, and this rat was excluded from the study.We performed tail-cuff SBP registration and collected 24-h urine samples for determination of protein excretion  with the rats in individual metabolic cages while fasting, as described , at weekNa and FEK were calculated using standard formulae. At the end of the terminal protocol, rats were sacrificed by exsanguination via the aorta, perfused with 0.9% NaCl via the aorta at a perfusion pressure 10 mmHg above terminal MAP, and tissues were collected. Organ weights were noted. Furthermore, kidney samples (post-Tx) were fixed in 4% paraformaldehyde for embedding in paraffin, or snap-frozen for histological and immunohistochemical evaluation.Terminal measurements were performed 6 weeks after Tx because one rat from the CD-CR group had to be euthanized as it reached the humane endpoint. Renal function was investigated under isoflurane anesthesia  as described . MAP, GFTI and GS were scored on PAS-stained, paraffin-embedded slides , 1998. S+) area in at least ten tubular fields and 50 glomeruli per kidney was determined using Adobe Photoshop CS5 Extended, version 12.0×32 . JG12+ area was corrected for glomerular area. ED1+ cells , iNOS+ cells  and CD163+ cells  were counted in glomeruli  and nonoverlapping tubular fields . TI score, GS, glomerular area, JG12 staining and ED1+ cell counts were performed in both donor (pre-Tx) and recipient (post-Tx) rats. Proliferative cells  were counted in glomeruli of the recipient's graft kidney (post-Tx). Anti-GFP/RECA  double staining was performed on 5 µm snap-frozen sections in the recipient's graft kidney, post-Tx, as described  was used to count all recipient-derived (eGFP+/ED1+) and donor-derived (eGFP−/ED1+) MQs in post-Tx kidneys. JG12/Ki67 double staining  was used to count proliferation in glomerular endothelium and peritubular capillaries (JG12+/Ki67+) in post-Tx kidneys. Analyses were performed manually or with ImageJ software, version1.46r .Peritubular and glomerular endothelial cells were stained with JG12 . The endothelial  in urine collected prior to termination as described , 2014. Tt-test, two-way ANOVA or two-way repeated measures ANOVA, and Newman-Keuls post hoc test were used where indicated. The two-way analysis allows us to determine donor effects , irrespective of the recipient and vice versa recipient effects , irrespective of the donor, as well as interacting effects of the donor and recipient on graft function and structure. The two-way repeated measures analysis allows us to compare the time-dependent changes in the graft using the contralateral donor kidney as baseline-biopsy (post-Tx versus pre-Tx), time-dependent changes in the recipient for functional variables, and finally interaction between graft and recipient on such time-dependent variables.Data are presented as mean±s.d. Student's


Full-thickness skin graft (FTSG) is a very versatile tool regularly used in small sizes. With the established knowledge of the graft take through the margins as well as wound bed, we extended the use of the FTSG for reconstruction of large-sized defects with satisfactory results. 2. Seven patients had chronic but healthy granulating wounds and 21 patients had fresh raw areas. Sizes of FTSG used varied between 130 to 452 cm2 of outstretched skin at donor sites. We used subgluteal skin crease in most of cases, though groin, upper medial thigh and medial arm aspects were also used.We presented our experience in wound reconstruction using FTSG in 28 patients. We selected cases with graft size equal to or more than 130 cmThere was no difference in graft take between fresh and chronic wound sites. Almost complete graft take was the rule in all cases apart from mild epidermal skin peeling in four cases and small spots of graft necrosis in three cases. Primary wound healing at donor sites was the rule with scar hypertrophy in six cases necessitating scar conservative care for few months.Use of FTSG for reconstructing raw areas in acute and chronic wounds has to be safely reconsidered regardless of wound size. It’s still a sound and reliable tool which can decrease the necessity of complex flap coverage. Subgluteal skin crease is relatively a new donor site to be considered for large grafts with primary direct closure.  The Scottish surgeon John Reissberg Wolfe (1824–1904) perfected the procedure of full-thickness grafting. Wolfe wrote a paper in 1875 contesting many of the accepted ideas regarding graft surgery and describing his method of reconstruction.3,4The donor site is usually hidden and can be easily closed directly. Skin at different parts of the body varies greatly in the terms of texture, color, appearance and thickness. All of these components had to be considered when choosing a donor skin area appropriate to a certain defect. Human skin varies in thickness based on its anatomical location, age, and sex of patients. Male skin is usually thicker than female skin in all anatomical locations. Children have relatively thin skin that progressively thickened until the fourth or fifth decades of life when it begins to thin once more.4 The principal of FTSG graft revascularization through the graft margins only was completely changed. It was proven that mechanisms of FTSG revascularization through the plasmatic imbibition, capillary ingrowth and revascularization could happen at the graft margins and bed as well.5,6 Full-thickness skin graft is often performed for defects of the face including the nose, eye lids, and ears and hands where contraction of the graft should be minimized; it should also be used when matching the skin graft’s color to the normal skin is important.7Some surgeons have bad experience with FTSG, since the graft is thick, it will need long time to heal, and it also has a higher risk of graft failure.8 Tissue expansion can be also practiced to augment the amount of skin harvest at the donor site with the advantage of primary closure preserved.8 Full-thickness skin graft consists of the epidermis and the entire thickness of the dermis containing almost all of the skin appendages which will not only serve as a cover for raw areas, but it will also preserve multitude of the specialized skin functions.Full-thickness skin graft can also be used to reconstruct the penile urethra in one or staged procedures with satisfactory results.In this article we presented our experience with large-sized FTSG (size more than 10×13 cm) for twenty-eight patients. Twelve patients were males and sixteen were females. Ages of patients were ranging from eight years to 74 years, and mean age was 30.8 years. The graft sizes were ranging from 13×10 cm to 25×14 cm with mean size of 18.7×11.7 cm. The patient’s clinical data are summarized in From those donor sites we can harvest skin and easily close primarily without prior tissue expansion in even graft dimensions as up to 25×20 cm from the groin and subgluteal areas, and up to 16×10 cm from inner aspect of arms and supraclavicular areas. These dimensions could sometimes be increased or decreased according to the patient body mass index and degree of skin laxity. We also extensively use the post auricular and distal forearm crease but for a smaller size defect; which was not included in this study.We usually used to meticulously circumvent the subcutaneous fat from the under surface of the graft either primarily during graft harvesting using the knife or after graft harvesting using scissors (defattening). All yellow fat must be trimmed until only the shiny white under surface of the dermis is visible; as fat is poorly vascularized and will prevent firm adherence between graft dermis and the recipient bed. Scissors for defattening have to be sharp to cut and not to crush the dermis to keep viable tissues for graft rapid revascularization. In this study we are highlighting the importance of excision of any residual white dermal remnants left at the donor site. This practice will decrease chances of hypertrophic scar formation in our experience. We always plane and succeed to close the graft donor site primarily with or without limited undermining. Harvesting of subgluteal large size FTSG could be accomplished easily while the patient lying supine, through flexing the hip and knee joints, and widely exposing the subgluteal region for measurements, graft harvesting and primary closure . We usedFull-thickness skin graft recipient site preparation has to be very meticulous. In chronic wounds the covering granulation tissue has to be healthy without debris, necrotic tissues, infections or biogenic membrane. Debridement has to continue until appearance of punctate bleeding through the healthy granulating wound surface, and without exposing bones, tendons, or joints . In chroThe real challenge with the large size FTSG is the good graft inset and immobilization on its bed and to prevent haematoma and seroma formation. In some cases we used to fix the graft using quilting sutures between the graft and its bed to ensure good contact and prevent shearing movements with rigid splitting in certain locations. We are always used to use the tie-over sutures for dressing to fix the graft at the recipient site. A non-adherent layer such as petrolatum-based dressing is necessary to facilitate easy separation of the dressings postoperatively. We not only depend upon the usual absolute heamostasis at the graft bed, but also thorough washing of the graft bed with normal saline by mean of wide-pored cannula throughout the procedure and directly before dressing application, that is to wash up any possible even minor haematoma collection. The first postoperative dressing was usually done after seven days in chronic and acute raw areas. Prophylactic antibiotics were given in pre and postoperative periods according to patients and wounds flora monitoring protocol; in cooperation with the infection control department. With subsequent dressing changes, topical fucidic acid ointment was used. For donor sites, topical scar care products were usually prescribed for few months. Graft take was complete in all cases a part from mild epidermal peeling at small areas