The diets consisted of sugarcane bagasse, ground corn, soybean hulls, cottonseed, soybean meal, mineral-vitamin core, and additives. The animals fed BEO35 had higher dry matter intake (P = 0.02) and daily weight gain (P = 0.02). The MON treatment improved feed efficiency (P = 0.02). The treatments BEO35 and BEO45 increased hot carcass weight (P < 0.01). Animals fed BEO presented greater carcass yield (P = 0.01), carcass gain (P < 0.01), rib eye area gain (P = 0.01), and final rib eye area (P = 0.02) when compared to MON. The MON25 treatment improved carcass gain efficiency (P = 0.01), final marbling (P = 0.04), and final subcutaneous fat thickness (P < 0.01). The use of MON reduced the fecal starch% (P < 0.01). Cattle-fed BEO increased rumen absorptive surface area (P = 0.05) and % ASA papilla area (P < 0.01). The MON treatment reduced the cecum lesions score (P = 0.02). Therefore, the use of BEO with 35 and 45% starch increases carcass production with similar biological efficiency as MON; and animals consuming MON25 improve feed efficiency and reduce lesions in the rumen and cecum.Feed additives used in finishing diets improve energy efficiency in ruminal fermentation, resulting in increased animal performance. However, there is no report evaluating the effect of BEO associated with exogenous α-amylase in response to increased starch content in feedlot diets. Our objective was to evaluate increasing levels of starch in the diet associated with a blend of essential oils plus amylase or sodium Monensin on performance, carcass characteristics, and ruminal and cecal morphometry of feedlot cattle. 210 Nellore bulls were used , where they were blocked and randomly allocated in 30 pens. The experiment was designed in completely randomized blocks in a 3 × 2 factorial arrangement: three starch levels , and two additives: a blend of essential oils plus α-amylase  or sodium Monensin . The animals were fed once a day at 08:00  The increase in performance associated with lowering the age at slaughter is often achieved through the use of high-concentrate feeds to meet energy requirements and maximize production in feedlot cattle , 2. The Feed additives used in finishing diets improve energy efficiency in ruminal fermentation, resulting in increased animal performance. Diets with high starch content promote high production of SCFA in the rumen, which leads to a drop in pH. The use of additives is important for controlling rumen acidification and manipulating the rumen fermentation process to maximize the utilization of dietary nutrients . Among tIn this context, this has instigated a search for natural alternatives to ionophores, such as essential oils and their blends (BEO), which act as growth promoters when added to animal feed diets . BEO areIt has been suggested that exogenous amylase improves feed efficiency by increasing nutrient utilization, resulting in improved animal performance . Some stHowever, based on the aforementioned information, there is no report evaluating the effect of BEO associated with exogenous α-amylase in response to increased starch content in a feedlot diet. It was hypothesized that BEO should be an alternative to MON in finishing feedlot diets, as well as that BEO associated with exogenous enzymes would further improve nutrient utilization and animal performance in high-starch diets, reducing fecal starch and cecum lesions score. Therefore, the objective of this study was to evaluate the increasing starch content in the diet associated with a blend of essential oils plus amylase or sodium monensin on performance, carcass characteristics, and ruminal and cecal morphometry of feedlot cattle.All the procedures involving the use of animals in this study were by the guidelines established by the São Paulo State University Ethical Committee for Animal Research, and approved by Ethical Commission in Use of Animals of the Innovation and Applied Science DSM Nutritional Products SA (number BR190313).2 of area, a water trough, and a collective bunk .The trial was conducted at the feedlot facility of the Center for Innovation and Applied Science in Ruminants, from DSM Nutritional Products , using 210 22-mo-old yearlings Nellore bulls (375 ± 13.25 kg). Animals were housed in 30 pens, with 120 mThe experimental design was performed in completely randomized blocks and the initial BW was utilized as a criterion for block formation, in a 3 × 2 factorial arrangement, in which were evaluated: 3 levels of starch  and 2 feed additives (BEO + α-amylase or sodium monensin), totaling 6 treatments: (1) MON25: 25% of starch + monensin; (2) BEO25: 25% of starch + essential oil and α-amylase; (3) MON35: 35% of starch + monensin; (4) BEO35: 35% of starch + essential oil and α-amylase; (5) MON45: 45% of starch + monensin; (6) BEO45: 45% of starch + essential oil and α-amylase. Each treatment had 5 replicates , with 7 animals/pen .® Ruminants DSM Nutritional Products Ltd., Basel, Switzerland) which contains thymol, eugenol, limonene, and vanillin  was included in the diet at a dose of 26 ppm. The blend of essential oil . The animals were submitted to a pre-adaptation period of 10 days to standardize their ruminal population and adapt to the facilities and management. Cattle were fed ad libitum once a day at 08:00 h, targeting 3–5% refusal, with free-choice water access to a water trough.At the beginning of the study, all yearling bulls were weighed, dewormed, and vaccinated  was obtained by increasing the proportion of Corn grain fine grind. During the experimental period, weekly samples were taken of the rations for the chemical analysis of dry matter (DM), crude protein (CP), ether extract (EE), and mineral matter (MM) according to AOAC , a step-up adaptation program diet of 14 days (day 0 to day 14), and a finishing period program of 89 days (day 14 to day 89).At the beginning of the experimental period (day 0), and 30 and 89 days of the study, the feed was withheld from bulls for 16 h before every body weight (BW) assessment. Consequently, average daily gain (ADG), and feed efficiency  were calculated at the end of the experiment. The DMI was calculated for each pen daily, by weighing the ration offered and refused before the next morning delivery and expressed in kilograms and as a percentage of BW. To estimate the net energy for maintenance and net energy for gain, the methods described by Lofgreen and Garrett , NRC 30 were usen = 6) was randomly chosen to be slaughtered as a reference to the evaluation of the gain composition in the function of the initial BW, and determination of the equation of adjustment of the BW in the function of the carcass weight . The remaining Nellore yearling bulls were harvested at the end of the study. The final BW was obtained at the feedlot before truck loading, and the cattle were then transported to a commercial abattoir. The hot carcass weight (HCW) was obtained after kidney, pelvic, and heart fat removal. The dressing percentage was calculated by dividing HCW by the final BW , according to Johnson and Combs . FeedingDuring the observation period, samples of diets and orts were collected after 24 h for future analyses of DM  and neun intake/n predicted intake, in which n = particle fraction retained on screens of 19 (long), 8 (medium), and 1.18 mm (short) and a pan (fine). Particle sorting values equal to 1 indicate no sorting. Those < 1 indicate selective refusal (sorting against), and those >1 indicate preferential consumption sorting for Leonardi and Armentano (On the day of observation of feeding behavior (day 71), samples of diets and orts were also collected from all pens for the determination of particle size distribution, which was performed by sieving using the Penn State Particle Size Separator  and reported on an as-fed basis as described by Heinrichs and Kononoff . Particlrmentano .Rumenitis evaluation was performed after bull evisceration, and all rumens were scored after washing. Rumen epithelium was classified according to the incidence of lesions (rumenitis and hyperkeratosis) as described by Bigham and McManus  based on2) of each rumen was collected from the dorsal cranial sac, and placed into a phosphate-buffered saline solution for future morphometric measurements according to Resende Júnior et al. (2) and scanned, and the mean papillae area (MPA) was determined using an image analysis system . The rumen wall absorptive surface area (ASA) was calculated according to Daniel et al.  were obtained between the 12th and 13th ribs of animals . The samples were identified, vacuum packaged, and frozen at −20°C for further analysis. The analyzes were performed at the Food Products of Animal Origin Laboratory .For meat quality analysis, 2.54-cm thick steak samples of For the shear force analysis, the samples were thawed, for 24 h at 2°C, and processed according to the American Meat Science Association (AMSA)  and Whee* is the chrome associated with luminosity , a* is the chrome that varies from green (–) to red (+), and b* is the chrome that ranges from blue (–) to yellow (+). Color values were considered the average of the three readings.The color was determined by the CIE Lab system . Color mLongissimus dorsi were used, and thawed the day before the analysis in a refrigerator at 2–5°C. Moisture was evaluated according to AOAC (method 39.1.02)  . The tot39.1.02) . Total p39.1.02) . Ash was39.1.02) .n = 30), and each treatment was replicated five times. Data were analyzed using the MIXED procedure of SAS  and the Tukey test to compare means. Tests for normality (Shapiro-Wilk and Kolmogorov-Smirnov) and heterogeneity of treatment variances (GROUP option of SAS) were performed before analyzing the data. The model also included fixed effects of treatments and their interaction. The block was considered a random effect in the model. Results were considered significant at P ≤ 0.05 level.The experimental design was performed