To compare drug survival of ixekizumab to other IL‐17 inhibitors (IL‐17i) and TNF inhibitors (TNFi) among patients with psoriasis (PsO) in a real‐world setting. Participants included adult PsO patients enrolled in the Corrona Psoriasis Registry who initiated ixekizumab, TNFi, or other IL‐17i between 16 March 2016 to 10 August 2019 and completed ≥1 follow‐up visit. Multivariable adjusted hazard ratios (HR) were calculated to estimate the risk for drug discontinuation in the ixekizumab group relative to the other drugs. Among the 1604 drug initiations, 552 initiated ixekizumab, 450 initiated TNFi, and 602 initiated other IL‐17i. Mean age was 51 years, 49% were women, and 52% were obese (BMI > 30). Ixekizumab patients had a higher proportion of patients with PASI >12 at drug initiation (24%) than TNFi (15%) and other IL‐17i (19%). Over a median of 11 months of follow‐up, 723/1604 (45%) drug discontinuations occurred. Persistence of ixekizumab, TNFi, and other IL‐17i at 24‐months were 68%, 33%, and 46%, among biologic‐naïve patients (n = 543), and 46%, 23%, and 36%, for biologic‐experienced patients (n = 1061), respectively. Ixekizumab patients had a 64% lower risk of discontinuation vs TNFi  and a 31% lower risk vs other IL‐17i  after adjustment for biologic experience and other covariates. HRs were similar when limited to patients with moderate‐to‐severe PsO  at initiation. In our study of real‐world patients with PsO, initiators of ixekizumab had more prolonged drug survival than both initiators of TNFi and other IL‐17i up to 2 years of follow‐up. In rheumatology, ixekizumab has been approved for active psoriatic arthritis, ankylosing spondylitis, and nonradiographic axial spondyloarthritis (nr‐axSpA).When investigating select categories of adverse events (AEs) , inflammatory bowel disease (IBD) , and serious hypersensitivity events among all patients exposed to ixekizumab in the Corrona PsO Registry, Grace et alCurrently, there are few studies comparing the drug survival for ixekizumab to that of other biologic therapies in real‐world patients with PsO. Two recent claims‐based studies of US patients demonstrated greater drug survival for ixekizumab vs adalimumabThe objective of the current study was to compare drug survival of ixekizumab to that of TNF inhibitors (TNFi) and non‐ixekizumab IL‐17 inhibitors (IL‐17i) among real‐world patients with PsO in the Corrona Psoriasis Registry.22.1This analysis used data from the Corrona Psoriasis Registry, a prospective, multicenter observational disease‐based registry launched in April 2015 in collaboration with the National Psoriasis Foundation, the design of which has been previously described.2.2The study population included 1604 patient drug initiations  that occurred between 22 March 2016  and 10 August 2019 . Initiations were required to occur at or after registry enrollment, initiators had at least one subsequent follow‐up (FU) visit, and an available index visit (defined as the visit coinciding with or within 4 months before initiation) at which baseline characteristics were assessed.2.3The primary outcome was drug survival, defined as persistent use of drug during follow‐up, allowing for a medication gap of ≤60 days. Drug survival time was defined from the date of drug initiation through the date of drug discontinuation. Patients persistent on drug at their last follow‐up visit were censored on the date of their last follow‐up visit.2.4Baseline characteristics included: sociodemographics ; PsO disease characteristics ; disease severity , body surface area (BSA), Investigator Global Assessment (IGA); prior systemic therapy (biologic and nonbiologic); concomitant PsO treatment ; and patient‐reported outcomes (PROs) .2.5Z score of a standard normal distribution and a threshold greater than 0.1 was the criteria used to represent a relevant imbalance between groups. Additionally, thresholds of 0.2, 0.5, and 0.8 are often used to indicate small, medium, and large effect sizes.d) > 0.10. Selection of variables was based upon examination of collinearity between candidate variables , to avoid multicollinearity in Model 2. The additional covariates selected for adjustment in Model 2 were race, education, work status, geographic region, smoking history, history of Crohn's disease, ulcerative colitis, indeterminate IBD, and other gastrointestinal (GI) disorders, history of infections, erythrodermic morphology, inverse/intertriginous morphology, nail morphology, IGA, PASI, concomitant topical therapy, patient‐reported overall itch/pruritis, and patient‐reported health state today (EQ‐VAS).Baseline characteristics were summarized descriptively for the treatment groups . Pairwise standardized differences were used to assess differences in characteristics between the treatment groups. A standardized difference effect size is equivalent to a Sensitivity analyses were conducted for the subset of initiations with moderate‐to‐severe disease activity at baseline . The proportional hazards assumption was evaluated for the models by examining Schoenfeld residuals3There were 1604 initiations among 1412 unique patients that met the inclusion criteria for analysis of drug survival . Duration of PsO was comparable between initiators of ixekizumab and non‐ixekizumab IL‐17i , while duration of disease for initiators of TNFi was shorter . Comorbid psoriatic arthritis was reported by dermatologists for 47% of initiators of ixekizumab, which was slightly lower than that reported for initiators of TNFi (51%), and non‐ixekizumab IL‐17i (54%). Compared with TNFi and non‐ixekizumab IL‐17i initiators, ixekizumab initiators had a higher proportion of patients with PASI >12  and severe IGA   , but not for patient global assessment of PsO . When evaluating WPAI domains, TNFi initiators had a higher mean overall percent of work hours affected by psoriasis (18.9 vs16.1) compared with ixekizumab initiators, although the effect size was small (d = 0.12). Ixekizumab initiators reported lower mean patient‐reported overall fatigue (VAS range 0‐100)  and more ixekizumab initiators (53%) had taken ≥2 previous biologics than patients on TNFi (17%) and non‐ixekizumab IL‐17i (40%).Table There were 723 drug discontinuations among all patient initiations. The median follow‐up time for those persistent at their last Corrona visit was 11 months , and for all initiations, including those resulting in discontinuation, was 8 months . Among biologic‐naïve patients, ixekizumab showed a drug survival trajectory that started diverging versus the non‐ixekizumab IL‐17i after the first 6 months of treatment, at which point ixekizumab began to demonstrate increased persistency out to 24 months Figure , though P < .001), and a 34% lower relative risk of discontinuation in comparison to the non‐ixekizumab IL‐17i group . Hazard ratios were like the observed data after adjustment in multivariable models. The adjusted hazard ratio from Model 2 for the ixekizumab group in comparison to the TNFi group was 0.36 , and for the ixekizumab group in comparison to other non‐ixekizumab IL‐17i group was 0.69   Table  and S2.P < .001), and for the ixekizumab group in comparison to the non‐ixekizumab IL‐17i group was 0.69 .In sensitivity analyses, estimates of drug survival and hazard ratios were similar within the subgroup of patients with moderate‐to‐severe disease activity at baseline Tables  and 4. A4Among patients with PsO in a large North American registry, ixekizumab demonstrated higher drug survival compared with TNFi and non‐ixekizumab IL‐17i therapies. Patterns of drug survival by drug group over 24‐months of follow‐up were similar in both biologic‐naïve and biologic‐experienced patients. Although ixekizumab initiators compared with TNFi and non‐ixekizumab IL‐17 initiators were more likely biologic‐experienced, obese, had a higher prevalence of nail morphology, worked full‐time, and had more severe disease, drug survival differences favorable to ixekizumab initiators remained even after adjusting for potential confounders. These factors, in particular previous biologic experience, often have a negative impact on drug survival. However, in our study, ixekizumab still showed better drug survival.Drug survival of TNFi such as adalimumabBecause line of therapy influences drug survival, analyses were stratified by biologic‐naïve and biologic‐experienced patients. In the current study, 66% (n = 1061) occurred among biologic‐naïve patients, and 34% (n = 543) of initiations occurred among biologic‐experienced patients. Although TNFi are still largely adopted as a first line biologic, among our biologic‐naïve patients, the TNFi group had the lowest drug survival, with 46% and 33% at 12 and 24 months, respectively. As expected, drug survival was worse among biologic‐experienced patients compared with biologic‐naïve patients, yet in both cohorts, ixekizumab had better survival than TNFi and non‐ixekizumab IL‐17i. Our 12‐month drug‐survival rates among biologic‐experienced patients of 65% for ixekizumab vs 34% for TNFi, and 54% for non‐ixekizumab IL‐17i initiators, are comparable to those recently reported for adalimumab The results from the current study, which found ixekizumab initiators experienced longer drug survival compared with TNFi and non‐ixekizumab IL‐17 initiators, are also consistent with studies that have investigated drug survival of ixekizumab in a real‐world setting. A small, retrospective, clinic‐based study conducted between 22 March 2016,  and 25 November 2018, found that 14 of 22 patients (64%) were still taking ixekizumab at the end of the study period and had been taking it for an average of 379 days.Despite the efficacy and safety profile described for the newer generation of biologics in the treatment of psoriatic diseases, Murage et alOur study is not without limitations. The data source is a North American Registry, and the results may not be generalizable to other regions. The data are subject to limitations inherent in all observational studies, such as the lack of randomization to patients for different biologics and the unknown patient factors that may be associated with access to care. Estimation of drug survival between treatments should also be cautiously interpreted when using nonrandomized data. Nevertheless, our study has important strengths. Data for the current study were collected across the United States and Canada from both academic and private practice dermatologists, and these patients are more likely to reflect the typical real‐world patient population than those in clinical trials. The duration and 2‐year follow up of the current study were reasonable and clinically meaningful. We were able to adjust for several potential confounders, including baseline disease severity, clinical outcomes, and treatment patterns, which are not captured in claims databases.5The current study from a large cohort of North American PsO patients demonstrates that over 2 years ixekizumab has a better drug survival compared with TNFi and non‐ixekizumab IL‐17i. The findings were consistent in both biologic‐naïve and biologic‐experienced patients. The current study data offer valuable evidence regarding drug performance and real‐life differences, which may be taken into clinicians' consideration when choosing between biologics.Benjamin Lockshin, Grant/Research Support: AbbVie, Regeneron, Lilly, Franklin, DermTech, Trevi, Dermira, Celgene, Novartis, LEO, Strata; Consultant: AbbVie, DermTech; Speakers Bureau: AbbVie, Lilly, Novartis, Regeneron, Sanofi; Angel Cronin, Employee of Corrona LLC; Ryan W. Harrison, Employee of Corrona LLC; Robert R. McLean, Employee of Corrona LLC; Laura Anatale‐Tardiff, Employee of Corrona LLC; Russel Burge, Employee/Stock, Eli Lilly and Company; Baojin Zhu, Employee/Stock, Eli Lilly and Company; William N. Malatestinic, Employee/Stock, Eli Lilly and Company; Bilal Atiya, Employee/Stock, Eli Lilly and Company; Mwangi J. Murage, Employee/Stock, Eli Lilly and Company; Gaia Gallo, Employee/Stock, Eli Lilly and Company; Bruce Strober, Investigator: AbbVie, Dermavant, Corrona, Dermira; Speaker for AbbVie, Lilly, Janssen, Ortho Dermatologics; Consultant (Honoraria): AbbVie, Almirall, Amgen, Arena, Aristea Therapeutics, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Dermavant, Dermira, Janssen, LEO Pharma, Eli Lilly, Kyowa Hakko Kirin, Meiji Seika Pharma, Novartis, Pfizer, GlaxoSmithKline, UCB, Sun Pharma, Ortho Dermatologics, Regeneron, Sanofi‐Genzyme; Scientific Director (consulting fee): Corrona Psoriasis Registry; Abby Van Voorhees, Advisory Board: Celgene; Consultant: Celgene, DermTech, Lilly, ICOS, Novartis, UCB, WebMD; Grants/Research Support: AbbVie, Celgene, Lilly, ICOS; Other: Merck.TABLE S1 Adjusted model 1 (prespecified covariates): hazard ratios from multivariable Cox proportional hazards regressionTABLE S2 Adjusted model 2 (prespecified covariates plus consideration of characteristics with standardized differences >0.10): hazard ratios from multivariable Cox proportional hazards regressionClick here for additional data file.


