The cyclin-dependent kinase 5 (CDK5) represents an unusual member of the family of cyclin-dependent kinases, which is activated upon binding to non-cyclin p35 and p39 proteins. The role of CDK5 in the nervous system has been very well established. In addition, there is growing evidence that CDK5 is also active in non-neuronal tissues, where it has been postulated to affect a variety of functions such as the immune response, angiogenesis, myogenesis, melanogenesis and regulation of insulin levels. Moreover, high levels of CDK5 have been observed in different tumour types, and CDK5 was proposed to play various roles in the tumorigenic process. In this review, we discuss these various CDK5 functions in normal physiology and disease, and highlight the therapeutic potential of targeting CDK5. The funKip1, p57Kip2 and p21Cip1 inhibit the kinase activity of cyclin-CDK1 and cyclin-CDK2 complexes, but they have essentially no effect on p35/p39–CDK5 kinase [Human CDK5 shares 58% and 62% sequence homology with human CDK1 and CDK2, respectively . Like ot5 kinase ,14. CollAlthough CDK5 is expressed ubiquitously, its activity was thought to be restricted to the nervous system. Indeed, CDK5 activators p35 and p39 are mainly expressed in post-mitotic neurons, explaining tissue-specific activity of CDK5 ,15. The Hyper-activation of CDK5 is seen in a number of neurodegenerative diseases where it is believed to play a causative role in the degenerative process. It is assumed that hyperactive CDK5 phosphorylates non-physiological substrates resulting in neuronal pathology. In Alzheimer's disease (AD), neuronal stress leads to calpain-dependent cleavage of p35 into hyper-stable p25 species, leading to hyper-activation of CDK5. Hyperactive CDK5 phosphorylates amyloid precursor protein (APP), tau and neurofilaments, and these events contribute to AD pathogenesis ,30. Hypea and 2a) and discuss the potential of targeting CDK5 for therapeutic intervention in human diseases.While most studies of CDK5 focused on the nervous system, its non-neuronal functions have been overlooked for a long time. However, there is an increasing evidence that CDK5 may play an important role outside the nervous system and that it may regulate a diverse array of functions, such as cell cycle progression, apoptosis, DNA damage response, metabolism, angiogenesis, myogenesis, immune function, cell migration, invasion and epithelial to mesenchymal transition ,34. Here12.2.1.b). Using mice reconstituted with hematopoietic progenitors from Cdk5−/− embryos, Pareek et al. [b). Consistent with this model, Cdk5−/− T cells were shown to be deficient in IL-2 production, leading to increased susceptibility of mice to infections [CDK5 has been postulated to regulate different aspects of the immune response ranging from survival and motility of lymphocytes to cytokine secretion by lymphocytes, neutrophils and macrophages b. Using k et al.  demonstrk et al. . Specifik et al.  , CDK5 inhibition had an additive effect on insulin secretion in both high-glucose and low-glucose conditions ,43.et al. have shown that in high-glucose conditions, CDK5 inhibits the binding of a transcription factor pancreatic duodenal homeobox 1 (PDX-1) to insulin promoter and promotes nuclear export of PDX-1. The inhibition of CDK5 activity in high-glucose conditions maintains the binding of PDX-1 to the insulin gene promoter, thereby increasing insulin production [Mechanistically, Ubeda oduction . Anotheroduction .et al. demonstrated that Cdk5 is activated in high-fat diet-induced obese mice. This leads to insulin resistance, a condition characterized by impaired response to insulin that results in elevated levels of blood glucose [In contrast with these studies, Daval & Gurlo  demonstr glucose . The aut glucose . Interes glucose .2.3.c). According to Liebl et al. [in vitro and in vivo. In addition, mouse cornea neovascularization upon FGF treatment was blocked following intraperitoneal administration of roscovitine. This effect was attributed to reduced lamellipodia formation in the leading edge of migrating cells due to disruption in the localization of Rac1 and its effector cortactin [Several studies proposed different roles for CDK5 in angiogenesis c. Accordl et al. , CDK5 prortactin . Anotherortactin .et al. [Merk et al.  proposedet al. .Epb41l5 and Csnk1g3 [Paradoxically, endothelial-specific Cdk5 ablation in mice led to hypervascularization of the yolk sacs, resulting in perinatal lethality. The authors found that Cdk5 ablation promoted non-productive angiogenesis characterized by an increase in vascularity and a decrease in tissue perfusion, a phenotype similar to that observed in mice with defective