in completely randomized blocks, and the initial BW was utilized as a criterion for block formation, in a 3 × 2 factorial arrangement of treatments (3 levels of starch and 2 feed additives). Pens were considered the experimental unit for this study (P < 0.01) and ADG (P = 0.01) on the first 30 days of the study, where the cattle fed BEO45 had greater values when compared with MON in the same levels of starch. Similarly, there is a significant interaction between starch level and additives for DMI (P < 0.01) and DMI expressed as %BW (P = 0.01) on the first 30 days of the study; and for final BW (P = 0.04), ADG (P = 0.02), DMI (P = 0.02) and DMI expressed as %BW (P = 0.05) at the end of the experimental period, where the cattle fed BEO35 had greater values when compared with MON35 and MON45. No effects of treatments were observed (P > 0.05) for G/F in the first 30 days of the study. However, at the end of the study, there was a significant linear decrease in G/F (P = 0.05) in response to increasing levels of starch . Moreover, cattle-fed MON improved G:F . Regarding fecal starch, there was a significant (P < 0.01) quadratic effect in response to increasing levels of starch, where cattle fed BEO had greater fecal starch content when compared with MON  and HCW gain (P = 0.01), where the cattle fed BEO25 and BEO35 had greater values when compared with MON35 and MON45. However, a significant interaction between starch level and additives was observed for final carcass gain efficiency , biological efficiency , final marbling , final subcutaneous fat thickness  and P8 rump fat thickness gain , where the cattle fed MON25 had greater values when compared to BEO25 and BEO45. Moreover, there was a significant linear decrease in dressing percentage (P = 0.01), carcass gains (P = 0.05), final P8 rump fat thickness (P = 0.01), rib-eye area gain (P = 0.03) and P8 rump fat thickness gain (P = 0.01) in response to increasing levels of starch. In addition, cattle fed BEO had higher dressing percentage , carcass gain , rib-eye area  and rib-eye area gain  when compared with MON.The results of carcass traits are presented in P < 0.01) and moisture (P = 0.02) in response to increasing levels of starch, where the highest value for cooking loss and the lowest value for moisture was obtained on a 35% starch diet. No effects of treatments were observed (P > 0.05) for chroma L, chroma A, chroma B, pH, marbling, shear force, ash, and protein , time spent resting (P = 0.02), and meal length (P =0.02). Regarding feed efficiency, the highest value was observed in cattle fed MON45, followed by MON35 and BEO45. For time spent resting, the highest values were observed for BEO35 and MON45. In addition, for meal length, the lowest values were observed for BEO45 and MON45. Regarding particle sorting, no significant treatment effect was observed (P > 0.05) for most of the feeding behavior and particle sorting variables evaluated. However, animals fed CR35 sorted (P = 0.02) more intensively for medium diet particles when compared to MON35.The results on feeding behavior are summarized in P < 0.01) in response to increasing levels of starch, where the highest value was obtained on 35% starch . Moreover, there was a significant linear increase in time spent eating (P < 0.01), meals per day (P < 0.01), eating rate of NDF (P < 0.01), rumination rate of NDF (P < 0.01), in response to increasing levels of starch. There was a significant linear decrease in DMI per meal (P < 0.01) and eating rate of NDF (P < 0.01) in response to increasing levels of starch.There was a significant quadratic effect for DMI (P < 0.01), time spent ruminating  and NDF intake  were affected by the type of additive, where cattle fed BEO obtained the best results. No effects of treatments were observed (P > 0.05) for the rumination rate of DM and drinking bouts.Besides that, DMI  in response to increasing levels of starch, where the highest value was obtained on 35% starch .The results on rumen and cecum morphometrics are presented in 2/cm2 of rumen wall, for BEO and MON, respectively; P = 0.05), papillae area  and papillae height  when compared to MON treatment. However, the supplementation with BEO increased the cecum lesions score , crypt/enterocytes ratio  and crypt/goblet ratio .Moreover, animals fed BEO obtained greater absorptive surface area  and exogenous enzymes may have the potential to replace ionophores, increase BW and ADG and contribute to improved beef production and animal performance , but theIn the present study, cattle fed BEO35 had the highest final BW and ADG, and DMI compared with treatments at MON; however, the results for feed efficiency were more satisfactory compared with treatments fed BEO regardless of starch content than when MON was used. These results could be related to the change in rumen fermentation pattern in response to essential oil treatment in combination with exogenous α-amylase, which may alter the acetate/propionate ratio in the rumen. However, results regarding DMI in response to supplementation with EO are inconsistent because several factors may influence the mode of action of this additive, such as the type of feed, breed, sex, and age, as well as the type of essential oil used in the blend , 52. EssIn this context, data on feeding behavior may help in the interpretation of DMI in response to BEO supplementation. However, evaluation of the feeding behavior in feedlot cattle supplemented with BEO is limited in the literature , 58. In Regarding monensin, it is well documented that the main effects of ionophores are a reduction in DMI and increased feed efficiency , 7, 33. In this context, starch-rich diets can increase the risk of rumen acidosis, as well as lesions in the ruminal epithelium, due to the greater rumen fermentation and consequently higher production of SCFA . The numHowever, treatment with BEO also increased the cecum lesions score and decreased the number of goblet cells. Considering the higher DMI observed, it may be that animals fed BEO had a higher rumen passage rate so that a greater amount of starch bypassed the rumen and some of this starch entered the cecum, which may lead to excessive fermentation in the cecum, increasing the production and accumulation of SCFA, and lowering the pH in the cecum. Thus, acidification of the intestinal environment may lead to an increase in lesions in the cecal epithelium and a decrease in the number of goblet cells and enterocytes because the intestinal epithelium is more sensitive to pH changes than the rumen . HoweverJust as SCFA concentrations and rumen pH cause changes in SCFA absorption rate , 66, SCFvia feces. Ionophore antibiotics have limited effects on the microbiota and intestinal fermentation of ruminants, as the effect of monensin is more likely to be observed in the rumen (P = 0.04) with BEO than with MON.In addition, an increase in fecal starch was observed in animals fed high starch levels, with cattle fed BEO35 having higher fecal starch, suggesting that the cecum was unable to digest the starch from the rumen and absorb SCFA, thereby increasing fecal starch. On the other hand, animals receiving MON had the lowest levels of fecal starch, which may be related to the effectiveness of this additive in influencing rumen fermentation, selection, and stimulating the growth of rumen bacteria, thereby preventing starch losses he rumen . There ahe rumen . In the he rumen . Howeverhe rumen  reportedThus, the manipulation of rumen fermentation and better feed efficiency, in response to additives use may affect carcass characteristics. In the present study, it was observed that cattle fed BEO35 had the best HCW values. However, the best results in final fat thickness were obtained when cattle were treated with MON25. As mentioned earlier, it was observed that increasing the starch content in the diet resulted in more meals per day and lower DMI per meal while trying to control rumen acidity. As a result, carcass fat deposition was negatively affected in cattle-fed MON because increasing starch content linearly decreased final fat thickness and daily fat gain. Better dressing percentage and carcass gain were observed in cattle fed BEO, which may be explained by the higher DMI observed in this treatment as a function of manipulation of rumen fermentation, and the higher carcass yields may be related to higher production of propionate in the rumen, which is an SCFA precursor of glucose in ruminants . TricariThe change in rumen fermentation pattern in response to the use of these additives affects the production of SCFA and, consequently, the acetate:propionate ratio. There are reports that glucose is the major contributor to fatty acid biosynthesis in intramuscular adipose tissue , 80. ConThe BEO increased DMI and enhanced overall animal performance but the effect was dependent on starch content, with no effect on feed efficiency. Animals receiving monensin improved feed efficiency and decreased lesions in the rumen and cecum in finishing feedlot cattle. Therefore, the blend of essential oil evaluated in the current study can be an alternative to replacing MON in finishing feedlot diets. Overall, cattle fed MON25 improved feed efficiency, whereas BEO presented better results on feedlot performance and carcass traits with 35 and 45% starch diets.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by São Paulo State University Ethical Committee for Animal Research, and approved by Ethical Commission in Use of Animals of the Innovation and Applied Science DSM Nutritional Products SA (number BR190313).TS and DM: conceived and designed the study, collected and complied, and analyzed data. TS, TA, VC, AP, LS, AS, MN, WS, and DC: investigation. CM and MA: provided intellectual input. TS, JS, and DM: provided intellectual input, drafted, and edited manuscript. All authors contributed to the article and approved the submitted version.


