In late autumn, the Kara Sea becomes covered by ice, which hinders in situ measurements at this area. As a result, the fate of the Ob-Yenisei plume below sea ice during winter and spring remains unclear. In this study, we report multiple in situ measurements performed in the Kara Sea shortly before and during ice-covered season. We demonstrate that late autumn convection in the plume shortly before ice formation significantly reduces friction between the plume and the subjacent sea. The subsequent formation of solid sea ice coverage isolates the plume from wind forcing. These two factors precondition the Ob-Yenisei plume to form an intense buoyancy-driven coastal current below sea ice. As a result, the plume advects eastward to the Laptev Sea through the Vilkitsky Strait during several months in November-February. Eventually, by late winter this huge freshwater volume disappears from the Kara Sea and contributes to freshwater content of the Laptev Sea. The obtained result improves our understanding of freshwater balance of the Kara and Laptev seas, as well as provides an important insight into the large-scale freshwater transport in the Eurasian Arctic, which remain largely unknown during ice-covered season.The Kara Sea receives ~ 1/3 of total freshwater discharge to the Arctic Ocean, mainly from the large Ob and Yenisei rivers. The Ob-Yenisei plume covers wide area in the central part of the Kara Sea during ice-free season (June–October) and accumulates ~ 1000 km Spreading and transformation of freshwater runoff governs many physical , chemical , and biological  processes in the Arctic Ocean6. In particular, it maintains salinity-driven density stratification and affects sea ice formation, which is of key importance for the Earth’s albedo and global climate9.River runoff to the Arctic Ocean  located in the Kara Sea and the Lena plume  located in the Laptev and East-Siberian seas are the largest plumes in the Arctic Ocean and among the largest in the World Ocean19 16. Second, watershed basins of the Ob and Yenisei rivers are extended several thousands of kilometers southward from the Kara Sea, as a result, temperatures within the Ob-Yenisei plume are greater than in saline seawater during the majority of the ice-free period31. Temperatures within the plume decrease from > 10 °C in July to 0.5–1 °C in late autumn. Temperatures below the plume are < 0 °C during the whole year.During ice-free season, the Ob-Yenisei plume has relatively stable area  in the central part of the Kara Sea bounded by sharp thermohaline gradient, because thermohaline characteristics of the Ob-Yenisei plume are significantly different from those of the surrounding sea. First, salinities within the plume are < 25–28, while salinities outside the plume are > 3233. This process requires specific wind forcing conditions, namely, strong and long-term southwesterly winds, which occur relatively rarely during ice-free seasons 29. Therefore, it results in eastward advection of only small share of the huge freshwater volume accumulated in the Ob-Yenisei plume, while the majority of this volume remains in the central part of the Kara Sea till the end of the ice-free season. Eastward advection of the Ob-Yenisei plume through the Shokalsky Strait located ~ 100–150 km northward from the Vilkitsky Strait  freshened buoyancy current, which reaches the Vilkitsky Strait and further inflows to the Laptev Sea with potential to merge with the Lena plumeIn situ measurements of thermohaline characteristics of the surface layer performed in the Kara and Laptev seas by a ferry-box system on 21–25 October 2020 revealed low salinities (< 28)  on 5 October 2020  propagation of freshened water from the Kara Sea to the Laptev Sea through the Vilkitsky Strait shortly before ice formation, (2) low salinities in the Vilkitsky Strait during October–January followed by high salinities during February–July, (3) high salinities in the central part of the Kara Sea from late January till late April. These results are evidences of eastward advection of the Ob-Yenisei plume through the Vilkitsky Strait during ice-covered season. The Ob-Yenisei plume was leaking from the Kara Sea to the Laptev Sea from early October till the middle of January.16, which are the earliest available measurements in this area, reveal absence of low salinities in the northeastern part of the Kara Sea. Second, regular accumulation of freshened surface layer in the northeastern part of the Kara Sea would strongly affect seafloor sediments in this area, which was not observed by previous studies35. Nevertheless, this question could be properly solved only by in situ measurements, which is a part of our planned future work.We presume that in late January this processes finished because the whole plume was transferred either eastward to the Laptev Sea, or northward to the central part of the Arctic Ocean . Nevertheless, we lack in situ measurements during winter and spring in the northeastern part of the Kara Sea, in particular, at the area between the Gulf of Ob and the Vilkitsky Strait. As a result, some freshwater volume from the Ob-Yenisei plume could possibly remain in winter and spring in the northeastern part of the Kara Sea. However, there are indirect evidences that the whole plume leaks through the Vilkitsky Strait to the Laptev Sea. First, in situ measurements in August31 recently described abrupt transformation of vertical structure of the Ob-Yenisei plume shortly before ice-covered season caused by autumn convection. Atmospheric cooling of the freshened surface layer in the Kara Sea in late autumn results in, first, vertical homogenization of the Ob-Yenisei plume and, second, formation of a very sharp vertical salinity gradient at the plume-sea interface. It significantly affects dynamics of the Ob-Yenisei plume once it becomes isolated from wind forcing after the beginning of the ice-covered season. Formation of a very sharp vertical salinity gradient at the bottom boundary of the Ob-Yenisei plume dramatically reduces friction between the plume and the subjacent sea. Before convection starts, salinity at the plume-sea interface increases from 20 to 30 at vertical distance of ~ 5–10 m, while convection reduces this distance to 1–2 m 1/2, where Ri is the Richardson number equal to the relation between buoyancy frequency and vertical velocity shear36. Autumn convection increases vertical density gradient, and, therefore, the Richardson number changes from 1.1 to 8 indicating transition of the plume dynamics to the buoyancy-driven alongshore flow. Vertical eddy viscosity reduces from 8.8·10–4 to 5.9·10–4, i.e., friction at the plume-sea interface decreases by 1.5 times.Osadchiev et al.37. Conservation of potential vorticity of the geostrophic flow is described by the equation Due to the lack of direct velocity measurements in the Vilkitsky Strait, we use a model of two-layer buoyancy-driven coastal current to estimate the alongshore flow velocity of the Ob-Yenisei plume once it becomes isolated from wind forcing38. The main advantage of this model is that it is simple and can provide estimations of average velocity without usage of complex and computationally expensive numerical models. However, a disadvantage is that the model is one-dimensional and only depends on cross-shore dimension, i.e., distance from the shoreline, so it cannot account for vertical effects like drag from sea ice. The coastal current model could be used to estimate the average coastal current velocity based on measured density gradients and depth of the plume. However, the effect of drag from the sea ice cover on the flow needs to be added. We are studying a coastal current during the ice season, so we reproduce the influence of landfast ice cover. We parameterize ice cover to act as a solid motionless plate39. Below ice is a two-layered flow, with fresher water over denser water. We assume steady laminar flow in the upper layer, with no mixing between layers. The interface between the layers is assumed to be at rest40. Laminar flow between plates has a parabolic velocity profile The coastal current model used in this study is based on41 and Savelieva et al.42 described multiple in situ measurements performed in the northeastern part of the Shokalsky Strait (the Baranov Cape at the Bolshevik Island of the Severnaya Zemlya Archipelago). They report that salinity in the surface layer exceeded 30–31 during January–May in 2014–2016 and 2019. This result reveals that advection of the Ob-Yenisei plume from the Kara Sea to the Laptev Sea occurs only through the Vilkitsky Strait, this flow does not passes through the Shokalsky Strait. This result is indirect evidence that northward advection of the Ob-Yenisei plume to the central part of the Arctic Ocean does not occur at least in the northeastern part of the Kara Sea, otherwise it could be registered in the Shokalsky Strait. However, this question should be addressed primarily on the base of specific in situ measurement in the northern part of the Kara Sea performed during the whole ice season. To the extent of our knowledge, these measurements were not yet performed, but are within the scope of our future work.The Kara and Laptev seas are connected by two straits, namely, 50–80 km wide Vilkitsky Strait in the south and 15–40 km wide Shokalsky Strait in the north  will pass through the Vilkitsky Strait (with average width of 65 km) during 110–140 days. This estimation is in a good agreement with the in situ measurements at the mooring station in the Vilkitsky Strait, which demonstrated presence of the plume in the straight during 3–4 months from the middle of October till January–February.Once we estimate average velocity of eastward alongshore flow of the Ob-Yenisei plume below sea ice as 0.32 m/s, the whole plume  is similar to total river runoff to this sea during summer-autumn freshet period. On the opposite, freshwater volume accumulated during ice-free season in the Laptev and East-Siberian seas (1500–2000 km3) is much greater than total annual river runoff to these seas (~ 1000 km3)44. This difference could be explained by the advection of the Ob-Yenisei plume from the Kara Sea to the Laptev Sea during winter and spring.Previous in situ measurements in the Eastern Arctic demonstrated that freshwater volume accumulated during ice-free season in the Kara Sea  at 0.2-m spatial resolution. Salinity measurements in the surface layer (at the depth of 4 m) were performed by a ferry-box system equipped by thermosalinograph (SBE21) every 15 s.Temperature and salinity measurements in the Vilkitsky Strait at the depth of 30 m during ice-covered season were performed at the mooring station equipped by a CTD logger (centi-CTD StarOddi). The mooring station was installed at the distance of 5 km from the Cheluskin Cape at the most narrow part of the Vilkitsky Strait and performed measurements from October 2020 to July 2021. The measurements were performed every 6 h.Salinity measurements in the surface layer (at the depth of 4 m) in the Kara Sea during ice-covered season were performed by a ferry-box system equipped by salinity logger (centi-CT StarOddi). The ferry box was installed on the ice-breaking container vessel “Monchegorsk”, which crossed the Kara Sea from the Kara Gates Strait to the Yenisei Gulf on 24–29 January and 16–24 March 2021 and from the Zhelaniya Cape to the Yenisei Gulf on 17–22 April 2021. The measurements were performed every 1 h.57.Wind forcing conditions were examined using ERA5 atmospheric reanalysis with a 0.25° spatial and hourly temporal resolution