In living donor liver transplantation (LDLT) for patients with Budd‒Chiari syndrome (BCS), there are several concerns about reconstruction of the inferior vena cava (IVC) and hepatic veins. Herein, we report the case of a patient with BCS who underwent LDLT with right posterior segment graft (RPSG) and patch plasty for reconstruction of the hepatic venous outflow, using the patient’s own superficial femoral vein (SFV).A 19-year-old man, who was diagnosed with primary BCS, underwent LDLT. His main hepatic veins were totally obstructed, and membranous stenosis was seen in the IVC. The LDLT donor was his mother; however, liver volumetric analysis showed that only her RPSG was appropriate. In the recipient surgery, 16 cm of the left SFV was harvested and was cut longitudinally and opened. The right hepatic vein (RHV) of the RPSG was anastomosed to the sidewall of the SFV graft. After explantation of native diseased liver was completed, the stenotic and thickened wall of the IVC was widely resected, and a large anastomotic orifice was created. Patch cavoplasty was performed with the RHV‒SFV graft patch. After portal reperfusion started, hepatic venous outflow was satisfactory, and there was no venous graft congestion. Both his postoperative course and his long-term course after discharge were uneventful.In LDLT for BCS patients, ingenuity is required for the reconstruction of venous outflow. The SFV patch can be safely harvested from liver transplant recipients and is suitable for venous reconstruction. In addition, RPSG is an alternative type of liver graft for LDLT if a conventional right- or left-lobe graft cannot be used. Budd‒Chiari syndrome (BCS) is a rare disease involving obstruction of the hepatic venous outflow at various levels from the small hepatic vein to the suprahepatic lesion of the inferior vena cava (IVC) , 2. This3/ml, 56.2%, respectively). His Child–Pugh score was 11 (grade C), and his Model for End-stage Liver Disease score was 14. He was considered to have no blood disorders that could cause coagulation abnormalities, such as abnormal protein C and S activities. Accordingly, he was diagnosed with primary BCS and decompensated liver failure. Endovascular treatment for the main hepatic veins was considered; however, we recognized that it could be impossible to restore the patency of the hepatic vessels, and we did not perform the preoperative angiography. In addition, his esophagogastric varices were severe. For these reasons, we decided to perform LDLT 5 months after referral.A 19-year-old man with no medical history or relevant family history was referred to our hospital because of elevated liver enzyme levels and abdominal distention. An abdominal computed tomography (CT) scan showed that his three main hepatic veins were completely obstructed and that the suprahepatic IVC had stenosis involving a membranous-like structure Fig. . The patDonor candidates for LDLT were limited in this case. The eventual donor was his 46-year-old mother, who had no notable medical history and whose blood type was O Rh (+), identical to that of the patient. Based on preoperative CT investigation, we calculated that her estimated whole liver volume was 1047 ml. Her estimated left-lobe graft volume, estimated graft-to-recipient weight ratio (GRWR), and estimated ratio of liver remnant were 303 ml, 0.49, and 71.0%, respectively, which indicated that the size of the left-lobe graft was insufficient for the patient. On the other hand, in the right-lobe graft without the middle hepatic vein, the estimated graft volume was 734.2 ml, and the estimated GRWR was 1.19, which were sufficient for the patient. However, the estimated liver remnant was only 285.7 ml (31%), which was not applicable for our donor selection criteria. Under these mismatches, neither left-lobe graft nor right-lobe graft could be selected. For these reasons, we considered her RPSG, which could provide sufficient liver volume for the patient (estimated graft volume was 609 ml and GRWR was 0.99), and a safe residual liver volume for the donor (estimated ratio of liver remnant was 38.1%). There were no identified abnormalities of the portal vein, hepatic artery, bile duct, or hepatic vein in preoperative CT and MRI.Donor hepatectomy was performed with a mid-line incision, and liver mobilization was performed using a laparoscopy-assisted technique. During mobilization, the root of the middle hepatic vein and the right hepatic vein (RHV) were identified. Then, anterior and posterior branches of the hepatic artery and portal vein were skeletonized. The transection line was marked on the liver surface according to the RHV and to the demarcation line when the posterior branch of the portal vein was temporarily clamped. Following a hanging maneuver, parenchymal transection was performed using a cavitron ultrasonic surgical aspiration system without occlusion of portal vein inflow. After transection of the liver parenchyma was completely finished, the biliary duct and portal vein of the right posterior branches were cut. Because the right posterior branch of the hepatic artery was thin and it was considered that anastomosis of this artery with recipient hepatic artery increased risks of artery-related complications, the donor’s right anterior branch of the hepatic artery was sacrificed after the artery blood flow in the anterior liver segment was detected by Doppler ultrasound with temporary clamping of the right hepatic artery, and then the right hepatic artery was cut at the root. Finally, the inferior right hepatic vein (IRHV) and RHV were cut, and the liver graft was harvested. The RPSG was flushed with 1000 ml of University of Wisconsin solution from the portal vein. The actual RPSG weight was 570 g.Recipient surgery was started simultaneously with donor surgery. Prior to the abdominal incision, 16 cm of the SFV was harvested from his left leg for reconstruction of the IVC and hepatic vein. During the operation, there were approximately 3500 ml of ascites, and dense adhesions were seen around the IVC. The native liver was dark and hardened because of the liver congestion. The common bile duct, right and left hepatic arteries, and portal veins were cut, and then all three major hepatic veins, which were thickened and scarred, were cut while preserving the recipient IVC. Explantation of native, diseased liver was completed. The diseased liver weight was 1765 g, and the roots of three major hepatic veins were completely occluded.At bench surgery, a longitudinal incision was made from the caudal side of the SFV graft, followed by ligation of the cranial side of the SFV graft. The graft RHV and IRHV was anastomosed to the sidewall of the SFV graft for patch plasty of the IVC Fig. a, b.Fig.For graft implantation, the infrahepatic IVC was mobilized and exposed, and then cross-clamping was performed. Venoveno bypass was not used during graft implantation because the collaterals were well developed, and the hemodynamic parameters were stable after IVC clamping. The thickened anterior suprahepatic IVC was longitudinally cut and opened, and the stenotic lesion of the IVC was identified Fig. a. There The donor had no complications and recovered rapidly, and was discharged at postoperative day (POD) 9. The recipient’s postoperative course was also uneventful. Daily Doppler ultrasound revealed patency of the RHV‒SFV graft without venous congestion of the liver, and postoperative CT imaging clearly showed the graft RHV and IRHV with no stenosis in the IVC Fig. a, b. At Budd‒Chiari syndrome is a rare disease and is characterized by an obstruction of the hepatic venous outflow tract, which is located at the level of the small or large hepatic veins, or on the suprahepatic portion of the IVC. This hepatic venous obstruction increases hepatic sinusoidal pressure followed by portal hypertension, which causes liver injury and eventually liver cirrhosis , 2. DiffFor patients with BCS, endovascular treatments, such as angioplasty, stenting, and local thrombolysis, are first conducted. In patients with liver cirrhosis and portal hypertension, a transjugular intrahepatic portosystemic shunt or surgical venous shunt should be considered to decrease the portosystemic gradient . AdditioIn DDLT for patients with BCS, suprahepatic caval resection and replacement of the IVC are considered a standard and adequate procedure. In contrast, because the deceased donation rate is low and there is a critical shortage of liver grafts, many patients with BCS in Asian countries undergo LDLT. In LDLT for patients with BCS, the key considerations in surgical procedures are the management of stenosis and occlusion of the IVC and reconstruction of the graft hepatic veins . Due to Additionally, this case underwent LDLT with a RPSG for BCS patient, which has not been reported previously. In adult-to-adult LDLT, right-lobe graft and left-lobe grafts are mainly used. Because of the limited number of donor candidates in many Asian countries, we frequently encounter problems related to an adequate graft size between recipients and donors. Insufficient volume of the left-lobe graft would lead to a small-for-size graft syndrome in recipients. In contrast, the right-lobe graft usually satisfies the liver volume in recipients. However, if donation of the right-lobe graft would induce too small remnant liver volume for the donor, for example, if the remnant liver volume is less than 30%, safe donation is not possible . AlthougThis case report describes a patient with BCS who underwent LDLT with RPSG and patch plasty for reconstruction of the hepatic venous outflow using an SFV graft; this approach has not been reported previously. In LDLT for patients with BCS, marked ingenuity is required to perform optimal treatment under limited conditions. The SFV patch can be safely harvested and is suitable for venous reconstruction.