appeared in first dressing in four cases, which was managed conservatively with non-adherent gauze and topical antibiotic ointment on daily dressing for less than one week . Small sIn wide healthy granulating chronic wounds we successfully applied the FTSG, after meticulous wound debridement, with removal of all debris and securing heamostasis. Graft take was complete for all cases except in three cases in which there were small islands of graft loss due to small subcutaneous hematomas. In those three patients, bed-side wound debridement and regular dressing changes ended with epithelialization and complete healing within two weeks. There were no differences in graft take between acute and chronic wounds, provided that meticulous debridement and absolute heamostasis was done. Whatever the site and size of the grafts; early postoperative erythema or hyperpigmentation were observed in donor site scar. It was improved by time until regaining its original skin color almost within three to six months.2 depending on the established facts of graft take through plasmatic imbibition, capillary ingrowth and revascularization obtained from wound margins and healthy wound beds as well.5,6 Through our practice, we respect variations of skin at different parts of the body varies in terms of color, texture, thickness and appearance.9In this retrospective study, we expanded the use of FTSG to almost unlimited dimensions confirming its reliability and versatility as a reconstructive tool for wound coverage; even though it is very wide. We used to present cases with raw areas up to 452 cm2-4 We share the consensus of superiority of full-thickness over split-thickness skin graft, as they undergo minimal secondary contracture and maintain their robustness in less likelihood of graft trauma with more uniform texture.9-12In reconstruction of small areas particularly at the face we have to do our best for skin matching in selecting donor sites, but this golden rule can be violated with the relatively large-sized defects. In our cases we always insist to close the donor sites directly in a linear manner. We share the opinion of many authors regarding the precautions on graft harvesting, application and fixation.13 This is applied to all types of skin grafting operation either split or full thickness. In our series with these large-sized defects, this precaution is amplified and dealt with extensively to decrease any possible morbidity. The importance of immobilization of the graft on its bed for the early postoperative days is stressed. We employed many techniques like tie-over dressing and, with these exceptionally large-sized grafts, we occasionally used quilting sutures for fixing the graft on its bed. This helps to fix the graft and reduce shearing forces.11The healthy wound bed without any clinical signs of infection is an important precaution before taking the decision of grafting any raw area.In some cases, we used external splints for further immobilization of the grafted areas. The hidden donor site of the subgluteal crease was preferred. This was the back-hoarse area in our study due to its great advantage to give us a large sized skin graft with minimal and most hidden donor site morbidity. The possible two disadvantages are disturbance of subgluteal sulcus in some cases with poor planning. The second is that it has a thicker skin compared to other common donor sites. Although we found that thick skin was advantageous in certain clinical scenarios. Excision of the subcutaneous fat directly beneath the subgluteal crease is suggested to facilitate primary donor site closure and to recreate the crease back. The use of the subgluteal donor site can provide up to 15×3 cm graft in adults with possibility of direct closure of the donor site. After limited removal of subcutaneous fatty tissues, it heals without any noticeable changes of the gluteal contour. We cannot declare that there is only limited sites of donor skin that can be closed directly as it used to be believed in harvesting FTSG. The subgluteal crease area can supply the largest hairless FTSG at a most hidden and directly closed site. In this article we introduce this relatively new donor site of FTSG with new application as FTSG is the second step in reconstructive ladder.The aim of this article is to reintroduce a simple reconstructive surgical technique well known by all plastic surgeons, safely into a new horizon of application dimensions, with almost unlimited extents, expecting sound healing with satisfactory function and aesthetic results. The use of FTSG with these large dimensions could be of help in some clinical situations in which the alternative complex reconstructive tool, may not be easy from either the patient general or local conditions. It could also be of help in some centers with shortage of facilities for more complex flaps either local, regional, or free.


Bran extracts (70% aqueous acetone) of specialty sorghum varieties  were used to investigate the effects of sorghum phenolic compounds on starch digestibility, Estimated Glycemic Index (EGI), and Resistant Starch (RS) of porridges made with normal corn starch, enzyme resistant high amylose corn starch, and ground whole sorghum flours. Porridges were cooked with bran extracts in a Rapid Visco-analyser (RVA). The cooking trials indicated that bran extracts of phenolic-rich sorghum varieties significantly reduced EGI, and increased RS contents of porridges. Thus, there could be potential health benefits associated with the incorporation of phenolic-rich sorghum bran extracts into foods to slow starch digestion and increase RS content. Daviet al.  found thsorghums . Jankows [et al.  reported [et al.  provided [et al.  comparedRecently, we demonstrated that phenolic-rich specialty sorghum brans decreased starch digestibility of endosperm porridges, regardless of endosperm hardness, except for bran from black sorghum, which may have been due to its unique bran structure . It woulBrans of the specialty sorghum varieties are a more dense source of total phenols and condensed tannins than the whole sorghum grains . Extractp < 0.05) lower breakdown and setback in the Rapid Visco-analyser (RVA) test  test , suggestp < 0.05) decreased overall corn starch digestion. Black bran or black with tannin bran extracts had similar effects on corn starch digestibility, while the extract from tannin bran dramatically reduced corn starch digestibility (p < 005) higher starch digestibility than corn starch porridge  increased RS contents of corn starch porridges, compared to extracts from white sorghum bran  decreased overall starch digestibility, EGIs and increased RS contents of high amylose corn starch porridges. The largest reduction in starch digestibility  lower starch digestibility than whole white corn flour porridges. Phenolic-rich whole specialty sorghum porridges cooked with distilled water had lower (p < 0.05) starch digestibility after 180 min of digestion than whole white sorghum porridges  decreased starch digestibility, EGI and significantly (p < 0.05) increased RS contents of whole sorghum porridges compared to whole white corn flour porridge cooked with corn bran extract (p < 0.05) decreased starch digestibility, EGI and significantly (p < 0.05) increased RS contents of the porridges  lower starch digestibility than whole corn porridge. Whole tannin, black, and black with tannin sorghum porridges cooked with distilled water had significantly (p < 0.05) lower starch digestibility than whole white sorghum porridges, again demonstrating the dependence upon tannins and anthocyanins in these grains to reduce the rate of glucose release from starch in the grain.Similar to what has been shown previously ,30,31,32et al.  observedSome assumptions are that during cooking condensed tannins are either destroyed or structurally modified. Dlamini  and Portp < 0.05) decreased starch digestibility of whole sorghum porridges (p < 0.05). One hypothesis could be that the high amounts of phenols in aqueous medium would increase alpha amylase inhibition and promote phenolic compounds interactions with soluble gelatinized starch and denatured protein during and after cooking. Through these mechanisms the bran extracts may have further decreased starch digestibility, EGI, and increased RS contents in whole sorghum porridges.Specialty sorghum bran extracts significantly , air-dried, manually cleaned, glumes removed, and the cleaned grain was stored at −20 °C. Tannin sorghum varieties were high-tannin sorghum  and black with tannin sorghum . Non-tannin sorghum varieties were white food-type sorghum  and high anthocyanin black sorghum . Homogenous 10 kg batches from each sorghum variety were prepared from 50 kg of mother batches to use in the analyses described below.A whole white corn  and a commercial corn starch (Argo) were used as controls. High amylose corn starch  was obtained from National Starch & Chemical . Whole white corn was milled to pass through a 1 mm screen using a UDY cyclone mill . The samples were stored at −20 °C until used for analysis.Whole sorghum grains and whole white corn were milled to pass through a 1 mm screen using a UDY cyclone mill (Model 3010-030). Brans of sorghum varieties were obtained by decorticating 4-kg batches in a PRL mini-dehuller . The bran was then separated with a KICE grain cleaner . The bran  was further milled to pass through a 1 mm screen using a UDY cyclone mill (Model 3010-030) and stored at −20 °C.g for 20 min and supernatants were collected. Acetone (reagent grade) was removed under vacuum at 30 °C, and total of 27 mL aqueous extracts were collected from each bran to cook the porridges. These aqueous bran extracts were added to corn starch, high amylose corn starch, and whole sorghum flours to make porridges. Extracts were kept at −20 °C in the freezer until the next day, and were brought to room temperature to make porridges. Tannin and phenol determinations were conducted on bran extracts and bran residues after extraction to obtain % extract recovery, which was calculated as below:The milled brans (3 g) were extracted