Notch signalling . The sam Csnk1g3 .2.4.et al. demonstrated that at an early stage of muscle development, protein kinase Cζ (PKCζ) phosphorylates p35 on Ser-33. This allows calpain-mediated cleavage of p35 to a more stable p25 form, resulting in the activation of CDK5 [Early indication that CDK5 is involved in myogenesis came from the study of Xenopus embryos. These studies demonstrated that Cdk5, together with the Xenopus orthologue of p35, Xp35.1, regulates the expression of transcription factors involved in myogenesis, such as MyoD and myogenic regulator factor 4 (MRF4). Inhibition of Cdk5 activity by injection of a dominant-negative Cdk5 construct into early Xenopus embryos resulted in decreased expression of MyoD and MRF4 and disruption of somitic muscles . Since t of CDK5 . Active  of CDK5 ,55. Conv2.5.et al. demonstrated that in alpaca melanocytes, depletion of Cdk5 decreased melanin production by downregulating the expression of essential components of the melanogenesis pathway, such as transcription factor paired box 3 (PAX3), melanocortin receptor type I (MC1R) and tyrosinase (TYR) [in vivo depletion of Cdk5 resulted in lighter coat colour and polarized distribution of melanin. In addition, they observed an abnormally thickened epidermis and ascribed this phenotype to decreased levels of keratin 10 upon Cdk5 depletion [Recently, CDK5 was shown to be essential for normal epidermal development and melanogenesis. Dong se (TYR) . The autepletion . While t2.6.CDK5 was shown to inhibit the motility of corneal epithelial cells by regulating stress fibre formation and contraction in migrating cells . Accordi3.CDK5 gene [et al. [et al. [et al. [CDK5 gene to increased susceptibility to lung cancer in the Korean population [Increased expression of CDK5, p35 or p39 and the resulting hyper-activation of CDK5 have been reported in pancreatic, medullary thyroid, non-small cell lung, small cell lung, colorectal, liver, breast and ovarian cancers, glioblastoma multiforme, multiple myeloma and mantle cell lymphoma ,59–66. IDK5 gene ,68. HighDK5 gene ,62,69–71 [et al.  and Cao  [et al.  proposed [et al.  reportedpulation  and highpulation .a). In the following section, we will discuss these different findings.CDK5 has been implicated to play a role in various aspects of tumorigenesis and tumour progression a. In the3.1.et al. [in vitro cultured MTC, prostate, colon, colorectal and liver cancer cells, as well as proliferation of tumour cells in xenografts in vivo [b).The function of CDK5 in cancer cell proliferation remains controversial. Pozo et al.  reportedet al. . Consist in vivo ,62,77–79 in vivo ,80,81, w in vivo ,82,83, Bet al. demonstrated that MTC tumours arising in p25 transgenic mice displayed increased phosphorylation of RB1 at Ser807/811 as well as increased expression of an E2F target, cyclin A as well as Cdk2. Turning off p25 expression in these mice decreased the phosphorylation of RB1 at Ser 807/811 and decreased expression of Cdk2 and cyclin A [et al. [CDK5 was postulated to phosphorylate the retinoblastoma protein (RB1). The classical consequence of RB1 phosphorylation by cyclin–CDK complexes is the release of E2Fs transcription factors and the resulting transcription of genes required for cell cycle progression . Pozo etcyclin A . A similcyclin A . Analogo [et al. ,85.Other groups also reported that inhibition of CDK5 activity in MTC and prostate cancer cell lines decreased cell proliferation. This effect was attributed to the ability of CDK5 to phosphorylate STAT3 at Ser727, and upregulate the expression of STAT3 target genes, c-Fos and JunB. The authors reported that chemical inhibition of CDK5 activity decreased STAT3 Ser727 phosphorylation and diminished expression of c-Fos and JunB ,86,87.et al. proposed that CDK5 is required for proper mitotic spindle assembly and chromosome alignment during mitosis. This function was attributed to phosphorylation of FAK by CDK5 at Ser732. Phosphorylated FAK localizes to the microtubules of the spindle and facilitates microtubule depolymerization. The inhibition of CDK5 activity with roscovitine decreased the phosphorylation of FAK at Ser732 and impaired mitotic spindle assembly and correct chromosome alignment during mitosis [Rea  mitosis .et al. proposed that CDK5 promotes proliferation of prostate cancer cells by regulating p53 and p21Cip1 levels via an indirect mechanism. Specifically, they reported that CDK5 phosphorylates AKT at Ser 473, thereby activating its kinase activity [Cip1. Consistent with these observations, several groups reported that siRNA-mediated depletion of CDK5 led to increased p53 and p21Cip1 levels, resulting arrest of cells in G1 phase of