Musa acuminata  and Musa sapientum L.  growing in Chiang Rai (Thailand) were used for extraction. The yield of the starches was 16.88% for Hom Khieo (HK) and 22.73% for Namwa (NW) based on unripe peeled banana fruit. The amylose contents of HK and NW were 24.99% and 26.23%, respectively. The morphology of starch granules was oval shape with elongated forms for large granules and round shape for small granules. The HK and NW showed B-type crystalline structure and the crystallinities were 23.54% and 26.83%, respectively. The peak temperature of gelatinization was around 77 °C and the enthalpy change (ΔH) was 3.05 and 7.76 J/g, respectively. The HK and NW banana starches showed 1.27 ± 0.12 g/g and 1.53 ± 0.12 g/g water absorption capacity, and 1.22 ± 0.11 g/g and 1.16 ± 0.12 g/g oil absorption capacity, respectively. The swelling power of the banana starches was 17.23 ± 0.94 g/g and 15.90 ± 0.15 g/g, respectively, and the percentage of solubility in water showed 26.43 ± 2.50 g/g and 20.54 ± 0.94 g/g, respectively. The banana starches showed very poor flow character. The HK and NW starches have the potential to be used in powder base preparations with no effect on the sensory texture of the product at 15% w/w maximum.Unripe banana fruit of  Musaceae are monocotyledon herbaceous plants that represent hybrid-polyploid complexes of Musa acuminata (AA) and Musa balbisiana (BB), as diploids, triploids, and tetraploids [M. balbisiana (B genes) tend to be more tolerate and produce more starch than bananas with high proportion of M. acuminata (A genes) [Starch is a natural polysaccharide which is a semi-crystalline structure consisting of amylose and amylopectin. Amylose is a straight chain, whereas amylopectin has a branched chain structure. Starch can be extracted from various plant sources, especially tropical plants such as cereals, tubers, and some unripe fruits which have polysaccharide contents are in the range of 60–90% ,2. Bananraploids . GeneralA genes) . The maiIn Thailand, bananas are called “Kluai” and the varieties that widely cultivated in Thailand are Kluai Namwa (ABB group), Kluai Hom (AAA group), Kluai Khai (AA group) and Kluai Lep Meu Nang (AA group) . Most baM. acuminata (AAA group) [sapientum subgroup in the ABB group, was investigated alongside, and compared to, the HK starch in terms of yield and properties. Additionally, the utilization of starch as an ingredient in cosmetic formulation was also described.Recently, we have preliminarily reported the basic properties such as morphology, amylose content, crystalline structure and the thermal properties of A group) . In thisM. paradisiaca (AAB) and M. acuminata (AAA) were 43.8% and 11.8%, respectively. Chávez-Salazar et al. [M. paradisiaca (AAB), and M. acuminata (AAA) were 32.33% and 18.73%, respectively. The variety and texture of fruit affect the yield of starch, lower firmness results in lower yield than higher firmness, which is related to the stage of ripeness [The percentage yield of HK and NW that was obtained from extraction with 1% sodium sulfite was 16.88 and 22.73%, respectively, calculated based on fresh peeled banana. It can be seen that NW (Musa ABB) had higher yield than HK (Musa AAA), as it has been reported that bananas with high proportion of B genes tend to produce more starch than that with high proportion of A genes . Howeverr et al.  reportedripeness .The starches showed light yellow color  with finStarch is a polysaccharide, consisting of amylose and amylopectin. In general, common starch constitutes of 20–30% amylose and 70–80% amylopectin . AmyloseThe percentage of moisture content of HK and NW were 13.10 ± 0.10% and 13.40 ± 0.40%, respectively. The moisture content was similoar to that found by Barros Mesquita et al.  which reThe HK and NW starch granules exhibited various sizes and shapes. The large size of both HK and NW starches was an oval shape with elongated form. The average size of HK was around 39 µm, which was close to that of NW (37 µm), while the smaller size of HK and NW was round shape with a size around 10 µm. The HK and NW granules had a higher proportion of large sizes than smaller sizes. The appearance of granules from the SEM showed that their surfaces were smooth and dense .The banana starch granules were similar to the previous studies of various cultivars which were irregular elongated oval shape with dense and smooth surfaces ,27,28,29The morphology of starch depends on the botanical source that affects the shapes and size of starch granules. Starch granules of corn are angular-shaped with 1–7 µm for small granules and 15–20 µm for large granules. Rice starch granules are pentagonal with angular-shapes and the granules sizes range from 3 to 5 µm. Whereas potato starch granules are oval and irregular shapes with 1–20 µm for the granule size. The SEM images of potato starch granules are smoother than those of corn and rice starch granules . In this−1 (O-H stretching); 2900 cm−1 (C-H stretching); 1650 cm−1 (C-O associated with OH group); 1424 cm−1 (CH2 symmetric deformation); 1370 cm−1 (C-H symmetric bending); 1160 cm−1 (C-O-C asymmetric stretching); 1082 cm−1 and 1000 cm−1 (C-O stretching); and 930 cm−1, 860 cm−1, and 767 cm−1 (C-O-C ring vibration of carbohydrate). The FT-IR spectra obtained from this study were similar to those from a previous report [The FT-IR spectra of HK and NW starch samples are shown in s report .θ of about 15°, and 23° and small peaks at about 17° and 18°. However, the strong peaks of B-type presented at about 17° and small peaks at about 15°, 20°, 22°, and 24° with a characteristic peak at about 5°. The peaks of C-type starch were a mixture of A-type and B-type at 2θs of about 17° and 23° and small peaks at 5° and 15° [Starch is a semi-crystalline material affected by amylose content and amylopectin chain length that consists of amorphous and crystalline regions. The crystalline region is parallel of glucan chains (amylopectin) that are closely packed and ordered, whereas the amorphous region is the linear region (amylose). The amylose content directly affects the crystallinity degree of the starch, such that when there is a lack of amylose content, the crystallinity degree increases, whereas the longer chain amylopectin forms have a more stable crystalline structure ,33. Gene and 15° ,35,36.θs of about 15.12°, 17.07° and a doublet at 22.13° and 23.64°, with weak diffraction peaks at 5.31° and 19.83°, with 23.54% crystallinity. The NW was identified by major diffraction peaks at 2θs of 15.30°, 17.22°, and a doublet at 22.70° and 23.90° with weak diffraction peaks at 5.76° and 19.81°, with 26.84% crystallinity. From the X-ray pattern of the HK and NW starch granules, they were determined to be B-type starches.The X-ray diffraction pattern of HK was similar to that of NW starches . The HK The major difference between A- and B-types of starch granules is the arrangement of double helices. A-type starches form dense packing with 4 water molecules, whereas B-type starch is more open causing more water molecules (36 water molecules) to be located in the center of a hexagonal packing of helices. For this reason, it is indicated that the A-type is more stable and requires a higher temperature than B-type starch for gelatinization ,34,37.The differential scanning calorimetry (DSC) and gelation methods were used to determine the thermal properties of banana starches. The thermal properties from DSC were measured in terms of transition temperature; onset (To), the starting point of starch gelatinization; peak (Tp), the point where a complete loss of crystalline occurs; and end set (Te), the final temperature required to complete the gelatinization. The enthalpy (ΔH) is consistent with the changes of energy during melting of the crystallites and measures the crystallinity or damage in the starch structure before gelatinization occurs ,39,40.The HK banana starch has an onset gelatinization temperature (To) of 74.52 °C, a peak gelatinization temperature (Tp) of 77.97 °C, and an endpoint gelatinization temperature (Te) of 80.37 °C, with gelatinization enthalpy (ΔH) of 3.05 J/g. The NW banana starch has an onset gelatinization temperature (To) of 73.64 °C, a peak gelatinization temperature (Tp) of 76.98 °C, and an endpoint gelatinization temperature (Te) of 80.69 °C, with gelatinization enthalpy (ΔH) of 7.76 J/g . The resw/v) at different temperatures  and it was found that all samples at temperatures lower than 80 °C with low concentrations (0.5–6% w/v) did not show gel formation and high concentrations (8–14% w/v), showed some of gel forming. At 80 °C, the gelation of HK and NW starches are shown in w/v showed some gel forming. At 8% w/v the starch presented the gel formation stage. The banana starch showed complete gel formation at 10 to 14% w/v. The results indicated that the gelatinization temperatures of HK and NW obtained from the DSC method were related to those found by the gelation method.According to Mar et al.  the highThe water absorption capacities (WAC) of HK and NW were 1.27 ± 0.12 g/g and 1.53 ± 0.12 g/g whereas the oil absorption capacities (OAC) of HK and NW were 1.22 ± 0.11 g/g and 1.16 ± 0.12 g/g, respectively. The WAC represents the ability of a substance to associate with water under a limited water condition. Agnes et al.  reportedThe swelling power and solubility of HK and NW were directly related to increases in temperature. Both HK and NW starches showed a two-stage process. The initial stage was in range of 60–70 °C where the starches exhibited low swelling, then a significant increase showed above 70 °C, which is in agreement with previous studies ,49,50. AAmylose content maybe one of the factors affecting the swelling power of HK and NW starches due to the lower amylose content of HK causing the higher swelling power than the NW starch. HK and NW banana starch solubility also increased with temperature, reaching 26.43 ± 2.50% and 20.54 ± 0.94% at 90 °C, respectively b. The HKThe starch granule is a water-insoluble compound that can be hydrated at high temperatures. The disorder of starch crystallinity from heating can hydrate and swell the starch granules . The sweThe flow character of HK and NW banana starches was obtained from examination and compared with the compressibility index (CI) and Hausner ratio (HR) chart . The HK Currently, the trend of using natural materials in the cosmetic industry is on the rise. Consumers demand natural ingredients to replace synthetic ingredients because synthetic ingredients can have possible negative effects on health. For this reason, to satisfy the consumer, industry needs to develop cosmetics based on natural ingredients ,63. AccoBody powder and compact powder containing HK or NW in a range of 5 to 20% were prepared. It was found that the products with a starch component in the range of 5 to 15% provided a smooth and soft touch similar to that of the base formulations whereas at higher than 15%, it has a less smooth and soft touch. The 15% HK and NW body powder showed a light-yellow color  with ligM. acuminata; AAA) and Namwa  banana cultivars were used. The unripe bananas were peeled and cut into small cube pieces then placed into a blender and 1% w/v of sodium sulfite solution was added to immerse the banana in a ratio of 1:2 w/v. The samples were blended in the blender at maximum speed for 10 min then filtered with 200 mesh sieving and white cloth to remove the pulp. After being sieved, the samples were centrifuged at 8500 rpm for 5 min to precipitate the starch. The precipitated starches were dried at 40 ℃ in a hot air oven overnight. The dried banana starches were ground by using mortar and pestle then sieved with a 125 µm mesh. The percentage of yield of banana starch was calculated asThe extraction method for the banana starches was a slight modification of the method used by Bello-Pérez et al. . Two varThe color of Hom Khieo (HK) and Namwa (NW) starch was measured by colorimeter . The color was determined by the international color scale  which is in terms of CIELAB .Total amylose content was determined by using the colorimetric method described by Kaufman et al. . The staThe moisture content of the starch samples was determined by using a moisture analyzer . One gram of each starch sample was placed into the moisture analyzer in condition of 100 °C for 1 min then the percentage of moisture content was recorded.The morphologies of HK and NW banana starches were determined by using a scanning electron microscopy (SEM) with SEI detector. The starch samples were placed on double-side adhesive carbon tape and coated with a gold layer. The starch sample granules were measured at 1000× magnification. The sizes of HK and NW starch granules were determined by using TESCAN Essence software .