Intraoperative OCT is an innovative and promising technology which allows anterior and posterior segment ocular surgeons to obtain a near-histologic cross-sectional and tomographic image of the tissues. Intraoperative OCT has several applications in ocular surgery which are particularly interesting in the context of corneal transplantation. Indeed, iOCT images provide a direct and meticulous visualization of the anatomy, which could guide surgical decisions. In particular, during both big-bubble and manual DALK, the visualization of the relationship between the corneal layers and instruments allows the surgeon to obtain a more desirable depth of the trephination, thus achieving more type 1 bubbles, better regularity of the plane, and a reduced risk of DM perforation. During EK procedures, iOCT supplies information about proper descemetorhexis, graft orientation, and interface quality in order to optimize the postoperative adhesion and reduce the need for re-bubbling. Finally, mushroom PK, a challenging technique for many surgeons, can be aided through the use of iOCT since it guides the correct apposition of the lamellae and their centration. The technology of iOCT is still evolving: a larger field of view could allow for the visualization of all surgical fields, and automated tracking and iOCT autofocusing guarantee the continued centration of the image. Anterior segment OCT (AS-OCT) was described for the first time in 1994 by Izatt et al. as an essential tool for the clinical diagnosis and follow up of many corneal pathologies [Standard OCT systems were unsuitable for a surgical setting due to their large dimensions and the traditional sitting position required for image acquisition, which was not practical for supine patients. The introduction of portable OCT systems allowed for the introduction of this tool into the operating room. The first two iOCT systems developed were the Bioptigen EnVisu  and the Optovue iVue  ,4,5,6,7.A major advance was achieved with the introduction of microscope-integrated OCT (MIOCT), which could finally enable the acquisition of real-time intraoperative OCT sections and the visualization of the instrument–tissue interaction ,11. Two At first, iOCT was employed in vitreoretinal surgery, focusing on macular hole ,12,13, vIn this narrative review, we describe the application of iOCT during different types of corneal lamellar surgery and provide a useful and complete guide for both novice and expert corneal surgeons.Deep anterior lamellar keratoplasty (DALK) is the gold standard for the treatment of diseases of the anterior cornea, such as corneal ectasia, anterior stromal leucomas, and stromal dystrophies. In terms of improved visual acuity, DALK is associated with better outcomes than penetrating keratoplasty thanks to lower incidences of post-operative astigmatism and rejection ,26. MoreThere are several applications of iOCT, aimed mostly at avoiding the conversion to PK. First, it allows for the assessment of the corneal and anterior segment architecture, thus determining corneal thickness and regularity, and for the detection of the presence of anomalies such as Descemet’s membrane (DM) rupture or peripheral anterior synechiae. The definition of corneal anatomy aids in making decisions about the depth of trephination, which can be identified by a vertical hyperreflective band along the anterior stroma in the peripheral cornea, where the cut was made. In their study, Santorum et al. suggested an intended stromal bed of 150 μm within the posterior corneal surface, measured with the built-in caliper tool of the intraoperative OCT software  . HoweverIn BB-DALK surgery, the second step is the insertion of a cannula or needle into the stroma to create an air bubble. iOCT allows for the real-time visualization of the instrument, which can aid in guiding the insertion to the desirable depth and in the right direction. However, the visualization of the layers under the cannula can be masked by the hyperreflectivity of the instrument itself, hampering the correct assessment of the thickness of the tissue. In order to obtain the measurement of the residual stromal tissue, it is possible to remove the cannula and then, by acquiring a longitudinal iOCT scan, observe the presence of a stromal pocket, which is visible as an hyperreflective line along the posterior stroma . AlternaThe proper identification of the residual stromal thickness under the cannula is useful for predicting the probability of achieving the big bubble. In 2013, Scorcia and colleagues observed how the distance between the cannula tip and the DM was significantly smaller (90.4 ± 27.7 μm) in cases in which a big bubble was achieved than in a group of unsuccessful surgeries (136.7 ± 24.2 μm) . Indeed,The next step is the creation of the big bubble itself is to obtain a cleavage plan of the posterior stroma. The successful formation of the bubble is visualized on iOCT scans by observing the separation between the corneal stroma and DM, visible as a clear space between the internal concave surface and the external convex surface . iOCT isAlthough BB-DALK is the preferred technique for performing anterior lamellar keratoplasty, there are few situations in which a bubble cannot be achieved or is associated with a high risk of perforation. Specifically, in cases in which corneal opacities do not allow for the visualization of the depth of the stromal involvement, or cases in which the bubble could easily provoke DM ruptures , the majority of corneal surgeons prefer to perform manual stromal dissection.Intraoperative OCT could be helpful during the multiple surgical steps of DALK by stromal dissection. Firstly, iOCT permits the determination of the depth of the opacity and therefore the type of ALK to perform. Variations in corneal thickness at different points may be visualized in irregular or ectatic corneas, helping surgeons decide on a safe depth of initial trephination . In otheiOCT is especially useful because it allows the surgeon to better control the incision depth and assess the uniformity of the dissection plane to optimize visual outcomes, especially when coaxial microscopy does not offer an excellent evaluation of the depth of a corneal incision ,36. In aMoreover, the use of OCT enables surgeons to attempt manual dissection in the case of an emphysematous opaque cornea after a failed big bubble attempt: the opaque bubble layer hampers visualization during subsequent dissection; thus, iOCT can help surgeons perform a safe manual dissection and minimize the risk of DM perforation .During manual stromal dissection in particular, sharp instruments inserted too deeply into the stroma can cause a DM rupture. In these cases, iOCT can detect the location of the rupture and guide further dissection. When a DM rupture is present, it can be advantageous to inject air into the anterior chamber to keep it formed. However, in cases of narrow iridocorneal angles, the air can be misdirected into the posterior chamber, causing an iris protrusion, which can be readily detected by the iOCT .Once the proper plan is achieved, it is possible to proceed with the graft placement. In this step, iOCT might be useful in detecting residual interface fluid, allowing the surgeon to evacuate it by applying moderate massage on the corneal surface, leading to a reduced risk of a double anterior chamber in the postoperative period. After lamellar dissection, iOCT aids in the assessment of graft thickness, graft–host apposition, and interface regularity, as well as in the evaluation of any residual stromal pathology or disparities in the graft–host sizing .Finally, iOCT can aid in the management of post-DALK Descemet’s membrane detachment (DMD) by determining the localization of maximal DMD, managing an iOCT-assisted injection of isoexpansile gas SF6  into the anterior chamber, guiding the location for venting incisions to drain the interface fluid, and confirming the apposition of the grafts .Descemet Stripping automated endothelial keratoplasty (DSAEK) is one of the leading procedures for the management of corneal endothelial dysfunction . CompareDuring graft preparation, either one or two consecutive passes of a microkeratome blade allow for the creation of a very thin lenticule in a procedure known as UltraThin-DSAEK (UT-DSAEK), which has become the standard modern approach to DSAEK ,45,46. IThe iOCT can also be useful for visualizing the DM during descemetorhexis, as well as for locating DM residues and guiding their removal under direct visualization, even in cases of poor visibility due to corneal edema or scarring ,52,53,54One of the most significant contributions of iOCT comes at perhaps the most crucial step of DSAEK: the apposition of the DSAEK lenticule to the recipient stroma. The assessment of the graft attachment and the evaluation of the residual interface fluid between the graft and the recipient stroma are readily available to the surgeon using iOCT. The routinary use of iOCT could, for instance, supersede the use of venting incisions, which have been developed to preemptively drain any potential residual fluid in the graft–stroma interface. Under direct iOCT visualization, residual fluid can be expressed out of the interface through focal manipulation and sweeping of the cornea ,53,55. RThe iOCT can also aid in positioning the graft in cases of uneven posterior corneal surfaces, such as in cases of a previous penetrating keratoplasty in which the posterior lip of the trephination margin could hamper the successful attachment of a DSAEK graft. If a focal irregularity of the posterior corneal surface is detected, the graft can be recentered far from the irregular area .Finally, iOCT’s role could be even more prominent in times in which nanothin (<50 μm) DSAEK is gaining popularity: in complex eyes at a higher risk for rejection and graft detachment, nanothin DSAEK offers comparable results to Descemet membrane endothelial keratoplasty (DMEK) with lower rates of complication, and its results are enhanced by the use of iOCT for correct positioning and orientation ,59 Figu.Although it is steadily gaining popularity, Descemet membrane endothelial keratoplasty (DMEK) is still considered to be a difficult technique characterized by a steep learning curve ,61,62. iOne of the most useful applications of iOCT in DMEK surgery is the possibility of confirming the correct orientation of the graft in real time, with the endothelial side facing the anterior chamber ,66,67,68At present, the orientation must be confirmed qualitatively by the surgeon based on the rolling and unfolding properties of the graft, as shown by iOCT . HoweverAs in DSAEK surgery, iOCT during DMEK can be helpful not only to check the configuration and orientation of the graft but also to visualize areas of synechiae, remnants of Descemet membrane, the presence of fluid or folds at the graft–stromal interface, and to center the graft ,64,75,76The idea of asymmetrically shaping grafts in order to take advantage of the specific features of each layer of the cornea is not new ,78,79. TA variety of technique for preparing mushroom grafts have been described, from manual trephination to femtosecond-laser-assisted mushroom PK ,84. In 2Although no publication in the literature thus far has focused on the use of iOCT in mushroom PK, its unique ability to examine the profile of asymmetric cuts and their relationship with the recipient tissue in real time can surely be of great help to the surgeon. In Mushroom PK is only one of the possible asymmetric graft configurations; the most well-known other configurations are the top-hat, the anvil, and the zig-zag configurations. Each exploits the benefits of their particular shape and is tailored to the need of the individual diseased recipient corneas ,87. AsymTo date, the most important limiting factor of a wider adoption of iOCTs is the entry cost of the device. The use of OCT in the operating room has transitioned from handheld OCTs to microscope-mounted OCTs and finally to microscope-integrated iOCTs, which have largely superseded earlier devices. This means that the entry cost for a state-of-the-art iOCT also includes, in more cases than not, the purchase of an integrated microscope.The DISCOVER and PIONEER studies provided solid evidence of the utility of iOCT in modern eye surgery, but no large clinical randomized trial has definitely proven the superiority of iOCT to microscopy alone ,11.It is reasonable to think that with the increased adoption of these devices, their cost will also be mitigated . The widFurthermore, iOCTs are still evolving devices and, in addition to decreasing costs, their technical specifications are also expected to improve. Available iOCT devices currently employ a central wavelength of 840–860 nm and are capable of axial resolutions between 2.5 and 5 µm in the order of high-resolution posterior segment OCT . HoweverFurther developments of iOCT would also include even larger fields of view than what is currently available , which could allow the surgeon to easily monitor the entire surgical field, from the center of the cornea to the periphery of the anterior chamber, in one glimpse. Moreover, improvement of the automated tracking of surgical instruments and iOCT autofocusing on the corneal structure of interest, which may escape the field of view during surgical movements, are warranted .Finally, with the goal of creating an all-in-one platform, microscope-integrated iOCT could be augmented with automated image analysis, providing useful insights on what is happening in the surgical field in real time, such as the analysis of graft orientation , or with


Correction to: Journal of Clinical Immunology10.1007/s10875-023-01547-yDue to typesetting mistake, the labels above the panel Figure 2B got duplicate data.The original version has been corrected.