Allergic reactions are mediated by human IgE antibodies that bind to and cross-link allergen molecules. The sites on allergens that are recognized by IgE antibodies have been difficult to investigate because of the paucity of IgE antibodies in a human serum. Here, we report the production of unique human IgE monoclonal antibodies to major inhaled allergens and food allergens that can be produced at scale in perpetuity.in vitro by comparing β-hexosaminidase release from a humanized rat basophilic cell line.The IgE antibodies were derived from peripheral blood mononuclear cells of symptomatic allergic patients, mostly children aged 3–18 years, using hybridoma fusion technology. Total IgE and allergen-specific IgE was measured by ImmunoCAP. Their specificity was confirmed through ELISA and immunoblotting. Allergenic potency measurements were determined by ImmunoCAP inhibition. Biological activity was determined n = 33) were derived from 17 allergic patients with symptoms of allergic rhinitis, asthma, atopic dermatitis, food allergy, eosinophilic esophagitis, or red meat allergy. The antibodies were specific for five inhaled allergens, nine food allergens, and alpha-gal and had high levels of IgE  with ratios of specific IgE to total IgE ranging from <0.01 to 1.39. Sigmoidal allergen binding curves were obtained through ELISA, with low limits of detection (<1 kU/L). Allergen specificity was confirmed through immunoblotting. Pairs of IgE monoclonal antibodies to Ara h 6 were identified that cross-linked after allergen stimulation and induced release of significant levels of β-hexosaminidase (35%–80%) from a humanized rat basophilic cell line.Human IgE monoclonal antibodies (Human IgE monoclonal antibodies are unique antibody molecules with potential applications in allergy diagnosis, allergen standardization, and identification of allergenic epitopes for the development of allergy therapeutics. The IgE antibody probes will enable the unequivocal localization and validation of allergenic epitopes. Thegillosis . Here, wgillosis .in vitro IgE testing, and/or skin prick tests to inhaled allergens or food allergens  were obtained from 17 allergic subjects, aged 3–63 years, recruited from within the Vanderbilt University Medical Center (VUMC). The subjects diagnoses were based on clinical history, llergens . The prohttps://insight.jci.org/articles/view/123387/sd/1 (accessed 110923) and can be downloaded as a PDF file. Briefly, PBMC were cultured for 7 days with irradiated NIH3T3 cells expressing CD154, B-cell-activating factor, and IL-21 to selectively expand B cells. After screening for IgE through ELISA, IgE-producing B cells were fused with non-secreting myeloma cells and cloned using single-cell flow cytometry. The hIgE mAb generated using this B-cell culture system and hybridoma methodology were not created by artificial class switching. Screening for specificity only occurred after all IgE lines were fused. The hIgE mAb were screened for allergen specificity through ELISA by using microtiter plates coated with ∼1 µg/well of purified natural or recombinant allergens: nDer p 1, nDer p 2, nFel d 1, nCan f 1, rCan f 6, nAra h 1, nAra h 2, nAra h 3, nAra h 6, nBos d 5, nBos d 11, nGal d 2, nGal d 4, rAna o 3, and rJug r 1. The bound hIgE mAb was detected with peroxidase-labeled mouse anti-human IgE. The hIgE mAb were purified by affinity chromatography using immobilized monoclonal anti-IgE antibody omalizumab. The purity of the hIgE mAb was assessed by SDS-PAGE under reducing conditions.Detailed cell culture methods for producing hIgE mAb from PBMC have recently been published , 21, 22.The concentration of total IgE and allergen-specific IgE was determined through ImmunoCAP  , 23. TheThe IgE concentration of the purified hIgE mAb was initially determined by total IgE ImmunoCAP using highly diluted samples . Once the concentration of each purified hIgE mAb stock was determined, samples were diluted to a concentration of ∼50,000 kU/L and re-assayed at 1/100–1/10,000 dilutions. The samples were assayed for the concentration of the allergen-specific IgE antibody by ImmunoCAP, and the ratio of total IgE to allergen-specific IgE was compared.The specificity of the hIgE mAb was further assessed by ELISA using microtiter plates coated with 100 ng/well of purified allergen. Doubling dilutions of the hIgE mAb were prepared over a three-log range (0.1–100 kU/L) to form dose–response curves. The bound hIgE mAb was detected using peroxidase-labeled goat anti-human IgE .For immunoblotting, purified natural or recombinant allergens, cetuximab, and extract samples were run on SDS-PAGE under reducing or non-reducing conditions. Allergens were transferred to PVDF membranes and incubated with a hIgE mAb at 1–4 µg/ml for 1 h. The membranes were washed and incubated for 30 min with alkaline phosphatase-conjugated mouse anti-human IgE Fc  and Strep-Tactin . They were developed with chemiluminescent substrate prior to chemiluminescence imaging.ImmunoCAP inhibition was performed according to the manufacturer's instructions. Pools of the hIgE mAb for Ara h 1 and Ara h 2 and for Der p 1 and Der p 2 were compared with human serum pools from patients allergic to peanuts and to house dust mites, respectively. Pools were diluted to ∼10 kU/L for peanut and house dust mite ImmunoCAP, respectively. The pool of hIgE mAb for peanuts comprised six Ara h 2 hIgE mAb  and a single Ara h 1 hIgE mAb (4G4). The pool of hIgE mAb for house dust mites used a single Der p 1 hIgE mAb (2L11) and three Der p 2 hIgE mAb . Sera from patients allergic to peanuts were provided by Dr. Stephen Dreskin of the University of Colorado, Denver, CO, USA . Sera frβ-Hexosaminidase release was used as a marker of allergen-induced degranulation. Briefly, RBL-2H3 cells were sensitized with hIgE mAb pairs to Ara h 6 at 5–30 kU/L. After overnight sensitization with hIgE mAb, the cells were stimulated with 50 ng allergen. Untreated cells and cells sensitized with hIgE mAb but not stimulated with allergen were used as controls. For data analysis, the average background signal was subtracted from each value, the percentage of maximal lysis was calculated, and min–max normalized values were plotted. Results were observed from three independent experiments. An ordinary one-way ANOVA with a Tukey's mean multiple comparisons analysis was used to determine significant differences between the mediator release produced by all the combinations.The functional activity of the hIgE mAb was assessed by using humanized rat basophilic leukemia (huRBL) cells transfected with human FcɛRI for passive sensitization with hIgE mAb (RBL-2H3 cell line) , 27. β-Hn = 10) to female (n = 7) donors. The research subjects had total IgE levels of 351 to >5,000 kU/L, together with high levels of allergen-specific IgE (65 to >100 kU/L) and/or strongly positive skin prick tests to allergen extracts  and four adults (aged 27–63 years), with a preponderance of male  showed homogeneous heavy- and light-chain bands of 75 kD and 25–30 kD, respectively. Two hIgE mAb, to alpha-gal (16D9) or Ara h 1 (3B10), showed truncated heavy chains at ∼50 kD either alone or in addition to the 75 kD heavy chain. The IgE mAb 16D9 bound to alpha-gal and to FcɛRI by ELISA. Despite the presence of only the truncated form in 3B10, this antibody did specifically recognize Ara h 1. However, it did not bind to FcɛRI (data not shown), which indicated that this band with lower MW than expected for a heavy chain possibly results from a truncated form that lost its capacity to bind the IgE high-affinity receptor, while keeping its capacity to bind the allergen.This paper describes the IgE-binding activity and biological activity of 33 hIgE mAb directed against five inhaled allergens  and against nine food allergens  and alpha-gal. Multiple hIgE mAb were generated against several allergens such as Der p 1, Der p 2, Fel d 1, Ara h 1, Ara h 2, Ara h 3, and Ara h 6 and alpha-gal. Following monoclonal anti-IgE affinity purification, yields of hIgE mAb were typically 2–30 mg, and purity was >90% under reducing conditions on SDS-PAGE . Most ofThe total IgE measurements of purified hIgE mAb ranged from 53,450 to 1,702,500 kU/L . To stanSome hIgE mAb to peanut reacted strongly with allergen extract but displayed low IgE binding to the purified allergen, suggesting that the recombinant allergen(s) coupled to the cellulose solid phase of the ImmunoCAP may lack certain IgE epitope(s). This was investigated by comparing hIgE mAb binding to purified natural and recombinant allergens using the streptavidin CAP , 29. PurThe hIgE mAb showed a strong binding to purified allergens in ELISA with dose–response curves from <1 to 100 kU/L . In mostTo evaluate whether the pools of hIgE mAb could replace variable patient serum pools for potency assessment of immunotherapy extracts in standardization protocols, both were compared by ImmunoCAP inhibition targeting house dust mites and peanuts. Extracts of both allergen sources gave very similar inhibition curves using the pools of monoclonal antibodies and the pools of the human serum .ε receptor that was transfected into the RBL cell line upon allergen stimulation to induce degranulation. The mediator release data for hIgE mAb pairs to Der p 2, Fel d 1, and Ara h 2 was recently published  whether the hIgE mAb were directed against different epitopes and (ii) whether they could cross-link the high-affinity human IgE Fcublished . For eacβ-hexosaminidase release using several combinations of the hIgE mAb  were analyzed for basophil mediator release. Dose–response curves showed 30%–90% hIgE mAb . Two of in vitro studies, but the synthetic antibodies  either used murine variable regions or did not necessarily have the natural pairing of human heavy and light chains that formed the IgE response in allergic people . Patients were randomly enrolled in these studies based on history, symptoms, and positive skin test to given allergens, rather than being targeted cohorts of a given age range or allergic sensitivity. In the future, pediatric and adult populations could be targeted for certain allergens, e.g., foods  or inhaled allergens , to obtain a broader spectrum of the hIgE mAb. This would enable linear or conformational epitopes on food allergens to be investigated .The hIgE mAb have several applications in the investigation of allergic diseases: first, as potential replacements for human sera and, second, as reference material(s) in allergy diagnostics. Access to the panels of the sera from allergic patients presents a significant barrier to innovation for diagnostic test developers . The hIgSince the discovery of IgE, standardization of serologic measurements of IgE has relied on the World Health Organization’s human serum references for measuring the IgE levels for clinical diagnosis. The IgE tests produced by different manufacturers show variability and are not interchangeable . The hIgThe unique value of the hIgE mAb to define allergenic epitopes recognized by allergic patients, to answer the question about what constitutes an allergenic epitope, will have applications in allergy therapeutics. A recent clinical trial demonstrated the efficacy of allergen-specific monoclonal antibodies to Fel d 1 to treat mild asthma . Mutatio