for 3 h using 30 mL of 70% aqueous acetone with constant shaking at low speed in an Eberbach shaker  in quadruplicate. The extracts were centrifuged at 769 × g. A 1 mL volume of the supernatant was mixed with 5 mL vanillin reagent . The vanillin reagent (reagent grade) contained 4% concentrated HCl and 0.5% vanillin in methanol. The reaction was carried out in a 30 °C water bath for 20 min, and absorbance read at 500 nm after 20 min. Blank determinations were done to compensate for the color of the samples, by replacing the vanillin reagent with 4% HCl in methanol. The standard used was catechin ; tannin content was expressed as mg catechin equivalents per g (mg CE/g).A modified vanillin-HCl method  was usedet al. [et al. [The Folin Ciocalteu method of Kaluza et al. , as modi [et al. , was useet al. [The pH differential method of Fuleki and Francis  with modet al.  was usedCorn starch , high amylose corn starch , and ground whole sorghum  were cooked with extracts of bran from white, tannin, black and black with tannin sorghum varieties. Corn starch and high amylose corn starch cooked with only distilled water served as controls. Flours of whole sorghum and white corn cooked with distilled water were used as controls for whole flour porridges.p < 0.05) decreased starch digestibility.The samples above were cooked in 12 mL of the aqueous suspension of bran extracts in a Rapid Visco Analyser  interfaced with a personal computer equipped with Thermocline software. A programmed heating and cooling cycle was used, in which the mixture was held at 50 °C for 1 min, heated to 95 °C in 7.5 min at the rate of 6 °C/min, held at 95 °C for 5 min before cooling to 50 °C in 7.5 min and holding at 50 °C for 1 min. The porridges were cooled to 50 °C for 5 min, and left at room temperature for 10 min. An additional 13.1 mL of the aqueous suspension of bran extracts  was added to the cooked porridge for the second cooking using the described cooking profile above. The porridges were cooled to 50 °C for 5 min, and left at room temperature for 10 min before further analysis. Preliminary studies conducted showed that double cooking of corn starch significantly  equivalent wet porridges (as ready to eat) were homogenized in 10 mL of a HCl-KCl mixture (pH = 1.5) for 1 min using a Polytron homogenizer  with controlled speed (level 4). The samples were incubated at 40 °C for 60 min in a shaking water bath with 0.2 mL of a solution containing 1 mg of pepsin from porcine gastric mucosa . Fifteen mL of Tris-Maleate buffer (pH 6.9) was added to adjust pH. Then another 5 mL of Tris-Maleate buffer containing 2.6 UI of α-amylase from porcine pancreas  was added. The flasks were placed in a water bath at 37 °C with agitation. Aliquots (0.1 mL) were taken every 30 min from 0 to 3 h. α-Amylase was inactivated by immediately placing the tubes in a boiling water bath for 10 min with vigorous shaking every 30 s. Then, 1 mL of 0.4 M sodium-acetate buffer pH = 4.75 and 30 μL of amyloglucosidase from Aspergillus niger  were added. The samples were incubated at 60 °C for 45 min to hydrolyze the starch into glucose. Finally, the glucose concentration was measured using a glucose oxidase-peroxidase kit . The experiment was repeated two times for each sample.Potentially available starch content was assessed by following the multienzymatic protocol of McCleary et al. . The pro [et al.  was usedet al. [The rate of starch digestion was expressed as a percentage of total starch hydrolyzed at different times . The digestion curves were adjusted to the following non-linear equation established by Goñi et al.  to descrC is the percentage of starch hydrolyzed at time t (min); C∞ is the equilibrium percentage of starch hydrolyzed after 180 min; and k is the kinetic constant. The variables C∞ and k were estimated for each sample using SPSS for Windows 11.5.where Hydrolysis Index and Estimated Glycemic Index (EGI):From the digestion curves obtained during starch hydrolysis, the area under the hydrolysis curve (AUC) was calculated for each sample using the equation:C is the percentage of starch hydrolyzed at time t (min); C∞ is the equilibrium percentage of starch hydrolyzed after 180 min; and k is the kinetic constant. The hydrolysis index (HI) was obtained by dividing the area under the hydrolysis curve of each sample by the corresponding area of a reference sample  [where tf is the final time (180 min) and to is the initial time (0 min); I = 100) . Finallyet al. [g and the supernatant discarded. The residue is treated with 2M KOH for 1 h to solubilize resistant starch and the sample is then incubated with amyloglucosidase before glucose content is determined using a glucose oxidase assay . RS was calculated as glucose (mg) × 0.9 on dry basis.RS content in the food was measured using the method described by Goñi et al. . In briep = 0.05) were computed.The data were analyzed using one way analysis of variance (ANOVA) using SPSS v 11.5 software . Differences among treatments were detected using a Duncan’s test with a confidence level of 95% (α = 0.05). For physical and chemical characteristic of sorghum varieties, significance of differences within varieties and mean values of the cultivars, analysis of variance (ANOVA) and least significant differences  affected the rate and extent of starch digestibility of porridges. Significantly (p < 0.05) lower EGI and higher RS values were observed in porridges made with bran extracts containing high levels of phenolic compounds. These results demonstrate that the sorghum phenols, present mainly in the bran, have potential applications in food and pharmaceutical industries to decrease health problems related to type 2 diabetes and to reduce weight because of their ability to reduce starch hydrolysis.Overall, the phenol-containing extracts of specialty sorghum varieties used in this study significantly (


Pancreatic amylase activity and total 13C hepatic enrichment were determined in females immediately before and 4 h after administration of the starch bolus. For both sexes, BW, BC, and basal EE and RER were not affected by the type of starch, but dEI and hydrogen production were increased by the LDD. Only in females, total carbohydrate oxidation and starch-derived glucose oxidation in response to the starch bolus were higher in LDD versus HDD mice; this was not accompanied by differences in amylase activity or hepatic partitioning of the 13C label. These results show that starch digestibility impacts glucose metabolism differently in females versus males.Starches of low digestibility are associated with improved glucose metabolism. We hypothesise that a lowly digestible-starch diet (LDD) versus a highly digestible-starch diet (HDD) improves the capacity to oxidise starch, and that this is sex-dependent. Mice were fed a LDD or a HDD for 3 weeks directly after weaning. Body weight (BW), body composition (BC), and digestible energy intake (dEI) were determined weekly. At the end of the intervention period, whole-body energy expenditure (EE), respiratory exchange ratio (RER), hydrogen production, and the oxidation of an oral  The digestibility of dietary starch can influence metabolic health. This has been extensively studied in rodents, where lowly digestible starches have led to lower body weight (BW) and adiposity and normal glucose homeostasis compared to highly digestible starches . In humaA recent meta-analysis of rodent studies showed that consuming lowly digestible starches results in similar BW and fat mass (FM) compared to highly digestible starch feeding in female mice and rats . In contIn this study, we tested the hypothesis that exposure to an LDD compared to an HDD during the immediate post-weaning period leads to a better capacity to oxidise dietary carbohydrates, specifically the starch molecule, in female but not in male mice. Additionally, we explore whether small intestinal amylase levels or the partitioning of starch-derived glucose to the liver compartment could explain any potential sex differences in starch oxidation.All mice  were individually housed in Makrolon II cages with wood chips and wood shavings as nesting material, at 23 ± 1 °C, 50 ± 5% humidity, on a 12 h light/dark cycle (6.00 AM lights on). Unless otherwise indicated, mice had ad libitum access to food and water.n = 15 females, n = 8 males) or an HDD  for the rest of the study. These two diets only differed in the type of starch included. The experimenter was not blinded to the experimental diets. Animal welfare was monitored daily by visual inspection. BW, body composition , and food intake (FI) were determined weekly. At the end of PW 6  or during PW 7 , mice were re-stratified by BW and assigned to be sacrificed in either the fasting state or after ingestion of a 13C-labelled starch bolus (described below). Additionally, a subgroup of the mice assigned the 13C starch bolus was transferred to an extended indirect calorimetry (InCa) system for measurements of whole-body metabolism and total and starch-derived glucose oxidation (described below). The study was approved by the CCD/IvD 2017.W-0024.002 and performed in accordance with European Union (EU) directive 2010/63/EU.Adult mice (9–23 weeks old) on a chow diet  were time-mated and their offspring cross-fostered within 24–48 h postnatal to produce standardized litters of 6 pups and a sex ratio of 3:3 or 4:2. At the end of postnatal week (PW) 3, male and female offspring were weaned and individually housed and stratified by BW to receive either an LDD . The exact composition and digestible energy density of the diets are shown in The chow diet  for breeding and lactating dams consisted of 24, 60, and 16 energy% protein, carbohydrate, and fat, respectively. The semi-purified LDD and HDD contained 20, 55, and 25 energy% protein, carbohydrate, and fat, respectively, and were based on the BIOCLAIMS standard diet . The LDD−1 mixture of uniformly 13C-labelled potato starch  in phosphate buffered saline (PBS) was heated to 90 °C for 15 min and added to a 120 mg mL−1 suspension of non-labelled amylopectin maize starch  in PBS. The resulting mixture