In lower extremity reconstruction, the recipient vessel often requires long-range mechanical dilation because of extensive vasospasm or plaque formation induced by concomitant atherosclerosis. While a forceps dilator can be used to manipulate and dilate vessels approximately 1 cm from their end, a DeBakey vascular dilator can dilate long-range vessels. The authors successfully performed free flap reconstruction of the lower extremity using the DeBakey vascular dilator. Of the two patients who underwent lower extremity reconstruction, one had extensive vasospasm, and the other had plaques in the recipient arteries. Irrigation with 4% lidocaine and dilation of the lumen with a forceps dilator were insufficient to restore the normal arterial blood flow. Instead, a DeBakey vascular dilator with a 1-mm diameter tip was gently inserted into the lumen. Then, to overcome vessel resistance, the dilator gently advanced approximately 10 cm to dilate the recipient artery. Normal arterial blood flow was gushed out after dilating the vessel lumen using a DeBakey vascular dilator. The vascular anastomosis was performed, and intravenous heparin 5000 IU was administered immediately after anastomosis. Prophylactic low-molecular-weight-heparin  was administered subcutaneously to both patients for 14 days. The reconstructed flap survived without necrosis in either patient. The relatively low success rates of free flap surgeries in the lower extremity are due to the concomitant peripheral vascular disease, atherosclerosis, and vasospasm that are common in the lower extremity.Despite preoperative preparations for these risk factors, microsurgeons occasionally encounter insufficient blood flow in the recipient vessel of the lower extremity during surgery. Consequently, vessel manipulations such as intraluminal dilation of the recipient vessel, administration of topical vasodilator, and heparinized saline infusion may be performed to restore blood flow.Video 1The recipient vessel was first dilated using a forceps dilator. However, this was insufficient to restore normal arterial blood flow.Video 2A DeBakey vascular dilator was inserted and gently advanced into the recipient vessel. Normal arterial blood flow was gushed out after long-range dilating of the vessel lumen using the DeBakey vascular dilator.2-sized skin paddle was harvested to cover the defect. The right ATA was cut for microvascular anastomosis and normal arterial blood flow was confirmed. However, the blood flow weakened after a while because of vasospasms. Irrigation with 4% lidocaine and heparinized saline infusion was repeated at the end of the recipient vessel. Although the recipient vessel end was manipulated using a forceps dilator, normal blood flow was still not restored , which was to be used as the recipient vessel, was intact. After extensive resection, the right anterolateral thigh (ALT) flap containing a 7 × 7 cmestored .Consequently, a DeBakey vascular dilator with a 1-mm diameter tip was inserted into the recipient vessel lumen . The De2-sized skin paddle was harvested to cover the defect. The side branch of the left posterior tibial artery (PTA) was cut for use as the recipient vessel during surgery. Weak blood flow in the recipient vessel was immediately confirmed owing to the plaque formation inside the lumen. Next, 4% lidocaine irrigation, heparinized saline infusion, and intraluminal dilation using a forceps dilator were conducted. However, normal arterial blood flow still was not gushed out. Then, a DeBakey vascular dilator with a 1-mm diameter tip was gently inserted into the lumen of the side branch of the left PTA and advanced enough to reach the main branch of the left PTA. After this, normal arterial blood flow was gushed out through long-range dilation of the recipient vessel, and end-to-end microvascular anastomosis was performed. Heparin 5000 IU was administered intravenously immediately after the anastomosis, and prophylactic low-molecular-weight-heparin was administered for 14 days. There were no postoperative complications such as necrosis of the distal flap.A 58-year-old male patient with hypertension and a cerebrovascular accident was scheduled to undergo extensive resection for malignant melanoma of the left heel. Preoperative three-dimensional angiography computed tomography revealed that atherosclerotic changes accompanied the lower extremity vessels. However, the patient did not undergo vascular interventions, such as percutaneous transluminal angioplasty, in advance. After extensive resection, the left ALT flap containing a 7 × 9 cmDeBakey vascular dilators are commonly used during cardiovascular surgeries to confirm and dilate coronary artery stenosis or dilate the lumen of the harvested internal mammary artery (IMA) to resolve arterial vasospasms.The choice of the recipient vessel in lower extremity reconstruction is limited, and concomitant atherosclerosis or diabetes mellitus makes the selection particularly difficult due to the narrowing vessel lumen.Normal blood flow can usually be restored by dilating the short-range lumen of the vessel with a forceps dilator.The intimal layer of atherosclerotic vessels is fragile and easily damaged due to vessel manipulation and suturing.In free flap surgeries of the lower extremity, conventional manipulation with a forceps dilator alone is occasionally insufficient to restore the blood flow of the recipient vessel. Hence, if mechanical dilation of a long-range recipient vessel is required, a DeBakey vascular dilator can be used to overcome this limitation.


Material and Methods. An artificial mandibular right molar tooth mounted on a typodont was prepared for a zirconia crown using the standard method. The study sample consisted of 30 zirconia crowns  milled using an (iMes-iCore) milling machine. Each group of 10 crowns was designed with 30–50 and 70 μm spacer thicknesses. The vertical marginal adaptation at the center of the four different planes  was measured under a microscope at 40x magnification. A one-way analysis of variance test was used for statistical analysis. Results. The mean of Group 30 was 27.45; of Group 50 was 22.22; and of Group 70 was 22.90. There was no statistically significant difference between the groups (p ≥ 0.5). Conclusions. The increase in the cement space up to 70 μm did not influence the vertical marginal adaptation of the monolithic zirconia crowns.The long-term clinical success of indirect restorations highly depends on their marginal integrity. The cement space is an element that might affect the marginal integrity, but it can be altered during the configuring of the computer-assisted designing/computer-aided manufacturing (CAD–CAM) restoration. However, there is controversy in the literature regarding the effect of the cement space on the precision of zirconia crown marginal adaptation. The aim of this study was to measure the vertical marginal discrepancies between different cement thickness settings for CAD–CAM monolithic zirconia restorations.  Porcelain fused to metal (PFM) fixed dental prostheses used to be the gold standard for the restoration of damaged or missing teeth to reestablish their function and esthetics . The intTraditional zirconia ceramic restorations have an opaque white appearance, which compromises their aesthetics and makes it essential to veneer them with feldspathic porcelain . HoweverMonolithic zirconia-based ceramics are an appropriate solution to restore posterior teeth to overcome the clinical complications associated with veneered zirconia. These restorations are produced using CAD/CAM processes and, thus, require fewer steps and less time –16. TheySeveral variables may reduce the longevity of monolithic zirconia restorations, such as improper crown design, poor tooth preparation, unbalanced load orientation, fabrication techniques, and excessive marginal discrepancy. The cause of failure is typically multifactorial and difficult to determine –25. ThusThe marginal adaptation of a monolithic zirconia crown could be affected during any of the several steps in the CAD–CAM production process. Intraoral scanner precision, CAD software coherence, CAM fabrication procedures optimization, and dental technicians' postmilling adjustments are essential for setting the prosthesis marginal adaptation , 30, 31.μm) had no substantial influence on the marginal adaptation of the posterior zirconia restorations [Several studies have examined the effect of the cement space on the precision of zirconia crown marginal adaptation, but the results have been contradictory. One study found that different cement spaces ; Group 2, had 10 crowns designed with a 50-μm spacer thickness (the teeth were coded from 0.5 to 9.5); and Group 3, had 10 crowns designed with a 70-μm spacer thickness (the teeth were coded from 0.7 to 9.7) [The sample was divided into three groups: Group 1, had 10 crowns designed with a 30- to 9.7) , 39.Before preparation, a putty index was used to achieve greater accuracy. The index was made via the conventional method that uses the condensation silicone putty material . The catalyst and the base were manually mixed at a 1 : 1 ratio until a uniformly colored mass was obtained. An artificial maxillary right molar tooth (#17) was mounted on a typodont model  and prepared using round-end tapered diamond bur following the recommended guidelines: occusal reduction 1.5 mm, axial reduction  1.2–1.4 mm with 2 mm reduction preparation of the functional cusps, rounded internal line angle, tapered walls with 10°–20° of total occlusal convergence, and uniform 1 mm wide rounded shoulder finish line . The preμm and the cement space was set at 25 μm around the margins for all groups. The files then were randomly distributed with an additional cement space starting 1 mm above the finish lines in 10 crowns in Group 1 with a 30-μm spacer thickness; in the 10 crowns in Group 2 with a 50-μm spacer thickness; and in the 10 crowns in Group 3 with a 70-μm spacer thickness. The Zirconia blocks  were milled using a CORiTEC 250i Loader Pro system  then fired following the manufacturer's instruction in a Zercomat furnace .All 30 scans were saved as standard tessellation language files and imported into CORiTEC design software  to be designed following the manufacture instruction for zirconia crown fabrication . In the The crowns were numbered, and each crown from each group was seated on the die to evaluate its vertical marginal discrepancy. Neither cement medium nor any other material was used to seat the crowns on the prepared tooth. The vertical marginal discrepancy was measured from four points at the center of each axial surface  under a stereomicroscope, which is a reliable and accurate tool for assessing the cement thickness at the margins. The measurements were taken at the middle of each aspect under 40x magnification. A ruler was attached to the die to facilitate the calibration of the measurements on the stereomicroscope Figures . To stanμm) was 27.45, and its standard deviation was 19.84. The mean vertical marginal discrepancy of group 2 (cement space 50 μm) was 22.22, and its standard deviation was 17.19. The mean vertical marginal discrepancy of group 3 (cement space 70 μm) was 22.90, and its standard deviation was 12.82 . None of the vertical marginal discrepancy locations in all three groups was over the clinically acceptable levels (100–120 μm)The results of the one-way ANOVA test showed that different cement space values did not significantly affect the vertical marginal discrepancy values of the tested crowns (μm) did not significantly affect the vertical marginal adaptation of the restorations. Therefore, the null hypothesis was rejected. Theoretically, cement space designation is a dilemma. A narrow cement space may achieve a smaller vertical marginal gap of indirect restorations but would provide difficulty to seat in clinical practice. A wider cement space provides more easier seating but might cause microleakage and loss of restoration retention [μm at the margin and 50 μm 1 mm above the margin