Peptide profiling by LC-MS QTOF and in silico interaction with target enzymes showed that only four derived bioactive peptides from QPHs could bind in the active site of CEase, whereas twelve peptides could bind in the active site of PL. Peptides QHPHGLGALCAAPPST, HVQGHPALPGVPAHW, and ASNLDNPSPEGTVM were identified to be potential CEase inhibitors, and FSAGGLP, QHPHGLGALCAAPPST, KIVLDSDDPLFGGF, MFVPVPH, and HVQGHPALPGVPAHW were identified as potential PL inhibitors on the basis of the maximum number of reactive residues in these bioactive peptides. In conclusion, QPHs can be considered as an alternative therapy for the treatment of hypercholesterolemia.Hypercholesterolemia remains a serious global public health concern. Previously, synthetic anti-hypercholesterolemic drugs were used for ameliorating this condition; however, long-term usage presented several side-effects. In this regard, natural products as an adjunct therapy has emerged in recent times. This study aimed to produce novel bioactive peptides with anti-hypercholesterolemic activity (cholesterol esterase (CEase) and pancreatic lipase (PL)) from quinoa protein hydrolysates (QPHs) using three enzymatic hydrolysis methods  at 2-h hydrolysis intervals . Chymotrypsin-generated hydrolysates showed higher CEase (IC The rise in diet-related metabolic disorders such as hyperlipidemic or hypercholesterolemia, cardiovascular diseases, hypertension, and other related metabolic diseases has been identified as the major cause of mortality globally, and thus has become a serious public health issue. The consumption of processed food and animal products has been associated with the occurrences of these health issues. Cholesterol esters are normally found in the human diet, which the human intestine cannot sufficiently absorb, thereby producing free fatty acids and cholesterol that elevate the level of blood cholesterol. It is a known fact that rising levels of cholesterol and triglycerides in the blood serum results in hyperlipidemia or hypercholesterolemia . AccordiNowadays, generating bioactive peptides from plant-based proteins have gained more attention owing to their biological and functional activities, which is highly significant in alleviating chronic diseases, as well as in promoting human health. Moreover, the effectiveness of the derived peptides depends on their sequence, structure, and inherent amino acid composition. Quinoa proteins are a very good source of essential amino acids . Apart fSimilarly, several bioactive peptides from protein hydrolysates have demonstrated the capacity to inhibit the digestion of dietary lipids in in-vitro and cultured cell assays . PeptideSeveral scientific investigations have shown that natural peptides found in plant proteins can inhibit cholesterol absorption or HMGCoAR activity . HoweverAccordingly, quinoa protein isolates were hydrolyzed using three different enzymes  in order to get a diverse sequence of peptides which might be potential inhibitors of CEase and PL. The generated quinoa hydrolysates were characterized biochemically via the degree of hydrolysis, and their CEase and PL inhibitory potentials were determined. Furthermore, peptides generated from quinoa protein isolates were characterized and identified using LC-MS-QTOF. A molecular docking approach was carried out to elucidate the molecular binding mechanism between potent anti-hypercholesterolemia peptides with the target enzymes (CE and PL) through the in silico structural activity relationship.Streptomyces griseus), α-chymotrypsin , and bromelain , HPLC grade reagents (acetonitrile and methanol), and other chemicals such as formic acid p-nitrophenyl butyrate, o-phthaldialdehyde, trizma base, β-mercaptoethanol, SDS, and sodium tetra-borate were procured from Sigma Aldrich . Other analytical grade reagents used were purchased from the UAE .Quinoa seeds used in this study were purchased from the local market . Enzymes such as protease  maintained at a pH of 10–11 and was stirred for 2 h on an electric shaker (24 ± 2 °C), and the resulting slurry was stored at 4 °C overnight. The slurry was homogenized for consistency and centrifuged twice at 4 °C at a speed of 10,000× g for 20 min after overnight storage. Whatman No. 1 paper was used to filter the obtained supernatant, and the pH of the filtrate reduced to 4.5 using the pH drop method with 1.0 mol L−1 HCl solution to precipitate quinoa protein. Afterwards, precipitated proteins were separated by centrifuging at 4 °C for 10 min at the speed of 10,000× g, and were subsequently washed twice with deionized water. The pH of the resultant pellets was adjusted to pH 7.0 (with 1.0 mol L−1 NaOH) and stored at −20 °C until further use. The Kjeldahl method was adopted to determine the protein content of quinoa protein isolate (QPI) slurries, which was found to be 44%.Firstly, quinoa seeds were oven dried (40 °C for 3 h), dry-blended , and passed through a screen (3 μm pore size) to obtain a fine powder. The methodology described by  as adopt® homogenizer IKA®-Werke GmbH, Staufen, Germany). The protein content of the slurry was set to 4% with deionized water, and the content was divided into four batches for treatment with bromelain, chymotrypsin, and protease, respectively, while the last part was kept as control. Then, 1 mol/L NaOH and 5N HCl were used to adjust the pH of the treated protein slurries for the bromelain enzyme to 7.0, the chymotrypsin enzyme to 7.8, and the protease enzyme to 8.0, respectively. Furthermore, enzyme concentration was pre-calculated, suspended in 1 mL of distilled water resulting in a 1% enzyme/substrate ratio, and subsequently added to the QPI slurry. The samples were then distributed into 50 mL falcon tubes in triplicate for each period of hydrolysis and incubated (water bath) for up to 6 h at the speed of 100 ramps/min at a controlled temperature of 50 °C. Samples were taken every 2 h from all enzymatic incubations, and the reaction was inactivated at 95 °C for 600 s. Generated QPHs were centrifuged at 4 °C at a speed of 10,000× g for 10 min, and supernatants were collected and stored at −20 °C for further analysis. All of the analyses were performed within two weeks of the production of hydrolysates.The methodology as described by  was usedThe previously published o-phthaldialdehyde (OPA) methodology of  was usedtoth = total number of peptide bonds per protein equivalent; and h = number of hydrolyzed bonds, and calculated using h = (SerineNH2 − β)/α. Where toth, β, and α, β and toth values for quinoa proteins were used as 8.0, 0.40, and 1.00 mEq/g protein, respectively.Herein, p-nitrophenyl butyrate (25 μL) were mixed with each sample (50 μL) in a sodium chloride: sodium phosphate buffer , and then incubated in a 96-well microplate reader. The quantity of the resultant reaction was modified with the above buffer to 150 μL and incubated (37 °C for 30 min). After the incubation period, the released p-nitrophenyl for each sample was measured on a plate reader  at 405 nm. The PL-inhibitory activity was calculated using the equation belowThe PL-inhibitory activity of generated hydrolysates was measured using the methodology of . Here, PA = the absorbance of control; B = the absorbance of control reaction blank; C = the absorbance of sample, and D = the absorbance of the sample blank. The IC50 values were determined by plotting the percentage inhibition against the concentration of the test compound and expressed in mg (eqv protein)/mL.Here: p-nitrophenyl butyrate in sodium phosphate (100 mM): NaCl buffer  was placed in a 96-well microtiter plate. Subsequently, the mixtures were incubated with 50 μL of porcine pancreatic CEase (5 μg/mL) at 37 °C for 180 min. After that, the p-nitrophenol released from the enzymatic hydrolysis of p-nitrophenyl butyrate was read at 405 nm. The percentage CEase inhibition was determined using Equation (2) above. The IC50 values were determined by plotting the percentage inhibition against the concentration of the test compound and expressed as mg (eqv protein)/mL.The CEase-inhibitory activity was determined as per the procedure documented by . Brieflyv/v) formic acid with a flow rate of 15 µL/min. The mobile phase gradient of the HPLC system was as follows: (a) 0–5 min, 10% B; (b) 5–115 min, 10–95% B, (c) 115–120 min, 95% B, (d) 120–135 min, 95–10% B, and (e) 140–150 min, 10% B. The electrospray ionization-quadrupole-time-of flight system (ESI-QTOF) condition was: (a) mass range: 70–2000 m/z; (b) collision energy: 6V/100 Da (offset-2); (c) flow rate: 15 µL/min; (d) ion spray sources: 3.5 kV; (e) drying gas: Nitrogen at a temperature of 350 °C with a flow rate of 10 L/min; (f) nebulizer pressure: 3 psig; (g) fragmentor voltage: 110 V; and (h) fragmentation mode: collision induced dissociation (CID). Data generated from the mass spectrometry approach were analyzed using PEAKS studio version 6.0 [For the identification of peptides in selected hydrolysates, QC-6 (chymotrypsin-generated at 6 h hydrolysis), LC-MS Q T-O-F was conducted as described previously by . The idesion 6.0 . The prehttp://bioware.ucd.ie/, accessed on 26 July 2022) [Potential bioactive peptides were screened using the PeptideRanker web server (ly 2022) . Peptidehttp://pepsite2.russellab.org (accessed on 16–19 August 2022) [http://www.rcsb.org/pdb/ (accessed on 16–19 August 2022). Peptide inputs were then entered jointly with a protein receptor in PDB format. The best interaction of peptides with enzymes showing p-values < 0.05 and the interaction with key hotspots on enzymes playing a significant role in enzyme inhibition were taken into consideration for further analysis.The preliminary insight into the molecular mechanism of peptides for their PL and CEase inhibitory activities were explored through an in silico docking investigation using the online program Pepsite2, accessible at The 3D structures of human and bovine CEase, PDB IDs 1F6W and 1AQL respectively, and PL (PDB IDs: 1LPB and 1ETH) were obtained from the Protein Data Bank (PDB) . These pThe active sites of CEase (PDB IDs: 1F6 W and 1AQL) and PL (PDB IDs: 1LPB and 1ETH) that are directly involved in ligand-binding were identified from the literature The active site of lipase is located in the N-terminal domain and their catalytic activity is mediated by the catalytic triad Ser152, Asp176 and His263 Similarly, CEase also shares a comparable catalytic triad composed of Ser194, Asp320 and His435. For the docking analysis, a receptor grid suitable for peptide docking was generated for the minimized protein structures enveloping the active site residues. No constraints were applied when default parameters for van der Waals scaling factor (1.00) and charge cut-off (0.25) were used. The OPLS 2005 force field was used for structural representation .The Peptide Docking panel of Schrödinger Maestro  was emplThe binding free energy of the best docked poses obtained after peptide docking were evaluated using the MM-GBSA approach. Binding free energy calculations were performed using Schrödinger Prime with OPLS 2005 force field combined with VSGB 2.0 implicit solvent model. The peptide was minimized, and the receptor was treated as rigid for the MM-GBSA calculations.p-value of 5% was defined as statistically significant (p < 0.05).All hydrolysates were produced in three batches representing three replicates. All analyses were carried out in triplicate. Data analysis was performed with SPSS 24.0 statistical software  using one-way analysis of variance. Means separation was conducted using Tukey’s multiple range test, and a The DH and enzyme specificity are among the measurable factors to be controlled because they define the biological activities of the resulting peptides. Studying the hydrolysis of protein reveals the capability of an enzyme to degrade proteinaceous substrate, and this serves as a pointer for their proteolytic reaction . NoteworIn this work, the effects of different enzymes  at 2 h-interval reaction periods on the DH of quinoa protein hydrolysates (QPHs) were reported .p < 0.05). These results are comparable with those obtained by [The results obtained showed a characteristic similar hydrolysis pattern among all the enzymes, where the DH values significantly increased with the progression of hydrolysis time up to 6 h . Bromelain-produced hydrolysates after 6 h (QB-6) showed an intermediate DH value of 51.91%, and the lowest protein hydrolysis was demonstrated in both bromelain-produced hydrolysates (QB-2) and chymotrypsin-produced (QC-2) after 2 h of bio-catalysis (p > 0.05). In general, chymotrypsin was found to be the most effective enzyme in hydrolyzing quinoa proteins as opposed to the other two proteolytic enzymes (bromelain and protease). The hydrolytic pattern of chymotrypsin indicates greater proteolytic efficiency of the enzyme, which may be attributed to the size and action of the released peptides. As a result, the abundance of small-sized peptides with enhanced effectiveness against metabolic markers could be produced from chymotrypsin at prolonged hydrolysis periods. This is consistent with previous studies that suggested that higher DH due to disintegration of more peptide bonds could be attributed to the higher activity of the enzyme [Based on the enhanced DH values with progressive hydrolysis time, chymotrypsin-produced hydrolysates after 6 h (QC-6) displayed the highest value of 66.05%. However, it was not noted that the DH value obtained in protease-produced hydrolysates after 6 h (QP-6) was not significantly different from the DH value of QC-6h (e enzyme .p > 0.05) of protease-generated hydrolysates did not show a very rapid increase in the displayed