−1 with a resolution of 4 cm−1 and 16 scans, at room temperature [The FT-IR spectra of sample starches were recorded by spectrophotometer  using the potassium bromide (KBr) pellets method. The finely ground samples were mixed with dry KBr then pressed under high pressure to form 7 mm pellets then the sample was measured in the range of 4000–400 cmperature .θ range with a 0.01° step size and a measuring time of 10.0 s per point. The measurements were made at ambient temperature. The percentage crystallinity was calculated from the following equation [The crystallinity was measured by using an X-ray diffractometer  with a slightly modified version of the method used by Cheetham and Tao . The HK equation .(3)CrysThe thermal properties of the samples were analyzed by using differential scanning calorimetry with slightly modified version of the method used by Carmona-Garcia et al. . Seven mw/v in test tubes with distilled water. The suspensions were mixed with a vortex until homogenous. The test tubes were heated in a water bath at 55, 65, 75, and 80 °C for 30 min and cooled down with cold running tap water then cooled at 4 °C for 2 h. The least gelation concentration was determined by the concentration when the test tube was inverted, and the sample did not slip or fall-down.Gelation of starch samples was determined by a slight modification of the method used by Lawal and Adebowals . Starch A slightly modified version of the method used by Agnes et al.  was usedw/v of starch solution was prepared with distilled water in centrifuge tubes and water loss was prevented by covering the tubes with plastic caps. The tubes were then heated at 60, 70, 80, and 90 °C for 30 min with shaking every 5 min. After heating, the samples were centrifuged at 8000 rpm for 5 min. The supernatant was decanted, then both of supernatant and precipitate were dried at 100 °C overnight. The swelling power and solubility of the samples were determined by the following equation.Swelling and solubility of the starch samples were determined by a slight modification of the method used by Carmona-Garcia et al. . 1% w/v Measurement of the bulk, and tapped densities followed the method of Olayemi et al. . Fifty gThe bulk and tapped densities were used to calculate the compressibility index and the Hausner ratio to provide a measure of the flow properties ,73.(8)HTwo powder formulations (body powder and compact powder) were prepared by varying the concentration of HK and NW starches to find the highest concentration suitable for the formulations. Body powder was composed of 5 to 20% HK or NW banana starch, talcum, magnesium carbonate, kaolin, zinc stearate, and zinc oxide. 1% of phenoxyethanol as a preservative was added to formula. Compact powder was composed of 5 to 20% HK or NW, talcum, kaolin, zinc stearate, zinc oxide, titanium dioxide coated mica, magnesium stearate, and magnesium carbonate. Titanium dioxide, red iron oxide, yellow iron oxide, and black iron oxide as a colorant, 0.5% of capric/caprylic triglyceride, 1% of mineral oil, and 1% of isopropyl myristate as binding agents, and 1% of phenoxyethanol as a preservative were added to formula.w/w maximum.The banana starch of Hom Khieo (HK) and Namwa (NW) cultivars were successfully extracted with 1% sodium sulfite with a high yield based on unripe peeled banana fruit. The starches showed light-yellow color with a fine powder. The morphology of both starches was oval shape with elongated forms for large granules and round shapes for small granules. The amylose content of HK and NW were 24.99% and 26.23%, respectively. Each starch showed the B-type crystalline structure and the gelatinization temperature was around 77 °C. The banana starches showed very poor flow character and provide smoothness when applied on skin. The HK and NW banana starch were used in powder formulations to provide a smooth feel and good spreading properties. The starch has the potential to be used as a substitute for talcum in powder base preparation with no effect on sensorial properties at 15%


Starch is the most abundant storage carbohydrate and a major component in pea seeds, accounting for about 50% of dry seed weight. As a by-product of pea protein processing, current uses for pea starch are limited to low-value, commodity markets. The globally growing demand for pea protein poses a great challenge for the pea fractionation industry to develop new markets for starch valorization. However, there exist gaps in our understanding of the genetic mechanism underlying starch metabolism, and its relationship with physicochemical and functional properties, which is a prerequisite for targeted tailoring functionality and innovative applications of starch. This review outlines the understanding of starch metabolism with a particular focus on peas and highlights the knowledge of pea starch granule structure and its relationship with functional properties, and industrial applications. Using the currently available pea genetics and genomics knowledge and breakthroughs in omics technologies, we discuss the perspectives and possible avenues to advance our understanding of starch metabolism in peas at an unprecedented level, to ultimately enable the molecular design of multi-functional native pea starch and to create value-added utilization. Starch or amylum is a polysaccharide synthesized in plants; it remains at the very epicenter of the food and feed chains, as well as a renewable source of industrial raw materials . Starch Pisum sativum L.) belongs to the legume family and is a major pulse crop with a production yield of 14.2 million tons globally in 2019 ). Known as a healthy pulse crop, peas have historically been part of the human diet as they are an excellent source of protein, starch, fiber, antioxidants and micronutrients. More recently, peas have gained considerable attention due to their potential health-promoting benefits in reducing the risk of chronic health conditions [https://www.alliedmarketresearch.com/pea-protein-market (accessed on 31 July 2021)). Consequently, the volume of pea starch will dramatically grow at a rate double that of pea protein. The explosion in demand for pea protein has led to a surplus of pea starch, which poses significant challenges to the pea processing industry. This highlights the importance of increasing the value-added utilization of pea starch. The successful utilization of starch in various applications depends largely on its physicochemical and functional properties such as gelatinization/pasting, viscosity and swelling power [Field pea  into uridine diphosphate glucose (UDP-Glc) and fructose to provide a carbon skeleton for the synthesis of adenosine 5′-diphosphate-glucose (ADP-Glc), the soluble precursor for starch biosynthesis . The chaPlants produce starch by converting glucose 1-phosphate (Glc-1-P) to ADP-Glc and then liberating the glucosyl unit to a growing glucan chain. These processes involve the coordinated actions of four major classes of enzymes: ADP-Glc pyrophosphorylase (AGPase), starch synthases (SS), including soluble starch synthases (SS) and granule-bound starch synthases (GBSS); starch branching enzymes (SBE); and starch de-branching enzymes (DBE) .In starch-storing organs, such as the endosperms of cereal grains and the embryos of legume seeds, sucrose derived from photosynthesis in the leaves is remobilized to provide a substrate for starch synthesis . In pea Vicia faba L., the AGPase activity in developing cotyledons is insensitive to 3-PGA, while the activity from leaves is. However, the AGPase activities from both tissues are sensitive to Pi-inhibition [The generation of ADP-Glc is the first committed step which is catalyzed by AGPase, a heterotetrameric enzyme consisting of two large subunits encoded by small multiple gene families and two small catalytic subunits encoded by one or two genes . AGPase hibition . The smahibition . Variatihibition . rb locus in peas results in the absence of one of the polypeptides of AGPase and consequently leads to a 10- to 40-fold reduction in AGPase activity during embryo development  and it is undetectable in leaves. The pea lines with RNAi-mediated repression of the small subunit Psagps2 produced wrinkled pea seeds with a starch content reduction of 40–50% and 25% less amylose. The repression of Psagps2 caused the re-partitioning of carbon into other pathways and activated amino acid and storage protein synthesis. These complicated responses, including hormonal, metabolic and transcriptional changes, ultimately influenced seed metabolism and morphology [Psat5g006360) and AGPase_L2 (Psat4g008080), and two small subunits, AGPase_S1 (Psat5g110720) and AGPase_S2 (Psat2g005160), were retrieved from the reference genome assembly of the pea cultivar Caméor (https://urgi.versailles.inra.fr/Species/Pisum/Pea-Genome-project (accessed on 31 July 2021)). All of these AGPase subunits share a common core region—the NTP_transferase (nucleotidyl transferase) domain - that is indispensable for catalytic activity. In addition, they all share 11 conserved motifs. Although the DNA sequence of the two large subunits is longer than that of the small subunits, the length of the amino acid sequence is similar among all subunits. Another two genes (Psat5g016480 and Psat7g184120) share high similarities to AGPase subunits at the amino acid level. The predicted protein of Psat5g016480 contains an NTP_transferase domain and has 10 out of 11 conserved motifs. It is likely that the annotated Psat7g184120 does not represent the full-length gene and that an incomplete NTP_transferase domain was identified in this gene  unit from ADP-Glc to the non-reducing end of a growing glucan chain of amylose or amylopectin through the formation of a new α-1,4-glycosidic linkage. SSs have multiple isoforms with differential organ specificity as well as temporal regulation, and can be categorized into GBSS and soluble SS. Each individual isoform plays a unique role in the biosynthesis of amylose and amylopectin. GBSS is required for amylose biosynthesis within the granule matrix , while GBSS is the most abundant protein found within starch granules. Multiple isoforms of GBSS have been found in pea seeds, rice and maize endosperms and Arabidopsis leaves . GBSS isGBSS is largely involved in amylose synthesis via the processive mode of action, leading to a linear polymer of up to the degree of polymerization (DP) 6000. Furthermore, GBSS plays a role in amylopectin synthesis by extending the existing side chains of amylopectin and contributing to the formation of long glucans (extra-long chains) of amylopectin ,30,34. HSeveral duplications of GBSS during plant evolution have led to tissue-specific and biochemical specialization and diversity among paralogues . Two isolam) lack or contain a greatly reduced amount of GBSSI (The mutants and transgenic lines of many plant species with defective GBSSI have low amylose or have amylose-free (or waxy) starch . The lowof GBSSI . The lacrug5 locus in pea results in abnormal starch granule morphology and amylopectin structure with fewer chains of intermediate length and more very short and very long chains [Soluble SSs are incorporated within stromal and granular fractions found in the granule matrix as granule-associated proteins . Solubleg chains   and GBSSIb (Psat0s3561g0040) have a glycosyltransferase-5 (GT5) domain, a glycosyltransferase-1 (GT1) domain and 13 conserved motifs with a similar distribution. Each soluble SS has a conserved domain of GT5 and GT1 except that SSIII and SSV each has only GT5. Similarly, in maize, SSV has a domain of GT5 but not GT1 [Psat1g180640) is the longest isoform with three carbohydrate binding modules (CBMs) of family 53 (CBM53) domains located in tandem in its N-terminal that are not present in other SS isoforms. CBMs are non-catalytic domains with carbohydrate-binding sites connected to catalytic domains, which promote the affinity of the enzyme  with the substrates by increasing the concentration of the substrate on the surface of the enzyme [Putative genes encoding GBSS and SSI–SSV in pea were identified by a blast search against the pea reference genome sequence database . Similar not GT1 . The pute enzyme . SSIII ie enzyme . One suce enzyme . In maize enzyme .r (rugosus) locus led to the complete loss of SBEI activity in the developing pea embryos producing the wrinkled (r) phenotype, one of the traits studied by Mendel in peas [r) seeds have a reduced total starch and amylopectin content compared to round peas, as well as an altered starch granule morphology [r mutant seeds also show pleiotropic effects and complex metabolic fluctuations such as a higher level of free sucrose, more lipid, less legumin and a reduced seed longevity [Psat3g034640) and SBEII (Psat0s2670g0080) have a conserved CBM48 domain at N-terminal, an α-amylase (Amy) domain in the center, and an α-amylase_C (Amy_C) domain that are signature domains of SBE (Starch-branching enzymes (SBEs) are structurally related to the α-amylase superfamily of enzymes and determine the starch polymer structure. SBEs introduce α-1,6-branch points in glucan chains through the cleavage of internal α-1,4 bonds and the transfer of the released reducing ends to C-6 hydroxyls, and produce new non-reducing ends for glucan elongation by SS and starch phosphorylase . SBEs st in peas . SBEI co in peas . Wrinklerphology . This inongevity ,23. No mongevity . In peass of SBE . Of noteStarch debranching enzymes (DBEs) hydrolyze the α-1,6-glucan branches of amylopectin and these processes are required for the normal synthesis of amylopectin by trimming excess branches . These ePhaseolus vulgaris) seeds [ISA generally functions as a multimeric enzyme in heterometric or homometric complexes depending on the species, and consists of up to six composite peptides. In the dicots such as Arabidopsis, AtISA1 and AtISA2 form a heteromultimetric enzyme and both are required to be present for catalytic activity . Similars) seeds . Howevers) seeds . psat7g021920 and Psat4g161960) with a CBM48 domain and an Amy domain are also identified. Further studies are required to investigate their possible functions for a better understanding of whether they play a role in starch biosynthesis.Both types of debranching enzymes were identified in peas, suggesting their roles in starch metabolism in the developing embryo and in starch degradation during germination . Three grug4 locus result in the loss of most of the activity of Susy in the developing embryos of peas  have been isolated from peas. These isoforms have distinct expression patterns and kinetic properties. The mutant line rug4-c carrying a mutation at SuSy1 has only 5% of SuSy activity in the embryo, but with minimal pleiotropic effects on other enzymes in primary metabolism. The starch content of the SuSy1 embryo is reduced by 30%, while the cellulose content is unaffected. This finding suggests that SuSy1 in embryos is necessary for starch synthesis but not required for cellulose synthesis, and that different isoforms of SuSy may have distinct roles in carbon channeling [Vicia narbonensis, a close relative of peas, leads to an approximate starch reduction of 50% in cotyledons [Mutations at the  of peas . The rug embryos . Genes eanneling . These sanneling . In agretyledons . While ityledons . Studiesrug3 locus encodes a plastidial PGM. Five pea mutants at the rug3 locus exhibit a wrinkled phenotype, reduced amylose content, and a starch content of between 1% and 20% of the dry weight compared with 50% in the wild type  catalyzes the interconversion of Glc-1-P and Glc-6-P in the cytosol and plastids. The pea ild type . In pea osphates . Starch phosphorylase (SP), also called α-glucan phosphorylase, catalyzes the reversible transfer of glucosyl units from Glc-1-P to the non-reducing end of α-1,4-glucan chains with the release of phosphate. It may be involved in either synthetic or degradative reactions depending on the relative concentrations of the soluble substrates. Two distinct forms of SP, plastidial and cytosolic, exist in higher plants ,67. Mounhttp://www.cazy.org/Carbohydrate-Binding-Modules.html (accessed on 31 July 2021)) with variable substrate specificity and biological functions. Arabidopsis Protein Targeting to Starch 1 (AtPTST1) and AtPTST2 contain a CBM48 domain. AtPTST1 regulates amylose biosynthesis in leaves by mediating the binding of GBSS to the developing starch granules. Arabidopsis ptst mutants demonstrate a dramatic reduction in GBSS proteins in starch granules which leads to the production of amylose-free starch and a similar phenotype to other mutants lacking GBSS [CBMs are a type of non-catalytic structural domain of carbohydrate-active enzymes with carbohydrate-binding activity. They promote the association of the enzyme  with the substrates by bringing the enzymes into close and prolonged proximity with their substrates ,71. CBMsing GBSS . AtPTST2ing GBSS . OsGBP, ing GBSS . aWaxy (aWx) allele, and their interaction at the post-translational level might be part of a network of starch biosynthetic enzymes [Starch biosynthesis is a coordinated and complex process with many core enzymes and non-enzyme proteins involved. Many of these enzymes are regulated by different post-translational modifications and control mechanisms . Accumul enzymes . These oPost-translational redox modulation also regulates starch synthesis in plants. Several enzymes involved in transient starch metabolism in photosynthetic tissues are sensitive to redox modulation, although in vivo evidence is still lacking for most of them . Much feIt appears that the formation of the complexes of SS and SBEII are conserved within cereal endosperms and that they may function in the global regulation of starch biosynthesis and granule formation , althougOsPK2, significantly reduces grain weight and starch content, and alters starch physicochemical properties in rice [tar2 (tryptophan aminotransferase related2) produces small, wrinkled seeds with a reduced starch content. The activity of different starch synthesis enzymes and the expression of the corresponding genes are reduced in the mutants, indicating that auxin is required for starch accumulation in peas. A link between auxin and the signaling sugar trehalose 6-phosphate (T6P) has been identified recently, in which T6P acts as an upstream positive regulator of TAR2 and auxin to facilitate the sucrose-to-starch conversion [In addition to the genes in the starch biosynthetic pathway, other genes involved in seed development, cellular metabolic flux and carbon partitioning also play a key role in starch synthesis. For example, the loss-of-function of 6-phosphogluconate dehydrogenase (6PGDH) in the oxidative pentose phosphate pathway causes a severely defective kernel with a reduced starch accumulation in maize . The mut in rice . Recent  in rice . The auxnversion . T6P regnversion , but alsOsAGPL3, Wx, OsSSIIa, SBE1, OsBEIIb, and ISA2 [TaSUT1, TaSSIIIa, TaBEIIa, TaISA1, and TaBEIIb in yeast, and is thought to be a regulator of starch biosynthesis in hexaploid wheat [Shrunken-2 (sh2), Wx, SSI, BT1, and SBE1 in maize [TaSSIIa, and the SuSy gene TaSuSy1. It also indirectly regulates the expression of TaSPA, a transcriptional regulator of genes encoding LMW-GS and gliadins [A number of transcription factors (TFs) have recently been identified as regulators of some of the major genes involved in starch biosynthetic pathway in the endosperms of cereal crops. A basic leucine zipper transcription factor (OsbZIP58) in rice positively regulates starch content and composition, and the amylopectin CLD. OsbZIP58 binds directly to the promoters of and regulates the expression of six starch biosynthetic genes: and ISA2 . Similarand ISA2 . Moreoveand ISA2 . The traid wheat . These rin maize . A NAC Tin maize . In addiin maize . An endogliadins . Although previous efforts have been made to characterize the function of starch biosynthetic enzymes and to elucidate the process of starch biosynthesis in peas, it is important to note that the catalytic function of various isoforms of different enzyme classes have largely been deduced from mutant studies. However, single mutations can have pleiotropic effects since starch biosynthetic enzymes generally function as complexes. The pleiotropic phenotypes of the mutant might result from the disruption of enzyme complexes and their associations, and not just the mutated gene . Such plTo the best of our knowledge, no studies in peas have reported on non-enzyme proteins such as CBM containing proteins, and the factors that can regulate starch synthesis through their interaction with starch biosynthetic enzymes. Very limited transcriptome research has been reported on the developing pea seeds. A comparative transcriptomics study was conducted on garden peas and grain peas at two developmental stages, 10 and 25 days after pollination (DAP) . In thisPhaseolus vulgaris L.) [Glycine max and Glycine soja. However, all these QTL were detected only in a single environment, indicating the environmental effect on their expression [Vigna radiata L. Wilczek) [Starch content and composition are quantitative traits determined by multiple genes, and their expressions are environmentally dependent. Genetic mechanisms of starch metabolism have been well studied in major cereal crops, whereas little information is available in legume crops including peas. Quantitative trait locus (QTL) mapping and a genome-wide association study (GWAS) coupled with transcriptome profiling have been conducted in cereal crops to decipher the mechanisms regulating starch metabolism: starch content, composition, granule size, and functional properties ,109,110.aris L.) . Nine QTpression . One majWilczek) . AGPase_L1/S1/S2, GBSSI/II, SuSy2/3, SBEII, sucrose phosphate synthase (SPS), and Glc-6-P/phosphate-translocator, involved in starch metabolism, were amplified from fifty diverse Pisum sativum accessions. Four SNPs with one within each of the genes AGPase_L1 and GBSSI, and two within SBEII, were significantly associated with amylose concentration in two out of eight environments. Ten SNPs within the genes SBEII, SuSy2 and Sps were significantly associated with the total starch concentration in at least five out of eight environments [r, UGPase, AGPS2) showed a significant association with CLD in both greenhouse and field conditions. Not surprisingly, amylose content was associated with the r locus in both environments [A few attempts have been made to identify SNPs associated with seed starch and amylose content, and amylopectin CLD in peas. Ten genes, including ronments . Similarronments . In addironments . QTL werronments . Nine SNPisum species are diploid (2n = 14) with a genome size of about 4.45 Gb in the recently sequenced Pisum sativum L. cv. Caméor, although the variation in genome size across the genus remains unknown [Pisum has two or three distinguished species. The P. sativum complex, including cultivated P. sativum subsp. sativum and wild subsp. elatius Asch. & Graebn. which is native to the European-Mediterranean region and mid-northwest Asia, while P. fulvum Sibeth. & Sm. is only found in the Middle East and is distinguished from P. sativum by crossing barriers, dehiscent pods and seeds dormancy [P. sativum subsp. abyssinicum A. Br. is cultivated in Ethiopia and Yemen and has no wild counterpart. Compared with other subspecies, P. sativum subsp. abyssinicum A. Br. possesses several unique traits such as strongly serrate leaflets, a glossy seed coat and early flowering [P. fulvum and P. sativum subsp. abyssinicum form sister clades. P. sativum subsp. elatius falls between P. fulvum–P. sativum subsp. abyssinicum and cultivated P. sativum [All  unknown . Pisum hdormancy ,122. P. lowering ,124,125. sativum .http://florilege.arcadproject.org/fr/crb/proteagineux/crb-proteagineux, http://urgv.evry.inra.fr/UTILLdb (accessed on 31 July 2021)) [https://npgsweb.ars-grin.gov/gringlobal/descriptors (accessed on 31 July 2021)) holds 6827 pea accessions. The adoption of the Germplasm Resource Information Network  database by the USDA allows for online queries across multiple traits, including phenological and morphological traits; seed mineral nutrient composition and protein concentration; and biotic stress resistance. The USDA pea core collection of 504 accessions was assembled based on geographical origin and flower color, representing ~14% of all USDA pea accessions at the time of construction [Pisum species, the diversity studies have mainly concentrated on cultivated subspecies [Pisum elatius accession was identified as a double null mutant for two closely linked genes encoding the TI1 and TI2 seed protease inhibitors, demonstrating that pea wild relatives can be exploited for increasing the nutritional value of peas [Several pea germplasm collections have been assembled and curated worldwide. Currently, the largest pea germplasm collections are held by INRAE