and elsewhere resulted in the smallest gap of 53 μm compared with 68 and 85 μm in the other groups, where vertical marginal cement spaces were 25 μm, and internal cement space was set at 40 and 30 μm 1 mm above the margin, respectively.Ensuring vertical marginal adaptation of monolithic zirconia crowns is critical for the longevity of these restorations. In this study, the vertical marginal adaptation of CAD/CAM monolithic zirconia crowns with different cement space settings  for further investigation. Crowns were neither cemented nor stabilized on their respective dies with any medium; in clinical scenarios, crowns are usually cemented; however, this study did not consider the effect of different luting agents, cementation procedures, or discrepancies in placement on vertical marginal integrity. It intended to see whether the default cement space in the CAD–CAM software was produced accurately in the manufactured copings or not.The three different cement space settings were chosen based on previous studies , 39. Theoduction , 43. An μm) for vertical marginal discrepancy was not surpassed at any tested point in all three groups [μm in Group 30, 23.22 μm in Group of 50, and 22.9 μm in Group of 70). A narrow cement space value usually prevents the restoration from complete seating, thus increasing the vertical marginal gap. A larger cement space value is beneficial as it would improve the vertical marginal adaptation and reduce the need for internal adjustments. However, the vertical marginal gap should not exceed the recommended values (120 μm) to ensure a long-term success of the restoration. The resultant standard deviation can be attributed to errors in the production, milling, or crystallization, of the zirconia copings, errors in reading of the measurements, and possible seating inaccuracy.In this study, the clinically acceptable limit .Increasing the cement space from 30 to 50 Despite the limitations of this in vitro study, we were able to conclude the following:


Based on the results, pedicle or hemicortical freezing techniques are recommended in cases where these techniques can be applied.In this retrospective cohort study, predictive factors for graft survival were investigated in 123 patients who underwent reconstructions using a tumor-bearing frozen autograft after bone tumor resection of the extremities. The graft survival rates were 83.2% at 5 years and 70.2% at 10 years. Multivariate analysis using the Cox proportional hazards regression model revealed that BMI of ≥23.6 , tibia , and freezing procedure  were independently associated with graft survival. Based on the results, pedicle or hemicortical freezing techniques are recommended in cases where these techniques can be applied.Tumor-bearing frozen autografts have been widely used for reconstruction of bone defects caused by tumor resection. However, some patients undergo removal of the grafted bone due to surgical site infection, tumor recurrence, or fractures of the grafted bone. In this retrospective cohort study, predictive factors for graft survival were investigated in 123 patients who underwent reconstructions using a tumor-bearing frozen autograft after bone tumor resection of the extremities. To determine the independent predictors of graft survival, the association between various parameters and graft survival was investigated. The graft survival rates were 83.2% at 5 years and 70.2% at 10 years. Among the 123 frozen autografts, 25 (20.3%) were removed because of complications. In univariate analyses, male sex, BMI of ≥23.6, tibia, and chemotherapy were significantly associated with poor graft survival, whereas the pedicle/hemicortical freezing procedure was significantly associated with better graft survival. Multivariate analysis using the Cox proportional hazards regression model revealed that BMI of ≥23.6 (HR, 3.4;  The treatment of bone tumors involves tumor resection with appropriate surgical margins, chemotherapy, and radiation therapy. Reconstruction methods for bone defects after bone tumor resection are classified into endoprosthesis and biological reconstruction  ,5,6,7,8.Between January 1999 and August 2022, 139 patients with bone tumors in the extremities underwent tumor resection and reconstruction using a tumor-bearing frozen autograft. All patients or guardians provided written informed consent at the time of admission for inclusion in the scientific studies. This retrospective cohort study included 123 patients based on the following inclusion criteria: (1) histologically confirmed bone tumors, (2) located in the extremities, (3) reconstruction using a frozen autograft, and (4) follow-up period of ≥6 months . Five paThe associations among various parameters, including age, sex, body mass index (BMI), tumor location , tumor histology (primary or metastasis), chemotherapy, freezing method , type of implant , length of the frozen bone, operative time, intraoperative blood loss, and graft survival were investigated. Graft survival was defined as the time from the day of the operation to the day of removal of the tumor-bearing frozen autograft. This retrospective study was approved by the Medical Ethics Committee of our institute . Informed consent was obtained using the opt-out method, and the written informed consent was waived by the IRB. This study was registered in the Japan Registry of Clinical Trials. The work has been reported in line with the STROCSS criteria .The freezing procedure was performed using the following steps: freezing the tumor-bearing bone in liquid nitrogen for 20 min, thawing at room temperature for 15 min, and thawing in distilled water for 10 min . Freezinp < 0.05. The optimal cutoff levels of age, BMI, length of the frozen bone, operative time, and intraoperative blood loss were determined using time-dependent receiver operator characteristic analysis. Graft survival was calculated and compared using the Kaplan–Meier method with the log-rank test. To identify independent predictors of graft survival, multivariate analysis using the Cox proportional hazards regression model was performed with graft survival time as the dependent variable. All parameters with p < 0.05 in the univariate analyses were included in the Cox proportional hazards regression model. EZR statistical software  was used to perform all the statistical analyses.Statistical significance was set at The mean length of the grafted bone was 13.7 ± 6.1 cm. The 5- and 10-year graft survival rates were 83.2% and 70.2%, respectively. Among the 123 frozen autografts, 25 (20.3%) were removed because of complications, including infections in 12 (9.8%), local recurrences in 8 (6.5%), and fractures in 5 (4.1%). In patients who underwent graft removal, the mean time to graft removal was 49.3  months. p = 0.017). The 5-year graft survival rates in patients with BMIs of ≥23.6 and <23.6 were 68.4% and 87.1%, respectively (p < 0.001). The 5-year graft survival rates were 100% in the upper extremities, 84.4% in the femur, and 73.4% in the tibia. Graft survival in patients with tibial tumors was significantly lower than in those with tumors located at other sites (p = 0.034). The 5-year graft survival rates in patients who underwent chemotherapy and those who did not were 80.3% and 91.1% (p = 0.033), respectively. The 5-year graft survival rates were 100% in patients who underwent hemicortical freezing, 93.6% in those who underwent pedicle freezing, and 70.6% in those who underwent free freezing. The hemicortical/pedicle freezing procedure had significantly better graft survival than the free-freezing procedures (p = 0.001). The 5-year graft survival rates were 88.8% in reconstruction using plates, 85.1% in frozen autograft-prosthesis composite reconstruction, 71.1% in reconstruction using intramedullary nails, and 75.0% in other or combination use of implants. Although the use of intramedullary nails resulted in a lower graft survival rate, no significant difference was observed (p = 0.077). Age, tumor histology, length of the frozen autograft, operative time, and intraoperative blood loss were also not significantly associated with graft survival.In the univariate analyses, male sex, BMI of ≥23.6, tibial tumor, and chemotherapy were significantly associated with poor graft survival, whereas pedicle/hemicortical freezing procedures were associated with better graft survival . The 5-yp = 0.005), tibia , and hemicortical/pedicle freezing procedure  were independent predictors of graft survivals.To identify independent predictors for graft survival after reconstruction using tumor-bearing frozen autografts, male sex, BMI of ≥23.6, tibia, chemotherapy, and freezing procedure were included in the Cox proportional hazards regression models . MultivaIn studies on reconstructive surgery, various biological materials including skin, acellular dermal matrix , allograft, iliac or fibular graft, and devitalized bone graft have been reported ,18. AlthPrevious studies have reported an association between BMI and postoperative infection. In a meta-analysis study on the risk factors for postoperative infection after spine surgery, BMI and diabetes were associated with an increased risk of postoperative infection . In anotIn other studies, high failure rates were reported in patients who underwent reconstruction in the tibia ,36. In aThis study has several limitations, including the small number of study patients, heterogeneous sites, histology, implants, and reasons for graft removal. Because this study focused on patients who underwent reconstruction using frozen autografts, only 123 patients were included in the analyses. In this study, the reasons for removal of frozen autografts included infections, local recurrences, or fractures. Risk factors for each complication should be investigated in future studies. Furthermore, most of the study patients were Japanese, and the BMI and bone size of these patients may differ from those of patients of other races. To elucidate the predictors of graft survival in other races, further studies in various races are required.p = 0.005), tibia , and freezing procedure  were independent predictors of graft survival. Pedicle freezing and hemicortical freezing are recommended in cases where these techniques can be applied. In summary, the present study showed a BMI of ≥23.6 (HR, 3.4;