DH. However, after progressing the bio-catalysis reaction to 6 h, a significantly higher DH (p < 0.05) was recorded, as presented in The enzymatic hydrolysis of quinoa proteins using bromelain and chymotrypsin exhibited a rapid and steady increase in the DH values over a period of 6 h, an indication that the larger peptides that were released at a lower hydrolysis time were further disintegrated into smaller peptides as the hydrolysis time progressed. We hypothesize that a higher degree of hydrolysis due to the increased time of hydrolysis is often related to further enzyme activity on the reaction substrates. In contrast, the progression of hydrolysis-reaction from 2 h to 4 h , and the results are illustrated in 50 value of hydrolysates connotes high CEase inhibitory activity. Upon hydrolysis, the QPHs derived from bromelain showed an increased CEase inhibition, with progression of the bio-catalysis reaction from 2 to 6 h . Therefore, bromelain-generated hydrolysates with high CEase inhibitory activity can be developed at longer hydrolysis times. On the other hand, the CEase inhibitory potential of chymotrypsin and protease derived hydrolysates decreased with the increased bio-catalysis reaction time from 2 to 6 h.QPHs were analyzed for their ability to inhibit CEase. CEase is a known polymeric enzyme existing in the bile that initiates dietary cholesterol esters’ hydrolytic reaction, thereby releasing cholesterol and free fatty acids . Inhibit50 values of 0.51 mg/mL, 0.55 mg/mL, and 0.55 mg/mL after 2, 4, and 6 h of hydrolysis, respectively. However, there was no significant difference (p > 0.05) observed between hydrolysates generated at 4 h and 6 h hydrolysis time. Similar results were obtained by [50 values of hydrolysates with respect to different time periods could be explained with the ability of specific enzymes to either degrade or generate CEase inhibitory peptides during hydrolysis [From this study, it was evident that chymotrypsin-generated QPHs showed the highest CEase inhibitory activity with ICdrolysis . The prodrolysis .50 value of 1.01 mg/mL). A study of the CEase-inhibitory activity of amaranth protein recently reported in our lab also showed a significant low CEase-inhibitory effect of amaranth protein isolate in comparison to IC50 values recorded for hydrolysates [50 value of 0.51 mg/mL (chymotrypsin-derived for 2 h) was approximately two times lower than the IC50 value (1.01 mg/mL) of QPI, indicating that derived hydrolysates can therefore inhibit CEase two-fold more than their intact protein counterpart. Furthermore, the results suggested an overall high CEase inhibitory activity of QPHs compared to intact quinoa protein, which is evident from the lower IC50 values observed. This is similar to the study of Jafar, et al. [Unhydrolyzed quinoa protein, i.e., intact quinoa protein (QPI), showed the least CEase inhibitory activity  and un-hydrolyzed quinoa protein are depicted in 50 values varied from 0.90 to 10.4 mg/mL, implying a substantial ability to inhibit PL activity. The PL-inhibitory activity of all the hydrolysates (0.90–3.32 mg/mL) exhibited significantly higher inhibition than the QPI, indicating that hydrolysis efficiently increased the inhibitory activity of quinoa protein. Hydrolysate QC-6 demonstrated the highest PL-inhibitory activity (0.78 mg/mL), followed by QB-6, recording IC50 values of 0.90 mg/mL, respectively. All chymotrypsin and bromelain generated hydrolysates showed higher PL-inhibitory activities than the protease generated hydrolysates. The different inhibitory activity observed among hydrolysates generated from different enzymes  could be explained with the specificity and varying degree of hydrolysis among other factors of the individual enzyme [Pancreatic lipase is the primary enzyme that contributes largely to the digestion of dietary lipids, and the only effective way to adjust lipid absorption is by inhibiting the enzyme . The panp < 0.05) was observed in the pancreatic lipase inhibitory activity of QPHs upon hydrolysis with different enzymes at different hydrolysis times. A significant increase in the PL inhibitory activity of QPHs was observed with the increase in hydrolysis time progression, which is shown with lower IC50 values. For instance, chymotrypsin derived hydrolysate with the most potent PL-inhibitory activity IC50 values decreased from 2.90 to 0.78 mg/mL as the hydrolysis time increased. In the past, various studies have demonstrated an increase in the enzyme inhibitory potential of protein hydrolysates with the increase in the duration of hydrolysis. This implies that the progressive time of hydrolysis would sufficiently release peptides that might bind the active site of pancreatic lipase, thus inhibiting lipase enzymes. In addition, hydrolysates generated from chymotrypsin showed the maximum inhibitory activity in comparison to bromelain and proteases. This result upholds the potent characteristic of chymotrypsin generated hydrolysates, as reported by [On the other hand, a significant effect , it was further chosen for peptide identification by LC-MS Q T-O-F. Additionally, the ABTS radical scavenging and anti-hemolytic activities shown by this specific hydrolysate reported in a previously published work by .p < 0.05) implies that peptides derived from QC-6 significantly bound to the hotspot sites of CEase, resulting in non-competitive inhibition due to the modification in protein interaction networks or loss of enzyme activity that blocked its catalytic binding sites and substrate [p < 0.05) binding effect on CEase  binding effect to PL. In this study, the number of peptides derived from QC-6 was 35, and the number of amino acids ranged from 2 to 16. This value is comparable to the range reported by [p < 0.05) potent PL-inhibitory potential, binding up to 14 and 13 important amino acids, suggesting superior linkages to the target enzyme  bound to the active sites, only peptide ML interacted with a lower number of hotspots (four sites), and can be regarded as a poor PL inhibitor.Furthermore, these peptides have also demonstrated CEase inhibitory potential, as explained above, implying that these peptides have valuable anti-hypercholesterolemic properties. Other peptides  have shown a similar potent inhibitory potential binding up to 11, 11, and 12 important amino acids of PL, respectively. These peptides could be considered as potential PL inhibitors due to their maximum interaction with the binding sites of PL enzyme. Similarly, some peptides showed a high number of binding sites, thus possessing promising PL-inhibitory activity. Peptide HMCH could interact with nine bound residues, four peptides  with eight bound residues, and peptide DFLM interacting with six bound residues. Among all the peptides that significantly  were capable of binding to similar active sites. Similar binding spots for PL inhibitory activity from cumin seeds have been reported by , and camIt was observed that peptides that have the potential to bind a higher number of active sites of lipase were the hydrophobic amino acids. A majority of the peptides that exhibited the potential of binding a high number of binding sites of lipase consisted of hydrophobic amino acid residues such as proline and leucine. This is comparable to a previous study that showed proline and leucine to be the most active residue bound of lipase inhibiting peptides derived from pinto beans . In addiTo elucidate the binding pose and to identify the underlying intermolecular interactions of potential quinoa derived peptides in the active site of human and bovine CEase, molecular docking was performed. CEase is a member of the alpha/beta hydrolase fold family. In the human CEase protein, the active site region includes an oxyanion hole comprising Gly107-Ala108 and Ala195 residues, and a catalytic triad containing Ser194-His435-Asp320 residues; both regions are essential for the functional activity of the protein. The hydroxyl group of Ser194 functions as a nucleophile that facilitates the hydrolytic action . Apart fAmong the docked peptides, the top docked peptide HVASGAGPW fits perfectly in the active site and is stabilized by hydrogen and hydrophobic bonds. Interestingly, Gly107, Ala108, and Ala195 residues, of the oxyanion hole, and His435, from the catalytic triad, were involved in the strong binding of this peptide with human CEase. The peptide AHCGGLPY showed interactions with several residues surrounding the active site including Ala108. Similarly, other top-docked quinoa peptides such as LYNDWDLR, MFVPVPH and FSAGGLP have shown interactions with the residues involved in the active site . These iThe binding pose and intermolecular interaction of quinoa peptides with PL was elucidated using molecular docking simulations. GlideScore and calculated binding energy, based on MM-GBSA, for the best-docked poses as well as intermolecular interactions observed in the docked protein-peptide complex are provided in The binding mode of the best-docked peptide, HVASGAGPW, within the active site of PL was analyzed. Results indicated that the peptide docked to the enzyme recorded a GlideScore of −11.74 kcal/mol and an MM-GBSA binding energy of −46.02 kcal/mol. The other top docked peptides AHCGGLPY, FSAGGLP, LLPYH and MFVPVPH also exhibited good GlideScores and binding energies . It was All of the reported peptides exhibited interactions with critical amino acids in the catalytic binding pockets of the enzyme by forming several hydrogen and hydrophobic interactions . Similar50 values. Findings showed that QPHs effectively inhibited the two enzymatic markers responsible for hypercholesterolemia by disrupting the enzyme-substrate interactions at the enzyme hotspot site. Novel peptide ASNLDNPSPEGTVM was found to have the best potential to be a CEase inhibitor, while FSAGGLP, KIVLDSDDPLFGGF, and MFVPVPH were found to be the potential PL inhibitors. The peptides QHPHGLGALCAAPPST and HVQGHPALPGVPAHW we observed to be potential inhibitors against both CEase and PL. In silico data showed that Gly107, Ala108, Ala195, and His435 were involved in the strong binding of these peptide with human CEase. Thus, peptides generated from quinoa seed protein may have a valuable application in ameliorating hypercholesterolemia based on predictions using software tools and molecular docking studies. However, further studies using in vitro, in vivo and cell line assays should be carried out to validate the potential anti-hypercholesterolemic activities observed in this work.This study investigated the CEase- and PL-inhibitory potential of QPHs as a functional ingredient for producing nutraceuticals that can be used to manage hypercholesterolemia. This current study explored the generation of hydrolysates from quinoa proteins using three enzymatic approaches . The three enzymes explored produced hydrolysates with higher inhibitory activities towards CEase and PL compared to unhydrolyzed quinoa proteins. Nonetheless, chymotrypsin-generated hydrolysates at 6 h hydrolysis time exhibited higher CEase and PL inhibitory activity as confirmed by lower IC