the cell cycle. Huang et al. demonstrated that CDK5 can directly phosphorylate p21Cip1 and regulate p21Cip1 protein levels. The authors reported that phosphorylation of p21Cip1 Ser130 residue by CDK5 promotes proteasomal degradation of p21Cip1. This, in turn, activates CDK2 and accelerates cell cycle progression of cancer cells [Lindqvist activity . Active activity . This, ier cells .et al. [in vitro and in vivo in xenografts. The authors proposed that nuclear CDK5 binds to E2F1 and attenuates the E2F1–DNA interaction, thereby inhibiting cell proliferation. In addition, nuclear CDK5 can upregulate the levels of a CDK inhibitor p16INK4a, which further contributes to cell cycle arrest [In contrast with all these findings, CDK5 was postulated by others to act as a tumour suppressor in gastric cancers. Cao et al.  reportede arrest .3.2.The role of CDK5 in cancer cell survival remains unclear, with some groups reporting that CDK5 prevents while others proposing that CDK5 promotes apoptosis. Different molecular mechanisms were postulated to mediate the function of CDK5 in tumour cell survival. CDK5 was implicated to protect breast cancer cells against reactive oxygen species (ROS)-mediated apoptosis. The depletion of CDK5 using siRNA leads to prolonged opening of mitochondrial permeability transition pores (mPTP) by an unknown mechanism, which results in mitochondrial depolarization followed by an increase in ROS levels and ultimately caspase activation and apoptosis . Other aIn addition, CDK5 was postulated to regulate the survival of breast cancer tumour initiating cells possibly by phosphorylating Forkhead box Type O transcription factor 1 (Foxo1), thereby promoting its nuclear export. This, in turn, suppresses the expression of pro-apoptotic genes such as Bim, which represent Foxo1 transcriptional targets. According to this model, depletion of CDK5 increases the levels of nuclear Foxo1, which leads to increased expression of Bim protein and tumour cell apoptosis . Anotheret al. [In contrast to the above-described findings, Lee et al.  reportedet al. . Consistet al. . Similaret al. ,80,81.3.3.CDK5R1) or CDK5 genes or by inhibition of CDK5 activity with roscovitine [Ectopic expression of the retinoblastoma protein (RB1) in RB1-null cancer cell lines results in senescence-like phenotype . It was covitine ,100.3.4.et al. [Hsu et al.  have shoet al. . Other get al. ,89.3.5.c), but the outcome (stimulation versus inhibition of migration) as well as the molecular mechanism remains unclear. Depletion of CDK5 or inhibition of its activity in several in vitro cultured cancer cell lines resulted in reorganization of microtubules and the loss of cell polarity [et al. reported that CDK5 promotes migration and invasion specifically in K-Ras mutant pancreatic ductal adenocarcinoma. The authors demonstrated that mutant K-Ras stimulates the cleavage of p35 to a more stable p25 species. This leads to hyper-activation of CDK5 kinase activity, resulting in increased tumour cell migration and invasion. Inhibition of CDK5 by ectopic expression of dominant-negative CDK5 constructs, or treatment of in vitro cultured cells with roscovitine significantly decreased the migration and invasion of pancreatic cancer cells [. Feldmann et al. [et al. [in vitro cultured breast cancer cells with transforming growth factor β1 (TGF-β1) upregulated the expression of CDK5 and of p35 leading to an increased CDK5 activity. Furthermore, they proposed that activated CDK5 phosphorylates FAK at Ser 732 and Tyr397 residues, and this phosphorylation is important for increased expression of α-SMA and for migration and invasion of breast cancer cells [Several studies have implicated CDK5 in regulating migration and invasiveness of cancer cells ,65,102 c, but thpolarity . This wapolarity . Moreovepolarity . Eggers er cells . Feldmann et al.  reportedn et al. ,103–107.n et al. ,104, then et al. ,108,109. [et al.  postulater cells .et al. demonstrated that CDK5 inhibits pancreatic cell migration and invasion by phosphorylating EZH2 at Thr261. This promotes binding of phosphorylated EZH2 to the F-box and WD repeat domain-containing 7 (FBW7) protein, a substrate recognition component of Skp1–Cul1–F-box (SCF) ubiquitin complex, resulting in ubiquitin-mediated degradation of EZH2 [In contrast with these findings, several groups reached an opposite conclusion, namely that CDK5 inhibits cancer cell motility. This inhibitory function has been ascribed to the ability of CDK5 to phosphorylate a tumour suppressor protein DLC1 , a trans of EZH2 . EZH2, a of EZH2 . Convers of EZH2 ,112. Hen3.6.in vivo and in vitro [. Consistent with these findings, inhibition of CDK5 reduced tumour