The tailored design of drug delivery systems for specific therapeutic agents is a prevailing approach in the field. In this paper, we present a study that highlights the potential of our modified starch, Q-starch, as a universal and adaptable drug delivery carrier for diverse therapeutic agents. We investigate the ability of Q-starch/cargo complexes to target different organelles within the cellular landscape, based on the specific activation sites of therapeutic agents. Plasmid DNA (pDNA), small interfering RNA (siRNA), and phosphatidylinositol -trisphosphate (PIP3) were chosen as representative therapeutic molecules, acting in the nucleus, cytoplasm, and membrane, respectively. By carrying out comprehensive characterizations, employing dynamic light scattering (DLS), determining the zeta potential, and using cryo-transmitting electron microscopy (cryo-TEM), we reveal the formation of nano-sized, positively charged, and spherical Q-starch complexes. Our results demonstrate that these complexes exhibit efficient cellular uptake, targeting their intended organelles while preserving their physical integrity and functionality. Notably, the intracellular path of the Q-starch/cargo complex is guided by the cargo itself, aligning with its unique biological activity site. This study elucidates the versatility and potency of Q-starch as a versatile drug delivery carrier, paving the way for novel applications offering targeted delivery strategies for potential therapeutic molecules. Various bioactive molecules, including small-molecule drugs, antibodies, proteins, RNAs, and DNAs, have been widely studied for the last few decades ,2,3,4,5.Polysaccharides are long-chain polymeric carbohydrates found in animals, plants, and microbial sources; they serve as an energy source and storage in addition to their structural role ,26. VariModified alginate, for example, has been used to prepare microcapsules through self-crosslinking by a template method targeting the nucleus . AnotherIn light of the above, we wondered if a more general approach might be feasible. Could the same polysaccharide carrier serve different biological agents for targeting different intracellular organelles? In this study, starch was modified using a quaternary amine group to form a positively charged carrier ,54,55. TThe three complex types were characterized, and their cellular uptake was observed in human immortalized keratinocytes (HaCAT), basal cell carcinoma (BCC), and melanoma cell lines. Next, after verifying that the complexes were able to reach their target sites, their biological activity was examined in different relevant cell lines. The Q-starch/pDNA complex was analyzed by transfection of the green fluorescent protein (GFP) gene to the nucleus of HaCAT cells using confocal microscopy. Q-starch/siRNA complexes were examined using siRNA that targets the GLI2 gene in basal cell carcinoma (BCC) cells, overexpressing the GLI2 gene ,59. GLI2Quaternary starch synthesis was carried out as previously described by Amar Lewis et al. . BrieflyDTAF, the dialyzed product was freeze-dried and lyophilized for 72 h so that complete dryness was obtained.Q-starch molecules were labeled using 5- amino fluorescein (5-DTAF) to be visualized by fluorescent microscopy. A 100 mg amount of Q-starch was dissolved in 3 mL DDW. The pH was adjusted to 11–12 with 1 M NaOH, and the mixture was stirred  for 30 min at room temperature. Dissolved 5-DTAF (7.5 mg of 5-DTAF in 0.3 mL DMSO) was added to the Q-starch solution. The solution was stirred  at room temperature under light exclusion for 24 h. The solution was neutralized with 0.1 M HCl and poured into a 14 KDa molecular weight cut-off dialysis tube. The dialysis tube was placed in a vessel containing PBS solution (pH 7.4) for 72 h and then for 48 h in DDW (18.3 MΩ∙cm). To obtain purified labeled Q-starch1H NMR, and elemental analysis (EA). FTIR was obtained in a Thermo-Nicolet FTIR spectrophotometer (Model-Nicolet iS50 FTIR), and samples were prepared in the form of potassium bromide (KBr) pellets. NMR spectra were recorded on a 500 MHz Brucker spectrometer using D2O as a solvent. Nitrogen content (N% weight) in the Q-starch product was measured by the elemental analysis method using a Thermo Scientific FLASH 2000 NC Analyzer.Starch quaternization was confirmed by Fourier transform infrared spectroscopy (FTIR), Escherichia coli DHα and purified using a HiSpeed Plasmid Maxi Kit from QIAGEN. Noncoding siRNA NC5 (siNC5) and siRNAGLI2 were purchased from Integrated DNA Technologies Inc. . The siNC5 sense sequence was 5′-CAUAUUGCGCGUAUAGUCGCGUUAG-3′, whereas the siRNAGLI2 sense sequence was 5′CAGAAUGAGGCUCGUAAUGGUACCUUC-3′. PIP3 (P-3916) was purchased from Echelon Bioscience.Plasmid pEGFP-C2 encoding for green fluorescence protein (GFP) was amplified in our laboratory in Complexes of different cargos were prepared at N/P molar ratios of 1 to 3; N/P is the molar ratio between the positively charged amine groups on the quaternized starch (N) and the negatively charged groups  on the cargo (P). N/P ratios between 1 and 3 were characterized in order to determine the ratio at which the complex’s zeta potential and size were most suitable for entering the cell. All the cargo molecules, i.e., pDNA, siRNA, and PIP3, were dissolved in Dnase- and RNase-free water to concentrations of 330 μg/mL, 20 μM, and 0.6 μg/μL, respectively. Q-starch was dissolved in Dnase- and RNase-free water to a concentration of 0.4 mg/mL and added in aliquots to the different cargo solutions. For example purposes, the procedure for preparing Q-starch/cargo complexes at N/P 2 is presented as follows: For Q-starch/pDNA complexes, 27.2 μL of Q-starch 0.4 mg/mL was introduced into tubes containing 11.5 μL of 330 μg/mL pDNA solution. For Q-starch/siRNA complexes, 6.6 μL of Q-starch 0.4 mg/mL was introduced into tubes containing 1.0 μL of 20 μM siRNA solution. For Q-starch/PIP3 complexes, 4.2 μL of Q-starch 0.4 mg/mL was introduced into tubes containing 1.0 μL (0.6 mg/mL) of 60-seconds-sonicated (Cole-Parmer Ultrasonic Cleaner) PIP3 solution. The mixed solution was gently vortexed  and incubated at room temperature for 40–60 min to allow the complex to form by self-assembly. For different N/P ratios, the volume of starch solution was changed accordingly. The hydrodynamic radius of the complexes was measured by DLS. Q-starch/cargo complexes were prepared as described above, reaching concentrations of 10 μg/mL plasmid-DNA, 250 nM siRNA, and 250 nM PIP3 in Eppendorf tubes containing the complexes at a final volume of 1 mL. The spectra were collected using a CGS-3  goniometer. The laser was powered with 20 mW at the He-Ne laser line (632.8 nm). Correlograms were calculated by an ALV/LSE 5003 cross-correlator, oriented at 90°, for 10 s, 30 times, at 25 °C. Each sample was placed into a thin-walled cylindrical glass cuvette and then placed in a vat filled with toluene as the optical matching fluid. A sample of each complex was then measured. The zeta potential of the complexes, indicative of each complex’s surface charge, was determined by a zeta sizer. The Q-starch/cargo complexes were prepared as mentioned, reaching a concentration of 5 μg/mL plasmid-DNA, 50 nM siRNA, and 50 nM PIP3 in an Eppendorf tube at a final volume of 1 mL. Samples were transferred to a U-tube cuvette  for measurement of zeta potential using Zetasizer . Each free cargo sample, free Q-starch, and samples of complexes at different N/P ratios in a range of 1–3 were measured in an automatic mode at 25 °C. The Smoluchowski model was used to calculate the zeta potential. The morphology of the complexes, as well as confirmation of their size, was evaluated by cryo-TEM. The Q-starch/cargo complexes were prepared to reach final concentrations of 10 μg/mL of plasmid-DNA, 250 nM siRNA, and 250 nM PIP3 in Eppendorf tubes at a final volume of 1 mL. Specimens of Q-starch/cargo complexes were prepared on a copper grid coated with a perforated lacey carbon 300 mesh  under controlled temperature (cryogenic temperature). A 3 µL drop of the analyzed solution was applied to the grid and blotted with filter paper to form a thin liquid layer. The blotted samples were immediately and automatically plunged into liquid ethane at its freezing point (−183 °C) using Plunger (Leica EM GP). The specimens were transferred into liquid nitrogen for storage. Samples were analyzed using FEI Tecnai 12 G2 TEM at 120 kV with a Gatan cryo-holder maintained at −180 °C. Images were recorded on a slow-scan cooled charge-coupled device camera  at low-dose conditions to minimize electron-beam radiation damage. The recording was carried out using the Digital Micrograph software package.2 flask. The human immortalized keratinocytes (HaCAT) cell line, basal-cell carcinoma (BCC) cell line, and melanoma cell line (A375) were selected as in vitro models in order to study the complexes’ target sites and their biological activity in relevant cell lines. HaCAT cells were grown in MEM Eagle growth medium containing 4.5 mM glucose, 10% vol. fetal bovine serum (FBS), 1% vol. L-glutamine (2 mM), and 1% vol. penicillin-streptomycin (100 µg/mL penicillin and 100 µg/mL streptomycin). BCC cells were cultured in DMEM growth media containing 10% vol. FBS and 1% vol. penicillin-streptomycin. Melanoma cells were cultured in RPMI growth media containing 1% vol. penicillin-streptomycin, 1% vol. L-glutamine, 1% vol. sodium pyruvate solution, 0.025% vol. HEPES buffer 1M, 0.125% vol. Nystatin suspension, and 10% vol. FBS. All cells were grown in a 75 cmv/v ratio). All cell-culture procedures were performed inside a laminar-flow hood that was sterilized before each procedure by UV light and by wiping the working area with 70% ethanol.Splitting of cells was performed with 2–3 mL of trypsin-EDTA every 3–4 days into 3–4 flasks to prevent high cell confluence and overpopulation of the culture. Following splitting, the trypsin was neutralized with 10 mL of growth medium. Cells were then well pipetted to ensure a homogeneous suspension and counted by the Countess™ II FL Automated Cell Counter. Trypan blue stain was used for counting viable cells by mixing 20 μL of the cell’s suspension extract with 20 μL of 0.4% trypan blue solution (1:1 4 HaCat cells/well were seeded in an eight-well plate (Ibidi µ-Slide 8-Well Glass Bottom 80827) for 24 h prior to the beginning of the experiment. On the day of treatment, cells were washed twice with PBS and stained with one or more of the following dyes according to the manufacturer’s instructions: NucBlue ; ViaFluor488 ; or CellBrite™ . After staining, cells were washed twice with PBS, and 300 µL from each of the complexes was added to the cells to final concentrations of 4 mg/mL of pDNA, 100 nM of labeled noncoding siRNA (siRNA NC5Cy5), and 500 nM of PIP3. Cells were incubated at 37 °C and 5% CO2 for a total of 4 h. Images were taken after 4 h using ZEN software on the LSM-880 confocal system . A Plan-Apochromat 40×/1.4 oil DIC M27 and 10× objective were used, and excitation was carried out with solid-state and argon lasers, as well as T-PMT for transmission light detection by a 32-channel GaAsP array. Images were acquired with EF6 and EF7 filters. The intracellular paths of Q-starch/siRNACy5 complexes were determined in melanoma A375 and BCC cell lines, in addition to HaCAT cells.For the cellular uptake, live confocal microscope samples of 5∙10Cy5 complexes was carried out with a custom-designed labeled siNC5 . For Q-starch/pDNA and Q-starch/PIP3 complexes, detection of fluorescently labeled Q-starchDTAF was carried out (excitation at 488 nm). Q-starch/cargo complexes were prepared at an N/P ratio of 5 for Q-starch/pDNA and an N/P ratio of 2 for both Q-starch/siRNACy5 and Q-starchDTAF/PIP3. These ratios were found to be the optimal N/P ratios to achieve high biological activity for these cell lines, based on previous work performed in our lab [In order to track the complexes’ intracellular paths by microscopic means, it was necessary to label the cargo or the carrier with fluorescent dyes. Detection of Q-starch/siNC5 our lab ,50. Labe4 HaCAT cells/well were seeded in Ibidi µ-Slide 8-Well Glass Bottom culture plates and grown to attain ~80% confluence on transfection day. Complexes of Q-starch/pGFP at a final concentration of 4 mg/mL pDNA, according to the N/P ratio of 5, were prepared in a serum-free media. A total of 300 µL of complex solution was added to the culture plate and incubated for 4 h at 37 °C under a 5% CO2 atmosphere. After 4 h, the media were replaced with fresh media containing serum; immediately after this, live images of the cells were taken using live confocal microscopy (LSM-880) for the detection of GFP expression. Untreated cells were used as a negative control.The Q-starch/pGFP transfection was carried out as previously described by Sieradzki et al. . Twenty-5 cells/well to reach a confluence of approximately 60–70%. On the day of transfection, Q-starch/siRNA complexes, either with non-targeting siRNA (siRNA NC5) or with siRNA targeting GLI2 transcription factor (siRNAGLI2), were prepared at an N/P ratio of 2 (based on previous work [2 for 72 h. Untreated cells were used as a negative control. BCC cells were seeded in a 6-well plate for 24 h in DMEM growth media at a concentration of ~1.8 × 10ous work ). Cell mTM cDNA Synthesis Kit  in a vol. % ratio of 5:4:1 . RNA samples were prepared for PCR analysis as follows: 10 µL from the master mix was added to 20 µL of the RNA samples in a PCR tube to reach a concentration of 20 ng/µL RNA, then it was placed in a PCR  to obtain cDNA. Following incubation, a series of RNA extraction and purification steps were performed using the PureLinkTM RNA Mini Kit  to extract the cells’ RNA. Samples were then prepared using the QuibitTM RNA BR Assay