had a total starch concentration of 80 mg ml−1, of which 7.5% (w/w) was 13C labelled starch. Preparations were made fresh on the day of use and kept under constant stirring before administration to the mice.A 20 mg mL13C starch bolus, mice (including the subgroup studied in InCa) were food-restricted approximately 2 h before the dark phase by receiving 1.21 (SD 0.02) g of their corresponding post-weaning diet (LDD or HDD). The day after, approximately 2 h into the light phase (LP) when the mice were fasted, each mouse was administered 0.5 ml of the 13C starch preparation by oral gavage, representing a dose of 40 mg of total starch per mouse (0.6 kJ).On the day prior to the administration of the 2 consumption and CO2 production from which daily energy expenditure (EE) and respiratory exchange ratio (RER) were calculated. Locomotor activity, and food and water intake were also measured. Recently, we incorporated hydrogen (H2) and methane (CH4) sensors for gut microbiota activity measurements [13C starch bolus in the fasted state, as described above. InCa measurements continued for the next 24 h. For logistical reasons, gas sampling frequency was set at 20 min for males and 11 min for females. LDD and HDD animals were equally represented in each InCa measurement. Bedding was reduced to about 200 ml during InCa measurements to facilitate detection of voluntary locomotion by infrared beam breaks in the horizontal plane. BW and BC were determined directly before and after InCa measurement. The technological extensions of the InCa system and other operational settings and procedures have been described previously [Animals were acclimatised to the extended InCa system  for approximately 36 h. The following 24 h period was used for measurements of Ourements . After beviously ,13,14.2 and VCO2, based on Péronnet’s table of non-protein RER [Total glucose oxidation (TGO) rates were calculated from VOtein RER . Starch-13CO2 (formulas 1 and 2) is the 13C enrichment in expired CO2 in atom% calculated from 13CO2 and 12CO2 gas concentrations (delta ppm). In formula 2  is represented by at%13Cs and has a value of approximately 8.4 atom%, according to the declared isotopic and dry chemical purity of the 13C-labelled starch, and assuming a 13C enrichment of 1.1 atom% of the non-labelled maize starch based on measured values of maize-derived fructose [2 is the production rate of total CO2 obtained using the summed concentrations of 13CO2 and 12CO2 multiplied by the constant air flow. The molecular weight of glucose is 180.16 g mol−1, the volume occupied by 1 mol of CO2 in STPD is 22.29 liters, and 6 carbons per mol of starch-derived glucose were 13C-labelled. To express SGO rates as percentage of the dose of starch-derived glucose administered, the declared moisture content in the 13C-labelled and non-labelled starches and the release of 1.1 g of glucose per 1 g of dry starch [The at%based on ), the tifructose . VCO2 isy starch  were tak13C starch bolus (~5 h into the LP). No anaesthesia was used, to prevent effects on glucose levels. Glycaemia was determined in whole blood directly after sacrifice using a Freestyle glucose meter . The liver was excised, weighed, separated into lobes, and snap-frozen in liquid N2. The luminal content of the small intestine was gently pressed out, weighed, and snap-frozen. Samples were then stored at −80 °C.Mice were food-restricted by receiving about 1.21 g of their corresponding post-weaning diet (LDD or HDD) 2 h before the dark phase, as described above. On the following day, mice were decapitated either in the fasted state (~3 h into the LP), or exactly 4 h after administration of a Amylase activity in small intestinal contents was measured with a colorimetric method  according to the manufacturer’s instructions. The intra-assay and inter-assay CVs were 2.2 and 14.8%, respectively. Total amylase levels per animal were estimated by multiplying the activity in the sample by the weight of the small intestinal contents.13C starch into liver because this organ extracts about 30% of an enteral glucose load, particularly in fasting conditions [13C enrichment was measured by EA-IRMS, as previously published [2, followed by separation for measurement of the 13C/12C ratio by EA-IRMS. Results were expressed as delta over baseline (DOB) atom%, calculated from the algebraic difference between atom% 13C from individual mice that received a 13C starch bolus and the mean atom% 13C of mice on LDD or HDD that did not receive the 13C starch bolus and were sacrificed in the fasted state. Label recovery in the liver compartment was calculated from the dry weight of whole liver and the total C and 13C content in the samples.We focused on the partitioning of nditions ,19. Liveublished . Brieflyn = 6) was calculated to detect a difference of 0.0466 mg min−1 in maximal exogenous glucose oxidation with 80% statistical power at a two-sided significance level of 0.05 (G*Power v. 3.1.9.3) [n ≤ 6, a normal distribution was assumed. Equality of variances was tested with an F-test. Differences between LDD versus HDD mice in single outcomes at the end of the study  were determined separately per sex with a two-tailed unpaired Student’s t-test  with Welch’s correction when variances were significantly different, or with a two-tailed Mann-Whitney test . Time course data  were analysed separately per sex by a two-tailed unpaired Student’s t-test on the incremental area under the curve (iAUC), taking as baseline the average RER over approximately 1 h before administration of the 13C starch bolus. To locate specific time points showing statistically significant differences, time course data were analysed by repeated-measures (RM) 2-way ANOVA and, when the interaction between time and post-weaning diet was significant, followed by Bonferroni’s post hoc test for multiple comparisons. Sphericity was assumed. Two RER values that were not recorded due to opening of the cage were replaced by the average of the previous and next recordings. To compare the cumulative SGO in males versus females on LDD or HDD, and to compare parameters measured in LDD versus HDD females in the fasted state or 4 h after receiving the 13C starch bolus, a 2-way ANOVA was used. Statistical analyses, iAUC calculations, and data visualisation were performed in Prism v.5.04 . Statistical significance was set at P < 0.05.Sample size (3.1.9.3) , based o2) production over 24 h was approximately 2.9-fold higher in LDD versus HDD mice (4) production was undetectable.Both in males and females, BW, FM, lean mass, and fasting blood glucose concentrations were not different after three weeks of consuming the post-weaning LDD versus HDD . SimilarHDD mice . Methane13C starch bolus (P < 0.01). In contrast, fasting RER was similar between LDD and HDD females (0.678 (SD 0.016) and 0.677 (SD 0.024) respectively).For both sexes, LDD mice showed a lower RER compared to HDD mice from the time of food restriction up until the moment before administration of the ch bolus . Fasted 13C starch bolus, LDD and HDD males showed a similar increase in RER , and a main effect of the post-weaning diet , being significantly higher in LDD females at 33 and 44 min upon administration of the 13C starch bolus  and diet (P < 0.05), indicating that males oxidised starch-derived glucose more extensively than females and that overall LDD mice oxidised more exogenous glucose than HDD mice.Regarding the cumulative amounts of starch-derived glucose oxidised throughout the 4 h after administration of the 13C starch bolus showed that LDD mice had lower pancreatic amylase levels compared to HDD mice, as indicated by an overall effect of diet  versus 1.0845 (SD 0.0004) atom%, respectively; P = 0.053). In females fed the 13C starch bolus, LDD females tended to have a higher deposition of the 13C label in liver above baseline compared to HDD females four hours after administration of the starch bolus . It has been shown previously that adult male mice fed a low glycaemic index diet for three weeks have a better capacity for oxidation of ingested fat than mice on a high glycaemic index diet, but this was not true for exogenous glucose oxidation [13CO2 enrichment, but not of total CO2 production volumes), and the study did not include female mice. We therefore considered it important to focus on the oxidation of the starch molecule in both sexes using a quantitative method. While RER data alone is a well-founded approach to substrate oxidation analysis, the precise distinction of exogenous versus endogenous fuels can only be achieved by including 13CO2 analysis together with total CO2 determination. This method was particularly useful given the uncertainty about the true nature of the fuels oxidised in the fasted state prior to the administration of the 13C starch bolus, as discussed below. Furthermore, complementing the analysis of exogenous glucose oxidation rates with O2 measurements revealed that the starch bolus affected metabolic rate in LDD and HDD males differently  in our t starch ,33,34, de uptake , have al2 production  helped us recognise important effects of starch digestibility shared by females and males and those that are sex-dependent, and is testament to the value of extended InCa systems for the refinement of animal research. Further, the absence of significant effects of LDD versus HDD on BW and BC, and on H2 production, amylase levels, and total 13C label deposition in liver after ingestion of a starch bolus, suggests that the higher capacity of LDD females to oxidise starch stems from differences in hepatocellular metabolism or may lie in other organ systems.All in all, the use of InCa with additional gas sensors (Female mice fed a lowly digestible starch post-weaning diet developed a better capacity to oxidise starch-derived glucose compared to females on a highly digestible starch diet. This effect was only marginal in male mice. Our results suggest that starch digestibility could have different consequences for metabolic health in females and males and should be considered when formulating health recommendations for carbohydrate quality.