For polymer-processing extruders, designing screws via analytical computational models is helpful for reducing experimental costs. However, the current simulation programs cannot predict the melt quality at the screw tip with sufficient accuracy. There are a number of definitions of melt quality in the literature. This paper will review some of these definitions and present how melt quality can be assessed in subsequent work. In this paper, both the thermal and material homogeneity of the melt quality are examined for correlations with the screw performance index. If correlations exist with the screw performance index determined from direct experimental measurement data, these can be used as target values for developing a melt quality prediction model. In the field of polymer processing, polymers are plasticized so that they can be further processed into end products or semi-finished products. Polymers are often processed on screw machines, some examples of which are extruders and injection-molding machines. Each plastic is processed at least once on a single-screw extruder on its path from polymerization to becoming an end product . The cosFor this reason, the design of the extruder’s hardware, especially the extruder screw, plays a particularly important role in the subsequent cost efficiency. Since the process of screw design should not involve the use of prototypes for reasons of cost and time, the screw designer is left only with empirical values and simulations as reference points . At presIn order to simulate the extrusion process, it is necessary to theoretically describe the melting process in the extruder. Tadmor was the first to develop a mathematical model of the melt zone. The aim of this research is to establish correlations between process parameters, melt quality and product quality. The correlations to be used must first be investigated and evaluated to ensure their suitability for making predictions in a simulation model. Once suitable correlations have been determined, they are to be used to predict melt quality by means of the process parameters.This quality criterion should already be calculable and usable for the screw feeder. In this way, a targeted screw design can lead to improved process behavior and thus to higher throughput with consistent melt quality.In the following section, the three screw concepts used in this work are presented. Shear and mixing elements in combination with a three-section screw, barrier screws and three-section screws are investigated.Barrier screws are used to increase throughput. The increase in throughput is made possible by the increased energy input . A barriMaddock shear section and the Troester shear section [For good product quality, the melt must be in a materially and thermally homogeneous state while it is being processed in the die. The temperature should be constant over the entire cross-section, and there should be no differences in concentration between additives and fillers. To ensure this, shear and mixing sections are used at the screw tip. Usually, the shear section is mounted on the screw before the mixing section. . The fun section . Mixing  section . The sha section . In order to ensure a suitable polymer melt flow for processing, the melt can be assessed in terms of its quality on the basis of various criteria. Good melt quality is indicated by a uniform melt temperature distribution across the cross-section , as well as the uniform mixing of the melt  at a constant temperature and back pressure over time . AccordiMelting is an essential process in extrusion in which the polymer is completely converted into a molten state. If the energy input is too low and the melting process is therefore incomplete, solid particles will form within the melt and pass through the extrusion die. These solid particles, in turn, have a negative effect on the quality of the end product which is clearly visible, for example, when producing a film. As a rule, more solid particles occur when the screw speed and the associated throughput are increased . AccordiIt is necessary to ensure the melt is sufficiently homogenous, both materially and thermally, in the extrusion process. Thermal homogeneity is important for ensuring consistent material performance over the entire cross-section of the die, especially with respect to flow behavior. If this is not the case, different exit speeds can occur at the die exit, which can lead to insufficient dimensional accuracy . It is aIt is important that the melt at the end of the screw has as uniform a temperature as possible across the entire cross-section to ensure thermal homogeneity. One means of confirming this is to measure the temperature at different points in the cross-section and then calculate the standard deviation. A higher standard deviation indicates greater differences between the temperatures across the cross-section and signifies worse thermal homogeneity. A common method of determining the temperature across the cross-section is to use a measuring blade or measuring bar . The devStatements about the quality of the melt can also be made on the basis of fluctuations in the stationary pressure and the stationary temperature over time. A fluctuation in the pressure signal can indicate a variation in the throughput over time. This can lead to variations in the product’s geometry, for example. Typical causes of fluctuations in the back pressure are deep-cut metering zones, fluctuating throughputs during infeed or screw plugging. In addition to these effects, the pressure fluctuation also serves as a measure of the homogeneity of the melt . As a reIn addition to the back pressure fluctuation, the pressure gradient across the screw is relevant to achieving good extrudate quality. The screw should build up pressure continuously and have as few areas with negative pressure gradients as possible. This means that the air in the granulate is degassed in the direction of the hopper and that no air bubbles occur within the melt. In addition, the pressure flow, which counteracts the drag flow, improves the mixing effect and consequently the homogeneity of the melt .A typical challenge in extrusion is material damage due to molecular weight degradation . During Reductions in molecular weight are usually due to changes in the flow’s behavior and therefore viscosity. Therefore, it is a good idea to determine the melt flow rate (MFR) in the melt index analyzer . This siThe requirements placed on an extrusion line have changed significantly in recent years. In the past, the output was almost exclusively the decisive criterion, whereas today, the efficiency and flexibility of the systems play increasingly important roles . A high örner [According to Dörner , the criDruckCk, all criteria are weighted with the factor 1. Only the difference between max. pressure and the back pressure DruckCk is weighted with a factor of 0.5. In addition to the degree of fusion, the averaged melt temperature GMT is also a necessary condition. This must lie between specified material-dependent limits. If both conditions are fulfilled, the SPI can be determined according to Equation (1) [SPI: Screw performance index;Each criterion is divided into five classes from 0 to 4. Class 0 is the best class. In terms of the degree of melting, class 0 represents a completely melted melt. As the class increases, the number of solid particles at the die exit increases. The degree of melting is also a necessary condition for calculating the SPI. Beginning already from class 2, defects accumulate so regularly that the melt is inadmissible for further processing and the SPI drops directly to a value of 0. It should be noted that this is a subjective observation since the evaluation of the degree of melting cannot be measured directly but is based on empirical values. Analogous to the degree of melting, the maximum and minimum values of each criterion are analyzed for each combination of material, screw and process point. Between these two values, the five equal classes are spanned, and the screws and process points are then assigned to these classes. Each class is given a character, c, and its respective characteristic value in the index. In the next step, an average value is calculated for all classes. With the exception of the difference between the max. pressure and back pressure tion (1) .(1)SPI=The following chapter presents the materials and experimental equipment used in this work. In addition, the evaluation methods used to determine melt quality are presented.2–p5, Gefran, Seligenstadt Germany, IMPACT IEC 1000 bar) and five temperature sensors  along its entire length. After the last heating zone of the extruder, a temperature-controlled flange is mounted in which the pressure, pstar, is measured and the radial temperature profile at the screw tip is recorded. For this purpose, as described by Chung [The investigations were carried out on a single-screw extruder from Reifenhäuser Extrusion Systems GmbH , model RH034-45-28D/HS, in the technical center of Kunststofftechnik Paderborn . The extby Chung , five teC: Slope of the shear thinning viscosity (1 − n);n: Flow exponent.Two different polymers from the company LyondellBasell were used for the experimental investigations. The low-density polyethylene (LDPE) is called Lupolen 2420D and the name of the polypropylene is HP420M. The Carreau parameters at 230 °C for both materials are listed in Maddock shear section (SM1), and second, a spiral shear section (SM2) in combination with the same diamond mixing section are investigated. This results in a total length of 28.44 L/D. The temperature profiles for the two materials are shown in örner [In the investigations, three different screw concepts were examined, each with two different configurations that differ slightly in their geometry. The aim was to determine the influence of changes in screw geometry within a screw design on both the thermal and material homogeneity. The concepts investigated were the barrier and three-section screws without shear and mixing parts. The schematic screw configurations of the two barrier screws and three-section screws are shown in in örner . BarrierAs part of the investigations, the temperature and pressure curves recorded by the measuring computer were evaluated over the entire screw length and over time in the measuring flange. In addition, a dead-stop test was carried out in each case, and the thin section sample was subjected to a gray value analysis. The absolute values of the temperatures and gray values were not suitable due to the different temperature profiles and the different gray tones of the different materials. To characterize the material homogeneity, the averaged frequency distribution of the gray values is determined. s2 and the standard deviation gs were calculated. Both values represent the deviation of the individual gray values from the previously calculated mean. The goal of good blending is to achieve the lowest possible standard deviation. In Using this mean, the variance From the equation for the flow velocity of a power-law fluid in the axial direction of a pipe cross-section (compare Equation (4), ), the meiX (Equation (8)).Using the area of the circular ring (Equation (6)) and the total volumetric flow rate of a power law fluid in a circular cross-section (Equation (7), ) resultsn) and the geometric design of the measuring flange . The average melt temperature GMT is calculated using Equation (9).The weighting factor depends on both the material . However, further investigations should be carried out to validate this thesis.In order to support the results from the shear and mixed-part combinations, the standard deviations of the gray value and the SPI for the barrier screws and three-section screws for both materials were also evaluated. Two exemplary test series are shown in To investigate the correlation between the SPI and the standard deviation of the weighted average melt temperature, these were also plotted against the throughput. As an example, the standard deviation of the weighted average melt temperature for the two shear and mixing-part, SM1 (a) and SM2 (b), for the Moplen 420 M are evaluated in As a verification of this correlation, the results for the other screw designs were also examined. The objective of this study was to determine if there is a correlation between melt quality and the screw performance index. In this paper, the dependencies of the SPI on the standard deviations of the gray value and the weighted average melt temperature were investigated. The evaluations of the investigations in this paper clearly show that the SPI correlates with the thermal and material homogeneity of the polymer melt. If the standard deviation decreases, the SPI increases because the melt was better homogenized. So, it is possible to use the SPI as a target variable for the development of an analytical prediction model. In the prediction model, simulation results obtained using REX 17.1.0 software (a computer-aided extruder design), which was developed at Kunststofftechnik Paderborn (KTP), are to be used as input variables. With the help of artificial intelligence and machine learning, a prediction model will be developed in subsequent work.