France ) . In addiy 2021)) . The U.Struction . This cotruction . There ibspecies . The peabspecies  and tolebspecies , but alsbspecies . A Pisum of peas . DespiteThe advances of next-generation sequencing (NGS) technology and, consequently, the significant reduction in the cost of sequencing enable the construction of pea pan-genomes for multiple reference genomes representing different subspecies. The multiple reference genomes will enable more accurate identification of structural variations and allow for the capturing of all possible functional variants which cannot be achieved by using a single reference genome. The availability of the first annotated reference genome assembly for a pea cultivar ‘Caméor’  facilitaStarch has a semi-crystalline granular structure consisting of amylose and amylopectin. Although the relative location of amylose and amylopectin in native starch granules is still under discussion, it is generally accepted that amylopectin is in the semi-crystalline structure, while amylose is in the amorphous regions of the amylopectin blocklets . StarcheStarch granule size is an important factor for many applications and uniform granules are required for both food and non-food industrial applications . One exar, rug3-a, lam-c and rug5-a) and the double mutation (r/rb) in pea all result in substantial changes in the amylose–amylopectin ratio and characteristic changes in granule structure and function [A series of pea mutants have been used to characterize the changes of starch granule structure and the functionality as a result of the specific mutations in the starch biosynthetic genes. Four single mutations , physical , and enzymatic methods, or a combination of these methods, are currently applied to modify starch structure to improve the functional properties of pea starch . Furtherhttps://www.jic.ac.uk/research-impact/molecules-from-nature/impact/peas/the-history-of-pea-research-at-the-john-innes-centre/pea-starch-an-alternative-material-to-plastic-2/ (accessed on 31 July 2021)). Compared with conventional plastic polymers, the main drawbacks of starch-based biopolymers are hygroscopic and poor mechanical properties [Currently, most starch concentrates coming from pea fractionation are being used for food and animal feed, while there are increasing demands for pea starch to be used in non-food industrial applications. A comprehensive review was recently published on the industrial utilization of starches from pulse crops including peas . Increasoperties . Some meoperties . Pea staoperties . Pea staoperties . Starch operties . Starch operties . Nanoparoperties . These aThe starch biosynthetic pathway is complicated and involves carbohydrate metabolism and seed development. Significant progress has been made in our understanding of starch metabolism in cereal endosperms; however, this does not necessarily equate with the metabolism in dicot embryos. Therefore, caution should be taken when attempting to infer the roles of particular isoforms in different species. Current genetic evidence demonstrating starch biosynthesis in peas is limited to mutant studies. Nevertheless, it is important to note that the interpretation of the function and contribution of an enzyme may be biased as starch biosynthetic enzymes form and act in coordinated complexes. As such, the pleiotropic phenotypes of the mutants might result from the combined effects of the disruption of multiple enzymes, as opposed to a single enzyme. Sizable gaps remain in our understanding of the genetic and molecular details of starch metabolism in peas. In particular, there is very limited knowledge about the transcriptional regulation of starch metabolic genes in pea. It is essential to identify genome-wide gene co-expression networks regulating dynamic starch biosynthesis at different seed development stages and to elucidate how the genes coordinate and interact to produce starch granules with specific composition and structure, and the mechanisms of complex interplay between starch and protein synthesis. Our knowledge of the isoforms of starch biosynthetic enzymes is not yet complete. The availability of a reference genome sequence and upcoming pan-genomes of pea facilitates the identification of more isoform candidate genes and novel non-enzyme proteins involved in starch metabolism. Together with subsequent functional genomics approaches, the integration of multiple ‘omics’-level datasets will provide the tools instrumental to elucidate starch metabolism in pea seeds. Furthermore, as the more efficient protocols become available for pea tissue culture and the regeneration of whole plants, genome editing technologies such as CRISPR/Cas9-based systems will enable high-precision molecular breeding in peas with tailored starch.Pisum has tremendous untapped potential for the discovery of novel functional alleles in starch metabolic genes using GWAS, which will enable the identification of genetic variations that cannot be achieved by analyses of mutants. Such resources highlight the potential for diversifying pea starch composition, structure and thus, functional properties for applications of commercial significance.Over 65% of the output of a pea fractionation plant is a lower value product: starch. As new pea protein processors surface online, the volume of pea starch will dramatically grow at twice the rate of pea protein. Current uses for pea starch are limited to low-value commodity markets, mainly due to its less desirable and narrow range of functionality. The economics of a pea fractionation plant dictates creating higher-value starch opportunities (communications with industry). Therefore, major efforts are required to expand the functionality of pea starch for broad use in food and non-food industries. It is a necessary prerequisite for the value-added utilization of pea starch that more research is conducted to correlate composition, structure, and functional properties. Starch biosynthesis is a complicated biological process with multiple biosynthetic enzymes forming complexes. Therefore, the modulation of a single starch-synthetic gene may be insufficient to obtain starch with desirable functional properties. This highlights the need for applying an integrated systems approach to better understand starch metabolism at a more global level, to identify functional alleles in starch metabolic genes, and to characterize the extent to which the allelic variation contributes to the diversity of the functional properties of pea starch. The strategies that integrate genetic diversity, starch structure and functional properties have proved successful in the production of rice varieties with modulated starch characteristics, resulting in improved eating qualities . SimilarAltogether, the research advances in the aforementioned aspects will enable the targeted breeding and molecular design pea varieties to produce novel starch with improved and/or customized functionality suited to specific industrial needs while maintaining a high protein content in pea seeds. This will ultimately promote the utilization of natural starch for food and non-food industrial applications.


At the request of the Journal Editor(s) the following article has been retracted.International Journal of Immunopathology and Pharmacology 33: 1-12. DOI: 10.1177/2058738418821837.Wang J, Chen G, Shi T, Wang Y, Guan C (2019) Possible treatment for cutaneous lichen planus: An in vitro anti-inflammatory role of Angelica polysaccharide in human keratinocytes HaCaT. Significant similarities between this paper and a number of other papers have been identified, as well as indication of image and data manipulation suggestive of the use of a “paper mill”. These papers share common structures and wording yet do not share any common authors.The authors did not provide a sufficient response or evidence to refute these claims. As a result, the scientific accuracy of this paper is not reliable.Adhering to the international guidelines established by the Committee on Publication Ethics, the Journal has determined these are grounds for retraction.


A new surgical procedure for the treatment of primary bladder neck obstruction with maintenance of anterograde ejaculation is proposed. In place of monolateral or bilateral bladder neck incision, associated with a loss of ejaculation rate of up to 30%, the new surgical procedure consists of laser drilling the bladder neck with a number of holes and without muscle fiber disruption. The effect of this novel procedure has been studied numerically, with a simplified two-dimensional numerical model of the internal urethral sphincter, varying the position and the number of holes in the fibrotic region of the urethral tissue. Results show an improvement of the urethral sphincter opening by increasing the number of holes, ranging from about 6% to 16% of recovery. Moreover, a non-aligned position of holes positively influences the opening recovery. The concentrations of maximum principal strain and stress have been registered in the proximity of the interface between the physiologic and diseased sphincter, and in those regions where the radial thickness is significantly thinner. The effects on the first five patients have been included in the study, showing improvement in micturition, lower urinary tract symptoms, sustained ejaculatory function, and quality of life. Lower urinary tract symptoms (LUTS) in men are not solely correlated to benign prostatic hyperplasia. Men younger than 50 years often seek medical attention for their LUTS . PrimaryThe progress of novel surgical treatment procedures and the design of bioengineering medical devices are currently related to the investigation and knowledge of the macro-mechanical behavior of male urethral tissues and structure. Moreover, the development of computational models, corroborated by experimental characterization data, can improve the understanding of the biomechanical response of such tissues and be a support for surgical practices. Over the last few decades, the mechanical behavior of the majority of tissues and organs has been deeply studied  model allows us to understand the overall biomechanical behavior of the organic tissues and their possible interaction with biomedical stents and devices. An exhaustive research work has been carried out in ,19,20,21The present work aimed at performing a preliminary validation through a computational modeling approach of a new surgical procedure for the treatment of primary bladder neck obstruction, with preservation of anterograde ejaculation. The computational model is developed in two stages: (i) the opening of the fibrotic internal urethral sphincter subjected to an imposed internal pressure is simulated; (ii) the opening of the fibrotic internal urethral sphincter, after a new surgical procedure (see below), is numerically evaluated. A comparison between the strain and stress fields before (urethra with PBNO) and after surgery is performed, highlighting the efficacy of such surgical procedure in terms of the sphincteric opening deformation recovery. Moreover, a parametric analysis is carried out by varying the position and the number of holes in the fibrotic region of the urethral tissue, produced by the surgical laser. The numerical model will allow us to assess the reliability of the new surgical procedure for the treatment of PBNO. It is important to remark that the computational model is based on the clinical evidence coming from the particular adopted surgical procedure developed by the urologists of our research group.We developed a new surgical approach of PBNO with the idea of preserving the bladder neck/internal urethral sphincter anatomy and contraction, maintaining an anterograde ejaculation together with micturition improvement. We assumed that it might be enough relaxing the internal urethral sphincter and widely opening the bladder neck musculature through its full thickness drillings with a number of holes and without muscle fibers disruption. The latter can be achieved only using a small laser fiber. The suitable laser for this full thickness drilling was identified with the Thulium: yttrium-aluminum-garnet laser (TL). Indeed, TL operates at a 1940–2013 nm wavelength, in a continuous wave mode, which enables accurate cutting of the tissue, with no tissue disruption due to its optical penetration of only 0.2 mm (see ).The surgical procedure was performed using 0.9% saline solution and a continuous flow 26 Ch endoscope , mounted with a 12° optic and with a separate operative channel for the fiber. Laser energy was transmitted