Effects of extrusion cooking and low-temperature storage on the physicochemical changes and resistant starch (RS) content in cornstarch were evaluated. The cornstarch was conditioned at 20%–40% moisture contents and extruded in the range 90–130 °C and at screw speeds in the range 200–360 rpm. The extrudates were stored at 4 °C for 120 h and then at room temperature. The water absorption, solubility index, RS content, viscoelastic, thermal, and microstructural properties of the extrudates were evaluated before and after storage. The extrusion temperature and moisture content significantly affected the physicochemical properties of the extrudates before and after storage. The RS content increased with increasing moisture content and extrusion temperature, and the viscoelastic and thermal properties showed related behaviors. Microscopic analysis showed that extrusion cooking damaged the native starch structure, producing gelatinization and retrogradation and forming RS. The starch containing 35% moisture and extruded at 120 °C and 320 rpm produced the most RS (1.13 g/100 g) after to storage at low temperature. Although the RS formation was low, the results suggest that extrusion cooking could be advantageous for RS production and application in the food industry since it is a pollution less, continuous process requiring only a short residence time. Starch is a complex carbohydrate composed of glucose units consisting of amylose and amylopectin ,2 and isRS yields vary according to the hydrolysis process used; e.g., HCl 12%–25%) ,9, lacti%–25% ,9,Extrusion cooking has conventionally been used to obtain a variety of extruded products because it is a continuous process, it is versatile, and it shows short processing times, high productivity, and energy efficiency ,19,20,21The proximate composition of the native cornstarch is shown in 2 values.The influences of the extrusion temperature, feed moisture content, and screw speed on the physical and chemical properties of cornstarch are shown in The WAI is an indicator of the capacity of flour to absorb water, which depends on the availability of hydrophilic groups that bind to water molecules and on the capacity of macromolecules to form a gel . The valThe WSI is a parameter for measuring the degradation of starch. Lower WSI values indicate that less starch is degraded and that the extruded products contain fewer soluble molecules . Figure p < 0.05) affected both G′ and G″ for the EBS and EAS. p < 0.05) affected tan δ for both the EBS and EAS, as shown in The storage modulus, G’, is a measure of the amount of energy stored in materials and recovered during cycling, which is indicative of the solid or elastic characteristic of the materials. Another parameter that indicates the physical behavior of a system is the loss modulus (G″), which shows the liquid or viscous behavior ,43. The p < 0.05) affected the apparent viscosity (η) of the EBS and EAS. The apparent viscosity increased to a maximum of 31.36 Pa·s for the EBS containing 28.46% moisture and extruded at 113.52 °C and 271.8 rpm  formed in the EBS. The formation of RS in the EAS showed a similar trend and was significantly affected by the screw speed. The EAS containing 29.4% moisture and extruded at 101.6 °C and 286.4 rpm contained a minimum of 0.400 g/100 g of RS d becauseThe gelatinization enthalpy is a measure of the crystallinity of the amylopectin, indicating the quality and quantity of the starch crystals , and of The amylose leached out of the starch granules during extrusion cooking and bound to the surface of the extruded product, as shown in Canonical analysis  was applThe identified optimum conditions were verified by performing an independent experiment with the EAS. TM, Muscatine, IA, USA) used for this study was analyzed for moisture, protein, fat, crude fiber, and ash contents according to methods 934.06, 920.52, 920.152, 945.16, 940.26, and 962.09, respectively [The cornstarch . A RS assay kit including a standard resistant starch with 52% of RS was obtained from Megazyme . The other analytical-grade solvents and reagents used for the extractions were obtained from J.T. Baker .Starch whose initial moisture content was 10.9% was fed into an extruder at 15.97 kg/h d.b. and was adjusted to 20%, 25%, 30%, 35%, and 40% moisture contents and extruded through a 600-mm-long twin-screw co-rotating pilot scale , where the length to diameter ratio (L/D) = 20. The die diameter was 4 mm, and the screw configuration was specifically selected to create high levels of shear, the first section contained only conveying elements, with the next containing both conveying and kneading elements. Finally, the high-shear section contained conveying, reverse conveying, and kneading elements , accordiThe extruded cornstarch was milled , sieved with a 0.8-mm sieve, and re-sieved with an 80-mesh (177-micron) sieve to obtain uniform EBS and EAS particles, which were stored in plastic bags until the analysis.g for 15 min . The supernatant was decanted and evaporated at 100 °C until dry. The residue from the supernatant and the sediment remaining in the tubes after decanting the supernatant were weighed. The ratio of the sediment-forming solids to the soluble solids was measured as g water/g sample.The WAI and WSI were determined in triplicate according to the method described by Anderson et al. . BrieflyThermal analysis by differential scanning calorimetry (DSC) was used to determine the gelatinization temperature and the enthalpy of starch gelatinization of the EAS, following the method used by Hasjim and Jane . Two mg The RS content was determined using an enzymatic kit  and the enzymatic method used by Goñi et al. , which iThe rheological properties  were determined using a rheometer . Starch slurry containing 10% solids was used. According to the moisture content of each sample, a volume of water was added gradually, and the mixture was stirred in a beaker until a semifluid mass was obtained. The instrument was equipped with stainless steel parallel-plate geometry (40 mm diameter). The sample was covered completely with the plate, and excess sample was eliminated. All measurements were carried out at 25 °C and a 1000-micron gap. To determine the linear viscoelastic region, strain amplitude sweeps were run from 0.1% to 5% at a frequency value of 1 Hz. The analyzed samples presented similar response with 2% strain. The flow sweep was performed at a shear rate (γ) of 0.1 to 15 1/s thereby relating shear stress (σ) and shear rate (γ) to calculate the apparent viscosity (η) using Herschel Bulkley model  at shear−2 kg/cm2. The samples were examined using a scanning electron microscope  equipped with a secondary electron detector operated at an acceleration rate of 10 kV.The EBS and EAS samples, whose particle sizes <0.15 mm and whose moisture contents were 1%, were stuck to stubs and coated with a gold layer under high vacuum in a Denton vacuum evaporator (Desk II) set to 7.031 × 10iY is the predicted response, iX and jX are the input variables, 0b, ib, iib, ijb are regression coefficients of the intercept, linear effects, squared effects, and interactions respectively. The variable combination is shown in p < 0.05).A second-order, three-variable, five-level, central-composite design was used, resulting 20 treatments, which axial and central points were done by duplicate. The results were analyzed using response surface methodology . The feeExtrusion is an alternative method of producing RS. The starch moisture content and the extrusion temperature significantly changed the physical properties of the native cornstarch by increasing the WAI and WSI and decreasing the viscosity and enthalpy because of the ECP-induced damage in the starch. The EAS previously extruded at 120 °C and a screw speed of 240 rpm from starch containing 35% moisture contained the most RS (1.134 g/100 g). Despite not generating high RS content, the ECP produced 68% more RS than that which naturally occurs in native cornstarch with the added advantages of the ECP being a high-productivity, faster, energy-efficient, continuous process.