Sargassum macrocarpum, and the molecular mechanisms underlying ACE inhibition. Four fractions of S. macrocarpum were prepared using hexane, chloroform, ethyl acetate, and water as solvents and analyzed for their potential ACE-inhibitory effects. The chloroform fraction showed the strongest ACE-inhibitory effect, with an IC50 value of 0.18 mg/mL. Three meroterpenoids, sargachromenol, 7-methyl sargachromenol, and sargaquinoic acid, were isolated from the chloroform fraction. Meroterpenoids isolated from S. macrocarpum had IC50 values of 0.44, 0.37, and 0.14 mM. The molecular docking study revealed that the ACE-inhibitory effect of the isolated meroterpenoids was mainly attributed to Zn-ion, hydrogen bonds, pi-anion, and pi–alkyl interactions between the meroterpenoids and ACE. These results suggest that S. macrocarpum could be a potential raw material for manufacturing antihypertensive nutraceutical ingredients.Angiotensin I-converting enzyme (ACE) is an important blood pressure regulator. In this study, we aimed to investigate the ACE-inhibitory effects of meroterpenoids isolated from the brown alga,  Hypertension is a chronic health problem related to cardiovascular diseases, including myocardial infarction, arteriosclerosis, and stroke, and is affected by various factors, such as salt intake, smoking, stress, and obesity . Among tSargassum species contain a variety of active compounds such as sterols, sulfated polysaccharides, polyphenols, meroterpenoids, and terpenoids [Natural resources as alternative treatments for hypertension are gaining attention, and studies on herb-derived compounds are ongoing ,13. Marirpenoids , which prpenoids , anticanrpenoids , antimicrpenoids , advancerpenoids , and neurpenoids  propertirpenoids ,26. DespSargassum macrocarpum based on the analytical data results and evaluate their ACE-inhibitory effects and the molecular mechanism underlying ACE inhibition.This study aimed to isolate meroterpenoids from the brown alga, S. macrocarpum were tested for their potential ACE-inhibitory effects. The 80% aqueous methanol extract of S. macrocarpum inhibited ACE activity in a dose-dependent manner by 6.1, 15.7, 40.7, 62.7, and 74.5% at concentrations of 0.0625, 0.125, 0.25, 0.5, and 1 mg/mL, respectively : δH 6.46 , 6.30 , 6.22 , 5.96 , 5.55 , 5.12 , 5.07 , 2.58 , 2.24 , 2.11 , 2.11 , 2.05 , 1.65 , 1.64 , 1.56 , 1.55 , 1.33  : δC 173.4 (C-15′), 148.7 (C-6), 145.8 (C-7′), 144.9 (C-8a), 134.5 (C-4′), 132.4 (C-12′), 130.8 (C-3), 130.7 (C-8′), 126.5 (C-8), 125.0 (C-3′), 123.6 (C-11′), 123.1 (C-4), 121.5 (C-4a), 117.2 (C-7), 110.5 (C-5), 77.9 (C-2), 40.9 (C-1′), 39.2 (C-5′), 34.7 (C-9′), 28.3 (C-6′), 28.0 (C-10′), 26.0 (C-17′), 25.8 (C-13′), 22.8 (C-2′), 17.9 (C-14′), 15.9 (C-18′), 15.7 (C-16′)   ; 13C NMR (C-16′) ; HRESIMS23.2535) .1H NMR : δH 6.29 , 6.21 , 5.97 , 5.52 , 5.11 , 5.06 , 2.58 , 2.23 , 2.12 , 2.11 , 2.10 , 2.05 , 1.65 , 1.65 , 1.56 , 1.55 , 1.33  : δC 172.6 (C-15′), 147.0 (C-6), 145.7 (C-7′), 144.9 (C-8a), 134.5 (C-4′), 132.4 (C-12′), 130.6 (C-8′), 130.0 (C-3), 128.3 (C-8), 125.2 (C-3′), 123.7 (C-11′), 123.6 (C-7), 123.0 (C-4), 118.9 (C-4a), 110.0 (C-5), 77.8 (C-2), 40.9 (C-1′), 39.2 (C-5′), 34.7 (C-9′), 28.3 (C-6′), 28.0 (C-10′), 26.0 (C-17′), 25.8 (C-13′), 22.8 (C-2′), 17.9 (C-14′), 15.9 (C-16′), 12.3 (C-19′), 11.8 (C-18′)  (7-methyl sargachromenol (2): brown oils; , H-17′) ; 13C NMR (C-18′) ; HRESIMS37.2691) .1H NMR : δH 6.53 , 6.44 , 5.98 , 5.13 , 5.11 , 5.07 , 3.11 , 2.58 , 2.24 , 2.10 , 2.07 , 2.05 , 2.04 , 1.65 , 1.60 , 1.58 , 1.56  : δC 188.3 (C-3), 188.2 (C-6), 172.7 (C-18′), 148.7 (C-2), 146.1 (C-4), 145.6 (C-10′), 140.0 (C-3′), 134.8 (C-7′), 133.3 (C-5), 132.49 (C-1), 132.44 (C-15′), 130.7 (C-11′), 124.6 (C-6′), 123.6 (C-14′), 118.1 (C-2′), 39.7 (C-4′), 39.2 (C-8′), 34.7 (C-12′), 28.4 (C-9′), 28.0 (C-13′), 27.7 (C-1′), 26.5 (C-5′), 25.9 (C-17′), 17.9 (C-16′), 16.3 (C-21′), 16.2 (C-19′), 16.1 (C-20′)  (Sargaquinoic acid (3): brown oils; , H-16′) ; 13C NMR (C-20′) ; HRESIMS23.2535) .50 value was observed for sargaquinoic acid at a concentration of 0.14 mM, and IC50 value of sargaquinoic acid was 3-fold lower than that of sargachromenol, which was the weakest inhibitor among the three meroterpenoids. It has previously been found that plant phenolic compounds such as quercetin, kaempferol, rutin, apigenin, epicatechin, phloretin, and resveratrol exhibit ACE-inhibitory effects and have IC50 values of 0.415, 0.512, 0.472, 0.667, 1.381, 1.110, and 0.970 mM, respectively [50 value = 2.57 mM), triphlorethol-A , eckol , dieckol , and eckstolonol  [50 value = 0.1 mM) from Terminalla chebula tree [50 value = 0.076 mM) from rice bran [50 value = 0.0086 mM) from bitter melon seed [50 value = 0.064 mM) from Pea [S. macrocarpum exhibited a significantly weaker effect than that of lisinopril , enalapril , and captopril , currently the most widely used synthetic ACE-inhibitor [R- and S-isomers, and structures of meroterpenoids are described in R-isomer interacted with GLU376 , ALA354 , VAL379 , VAL380 , VAL518 , PHE527 , HIS353 , HIS383 , HIS513 , PHE457 , and TYR523  , VAL380 , VAL518 , HIS353 , HIS383 (carbon hydrogen bond interaction), HIS513 , HIS387 , and TRP279  , GLU376 , VAL380 , LEU161 , ALA354 , HIS353 , PHE512 , and TRP279  , LEU161 , HIS353 , HIS387 , HIS410 , HIS513 , PHE512 , TRP279 , ALA354 , GLU411 (carbon hydrogen bond interaction), CYS352 , and CYS370   and S2 (HIS353 and HIS513) active site pockets and that sargachromenol S-isomer forms with S1 (ALA354) and S2  active site pockets. In the case of 7-methyl sargachromenol, R-isomer forms with S1 (TYR523) and S2 (HIS353) active site pockets of ACE, and S-isomer forms with S1  and S2  active site pockets of ACE. In addition, sargaquinoic acid forms an active site with the S1 , S’1 (GLU162), and S2 (HIS353) active site pockets of ACE.Three meroterpenoids, sargachromenol, 7-methyl sargachromenol, and sargaquinoic acid, with different chemical structures and molecular weights were isolated. The ACE-inhibitory effects of the three meroterpenoids are shown in ectively . Additioula tree , Tyr-Serice bran , Val-Serlon seed , and Lysfrom Pea . A previnhibitor . Diet thnhibitor ,38. Addinhibitor . S1 (antnhibitor . Lisinopnhibitor . The mosraction) A. Sargacraction) B. 7-methraction) C. 7-methraction) D. In theraction) E. The moectively . Among td HIS387 . The mol50 values 0.44, 0.37, and 0.14 mM, respectively. Among the three meropterpenoids, sargachromenol and 7-methyl-sargachromenol do exist as R- and S-isomers, and molecular docking studies of the respective other isomers were performed. These other isomers showed the similar docking poses and binding energies with those of isomers. Molecular docking studies revealed that these meroterpenoids in the ACE active site contributed to the stabilization of the docking complex, and it was confirmed that sargaquioic acid contributed the most to stabilization. These findings provide a partial molecular explanation for the ACE-inhibitory properties of meroterpenoids isolated from S. macrocarpum. Moreover, meroterpenoids are promising for the discovery of potential candidates for the future industrial production of functional foods.In conclusion, among the various organic solvent fractions, the chloroform fraction exhibited the highest ACE inhibitory effect. Three meroterpenoids, sargachromenol, 7-methyl sargachromenol, and sargaquinoic acid, having ACE-inhibitory effects were isolated, with ICS. macrocarpum was collected from Jeju Island, South Korea. The alga was washed three times with tap water to remove salt and epiphytes attached to the surface, followed by careful rinsing with fresh water. The algae were stored in a medical refrigerator at −80 °C. Frozen algae were lyophilized and homogenized using a grinder prior to extraction. The ACE assay kit (ACE kit-WST) was obtained from Dojindo Laboratories .S. macrocarpum powder was extracted with 80% aqueous methanol at room temperature, and the liquid layer was obtained via filtration. The filtrate was evaporated to obtain the methanol extract, which was suspended in distilled water and partitioned with hexane, chloroform, ethyl acetate, and butanol. The chloroform fraction exhibited a stronger ACE-inhibitory effect than the other fractions. Thus, the compounds were extracted from the chloroform fraction using a silica gel column and medium-pressure liquid chromatography . Finally, the compounds were purified using preparative high-performance liquid chromatography , and the structures of the compounds were identified by comparing the nuclear magnetic resonance  spectral data and high-resolution electrospray ionization mass spectrometry  analysis results with those in the existing literature.The dried 50 value was defined as the concentration of extract and fractions (mg/mL), and compounds (µM) required for 50% reduction of the ACE activity.The ACE-inhibitory activities of the extract, fractions, and compounds were determined using an ACE kit-WST (Dojindo Laboratories). The assay was performed according to the manufacturer’s instructions. The absorbance was measured at 450 nm using a microplate reader . ICMolecular docking was performed using CDOCKER and Calculate Binding Energies tools in Discovery Studio 2022  to assess the binding positions of meroterpenoids within the active site of ACE. A docking mechanism based on CHARMm  was usedp < 0.05 was considered statistically significant.Data are presented as mean ± standard error. Statistical comparisons of the mean values were performed using analysis of variance, followed by Duncan’s multiple-range test using the SPSS software (Version 21). A


The objective of this research was to evaluate the suitability of whey permeate powder for ice cream. Three white mixes were formulated with equivalent total solids, fat, and carbohydrates, but different concentrations of lactose and added sugar. Vanilla ice creams contained either reduced lactose , standard lactose , or high lactose . Trained panelists evaluated 8 body and texture, and 6 flavor characteristics through 10 mo of storage. All ice creams maintained low mean scores (<4.0/15.0 cm) for crumbly, lacks freshness, nonfat dry milk flavor, and whey, and moderate mean scores (5.0–8.3/15.0 cm) for gummy, melt rate, melt viscosity, sweet, and vanilla flavor for 10 mo. In mo 1 and 10, consumers in Iowa  and in mo 4 and 6, consumers in Kansas  rated the acceptability of the ice creams. Overall acceptability, flavor, and texture acceptability for products did not significantly differ until mo 10, when HL mean scores decreased lower than SL ice cream mean scores. The lower scores are attributed to crumbly and sandy texture defects, noted by trained panelists, only for HL ice cream stored 10 mo. The research demonstrates that whey permeate powder can be used to produce ice creams of acceptable quality for up to 10 mo. WPP), a co-product from the production of whey protein concentrate (WPC) and whey protein isolate (WPI), has been reported to be one of the most cost-competitive sources of dairy solids on the market , the demand for high-lactose ingredients has not kept up. Though the clinical condition lactose intolerance is not common (~10% of the population), varying degrees of lactose maldigestion and malabsorption are common (~66% of the population) worldwide . AdditioAnother limitation of WPP utilization is its high lactose content (approximately 82%). The complex chemistry