through an 800-micron, front-firing laser fiber. The laser setting was 30 W . Surgery started with enucleation of the transitional zone 1 cm proximal the verumontanum to preserve both ejaculatory ducts and the musculus ejaculatorius . Once thInclusion criteria were LUTS lasting for at least one year and not responsive to medical therapy, International Prostate Symptom Score (IPSS) > 8, maximal urine flow rate (Qmax) less than 15 mL/s, and willing to maintaining anterograde ejaculation. Exclusion criteria were previous and concomitant surgery of the lower urinary tract. Age, prostate volume, IPSS, Male Sexual Health Questionnaire-Ejaculatory dysfunction (MSHQ-ED), Qmax, intraoperative, and postoperative data were gathered. Early complications were considered within 30 postoperative days.Both IPSS and MSHQ-ED are two validated and international questionnaires that are widely used in clinical practice. IPSS is a self-administered questionnaire consisting of 8 items that assess LUTS  and quality of life associated with LUTS . The oveThe first 5 men meeting inclusion criteria were enrolled in this pilot study. The study was approved by our local Ethical Board (DGEN 421/2017). Signed informed consent was obtained from all patients.The experimental evidence and the image analysis during surgery see  allowed The definition of a solid and numerical model is based on a simplification of the internal urethral sphincter geometrical features. The domain consisted of a two-dimensional geometry, reported in x, uy) at two points, as described in p = 1960 Pa prescribed on the internal boundary  may enhance the structural compliance of the fibrotic region. This effect is confirmed by the significant distortion of the holes in the deformed configuration, assuming D-, P-, or S-shaped profiles, indicated in As an example, the maximum principal strain and stress along the intraluminal edge for tests P0, H0, and H6 are reported in The above diagrams confirm that the peak values of the maximum principal strain and stress have been registered especially where the radial thickness is smaller. The highest principal stress can be found in proximity of the fibrotic interface. As expected, the case H6 is included between the completely sane urethra case (P0) and the diseased case (H0).As concerns the sphincter response subjected to different intraluminal pressure, A new surgical procedure for the treatment of PBNO has been proposed, consisting of drilling the bladder neck with a number of holes and without muscle fiber disruption. A simplified numerical model of the internal urethral sphincter has been used for preliminary validation of the procedure, testing different configurations of the holes.Numerical results showed that the drilling of holes improves the urethral sphincter opening, which increases with the number of holes. The use of a staggered configuration could positively help the opening recovery. The concentration of maximum principal strain and stress have been registered in the proximity of the interface between the physiologic and diseased sphincter and within the stiffer epithelium region, this suggests avoiding the drilling of holes in such regions in order not to compromise the valve integrity. Moreover, Finally, the insertion of a relevant number of holes (H6–H7) yields to an enhancement of the surgical procedure efficacy since the removal of much more fibrotic tissue promotes a more prominent deformation and distortion of the remaining material.The feedback from the first five patients confirms the expected improvement in micturition, LUTS, sustained ejaculatory function and quality of life.The accuracy of the present results can be considered satisfactory, in spite of the limitation that pertains to the two-dimensional numerical model configuration. This preliminary approach is also justified by the relevant computational effort determined by the high nonlinearity of the problem, related to material, geometry, and contact conditions.Another limitation was the fact that this work is a pilot study, thus involving a small number of enrolled patients. However, the low rate of complications and long-lasting functional results were promising and allowed us to carry on a further clinical study.


The use of blends of essential oils associated with α-amylase as a feed additive promoted the upregulation of enzymes such as triosephosphate isomerase, phosphoglycerate mutase, alpha-enolase, beta-enolase, fructose-bisphosphate aldolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), l-lactate dehydrogenase B, l-lactate dehydrogenase A chain, l-lactate dehydrogenase, and ATP synthase subunit beta, which promote the degradation of carbohydrates in the glycolysis and gluconeogenesis pathways and oxidative phosphorylation, support pyruvate metabolism through the synthesis of lactate from pyruvate, and participate in the electron transport chain, producing ATP from ADP in the presence of a proton gradient across the membrane. The absence of proteins related to inflammation processes (leukocyte elastase inhibitors) in the cecum tissues of animals fed essential oils and amylase may be because feed enzymes can remain active in the intestine and aid in the digestion of nutrients that escape rumen fermentation; conversely, the effect of monensin is more evident in the rumen and less than 10% results in post-ruminal action, corroborating the hypothesis that ionophore antibiotics have a limited effect on the microbiota and intestinal fermentation of ruminants. However, the increase in starch in these diets promoted a downregulation of enzymes linked to carbohydrate degradation, probably caused by damage to the cecum epithelium due to increased responses linked to inflammatory injuries.Diets for feedlot cattle must be a higher energy density, entailing high fermentable carbohydrate content. Feed additives are needed to reduce possible metabolic disorders. This study aimed to analyze the post-rumen effects of different levels of starch  and additives  in diets for Nellore feedlot cattle. The cecum tissue proteome was analyzed via two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and then differentially expressed protein  Dietary strategies are adopted to increase energy metabolism, mainly through the fermentation of carbohydrates in the rumen. This is mostly accomplished by supplying hexoses (glucose) from starch. Thus, with increased starch in the diet, physiological limits to animals’ ability to digest a large amount of fermentable carbohydrates in the rumen and ruminal escape increased.Among the limitations to enhancing meat production is its large energy requirement, which means that feedlot cattle have a higher net energy demand (NE)2, which are metabolized in the liver and provide the greatest source of energy for ruminants4. However, the use of large amounts of starch can lead to disorders and metabolic diseases due to the accumulation of organic acids in the ruminal fluid, especially acidosis and bloat (NASCEM5). Thus, feed additives that decrease harmful ruminal fermentation processes are employed, such as sodium monensin, which is a polyester carboxylic ionophore used in growth and finishing diets5. It acts bacteriostatically on Gram-positive ruminal bacteria but may leave residues in products of animal origin and result in microbial resistance6. Alternative additives have shown the potential to replace monensin, such as blends of essential oils associated with the exogenous enzyme α-amylase, which has led to demonstrable gains in performance and carcass weight, in addition to reducing hepatic abscesses and fecal starch in animals with high-starch diets9.In the rumen, the fermentation of glucose from starch occurs and it is converted mainly into volatile fatty acids (AGV) and lactate5. The rumen microbiota can digest around 70–80% of the starch consumed13; however, the digestion and absorption of post-rumen starch are partially impaired as enzymatic digestion via pancreatic α-amylase in the duodenum is limited to the small intestine15. Others have postulated that glucose cannot be absorbed and transported in large quantities from the lumen into the bloodstream due to insufficient levels of the glucose transporters SLGT1 and GLUT217, which favors the escape of some starch to the large intestine and increases the potential for the digestion and use of this starch in the cecum. Therefore, feedlot diets that offer increased amounts of energy due to high levels of concentrates18 can cause excessive fermentation in the cecum, contributing to the metabolizable energy ruminants can access20 but potentially resulting in hindgut acidosis, which may generate inflammatory reactions in the cecal epithelium. Large amounts of starch in the cecum may contribute to the fermentation of AGV, NH3, and lactic acid as well as a decreased pH5. Additionally, the cecum has limited buffering capacity compared with the rumen, where saliva and protozoa modulate pH fluctuations21. Feed additives that can increase the use of starch in the rumen, reducing starch escape to the intestines, as well as lower starch levels in feedlot cattle diets can avert the risk of cecal acidification. Most studies with feed additives focus only on the rumen effects; however, the effects of feed additives on the post-rumen digestive tract are important to understand.With high levels of starch in the diet, the rate of passage and post-ruminal digestion increasesUnderstanding how the digestion and absorption sites act when high proportions of starch are included in feedlot diets is essential. Due to the levels of starch in cattle diets and the effects on the extent of the gastrointestinal tract associated with different feed additives, this study aims to map the proteome of feedlot cattle’s ceca and to elucidate how protein expression acts on metabolism when different nutritional strategies are applied.θ) that the expressed spots are identical. When testing all classes, protein spots were differentially expressed, as shown in Table In the workspace, classes were created to analyze differences in protein expression; an analysis of variance (ANOVA) was used to test the hypothesis  cycle, respiratory electron transport, innate immune system, and the immune system were affected in cecum tissues by different feeding strategies Fig. . The datAdditionally, the differential expression indicates similar encoding enzymes in the glycolysis and gluconeogenesis pathways in cattle’s large intestines under different feeding strategies Fig. .Figure 2The expression values (5) Table  were gro5) Table  and ordel-lactate dehydrogenase (EC 1.1.1.27) compared with those treated with monensin; all of these proteins are enzymes catalyzing the synthesis of pyruvate, which is responsible for the degradation of carbohydrates. Animals fed diets with the highest level of starch tested (45%) exhibited greater synthesis of l-lactate dehydrogenase (EC 1.1.1.27), fructose-bisphosphate aldolase (EC 4.1.2.13), and phosphoglycerate mutase (EC 5.4.2.4). Meanwhile, animals fed the intermediate starch level exhibited a higher expression of triosephosphate isomerase (EC 5.3.1.1) and glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12).The animals fed low-starch diets (25%) associated with BEOα exhibited increased expression of pyruvate kinase (EC 2.7.1.40), beta-enolase (EC 4.2.1.11), triosephosphate isomerase (EC 5.3.1.1), and When evaluating the tested additives, we found that the use of BEOα with intermediate levels of starch (35%) resulted in greater expression of glycolysis intermediates, thus, this additive may have a greater effect on the post-rumen tract. A companion study (data under review) reported that the optimum level of dietary starch for cattle fed MON was 25%; however, the optimum level of dietary starch for cattle receiving BEO was 35%, commensurate with protein expression synthesis. In summary, feed intake decreased when MON- and BEO-fed cattle were fed more than 25% and 35% starch, respectively, which resulted in decreased average daily gain and proteins linked with carbohydrate degradation. The diets with 45% starch may have caused excessive ruminal fermentation that may have resulted in increased inflammation of the ruminal epithelium, agreeing with the proteins leukocyte elastase inhibitor. The literature reports that increasing levels of starch play an important role in reducing feed intake (observed in a companion study); however, this effect was more evident in cattle that were fed MON as it is a feed additive that depresses intake.spots of groups 25BEOα and 