growth and improved sensitivity to anti-angiogenic treatment in U87 glioblastoma and Lewis lung carcinoma xenografts [The formation of new blood vessels provides tumour cells with necessary nutrients and oxygen and facilitates tumour growth. Several studies postulated that CDK5 promotes tumour angiogenesis ,50,51. Ain vitro . Consist3.7.et al. demonstrated an increased levels of CDK5 and upregulation of CDK5 kinase activity upon exposure of cancer cells to DNA damage-inducing agents such as ionizing radiation, topoisomerase inhibitors or PARP inhibitors. CDK5 was shown to phosphorylate ataxia telangiectasia mutated (ATM) protein at Ser729, thereby activating the DDR pathway in hepatocellular carcinoma cell lines [There is increasing evidence that CDK5 may play a role in the initiation of DNA damage response (DDR) in cancer cells. Ehrlich ll lines ,114. Othll lines ,116 or bll lines ,118, or ll lines . All thell lines ,118,120.3.8.et al. [+ tumour-infiltrating lymphocytes, resulting in robust CD4+ T-cell-mediated tumour rejection [CDK5 may play a role in the anti-tumour immune response, by regulating the levels of programmed cell death ligand 1 (PD-L1) in tumour cells. Dorand et al.  demonstrejection .3.9.The emerging evidence implicating CDK5 in several pathological processes, such as neurodegeneration, diabetes and cancer, points to CDK5 as an attractive therapeutic target.Unfortunately, no CDK5-specific inhibitors are currently available. Therefore, most of the studies conducted to date employed non-specific pan-CDK inhibitors to inhibit CDK5, However, as we describe below, these compounds inhibit several other kinases, in addition to CDK5 .Table 1R)-roscovitine and olomoucine inhibit CDK2, CDK1, CDK7 and CDK9, in addition to CDK5 [N-(5-isopropylthiazol-2-yl)-3-phenylpropanamide (PJB) show increased selectivity for CDK5 and CDK2 over other CDKs [in vivo remains to be tested.Flavopiridol, often used to inhibit CDK5, represents an ATP-competitive inhibitor of CDK1, CDK2, CDK5 and CDK9  table 1table 1.  to CDK5 –123,125  to CDK5 ,130. Ano to CDK5  , specifically inhibits hyper-activation of CDK5 by p25, while having no effect on physiological activation of CDK5 by p35/p39 . An evene models . In addi [et al.  reported4.Over the last two decades, several non-neuronal functions of CDK5 have been proposed. It seems likely that in addition to development and normal physiology of the nervous system and pathogenesis of neurodegenerative diseases, CDK5 plays distinct roles in physiology and pathogenesis of several different tissues. However, most of the non-neuronal functions of CDK5 remain controversial. For instance, while several studies proposed that CDK5 acts as an oncogene and it promotes proliferation, migration and invasion of cancer cells, others reported that CDK5 functions as a tumour suppressor by promoting apoptosis or inhibiting proliferation, and that it impedes cancer cell migration and invasion. Many of these conclusions were based on experiments which employed treatment of cell lines representing different normal or cancer types with pan-CDK inhibitors , or empl


In this study, we designed high fiber cookie recipe without using additives by means of extrusion-based 3D printing. We aimed to relate printing quality and cookie physical properties with dough rheology and dietary fiber content depending on the flour  and fat type (olive oil or butter). The flour choice influenced all cookie quality parameters: baking loss, color, line height and width, and dietary fiber content. Results indicated that lower baking loss and better printing quality were obtained for cookie dough containing olive oil, which had higher viscosity and consistency coefficient compared with dough containing butter. Cookies with olive oil in which part of the oat flour was replaced with rye and carob flour were printed with high accuracy (≥98%), close to the ideal 3D shape. Overall, this study demonstrates the importance of selecting fat and particularly flour, as well as the extrusion rate on the quality and repeatability of 3D-printed cookies. Recently, extrusion-based 3D printing methods became commonly used in the food sector, where a mixture or melted material is continuously pushed from a nozzle by pressure and deposited on printer platform to obtain the desired 3D shape . CerealsThe rheological properties of the material are crucial for successful 3D food printing. A material for extrusion-based printing should display shear thinning behavior which is an indicator that the material can be extruded through the nozzle . PhysicaThe composition of food has a more relevant effect on its rheological properties, and consequently on the