of lactose limits its use in various applications, including ice cream. The hemiacetal structure enables the molecule to interchange between α- and β-anomers by mutarotation , which iThe surplus of lactose-rich ingredients necessitates research to increase its utilization. The objective of this research was to evaluate the suitability of WPP for ice cream. Three ice cream mixes were formulated  to contaISU) Creamery . Weighing and blending  of ingredients, batch pasteurization , and aging  occurred on d 1. One 67-kg batch of the 3.8% lactose mix and one 67-kg batch of the 7.8% lactose mix were made. The 5.8% lactose ice cream base mix was made by combining 29 kg of the 3.8% mix with 29 kg of the 7.8% mix.In March 2021, ice creams were manufactured in the Iowa State University (K-State) at −23°C in insulated cold packs containing dry ice, then stored in a freezer (−23°C) at the K-State Dairy Processing Plant .On d 2, vanilla flavoring  was added before batch freezing in a Taylor Dual-Barrel  ice cream machine. Soft ice cream was drawn (−7°C) into 3-gallon (3.8 L) cardboard  and 0.95-L containers , then transported to a freezer (−23°C) for hardening and storage. In April 2021, 3-gallon tubs of each ice cream were transported to Kansas State University  on quality characteristics of ice cream. References representing slight (0 to 5), definite (5.1 to 10), and pronounced (10.1 to 15.0) intensities of the characteristics were practiced using a 15-cm line scale. Ice creams were evaluated once or twice monthly. Trained panelists earned $5 for each training session and tasting session completed.This study was approved for the participation of human subjects by the ISU Institutional Review Board (IRB #20–227). Six experienced dairy products evaluators received a minimum of 4 additional hours of training, using established , and newNFG).This study was a cooperation between ISU and K-State, and ISU served as the primary institution. The ice cream study was one component of a larger study, designed to investigate the effect of dairy nutrition educational messages on inadequate dairy consumers . ResearcTen NFG sessions were conducted in Ames, Iowa, in April 2021; 6 NFG were conducted in Manhattan, Kansas, in July 2021; and 8 NFG were conducted in Manhattan, Kansas, in October 2021. Upon arrival, panelists were asked screening questions, handed folders with 3-digit codes, and physically distanced at sanitized tables that contained a napkin, bottle of water, sanitized pen, and cuspidor. All panelists were seated facing the facilitators, who conducted each NFG according to a script.In preparation for NFG, ice creams were removed from frozen storage at least 24 h before the tasting event, tempered to approximately −12°C in a freezer, and scooped into 59-mL plastic sample cups with lids . The ice cream sample cups were maintained in a cooler with foam refrigerant ice packs  in the rooms where NFG were held.The ice cream acceptability test was conducted at a running time of approximately 32 to 37 min into each NFG. Samples were distributed one at a time to panelists, in a randomized order in April and July NFG. However, due to a change in K-State's COVID-19 protocols, October panelists received all 3 ice cream samples, simultaneously, in Styrofoam  trays, which maintained the temperature of the ice cream during the tasting segment. Panelists rated their liking of appearance, sweetness, flavor, texture, and overall liking on a paper ballot that contained a 5-point hedonic scale, anchored with the terms dislike very much (1), dislike slightly (2), neither dislike nor like (3), like slightly (4), and like very much (5).After sensory evaluation, facilitators revealed the nutrition facts panels and ingredients statements for each of the ice cream samples tasted. The script included information about how the level of lactose in the 3 samples differed because of different dairy ingredients (WPC80 and WPP). It was explained that since lactose is not an added sugar, to balance carbohydrates in the mix, the ice cream with 3.8% lactose had the highest added sugar, the ice cream with a standard level of lactose (5.8%) had a moderate level of added sugar, and the ice cream with 7.8% lactose had the lowest added sugar. Panelists were given opportunities to ask questions.JAR) analysis was conducted .After analysis of the 1-, 4-, and 7-mo data, it was discovered that ice cream quality (trained panel) and acceptability (consumer panels) did not drop significantly between mo 1 and 7. Thus, it was decided to conduct an additional consumer panel and trained evaluation at mo 10 (February). No NFG were associated with the panel, and the panel was not limited to inadequate dairy consumers. A 6-point liking scale was utilized, wherein 1 corresponded to dislike very much, 2 = dislike moderately, 3 = dislike slightly, 4 = like slightly, 5 = like moderately, and 6 = like very much, so mo-10 data were transformed to match the 5-point scale used for the April, July, and October NFG for subsequent statistical analysis. Additionally, to provide insight related to sweetness acceptability noted in the NFG, instead of sweetness acceptability, a just about right (P < 0.05. Wilcoxon signed-rank tests (a = 0.05) were conducted on matched pairs to determine if NFG participants preferred any ice cream over another for any attribute.Untrained consumer data collected in Iowa and Kansas were entered into Excel (Microsoft Corp.), then uploaded into JMP Pro 16 . Statistical analyses of ice cream acceptability data and trained panel attribute intensity data were performed using one-way ANOVA with Tukey post hoc tests when Based upon descriptive analysis by trained panelists (n = 4 to 6), across the entire 10-mo study, the ice creams were representative of typical fresh premium vanilla ice cream with low (~50%) overrun. Quality characteristics were expected to deteriorate during storage; however, all 3 ice creams maintained low mean scores (<5.25/15.0 cm) for crumbly, icy, greasy, sandy, cooked, lacks freshness, NFDM flavor, and whey; moderate mean scores (6.5–8.5/15.0 cm) were maintained for density, gummy, melt rate, melt viscosity, sweetness, and vanilla .Table 2TP < 0.05). Simultaneously, trained panel crumbly and sandy mean scores increased for HL, and gummy mean scores increased for RL and SL ice creams (P < 0.05). The negative effect of the crumbly and sandy defects is visually shown in the principal component analysis  consumers . Howeveranalysis , whereinP < 0.005). On the other hand, panelists preferred SL and HL sweetness over RL ice cream sweetness (P < 0.05). The JAR test in mo 10 helps explain the finding (P = 0.05). These findings align with the trained panel results (data not shown): mean sweetness of RL ice cream was higher (P < 0.05) than the ice creams with SL or HL. The findings are not surprising, considering that lactose is less sweet than sucrose, and the RL ice cream had the most added sucrose.To determine if individual panelists preferred one sample over another, differences in individual acceptability scores were tested using the Wilcoxon signed-rank test (data not shown). Panelists liked the ice cream  made with the typical amount of lactose (SL) more than the RL and HL ice creams  with WPP (approximately $0.57/kg) in ice cream applications, at similar usage levels as described here, could save a producer hundreds to thousands of dollars per production run.In conclusion, the level of lactose, in otherwise similar formulations of ice cream, did not detrimentally affect the quality or acceptability of ice cream for at least 7 mo of storage. After 10 mo of storage, ice cream with HL exhibited sandy and crumbly defects, and was less acceptable than ice cream with RL or SL. Wilcoxon signed-rank matched pair testing revealed that consumers preferred the SL ice cream flavor, texture, and overall more the RL or HL ice creams, though mean scores for acceptability were similar. Taken together, the findings demonstrate the potential for acceptable ice cream to be made with WPP, a less expensive source of dairy solids than WPC80, even at lactose levels previously considered too high for acceptable ice cream.


PI3K/AKT signaling pathway plays important role in tumorigenesis of human cancer. Protein phosphorylation is crucial for signaling transduction of this pathway. PIK3CA, encoding the catalytic subunit p110α of PI3K complex, is one of the most frequently mutated oncogenes in human cancers. However, phosphorylation sites of PIK3CA/p110α and their underlying mechanism in tumorigenesis are largely unknown.Tyrosine phosphorylation sites of PIK3CA/p110α are identified with Mass-Spectrum. Crispr/CAS9 strategy is applied to generate Y317F and Y508F mutant knock-in cell clones. The growth and metastasis abilities of cells are evaluated in vitro and in vivo. Phospho-proteomics analysis and Western blots are used to demonstrate downstream signaling pathways of PIK3CA/p110α tyrosine phosphorylation. In vitro kinase assay is applied to identify the kinase of PIK3CA/p110α tyrosine phosphorylation.Tyrosine phosphorylation of PIK3CA/p110α is stimulated by growth factors such as EGF, HGF and PDGF. Two tyrosine residues, Y317 and Y508, are identified on PIK3CA/p110α. Either Y317 or Y508 phosphorylation is essential for tumorigenesis of CRC. Mutation at Y317 of p110α reduces the proliferation, migration, and invasion of cancer cells through Src-MLC2 pathway, while mutation at Y508 of p110α impairs AKT signaling. Moreover, Src interacts with and phosphorylates p110α.PIK3CA/p110α phosphorylation at Y317 and Y508 play important role in tumorigenesis of colorectal cancer through two independent pathways.The online version contains supplementary material available at 10.1186/s13578-023-01102-7. PI3K/AKT signaling pathway plays important role in tumorigenesis of human cancers . NormallPost-translational modifications (PTMs) are crucial for regulating PI3K/AKT signaling pathway, notably phosphorylation. Tyrosine phosphorylation of RTKs, or adaptor proteins such as IRS-1 facilitate their binding with p85 regulatory subunits of PI3K, and then bring PI3K complex on membrane . ActivatPTMs of several PI3K complex isoforms have been demonstrated. PI3K complex consists of catalytic subunit p110s and regulatory subunit p85s. p110s perform lipid kinase activity, and p85s stabilize and regulate p110s. Phosphorylation at tyrosine 688 relieves p85 inhibitory activity on p110s . PhosphoIn this study, we identified two tyrosine phosphorylation sites Y317 and Y508 in PIK3CA /p110α. Y317 and Y508 are essential for PI3Kα-mediated oncogenic function. Src is a tyrosine kinase of PIK3CA/p110α.To identify PTMs on PIK3CA/p110α, we firstly endogenously knocked in SBP-3×FLAG-6×HIS tag at the C-terminal of p110α in DLD1 cells (named as DLD1-p110α-SFH cells) and HCT116 cells (HCT116-p110α-SFH cells) by recombinant adeno-associated virus (rAAV)-mediated homologous recombination Fig. A and S1BPIK3CA/p110α is one of the most frequently mutated oncogenes and a key therapeutic target in colorectal cancer. To assess whether phosphorylation of p110α affects the CRC progression, we endogenously mutated Y317 or Y508 to F  on PIK3CA locus using CRISPR/Cas9 mediated genome editing strategy in colon cancer cells  Fig. C and S1DWe then examined the function of p110α Y317 phosphorylation in CRC tumorigenesis. Compared with their parental