45BEOα compared with those fed MON, leukocyte elastase inhibitor, a serine protease inhibitor that is essential for the regulation of inflammatory responses and limits the activity of inflammatory caspases, was not expressed22 aligning with the results of the above-mentioned authors, who reported reduced ionophore effects in the hindgut. When comparing diets with 25% or 45% starch, regardless of the additive used, leukocyte elastase inhibitor was expressed more, corroborating previous studies demonstrating that inflammatory injuries are caused by the increased use of concentrates in the diet24. Additionally, we observed reduced expression of proteins that participate in energy metabolism in animals on high-starch diets, which can damage the epithelium of the cecum.In the protein 9 observed a reduction in fecal starch using BEOα, suggesting a greater degradation of carbohydrates because the feed enzymes can remain active in the intestine and aid in the digestion of nutrients that escape rumen fermentation25. As Thomas et al.26 demonstrated, the effect of monensin is more evident in the rumen, mainly in the diversity of microorganisms, but less than 10% results in post-ruminal action, corroborating the hypothesis that ionophore antibiotics have a limited effect on ruminants’ microbiota and intestinal fermentation.In a similar study, Toseti et al.27, corroborating our identification of proteins linked to immune responses. Notably, this was not observed when assessing the full range of starch levels (25% vs. 45%) but the proteins involved in inflammatory responses were expressed more . In ruminants, a high concentration of starch enables the fermentation of carbohydrates in the cecum with lactate production, which increases glucose metabolism in the intestine and leads to the observed expression of the enzyme l-lactate dehydrogenase and its isoforms l-lactate dehydrogenase B and l-lactate dehydrogenase A, which synthesize lactate from pyruvate (UniProt29).Fructose-bisphosphate aldolase (ALDOB), an enzyme that converts fructose-1,6-bisphosphate to fructose 6-phosphate, catalyzed by triosephosphate isomerase (TPI), is a precursor of glyceraldehyde-3-phosphate (GA3P), which is acted upon by the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during glycolysis. Alpha-enolase (ENO1) and beta-enolase (ENO3) are isoforms of enolase that are involved in Step 4 of glycolytic metabolism. Phosphoglycerate mutase (PGM) is a catalytic enzyme that converts 3-phosphoglycerate to 2-phosphoglycerate and, finally, pyruvate kinase (PKM) synthesizes pyruvate in the last step of glycolysis  and approved by the Ethics and Use of Animals Committee of the São Paulo State University , under protocol no 0107/2019 and in compliance with ARRIVE  guidelinesBos taurus indicus) from the grazing system with an average body weight of ± 380 kg were used. The animals were randomly allocated to pens  with 12 m2/animal and collective troughs . The program for receiving the animals consisted of weighing, deworming, and vaccinating according to the annual prophylactic calendar. The animals underwent a pre-experimental adaptation period of 10 days to standardize their rumen population and allow them to adapt to the facilities and management. The diets were formulated with the LRNS system (large ruminant nutrition system31), level 2, meeting the nutritional requirements for daily weight gain between 1.5 and 1.7 kg/animal. Animals were fed for 92 days and diets were offered ad libitum twice daily at 8 a. m. and 3 p.m.The animal experiment was conducted at the feedlot facilities of the Innovation and Applied Science Center of DSM Nutritional Products . Nellore bulls (n = 210) (® with the exogenous α-amylase Rumistar®) as the factor. The sodium monensin  used was included in the diet at a dose of 26 mg/kg of dry matter. The blend of functional oils  containing thymol, eugenol, limonene, and vanillin32 and the exogenous enzyme α-amylase , referred to as BEOα were added to the diet at a dose of 90 mg/kg of dry matter and 560 mg/kg of dry matter, respectively. The pens were distributed in a randomized block design, totaling 6 treatments with 5 repetitions or 30 experimental units overall. The treatments were distributed within the blocks as follows: T1 (25MON), T2 (25BEOα), T3 (35MON), T4 (35BEOα), T5 (45MON), and T6 (45BEOα). According to the statistical model:A factorial 3 × 2 arrangement was used, with starch level  and additives  were determined by the equation: TDN = digestible CP + (digestible EE × 2.25) + digestible NDF + digestible non-structural carbohydrate (NSC). Crude protein was determined by assessing the nitrogen content of the samples with the Kjeldahl method33. The NDF concentration was assessed with the methodology described by Van Soest et al.34 and corrected for CP and ashes. Starch was determined by the equation: NSC = 100 − CP − EE − NDF − ash, where ash content was determined by incinerating samples at 550 °C for 2 h in a muffle furnace35. Physically effective neutral detergent fiber (peNFD) was determined according to Kononoff et al.’s methods36. Samples of diets were collected to determine particle-size distribution by sieving with the Penn State particle-size separator and reported on an as-fed basis.Dietary energy content was calculated according to the LRNS systemThe animals were transported to a commercial slaughterhouse where they were stunned by brain concussions with a captive dart gun. After bleeding hide removal and evisceration, cecum samples about 4 cm square were collected and washed with phosphate-buffered saline (PBS), transferred to 15 mL polypropylene bottles, and placed in liquid nitrogen (− 196 °C) for later protein extraction. Each pen was considered an experimental unit, so a pool of samples was made by homogenizing cecal tissue from animals given the same treatment; three animals per experimental unit (N = 5) were used, i.e., 15 animals/group or 90 animals total .37, using an analytical curve with a concentration range of 0–100 g/L of standard bovine albumin solution  at 100 g/L.To extract the protein fraction, the tissue was macerated with a mortar and pestle in the presence of liquid nitrogen. The extracting solution was added at a rate of 1 mL ultrapure water per 1 g tissue and then the samples were homogenized with an OMMI-BEAD RUPTOR4 cell disruptor  over three 30-s cycles. The samples were then separated into protein extracts and the supernatant was collected after refrigerated centrifugation (− 4 °C) with a UNIVERSAL 320R HETTICH . The proteins were precipitated in 80% (v/v) acetone , using 300 μL of supernatant and 600 μL of 80% acetone. The samples were stored at 2 °C for 1.5 h and then centrifuged at 14,000 rpm for 30 min; the supernatant was discarded and the protein pellet was solubilized in 1 mL of 0.50 mol/L NaOH . The protein concentrations were determined by the Biuret method38. At the end of the focusing period, the strip was balanced in two 15-min stages. First, 10 mL of a solution containing 6 mol/L urea, 2% SDS (w/v), 30% glycerol (v/v), 50 mmol/L Tris–HCl (pH 8.8), 0.002% bromophenol blue (w/v), and 2% DTT (w/v) was used to keep the proteins in their reduced forms37. In the second stage, a solution in which DTT was replaced with 2.5% (w/v) iodoacetamide was used to alkylate the thiol groups of the proteins and prevent possible reoxidation. After strip balancing, the second portion of the electrophoretic process (SDS-PAGE) occurred.For isoelectric focusing, about 375 µg of proteins from each group were applied to individual strips; the sample was resolubilized with a solution containing 7 mol/L urea, 2 mol/L thiourea, 2% CHAPS (m/v) , ampholytes at a pH of 3 to 10 at 0.5% (v/v) , and 0.002% bromophenol blue , in addition to 2.8 mg of dithiothreitol . Approximately 900 µL of mineral oil was added at room temperature for 12 h to rehydrate the strips and prevent evaporation and urea crystals. After this period, the strips were added to the EttanTMIPGphorTM3 isoelectric focusing system (IEF) . The electrical voltage used was established by the protocol described by Braga et al.41.The strip was applied to a 12.5% (w/v) polyacrylamide gel previously prepared on a glass plate (180 × 160 × 1.5 mm). The gel was placed next to the strip with a piece of filter paper containing 6 µL of a molecular mass standard , with proteins of different molecular masses . The strip and filter paper were sealed with 0.5% agarose solution (w/v) to ensure contact with the polyacrylamide gel. The run program was then applied at 100 V for 30 min and a further 250 V for 2 h. After the run period, the gels were immersed in a fixative solution containing10% acetic acid (v/v) and 40% ethanol (v/v) for 30 min. Then, the proteins were revealed with colloidal Coomassie G-250  for 72 h and removed by washing with ultrapure waterwww.gelifescience.com), which allows the estimation of the isoelectric points and molecular masses of the separated proteins and calculation of the number of spots obtained via gel electrophoresis. Three replicates of each gel were used to evaluate the reproducibility of each protein spot obtained in the replicates of the gels by overlaying the image from one gel over the other in the image treatment program42.The gels obtained  closest to the theoretical and experimental results. Among the proteins identified, 12 were classified as functional for this study as they are related to energy metabolism and inflammatory responses.The differentially expressed spots were characterized with LC–MS/MS after being subjected to tryptic digestion and peptide elution according to the methodology Shevchenko et al.43 described. The aliquots of the solutions containing the eluted peptides were analyzed to obtain the mass spectra with the nanoAcquity UPLC system coupled to the Xevo G2 QTof mass spectrometer . Proteins were identified by searching in the UniProt database (www.uniprot.org) within the Bos taurus species. Proteins were considered depending on their theoretical and experimental isoelectric points, molecular masses, and scores (> 60). After identifying FASTA sequences in the proteins, their sequences were analyzed with OMICSBOX software (BLAST2GO)44 and they were categorized by their molecular function, biological processes, and biochemical activities with gene ontology (GO).The protein spots. Only proteins with significantly altered levels were selected for identification by MS. The images were analyzed with ImageMaster Platinum software version 7.0, which establishes correlations (matching) between groups. For this correlation, three gel replicates were compared for volume, distribution, relative intensity, isoelectric point, and molecular mass in an analysis of variance (ANOVA) with a t-test to determine the significance of differentially expressed protein spots.The starch level and additive were the fixed effects analyzed in a factorial design; thus, the groups were compared through contrasts to verify differentially expressed protein spot intensity volume was normalized with total intensity volume . The normalized intensity volume values of individual protein spots were then used to determine differential protein expression among experimental groups. A heatmap showed the correlation coefficient of the spot expression values and, after checking the differentially expressed spots , the log2 FC values were used for hierarchical cluster analysis.Following the average mode of background subtraction, individual 47 and Reactome pathway enrichment analysis yielded similar results about the specific pathways affected, allowing the expressions of proteins encoding enzymes found in the database to be mapped.The same KEGG-IDs were used to analyze metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes function (KEGG pathways)In verifying the differential expression of the cecal proteome in cattle, our results show that the blend of essential oils associated with α-amylase incorporated as a feed additive for beef cattle increased the expression of enzymes at dietary starch levels of 25%, 35%, and 45% compared with monensin. The higher expression of proteins related to carbohydrate degradation that participate in glycolysis and gluconeogenesis depended on increased feed intake and reduced protein synthesis expression. The optimum starch level was 35% for both feed additives; higher concentrations of starch (45%) increased the expression of inflammatory responses and reduced the expression of proteins involved in energy metabolism, probably due to damage to the cecum epithelium.Supplementary Information.