In addition to proteins and/or oils, mature seeds of most legume crops contain important carbohydrate components, including starches and sugars. Starch is also an essential nutritional component of human and animal diets and has various food and non-food industrial applications. Starch is a primary insoluble polymeric carbohydrate produced by higher plants and consists of amylose and amylopectin as a major fraction. Legume seeds are an affordable source of not only protein but also the starch, which has an advantage of being resistant starch compared with cereal, root, and tuber starch. For these reasons, legume seeds form a good source of resistant starch-rich healthy food with a high protein content and can be utilized in various food applications. The genetics and molecular details of starch and other carbohydrate components are well studied in cereal crops but have received little attention in legumes. In order to improve legume starch content, quality, and quantity, it is necessary to understand the genetic and molecular factors regulating carbohydrate metabolism in legume crops. In this review, we assessed the current literature reporting the genetic and molecular basis of legume carbohydrate components, primarily focused on seed starch content. We provided an overview of starch biosynthesis in the heterotrophic organs, the chemical composition of major consumable legumes, the factors influencing starch digestibility, and advances in the genetic, transcriptomic, and metabolomic studies in important legume crops. Further, we discussed breeding and biotechnological approaches for the improvement of the starch composition in major legume crops. The information reviewed in this study will be helpful in facilitating the food and non-food applications of legume starch and provide economic benefits to farmers and industries. Triticum aestivum) and rice (Oryza sativa) (Vigna unguiculata) contains ∼25% protein  . For ins protein , whereas protein . In gene protein . In addi protein .Although most of the legumes are a rich source of protein, some legumes are also vital sources of carbohydrates . In ordeSolanum tuberosum L.) (7–20%) have very limited RS but release more glucose in the blood (Pisum sativum L.) is about 50% of its dry weight, whereas the SSC is very low in soybean (≤0.91%) (Medicago truncatula (≤1–2%) (n = 17) genotypes with varied starch concentration. The result indicated that the highest value of starch content was from IT183905 genotype with 1.4%, and the least starch content was from IT228277 with 0.24%. Previously, The cereals are important sources of calories in the human diet and provide feed for animals. They are worthy sources of starch. Commonly, starch is the major component of cereal grains, which consists of 60–80% starch. Foods from high-starch cereals such as maize (65–75%), wheat (60–75%), rice (80%), and potato (he blood . Convershe blood . However(≤0.91%)  and Medi (≤1–2%) . HoweverIn recent years, there have been steady increases in the production and consumption of legumes, mainly due to increased awareness about the nutritional and health benefits of legume-based food products. Soybeans differ from other widely grown legume crops, as they have high protein content, which, in combination with oil, forms an average of 60% of the total seed content. Although the expression of carbohydrate and starch biosynthesis genes in soybean seeds was continuously increased before full seed stage, it was later reduced significantly than genes related to fatty acid and oil synthesis . LikewisIn recent decades, increased awareness of the use of legume seeds as source of nutritional food and animal feed is of the highest importance for the development of legume crops. The legume or pulse family has nutritional compositions, and health benefits have been a hot topic in recent decades . For insCommonly, the prime nutritional fraction of carbohydrate is starch in legume seeds. Generally, amylose and amylopectin comprise about 98–99% of the dry weight of total starch. Amylose content as well as long chains of amylopectin extends the retrogradation and increases benefit in reducing the glucose released in the blood, exhibits prebiotic effects beneficial for colonic microflora, and eventually promotes a lower GI. It is well known that processing and cooking methods with legume crops produce an increase in the levels of RS, which has slow digestibility resulting in the positive impact of GI. It leads to better benefits in human diet to control type 2 diabetes . In general, of starch synthesis starting enzyme, AGPase is activated by 3-phosphoglycerate and inhibited by inorganic pyrophosphate (PPi). This AGPase enzyme induces the regulating reaction in amyloplasts by converting Glc-1-P and ATP to ADP-Glc and PPi. The basic component of starch, amylose, and amylopectin synthesis occurs through the activity of three major enzymes, that is, granule-bound SS (GBSS), SS, and SBEs. In addition, interactions of pullulanase (PUL) and isoamylase (ISA) debranching enzyme play an important role to determine the complex structure of starch, and they are also involved in starch breakdown during germination. In brief, this systematic interaction of multienzymes gradually leads to synthesized starch (Lotus corniculatus L.), which was reported to be involved in starch metabolism in Arabidopsis, with various differences in isoform. In addition, they are also observed in duplications of a number of starch metabolism genes in Lotus japonicus, including AGPase large subunit isoform 2 (APL2), SS II (SSII), GBSS, α-amylase 3 (AMY3), β-amylase 3 (BAM3), and cytosolic glucan phosphorylase (PHS2). Similarly, Vigna radiata (L.) Wilczek). In soybean, nonsense mutations in the putative coding region caused non-function of the SSIIb genes , chickpea (Cicer arietinum L.), mung bean, lentil, pea, and pigeon pea (Cajanus cajan) for RDS, SDS, and RS. The GI in this study was estimated based on the hydrolysis indices; and the RDS, SDS, and RS were estimated based on isolated starch sample (20 mg) from each legume. The lowest GI and RDS content observed therein were 44.2 GI and 4.2% RDS, respectively, in pigeon pea, whereas the highest was 10.9% RDS and 49.8 GI observed for chickpea. Similarly, mung bean had the lowest RS content, which was observed at 50.3%, and the highest RS 78.9% observed for pigeon pea, whereas the lowest SDS content was observed at 16.9% in pigeon pea and the highest at 40.0% in mung bean.Starches from different biological sources have different structural and polymer forms and, therefore, possess different physicochemical properties. These properties determine the function of the starch. Starch consists of amylose and amylopectin units, and their numbers and organization vary among different species. Legumes contain a 5% to 10% higher content of amylose starch than cereals, which commonly contain 25–28% amylose and 72–75% amylopectin . The chestarches . Legume Literature showing evidence in general legume starches poses the lower digestibility and shows mixed types of X-ray diffraction. Legume starches have a high level of SDS, which makes them more attractive than starch of cereal or tuber crops. This leads to more appeals to the consumers for nutritional and diet perspective to use legume starches as foods.in vitro dialysis system, processing treatments  have been shown to increase the rate of starch digestibility in the flours and processed germinating seedlings of chickpea, cowpea, and mung bean (Vigna radiata)  . Similarradiata) . HoweverPhaseolus vulgaris L.) on how environmental conditions affect starch granule morphology, low-molecular-weight amylopectin, polymerization, average chain length, amylose, and digestibility of starch content within cultivating locations  have an absence of sucrose synthase (SuSy) activity in cell types except for the phloem. However, the mutant plants showed normal cell-wall structure and cellulose content, sugar content, lipid content, and seed weight and synthesized normal amounts of leaf starch  ) was well established and functionally characterized for seed starch turnout, structure, and granular properties through mutagenesis approach , rug5 (SSII), and lam (GBSSI) loci straightaway affect polymer biosynthesis and degree of starch branching. This alteration leads to an immense increase in amylose content of genotypes with an r and rug5 loci from 60% to 75% and 43% to 52% in the total starch content, respectively. On the contrary, lam loci, which have been shown to lack SS activity, reduce the amylose content at 4–10%, which is significantly lower than in wild type on the basis of total starch (rb [ADP-Glc pyrophosphorylase L1 subunit (AGPL1)], rug3 ), and rug4 (sucrose synthase) loci. The other mutations in pea at “rugosus” loci are known to modify the shape of seeds from round to wrinkled and to exhibit pleiotropic effects on seed morphology (n = 92) for association mapping. They observed that the natural allelic variation in r, rb (AGPL1), and rug5 (SSII) was associated with chain length distribution (CLD) variation. From the reports, it indicates that the allelic difference at r locus has a great influence on pea phenotypes, which is associated with amylose content and amylopectin CLD that can produce the various forms of starch contents and structure via infusion of the genes at r and rb loci. Similarly, a study reported on another model legume species Lotus japonicus with a range of mutants for starch synthesis and accumulation. These isolated mutants are categorized as “synthesis mutants,” which are defective in a starch synthesis, which had a lower level of leaf starch, or breakdown “degradation mutants,” which had higher levels of leaf starch than do wild type. For instance, LjPGI1), phosphoglucomutase, (LjPGM1), AGPase large subunit isoform (LjAPL1 and LjAPL2), and Glc-1-P adenylyltransferase small subunit (LjAPS1), which lead to a huge reduction in starch contents or completely no starch due to lacking respective genes encoding enzymes. In addition, they evaluated L. japonicus population derived from the ethyl methanesulfonate (EMS) and further performed the mapping. They showed the relationship of AGPase and glucan water dikinase 1 (GWD1) enzyme with the starch-excess mutant