Kit , and RNA concentration was measured by the Qubit 3.0 Fluorometer . In line with the manufacturer’s instructions, a master mix solution was prepared on ice by gently mixing nuclease-free water, buffer, and enzymes from a qScript® Gene Expression Assay, INV/SM: GLI2:Hs01119974, AB-4331182, HPRT:Hs99999909, Rhenium) in a ratio of 6:10:1, i.e., 60 µL of nuclease-free water was mixed with 100 µL from the master mix and 10 µL of probe and primers assay. Then, 3 µL of cDNA sample was added to a well in a MicroAmp™ Fast Optical 96-Well Reaction Plate with Barcode , followed by the addition of 17 µL of stock solution, to reach a total volume of 20 µL in each well. Each group consisted of three different wells, resulting in three repetitions for each group. All gene expression values were normalized to the untreated cells control group, which was considered as 100% gene expression and normalized to an unaffected housekeeping gene HPRT.The produced cDNAs were subjected to real-time polymerase chain reaction (RT-PCR) testing  to determine gene silencing by quantifying GLI2’s mRNA expression. cDNAs were diluted from the produced PCR stock to 10 ng/µL (1:2 with nuclease-free water). A stock solution was prepared on ice by gently mixing nuclease-free water, master mix solution , and TaqMan probe and primers  for 24 h of incubation; 3. cells treated with 500 nM of free PIP3 for 3 h after 24 h of incubation with 10 µM of PI3K inhibitor (BYL719); 4. cells treated with 500 nM of Q-starch/PIP3 complexes for 3 h post 24 h incubation with 10 µm of PI3K inhibitor (BYL719).HaCAT cells were seeded at an initial concentration of 5∙10After treatment, the cell culture dish was placed on ice, and the cells were washed twice with ice-cold PBS. Lysis buffer was added to cells after the aspiration of the PBS. Adherent cells were scraped off the dish using a plastic cell scraper and transferred to microcentrifuge tubes. Then, the cells were centrifuged at 4 °C for 10 min at 14,000 rpm. The tubes were gently removed from the centrifuge and placed on ice; the supernatant was then removed and placed in a fresh tube kept on ice. Using the Bradford protein assay, the total protein concentration was measured. Amounts of 20 µL of each sample were placed in 96-well plates, and 180 µL of diluted (1:5 dilutions in DDW) Bradford solution (Bio-Rad Protein Assay Dye Reagent Concentrate) was added to each well. The plate was inserted into the Tecan Infinite M200 plate reader and analyzed at 595 nm. The samples were then diluted according to the resulting concentration and heated to 95 °C for 5 min. Equal amounts of protein, 10–20 µL , were loaded into the wells of the SDS-PAGE gel, along with a molecular-weight marker. The gel was then run for 1–2 h at 100 V. Then, the gel was placed between two transfer membranes , and air bubbles were gently pulled out. The cassette was inserted into the transfer blot machine, and the transfer was carried according to the manufacturer’s instructions. The membrane was then placed in a tray with a blocking solution consisting of 10 mL of TBST , and 0.5 g of BSA. Membranes were shaken gently with the blocking solution for 1 h at room temperature. The primary antibodies of actin, P-AKT 473, and total AKT  were added to the membranes at a concentration of 1:1000; the tray was placed in a cold room and tilted overnight. Membranes were then washed three times, for 5–7 min on each wash, with ~5 mL of TBST. Secondary anti-rabbit antibody conjugated to horseradish peroxidase  was added to 5 mL of blocking solution at a concentration of 1:20,000. Then, the secondary antibody solution was added to the membrane for 1 h of incubation at room temperature. After incubation, the membranes were washed three times with TBST for 5–7 min on each occasion. The membranes were dipped in an enhanced chemiluminescence solution (Cyanogen) and placed inside transparent nylon. The membranes were then inserted into the c300 Azure camera system , and images were captured.The positively charged carrier was obtained by substituting a quaternary positively charged amine group on the starch’s backbone using a basic pH environment, as described by Amar et al. . The subThe size, morphology, and surface charge properties of Q-starch/cargo complexes are all important parameters for achieving an efficient cargo delivery system. Because of this, and in line with previous studies ,55,56, tThe radii of the complexes were measured by DLS. As can be seen in The electrical potential of the complexes was measured using a zeta sizer. One of the barriers to entry of cargo into the cells is the electrostatic repulsion between the negatively charged cargo and the negatively charged cell membrane. By attaching the cargo to the positively charged carrier at different N/P ratios, it is possible to influence the overall charge of the complex and thus overcome this barrier. As we expected, and as shown in When comparing the complexes of siRNA and pDNA, notable differences in their charge can be observed. This disparity arises from the size discrepancy between siRNA and pDNA. As siRNA is considerably smaller, it binds more extensively to the positive nitrogen units on Q-starch, resulting in a less positive charge on the siRNA complex. Conversely, the larger size of pDNA leads to steric hindrances, limiting the binding of Q-starch’s positive sites to the negative sites of pDNA. Similarly, PIP3, which resembles siRNA in size, possesses a structure consisting of an inositol head and a hydrophobic tail. This unique configuration may hinder the connectivity between the sites of Q-starch and PIP3. Consequently, fewer positive sites of Q-starch bind to PIP3, thereby explaining the higher positive zeta potential observed in the complex with PIP3 as compared to siRNA.The size is affected in such a way that increasing the N/P ratio decreases the size of the complex. This can be explained by the compression of the Q-starch delivery system through stronger electrostatic bonds when increasing the N/P ratio.The morphology of the complexes was observed using Cryo-TEM. To assess our hypothesis regarding cargo-complex localization in the cell, it was essential to evaluate the cellular uptake and intracellular path of the different complexes. The Q-starch/cargo complexes’ cellular uptake was examined in the HaCAT cell line, where each complex’s type was prepared and incubated with the cells, as detailed in Cy5 complex’s cellular uptake at a concentration of 100 nM siRNA NC5Cy5 and an N/P ratio of 2. It can be seen  remained on the cell membrane. This result indicated that the cellular uptake of the complexes is not size-correlated and is probably affected by other properties of the complex.All the complexes in the cellular uptake experiments exhibited a positive zeta potential: 25.7 mV for Q-starch/pDNA; 9.95 mV for Q-starch/siRNA; and 18.5 mV for Q-starch/PIP3. The least-positive complex presented cellular uptake to the cytoplasm, while mid-range (Q-starch/PIP3) and high-range positivity (Q-starch/pDNA) was observed at the membrane and the nucleus, respectively. Thus, the preference of the complexes for entering different organelles in the cell cannot be explained by the difference in zeta potential.From this experiment, it can be assumed that the localization of the complex is cargo-dependent. That is, the entry of Q-starch/pDNA into the nucleus probably results from exposed pDNA at the surface of the complexes, which allows it to bind to DNA-binding proteins, chaperons, and nuclear-importing proteins, which allow movement toward the nucleus and further penetration into the nucleus through nuclear pores . siRNAs In addition to the size and charge of the complexes, the spatial structure of the complexes is not a the main affector for the targeted cellular uptake since all the complexes have a globular morphological structure. To determine if Q-starch might be used as a carrier for various therapeutic agents having different subcellular targets, the biological activity of the cargo was evaluated. We examined the biological activity in HaCAT and BCC cell lines for each of the complexes using a different assay for each complex.Plasmid transfection analysis was performed to determine the efficiency of Q-starch/pDNA complexes. The successful transfection of the plasmid would demonstrate the feasibility of using Q-starch as a suitable carrier for plasmids or other therapeutic molecules whose activities occur in the cell nucleus. Examined groups of free starch and free pDNA did not show emission at the suitable wavelengths of GFP. Because no expression of GFP was observed in the results for free pDNA shown in These results are proof of concept for further research of DNA-type cargo for therapeutic purposes in which Q-starch can be used as a suitable carrier. GLI2 complexes and Q-starch/siRNA NC5 complexes, both at an N/P ratio of 2 at a concentration of 35 nM, 72 h after incubation with the complexes. It can be observed that 72 h after transfection, Q-starch/siRNAGLI2 complexes successfully decreased mRNA levels to 37%, resulting in efficient silencing of the over-expressed GLI2 gene, i.e., 63% gene silencing compared to untreated cells, suggesting a significant influence of our delivery system and siRNA activity on the RNA mechanism. Gene expression analysis was performed to determine the efficiency of Q-starch/siRNA complexes as gene silencing agents. Decreasing the mRNA expression of the targeted genes of interest could shed light on the potential of the complexes as a therapeutic platform. To this end, transfection experiments were performed on the BCC cell line (TE-354.T) overexpressing the GLI2 gene, based on previous high-throughput screening assessing tumor-promoting pathways in our laboratory (results not shown) and on other previous studies ,74,75,76Western blot was performed to assess the effect of Q-starch/PIP3 on the phosphorylation of protein kinase B, also known as AKT. The activity of AKT was inhibited using PI3K inhibitors such as BYL719. After inhibition with BYL719, Q-starch/PIP3 or free PIP3 was added to assess whether exogenous PIP3 activated the inhibited AKT. The examination was on the phosphorylation site P-473, which was well studied and related to the activity of the AKT ,78,79. AThese results confirmed that the Q-starch/PIP3 complexes are located at the membrane and maintain the biological activity of PIP3. The external PIP3 activates the AKT pathway and thus can enhance migration, proliferation, and cell survival. These results demonstrate the suitability of Q-starch as a drug delivery system for therapeutic agents whose active site is the cellular membrane.In this study, we present an innovative approach utilizing a highly versatile Q-starch-based carrier capable of accommodating diverse cargo types, distinguishing it from other systems that are typically tailored to a specific cargo. Our findings demonstrate the adaptability of the Q-starch carrier, which enables targeted delivery to distinct cellular sites based on the unique activity mechanisms of different cargos. Notably, Q-starch/pDNA complexes specifically localize within the cell nucleus, Q-starch/siRNA complexes reside in the cytoplasm, and Q-starch/PIP3 complexes are situated at the cell membrane. Interestingly, each cargo exhibits its biological activity at its designated target site. Additionally, our study reveals that the cellular uptake of these complexes is not influenced by their size, morphology, or zeta potential, emphasizing the decisive role of the cargo itself in dictating the final localization within the cell.The significance and novelty of our research becomes evident as we shed light on the versatility of the Q-starch-based carrier. Our findings hold substantial implications, unveiling new possibilities for precise and efficient cargo delivery to desired cellular locations. This study paves the way for future investigations and potential breakthroughs in the targeted delivery systems of therapeutic molecules for different applications in various fields.