cells, Y317F mutation moderately reduced the proliferation and colony formation abilities of CRC cells including HCT116, DLD1, LoVo, and SW480 Fig. A-2C. Y31Next, we assessed the function of p110α Y508 on tumorigenesis. Although the isogenic cell colonies with homozygous Y508F mutations on PIK3CA locus could not be able to generate, heterozygous mutation of p110α Y508 already inhibited the proliferation, colony formation abilities of CRC cells in vitro and the growth of xenograft tumors in vivo Fig. A-S3E. MoTaken together, these results suggest that two tyrosine phosphorylation sites Y317 and Y508 are essential for p110α to promote CRC tumorigenesis.To determine how tyrosine phosphorylation of p110α Y317 affects tumorigenesis, we first detected the classic PI3K/AKT signaling including phosphorylation levels of AKT and its downstream substrates in parental cells and p110α Y317F KI mutant cells. It was observed that p110α Y317F mutation had no effect on phosphorylation of AKT and its downstream substrates, such as GSK3β, mTOR, p70S6K Fig. A. p110α To further identify the downstream signaling mediated by tyrosine phosphorylation of p110α Y317, we performed phospho-proteomics analysis Table . The difTo confirm whether oncogenic function of p110α Y317 phosphorylation in CRC was mediated by MLC2, MLC2 was overexpressed in HCT116 p110α Y317F KI mutant cells Fig. C. The reTo determine the mechanism of how p110α Y508 phosphorylation influences CRC progression, we also examined the AKT phosphorylation levels in parental cells and p110α Y508F KI mutant cells. Unlike tyrosine 317, mutation of Y508 on p110α decreased phosphorylation levels of AKT Fig. A. MoreovGPS 3.0 and NetPhos 3.1 were applied to predict the tyrosine kinases which might phosphorylate p110α at Y317 or Y508 Fig. A. The reAlthough it is well-known that post-translational modifications especially protein phosphorylation are essential for signaling transduction of PI3K pathway, whether protein phosphorylation occurs on PI3K catalytic subunit PIK3CA/p110α, one of the most frequently mutated oncogenes in human cancers, is still an open question. In this study, we identified two tyrosine sites on PIK3CA/p110α and demonstrated their function and underlying mechanisms in CRC.We clarified that PIK3CA/p110α underwent tyrosine phosphorylation. PI3Kα plays important role in tumorigenesis. Several tyrosine phosphorylation sites have been characterized in the regulatory subunits of PI3Kα, such as p85α and p85β. However, post-translational modifications on p110α are not well documented. In our data, p110α tyrosine phosphorylation was detected by a phospho-tyrosine monoclonal antibody pY-100. Moreover, p110α tyrosine phosphorylation could be stimulated by growth factors, such as EGF, HGF, and PDGF, suggesting their physiological relevance. With targeted phosphoproteomics approach, two tyrosine phosphorylation sites of p110α, Y317 and Y508, were identified. Indeed, several large-scale non-targeted phosphoproteomics data confirmed the existence of tyrosine phosphorylation of p110α. For examples, p110α Y508 phosphorylation has been found in HeLa cells and sarcoma cell lines after immunoaffinity enrichment of Tyr phosphorylated peptides , 26. p11We demonstrated that phosphorylation of p110α Y317 and Y508 were critical for CRC tumorigenesis. Although phosphoproteomics data uncovered that p110α had tyrosine phosphorylation, the function of p110α phosphorylation have not been studied yet. It is well-known that PIK3CA/p110α play oncogenic role in human cancer. Thus, we generated tyrosine sites mutation knocked-in isogenic cell clones to evaluate their function in tumorigenesis. The data showed that phosphorylation at either Y317 or Y508 was crucial for CRC progression. Interestingly, p110α Y317F mutation showed moderate or even inconsistent effect on proliferation, but significant and uniform inhibitory effect on tumor metastasis, suggesting that phosphorylation at p110α Y317 has a greater impact on tumor metastasis processes. This phenomenon might be explained by p110α Y317 phosphorylation-mediated downstream signaling, Src-MLC2 pathway. Src is a non-receptor tyrosine kinase that influences the migration and invasion of cancer cells by activating several signaling cascades on cytoskeletal recombination and cell adhesion . p-MLC2 Src phosphorylates PI3K regulatory subunit p85 at tyrosine 688  and clasUnfortunately, couple commercial p-p110α Y317 antibodies and our customized p-p110α Y317 or p-p110α Y508 antibodies were failed to specifically recognize p-p110α protein. High-quality phospho-p110α antibodies will not only help us to understand the signaling transduction pathway of phospho-p110α but also have potentially clinical application.In this study, we identify two tyrosine residues on PIK3CA/p110α that play important role in tumorigenesis of CRC. p110α Y317 phosphorylation influences Src-MLC2 pathway, whereas p110α Y508 phosphorylation regulates AKT signaling. Furthermore, Src is a potential tyrosine kinase for p110α. This study could broaden our understanding of the mechanisms underlying PIK3CA/p110α for CRC progression  using STR profiling and tested routinely to avoid Mycoplasma contamination .Colorectal cancer cell lines  were maintained in McCoy’s 5 A medium . Human embryonic kidney HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium . The medium was supplied with 10% of fetal bovine serum  and 100 U/mL penicillin plus 100 μg/mL streptomycin . Cells were incubated at 37℃ in a humidified atmosphere with 5% CODLD1 cells and HCT116 cells were endogenously tagged with SBP-3×FLAG-6×HIS at C-terminal of PIK3CA locus as described previously . Brieflyhttps://sg.idtdna.com/pages) and cloned individually into pX330 vector. Homologous arms of PIK3CA genome sequence containing Y317F or Y508F point mutations were cloned into the pAAV-loxP-Neo vector. Targeting vectors were co-transfected with gRNA vectors into indicated cell lines. G418-resistant clones were screened by genomic PCR and verified by cDNA sequencing. The primers for vector construction and PCR are listed in Table For CRISPR/CAS9-mediated PIK3CA/p110α Y317F or Y508F endogenous point mutation, guiding RNA in introns surrounding mutation sites were designed with IDT design tool (EGF (cat# 8916) and FGF  were purchased from cell signaling technology . PDGF-BB (cat# 100-14B) was purchased from Peprotech . HGF (cat# HZ-1084) was purchased from Proteintech . Insulin (cat# I9278) and Sodium orthovanadate  were obtained from Sigma . Src inhibitor Saracatinib (cat# S1006) was purchased from Selleck . pCMV backbones (Invitrogen) were used for gene expression in mammalian cells. PIK3CA/p110α was cloned into pCMV-3×FLAG vector. Src, Fyn, and Lyn were cloned into pCMV-3×Myc vector. Point mutations of Y317F, Y508F, or Y317F/Y508F on PIK3CA/p110α were generated with QuikChange Site-Directed Mutagenesis Kit . MLC2 and myr-AKT constructs were purchased from GeneCopoeia . The primers using for constructs cloning were listed in Table −ΔCt was used to calculate relative mRNA levels. Primers were listed in Table Total RNA was extracted using the TRIzol kit . 1 μg of total RNA was reverse-transcribed into cDNA using Prime-Script RT kit  according to the manufacturer’s instructions. Y317F or Y508F KI mutant colons were sequenced with primers surrounding Y317 or Y508 sites. The gene expression levels were measured by qRT-PCR with SYBR Premix EX Tag . Each experiment was performed in triplicate. Objective CT values were normalized to internal reference gene β-actin and 2Immunoprecipitation and immunoblotting were performed as described previously . Briefly3VO4, 50mM NaF). Cell lysates were immunoprecipitated with indicated agarose beads at 4℃ for 3 h. The beads were washed three times and boiled with SDS loading buffer. The antibodies-conjugated agarose beads were listed in Table For transfection-based immunoprecipitation assays, cells were transfected with indicated vectors and lysed in 1 mL of RIPA lysis buffer supplemented with complete protease inhibitor, PhosSTOP, and tyrosine phosphatase inhibitors . Baculovirus expressing 6×His tagged PIK3CA/p110α or p85α were prepared according to Bac-to-Bac baculovirus expression system . Sf-9 cell were infected with baculovirus of both 6×His tagged PIK3CA/p110α and p85α, and recombinant p110α were purified with Ni-NTA agarose as described previously . Src was2, and 7.5% normal SDS/PAGE solution (avoiding light). The samples were then dissolved in Phos-tag SDS/PAGE and electrophoresed at 30 mA for 3.5 h. Proteins were electrophoretically transferred to PVDF membrane at 15 mA in 4 degree overnight, then followed by regular immunoblotting.For in vitro kinase assay, 50 ng of purified 6×His-p110α/p85α complex proteins, and purified 6×His-Src proteins were incubated in 25 μl of 1×tyrosine kinase buffer  supplemented with 0.32 mM ATP at 37℃. After indicated times, the reactions were terminated by adding SDS loading buffer and boiling at 100℃ for 5 min. Phos-tag SDS/PAGE was made with 5 mM Phos-tag , 10 mM MnClIn vitro proliferation of CRC cells was measured using Cell Counting Kit-8 . Briefly, 2000 cells were seeded in 96-well plates and cultured for 5 days in 100 μl McCoy’s 5 A medium supplemented with 10% FBS. According to the manufacturer’s instructions, 10 μl per well of CCK-8 solution was added, and the absorbance values at 450 nm were measured for 5 consecutive days.4 CRC cells in 200 μl McCoy’s 5 A serum-free medium were seeded in the upper chamber, and 800 μl McCoy’s 5 A medium supplemented with 10% FBS was added into the lower chamber of the 24-well plate. After 48 h incubation, cells migrated through the membrane were fixed and stained by crystal violet. Average colony numbers were counted with randomized 3 ~ 5 images under microscope.Cell migration assays were conducted by 24-well plates and transwell filter chambers . 5 × 104 CRC cells in 200 μl serum-free McCoy’s 5 A were seeded in the upper chamber. The following steps were similar as described above.For Cell invasion assays, a matrigel chamber (BD Biosciences) were applied. 8 × 10Cells were grown in 12-well plates at 95% confluency. The linear wounds were scratched via a 200 μl sterile pipette tip. After washing with PBS to remove cell debris, adherent cells were incubated in medium with 10% FBS. The migrating distance were photographed every 3 h for 24 h.2)/2.All animal experiments were performed in accordance with protocols approved by the IACUC committee at Shanghai Medical Experimental Animal Care Commission. Subcutaneous xenograft tumor models were established as described previously . BrieflyFor in vivo tumor metastasis experiment, three million cells in 100 μL of serum-free McCoy’s 5 A were injected into nude mice through tail vein. Two to three months later, mice were sacrificed and dissected. The metastatic nodules in the lungs were counted and then stained with hematoxylin and eosin.t test (paired/unpaired) were used for statistical differences between groups, and Two-way analysis of variance (ANOVA) was used for comparing significant difference of multiple groups. P < 0.05 was considered statistically different , otherwise not significant (n.s.).All statical analyses were done using GraphPad Prism 5.0 software. Results were presented as mean ± standard deviation . The student’s Below is the link to the electronic supplementary material.Supplementary Material 1