phenotype. Moreover, the L. japonicus mutant studies focused on leaf starch content and information on other organs such as roots, embryos, and seeds. Generally, starch metabolism, physicochemical properties, and genetics are complex, and much less information is available across the legumes. Significant efforts in this regard have been made in some legume crops to improve seed carbohydrate composition through different approaches such as mutagenesis  . Moreoveapproach . Genes pl starch , whereasrphology . Recentlagenesis , quantitagenesis , and maragenesis . Such inRecent advancements in sequencing technologies and genotyping platforms have facilitated the genetic dissection of the simple as well as complex agronomic traits across plant species . Next-geqSSC8.1) for SSC with 12.3% phenotypic variation explained (PVE) (r = 0.6). This suggested that lines with high seed weight may contain high starch content, so these can be used as an indirect selection method. The region identified in mung bean for high SSC further can be explored to develop improved starch content in mung bean through MAS. Similarly, qSTR06_2 showed the highest PVE (9.1–11.3%). The reported results indicate that QTL has a significant environmental influence on the seed starch expression in the RIL population, and seed starch observed in the range of 0.11–1.39% showed continuous variation with transgressive segregation.The SSC is a quantitatively inherited trait and is influenced by growing environments . Very feed (PVE) . They haBreeding for the seed starch composition in legumes is a complicated process primarily due to the low variation in SSC. Hence, less focus has been given to identify loci, QTL, or genes identification for SSC. Moreover, it is necessary to identify the QTLs controlling the concentration of SSC using different genetic backgrounds across different environments. This can be simplified and accelerated using genomic resources, tools, and the dense genetic maps available for some of the legume species .Medicago truncatula seeds showed differential expression of about 45% of the functionally classified seed-regulated genes during seed maturation . These processes involve transcriptional, metabolic, biochemical, and physiological reorganization by several different pathways and the associated changes in the expression level of a number of genes . A combituration . These gVigna angularis), in which they observed differentially expressed genes for starch and fat content in adzuki bean and soybean. They have reported 27 starch biosynthesis genes in adzuki bean and 46 in soybean; but no significant variation in the ratio of the starch biosynthesis genes has been observed according to the χ2 test (P = 0.4135). However, the transcriptional level of starch biosynthesis and average transcription of individual starch biosynthesis genes in adzuki bean were significantly higher than in soybean at the mature seed stage. Such differences are thought to be caused by transcriptional abundance rather than copy number variations in the genes associated with starch and oil synthesis. Recently, phenotypic and deep sequencing of the transcriptome analysis performed in drought and salinity stressed chickpea by Recent transcriptome analyses have suggested a number of transcription factors (TFs) involved in seed development . Liu et M. truncatula, Arabidopsis, soybean, pea, chickpea, and adzuki bean . These tools, in addition to detailed experimental setups for desired metabolites, may be helpful for metabolic engineering efforts to enhance seed nutritional quality in soybean  Genetic and genomics method: Advances in next-generation technologies contributed to the significant reductions in the sequencing costs, because of which millions of SNPs were discovered in crop genomes (® SoyaSNP), cowpea (Illumina 60K iSelect BeadArray), peanut (58K Axiom_arachis SNP), pigeonpea (56K Axiom Cajanus SNP), and chickpea (50K Axiom®CicerSNP Array) ( genomes . In addiP Array) . This hi(2) Genetic engineering methods: Potential biotechnological strategies have been reviewed in the past few decades (AGPase) activity in plants, have been preferred to improve starch contents. The gene (AGPase) from Escherichia coli (glgC16) was transformed into potato plastids. The transgenic plants showed 60% more starch content in tubers than non-transgenic plants  and alfalfa (Medicago sativa) , which are genetically altered for AGP-antisense inhibition  . Manipultructure . Furtherhibition . The prohibition . In addihibition . This wa(3) Mutation breeding: The mutation causes sudden heritable change at the DNA level, which is not induced by genetic recombination and hybridization. Many mutants have been characterized in Arabidopsis that exhibit a starch excess phenotype and are mostly affected by enzymes involved in starch mobilization , Rural Development Administration, Republic of Korea.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Starch granule morphology is highly variable depending on the botanical origin. Moreover, all investigated plant species display intra-tissular variability of granule size. In potato tubers, the size distribution of starch granules follows a unimodal pattern with diameters ranging from 5 to 100 µm. Several evidences indicate that granule morphology in plants is related to the complex starch metabolic pathway. However, the intra-sample variability of starch-binding metabolic proteins remains unknown. Here, we report on the molecular characterization of size-fractionated potato starch granules with average diameters of 14.2 ± 3.7 µm, 24.5 ± 6.5 µm, 47.7 ± 12.8 µm, and 61.8 ± 17.4 µm. In addition to changes in the phosphate contents as well as small differences in the amylopectin structure, we found that the starch-binding protein stoichiometry varies significantly according to granule size. Label-free quantitative proteomics of each granule fraction revealed that individual proteins can be grouped according to four distinct abundance patterns. This study corroborates that the starch proteome may influence starch granule growth and architecture and opens up new perspectives in understanding the dynamics of starch biosynthesis. O-glycosidic bonds  [Starch is the main source of calories in human and livestock diets. It is also used in glue, bioplastic, and biofuel industries as well as a food additive. Starch is composed of two glucose polymers, amylose and amylopectin, and accumulates in the form of cold water-insoluble granules. Amylose and amylopectin are both constituted by α-1,4 glucan linear segments ramified through α-1,6  points) ,3. Starc points) .Starch synthesis involves several families of enzymes. Starch synthases (SSs) are responsible for the elongation of α-1,4 glucans . On the Other proteins observed in association with the starch granule are involved in starch degradation and its regulation. The glucan water dikinase (GWD) and the phosphoglucan water dikinase (PWD) add phosphate groups at C6 or C3 position of the glucose residues, respectively, to initiate starch degradation ,29. The Starch granules display a broad structural and morphological eterogeneity. This disparity is observed at the level of: (i) the botanical origin; (ii) the organelle and tissue localization within a given species; (iii) the individual cells of a given tissue; (iv) within the same plastid, where distinct granule populations can be observed ,41. In pStarch granules isolated from potato  tubers were analyzed for size and shape distribution by granulomorphometry . The graIn order to investigate the disparity of starch structure and composition according to the granule morphology, starch was fractionated sequentially with the use of nylon filters . Each fraction was then analyzed by granulomorphometry and scanning electron microscopy . Averagemax) of the amylopectin-I2 complex  [Each fraction was analyzed by shotgun mass spectrometry using 300 mg of starch. Starch granules were washed with 2% SDS to remove unspecifically bound proteins prior to gelatinization and isolation of internally trapped proteins. Peptides were obtained by the eFASP (enhanced filter-aided sample preparation) method and analyzed by UPLC-nano-ESI-MS/MS. Protein quantification was performed with the Maxquant and Perseus 1.6.0.2 software and a custom database to avoid quantification biases because of the redundant gene fragments present in the PGSC (potato genome sequencing consortium) databank . This daYP20-20) .In order to evaluate protein stoichiometry within starches of the different fractions, the abundance score  of each protein was expressed in fmol per mg of starch . RegardlA correlation analysis was also performed between individual protein concentrations and the other parameters measured in this study . SS1, 2 We investigated the heterogeneity of structure, composition, and morphology existing within individual starch samples isolated from whole potato tubers. While the morphological heterogeneity of starch is well-known and often relates to the botanical, tissular, and cellular origins , variatiIn order to relate the structural and morphological heterogeneity of starch to the corresponding metabolic pathways, we investigated the starch-bound proteome in the fractionated granule populations. Bottom-up proteomics allowed to identify all the proteins that we reported previously . Label-fAll solutions were prepared using ultrapure water purified with a MilliQ™ Academic system . All chemicals, biochemicals and solvents were purchased from Sigma-Aldrich  and used without purification.Solanum tuberosum  cultivated in the field in Villeneuve d’Ascq , France, between March and July 2014. Potato tubers (approximately 70 g) were washed with tap water and peeled prior extraction. The tubers were ground with a blender in 200 mL of ultrapure water. The tuber extracts were then filtered through a nylon net (100 µm mesh) and left for starch granule sedimentation during 3 h. The supernatant was then removed and sedimented starch was resuspended in 500 mL of ultrapure water. The starch suspensions were subsequently washed three times with 1 L of ultrapure water and stored in 20% ethanol at 4 °C.Starch granules were isolated from tubers of The starch granule size and shape were measured with an Ipac 1 granulomorphometer . The retained parameters for granule size were the ISO area diameter and granule shape with ellipsoid roundness. Before measurement, a numerical filter was applied. It excluded particles with a diameter smaller than 4 µm or higher than 100 µm. In addition, particles with an ellipsoid roundness lower than 46% and a diameter between 4 and 18 µm were also excluded. An average number of 3000 granules was analyzed.Granules were separated using nylon filters with a mesh width of 100, 50, 31, and 11 µm. The first filtration was realized with the 11 µm filter in order to isolate fraction A. The retained fraction was refiltered with a 31 µm filter to obtain fraction B. Then, the new retained fraction was filtered with a 50 µm filter. The eluted fraction was fraction C, and the retained fraction was fraction D. The fractionation quality was estimated by size measurement with an Ipac 1 granulomorphometer . The parameters used were the same as described above.Drops of dilute aqueous suspensions of starch granules were deposited on metallic stubs covered with C/Ni tape and allowed to dry. The specimens were coated with Au/Pd in a Baltec MED 020 sputter coater  and observed in secondary electron mode in a FEI Quanta FEG 250 microscope  operating at an accelerating voltage of 2.5 kV.