Reserve starch, the main component of durum wheat semolina, is constituted of two glucan homopolymers (amylose and amylopectin) that differ in their chemical structure. Amylose is mainly a linear structure formed of α-1,4-linked glucose units, with a lower polymerization degree, whereas amylopectin is a highly branched structure of α-1,4-chains linked by α-1,6-bonds. Variation of the amylose/amylopectin ratio has a profound effect on the starch properties which may impact the wheat technological and nutritional characteristics and their possible use in the food and non-food sector. In this work a set of genotypes, with a range of amylose from 14.9 to 57.8%, derived from the durum wheat cv. Svevo was characterised at biochemical and rheological level and used to produce pasta to better understand the role of amylose content in a common genetic background. A negative correlation was observed between amylose content and semolina swelling power, starch peak viscosity, and pasta stickiness. A worsening of the firmness was observed in the low amylose pasta compared to the control (cv. Svevo), whereas no difference was highlighted in the high amylose samples. The resistant starch was higher in the high amylose (HA) pasta compared to the control and low amylose (LA) pasta. Noteworthy, the extent of starch digestion was reduced in the HA pasta while the LA genotypes offered a higher starch digestion, suggesting other possible applications. Starch is the main carbohydrate in the endosperm of wheat grain, it is deposited in granules and makes up for 70–80% of flour weight. Starch composition includes two types of polyglucans, amylose and amylopectin, generally with a 1:3 ratio. Amylopectin is characterised by the presence of glucose residues connected through α--bonds and with considerably shorter chains and several α--branches, whereas the amylose is mainly linear and consists of glucose residues connected through α--linkages to form long chains with a low number α--branches [Amylose and amylopectin synthesis involve different classes of enzymes, with, a granule-bound starch synthase (GBSSI) involved in amylose synthesis, whereas amylopectin is produced by the coordinated action of different starch synthases , starch-branching enzymes  and starch-debranching enzymes of isoamylase and limit dextrinase-type (ISA and LD) (reviewed in ).The amylose/amylopectin ratio results in being a major contributor of the starch structure and its functional properties. The identification of genes involved in starch biosynthesis has enabled the possibility to manipulate the amylose/amylopectin ratio, through classical breeding, transgenesis, and mutagenesis ,4,5,6,7.Bread wheat possesses three GBSSI isoenzymes or waxy proteins, as they are commonly called, encoding genes that are present on the short arms of chromosomes 7A and 7D and on the long arm of chromosome 4A ,9. In duThe starch from waxy wheat can be used as a source of blending in the preparation of products where high water absorption, greater viscosities, high digestibility, and low retrogradation rate are desired. In particular, the use of waxy wheat flour or semolina allows modulation of the rate and the extent of starch retrogradation, with the result of increasing the shelf-life of baked products, retarding bread staling, improving the stability of frozen foods, and the palatability of baked and sheeted-wheat products ,13,14,15Starch digestibility plays an important role in human health . When foThe suppression of two genes has been found to be involved in increasing amylose content in cereals, the starch synthase IIa (SSIIa) and the branching enzymes IIa (SBEIIa), while both have been investigated in bread and durum wheat. The starch synthase IIa (SSIIa) is involved in the synthesis of intermediate amylopectin chains with regard to the degree of polymerisation (DP) 12–24 by elongating short chains (DP ≤ 10) of amylopectin . In duruIntrogression of the SSIIa null alleles in the durum wheat cultivar Svevo was obtained by Lafiandra et al.  with theSBEIIa gene. Sestili et al. [SBEIIa genes and increase the amylose content in the durum wheat cultivar Svevo up to 75% through an RNAi approach. The TILLING strategy was very effective in the identification of point mutations following EMS treatment and has been used by different authors to generate knockout mutants in SBEIIs genes in durum wheat [The other important target to obtain high amylose wheat is the i et al. , were abum wheat ,25,26. Sum wheat , reporteum wheat  used semGiven the number of studies described to modify the amylose content of durum wheat, there appear to be no studies comparing the impact on functional and starch digestion properties of a wide range in amylose content in a common genetic background, allowing for a better evaluation of the role of amylose content. Here dough properties, pasta quality, and in vitro starch digestion in pasta were evaluated in a set of starch durum wheat mutants with the same genetic background (cv. Svevo) but with different amounts of amylose (from 14.9 to 55%).−1) was split into three applications: the first was given before sowing as di-ammonium phosphate , the second when the first node was detectable above ground as urea , and the third 25 days later as ammonium nitrate . Each of the six genotypes are abbreviated as follows: Svevo is the control; Svevo LA = Svevo low amylose; Svevo Wx4A = Svevo waxy null 4A; Svevo Wx7A = Svevo waxy null 7A; Svevo SSIIa = Svevo starch synthase mutant IIa; Svevo SBEIIa = Svevo starch branching enzyme mutant IIa. Durum wheat lines Svevo Wx , Svevo SSIIa, and Svevo SBEIIa were previously produced ,23,26. TWheat was cleaned, conditioned to a water content of about 16.5% and left to moisten overnight. Standard milling was performed in a Buhler MLU 202 mill  with three breaking and three sizing passages . SemolinSpaghetti was prepared as previously described  but withStarch was isolated from semolina and used for analysis of viscosity using a Rapid Visco Analyser (RVA4) as described previously . Starch The incremental area under the digestion curves (AUC) was calculated. Duplicate digestions were performed on separate days. Ct is the percentage of starch digested at a given time (t), C∞ is the estimated percentage of starch digested at the end point of the reaction, and k is the starch digestion rate coefficient. In order to obtain the values of k and C∞, this equation can be transformed into a logarithm of slope analysis plot where there is a linear relationship between ln(dCt/dt) and k as follows:Starch digestion data were fitted to a first-order equation: k and C∞ are calculated from the slope (−k) and intercept (ln(C∞k)), respectively. p < 0.05. Data were checked for normality. Data were analysed using the statistical programme GenStat version 17.1.0.14713 with a generalised linear model and the means were tested for significant differences by the least significant difference statistic (LSD), using Glu-A1 null; Glu-B1 7 + 8) and low molecular weight glutenin subunits (data not shown) with differences only in their protein content arising during plant growth in the field  than the other samples (except Svevo SSIIa) that were all similar and took longer for the central starch core to disappear . A possiThe overcooking tolerance or resistance to firmness reduction is a good measure of cooking tolerance and pasta should resist being overcooked while still retaining al dente (having some firmness to the bite). The lower this value, the more tolerant the pasta is to firmness loss due to overcooking, which is desirable. It is clear that the Svevo LA had the worst overcooking tolerance losing a lot of its firmness after 10 min overcooking, consistent with a softer pasta typical of waxy wheat. For the Svevo LA we also find a significantly higher pasta stickiness peak height consistent with other reports  due to eThe starch pasting curves for the Svevo samples are shown in 1, k2 and extent of digestion, C∞ % I and C∞ % II , starch peak viscosity (~89%), and pasta stickiness area (~52%), while increasing the semolina water absorption (with the exception of Svevo LA) (~14.5%) and the pasta resistant starch (~66 fold). There was an indication that the dough properties of Svevo SBEIIa had weaker dough than Svevo but the HA pasta had acceptable pasta quality, with sensory analysis still needed to determine consumer preference for this kind of HA pasta with superior nutritional value. Importantly, the extent of starch digestion was reduced in the HA pasta, especially the Svevo SBEIIa, while Svevo LA offers a higher starch digestion more suited to other product applications.