λ = 0.1542 nm) using a Philips PW3830 generator  operating at 30 kV and 20 mA. Two-dimensional XRD patterns were recorded on Fujifilm imaging plates exposed for 2 h and read off-line with a Fujifilm BAS 1800-II bioimaging analyzer . Diffraction profiles were calculated as rotational averages of the 2D patterns.The starch granule fractions were placed in a chamber maintaining a relative humidity (R.H.) of 93% for 5 days. The powders were then poured into 0.7-mm (outer diameter) glass capillaries that were flame-sealed. All specimens were X-rayed by transmission in a Warhus vacuum chamber with a Ni-filtered CuKα radiation (maxλ) for each glucan-I2 complex.The protocol for amylose assay was adapted for Megazyme  K-AMYL kit ,52. Briev/v). The samples were incubated overnight at 42 °C, and diluted with 46 µL ultrapure water. After the first dilution, the sample was then rediluted 50 times with ultrapure water prior to injection in a Beckman Coulter PA800-plus Pharmaceutical Analysis System  equipped with a silicon capillary column . The capillary was rinsed and coated with Beckman Coulter N-linked carbohydrate separation gel buffer one-third diluted with ultrapure water prior to injection (15 s at 10 kV). The migration was performed at 10 kV during 1 h at 20 °C. The detection was made with a 488 nm solid-state laser module, and a laser-induced fluorescence (LIF) detector. The detection window was located 10 cm above the outlet of the capillary column.The amylopectin chain length distribution was measured by fluorescence-assisted capillary electrophoresis (FACE). Two milligram of starch from each fraction, in suspension in 250 µL of ultrapure water, was gelatinized by heating at 100 °C, with intermittent high-speed stirring in a vortex mixer, until the solution became clear. Then, 20 U of isoamylase and 1 U of pullulanase were added and the solution was incubated at 42 °C overnight in order to debranch amylopectin. Proteins were then precipitated by heating at 95 °C for 15 min, and the supernatant was recovered after centrifugation. The samples were desalted on Alltech™ Carbograph  solid-phase extraction (SPE) column, then freeze-dried. The dry samples were resolubilized in 2 µL of 1 M sodium cyanoborohydride in tetrahydrofuran (THF), and 2 µL 0.2 M 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) in 15% acetic acid (N-linked carbohydrate separation gel buffer prior to injection (10 s at 10 kV). The migration was performed at 10 kV during 10 min. The detection was made at 488 nm as described above.The phosphate content for each fraction was determined by FACE. The protocol was the same as that described in Verbeke et al. 2016. Briefly, 1 mg of starch from each fraction was submitted to acid hydrolysis with 100 µL of 2 M trifluoroacetic acid (TFA) at 95 °C during 1 h and 20 min. A total of 20 µL of the hydrolysate was then diluted with 200 µL of ultrapure water to stop the reaction. The sample was then dried with a SpeedVac™ Concentrator . The dry samples were then derivatized with APTS as described before. The derivatized samples were diluted with 96 µL of ultrapure water. A total of 15 µL of this first dilution was rediluted with 185 µL of ultrapure water prior to injection in a Beckman Coulter PA800-plus Pharmaceutical Analysis System with the same silicon capillary column as that described above. The capillary was rinsed and coated with the Beckman Coulter ®  membrane filter with a 50 kDa cutoff inside a 15 mL vial. The vial was centrifuged at 7500 rpm for 30 min in order to concentrate the samples. A total of 5 µL of each sample, diluted 10 times, was used to carry out a Bicinchoninic assay  following the manual instructions.The starch proteins were extracted from 300 mg of dry fractionated starch. Ten aliquots of 30 mg starch were then suspended in 500 µL of extraction buffer ; 50 mM dithiotreithol (DTT). Aliquots are then heated to 100 °C until gelatinization of the granules. After gelatinization, the samples were kept for an additional 10 min at 100 °C and centrifuged for 10 min at 13,000 rounds per minute (rpm). Supernatants containing proteins were rescued, pooled, and dropped on an Amicon® units  were incubated overnight with 5% v/v TWEEN-20 . Following incubation, the filter units were rinsed thoroughly by three immersions in ultrapure water. eFASP digestion is realized with 50 µg of samples estimated from the BCA assay. These samples were mixed in 50 µL of solubilization and reducing buffer A , 50 mM DTT, 200 mM ammonium bicarbonate (ABC)) overnight at 4 °C. Samples were centrifuged at 13,000 g at 20 °C for 15 min. Supernatants were mixed with 200 µL of SDS buffer exchange  and were transferred into Amicon® units. After 30 min of 13,000 g centrifugation, the filtrate was discarded, an additional 200 µL of exchange buffer A was deposited in each unit, and centrifugation resumed for 30 more minutes. This buffer addition/centrifugation step was repeated twice more. The reduced proteins were alkylated within the filter unit by adding alkylation buffer A  and incubating at 37 °C for 1 h with shaking in the dark. After 30 min of 13,000 g centrifugation, the filtrate was discarded, an additional 200 µL of exchange buffer A was deposited in each unit, and centrifugation resumed for 30 more minutes. This buffer addition/centrifugation step was repeated once more. After buffer exchange was performed with three exchanges with eFASP digestion buffer , 100 µL of eFASP digestion buffer was added to each Amicon® units, followed by 1 µg of trypsin (1:50 w/w). Digestion proceeded for 16 h at 37 °C. Peptides were recovered by transferring the UF filter to a new collection tube and spinning at 13 000 g for 20 min. To complete peptide recovery, filters were rinsed twice with 50 µL of 50 mM ABC that was collected by centrifugation. A total of 200 µL of ethyl acetate was added to the peptide containing filtrate and was transferred to a 2 mL tube. After that 2.5 µL TFA was added and was quickly vortex. White thread like precipitate may be visible for large quantities of peptides. Peptides precipitate were mixed with 800 µL of ethyl acetate and were centrifuged at 13,000 g for 10 min. The organic supernatant was discarded and this step was repeated twice more. The upper organic layer was carefully removed and discarded as much as possible without disturbing the boundary layer. Uncovered sample tubes were placed in a thermomixer at 60 °C, in a fume hood, for 5 min to remove residual ethyl acetate. Residual organic and volatile salts were removed by vacuum drying in a SpeedVac™ Concentrator . This step was repeated two times with 50% methanol. Ten micrograms of dry peptides are then resuspended in 0.1% (v/v) formic acid.A total of 50 µg of proteins are reduced, alkylated, and digested with eFASP Digestion protocol. Ultra-filtration filters from Amicon−1 with buffer A (5% acetonitrile and 0.1% formic acid) then separated on column  and separated with a linear gradient of 5–50% buffer B (75% acetonitrile and 0.1% formic acid) at a flow rate of 250 nL.min−1 and temperature of 45 °C. The total time for an LC MS/MS run was 270 min. MS data was acquired on Q Exactive plus Orbitrap mass spectrometer (Thermo Fisher Scientific) using a data-dependent top 20 method, dynamically choosing the most abundant precursor ions from the survey scan (350–1600 m/z) for HCD fragmentation. Dynamic exclusion duration was 60 s. Isolation of precursors was performed with a 1.6 m/z window and MS/MS scans were acquired with a starting mass of 80 m/z. Survey scans were acquired at a resolution of 70,000 at m/z 400 (AGC set to 106 ions with a maximum fill time of 100 ms). Resolution for HCD spectra was set to 70,000 at m/z 200 (AGC set to 105 ions with a maximum fill time of 200 ms). Normalized collision energy was 28 eV. The underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.4%. The instruments were run with peptide recognition mode , exclusion of singly charged and of unassigned precursor ions enabled.A nanoflow HPLC instrument  was coupled on-line to a Q Exactive plus Orbitrap mass spectrometer (Thermo Fisher Scientific) fitted with a nanoelectrospray ion source. One microliter of peptide mixture (corresponding to 500 ng of proteins) was loaded onto the preconcentration trap  using a partial loop injection, for 5 min at a flow rate of 10 µL.min−1 in order to obtain a result in fmol.mg of starch−1. Fold change values are obtained by normalizing the concentration of one protein on the concentration of this protein in the fraction A. LFQ values are transformed in log2 then filtered and used on Perseus 1.6.0.2 to realize statistical testing. For multiple comparisons, ANOVA analysis with a permutated based FDR correction (FDR = 0.05) was performed. For the comparison between two groups, t-tests were performed with P-values less than 0.05 were considered to be statistically significant (Proteins were quantified with Maxquant software 1.5.3.30 with custom starch bound protein databank  regroupinificant . The cor