retention of the printed structure than printing temperature . Raw matThe type and the amount of added fat has a strong effect on the desired rheological and textural properties of the dough . The addThe precision of 3D printing and the shape of final product are influenced by many technological factors: printing mechanism, material properties, printing process parameters, and postprocessing methods . The accIn addition, 3D printable material must form stable structure with self-supporting properties of the deposited layers during and postprocessing without slumping, spreading, or bridging . PostproThe main aim of this research was to investigate the effect of butter substitution with olive oil and the influence whole grain and carob flour on the rheological properties of the dough and finally on 3D printing accuracy and repeatability. In addition, the relationship between the printing process parameters and the rheological properties of the mixtures with the dimensions and accuracy of the 3D-printed forms was examined.For experiments we used four types of flour, two types of fat and floral honey purchased at local grocery stores . The used oat flour  contained 72.1 g/100 g carbohydrates, 9.9 g/100 g proteins, 5.5 g/100 g lipids, and 2.3 g/100 g fiber. Rice flour  had 79.6 g/100 g carbohydrates, 7.2 g/100 g protein, 2.2 g/100 g lipids, and 2.9 g/100 g fiber. Rye flour  was composed of 77 g/100 g carbohydrates, 9 g/100 g protein, 0.74 g/100 g lipids, and 11 g/100 g fiber. Carob flour  contained 81.4 g/100 g carbohydrates, 5.3 g/100 g protein, 0.5 g/100 g lipids, and 26.3 g/100 g fiber. Butter contained 82 g/100 g lipids, whereas olive oil had 91 g/100 g lipids.To determine the dough recipe for 3D printing, using a slightly modified method according to Lipton et al. , the ratThe dough samples were fabricated using the extrusion-based 3D printer  with a syringe-extrusion-type plunger (volume of 20 mL). Before printing, the mixture was homogenized. The desired 3D shape of a flower (with 12 layers and layer height of 0.4 mm) was selected from software Cura 15.02.1. to examine the ability of dough to form accurate and stable structure. The 3D printing process was adjusted at temperature of 25 °C, print speed of 25 mm/s, nozzle diameter of 2.0 mm, and nozzle height of 2.0 mm.−1. In addition, the consistency coefficient (Pasn) and flow behavior index were calculated.The rheological properties of baking dough with different compositions were characterized using a RM 100 Plus Viscometer  according to the Standard ISO 2555:2018 in mPas. The goal was to determine the rheological parameters at the same temperature. However, due to too high torsion during the determination of rheological parameters at 25 °C, measurements were performed at 30 °C. The temperature of samples was maintained at 30 ± 1 °C throughout the test using a Julabo F 33 refrigerated-heating circulator. The apparent viscosity (Pas) was measured at the shear rate 5 sThe extrusion rate for each 3D-printed cookie was determined according to Equation (1) , and it 1 is the weight of printed sample before and m2 after baking and cooling.Ten 3D shapes printed in parallels of the same mixture (5 cookies out of one syringe) were baked in a convection oven , at 180 °C, for 3 min. The weight of the baked 3D-printed forms was determined after 30 min of cooling at ambient conditions using Sartorius A 120S analytical balance (reading 0.001 g). Baking loss was quantified by measuring the weight of the 3D-printed forms before and after baking according to the Equation below (2):The weight of all printed shapes from each syringe filling was determined to calculate the utilization rate of the dough during 3D printing process for each mixture. The utilization rate indicates what percentage of total mixture input in 3D printer syringe entered into final product—cookie dough. Dough utilization rate was calculated as the ratio of the sum of weight of forms printed from one syringe to the initial weight of mixture inserted in the syringe.3/s), v—print speed (mm/s), a—width (mm), and c—value defined as ratio of line height over width.The total height of baked cookies was determined at 4 positions a for eacThe line spreading  calculated as the line height/width ratio is later represented as the normalized value to ideal.Since the printed and baked forms differed, the shape accuracy of each printed form from the desired one was quantified by analysis and comparison of binary photographs  of the finished shapes . The las*, a*, and b* were determined on the bottom of the cookie sample where the area was the largest .The moisture content of cookies was determined according to AACC method 44-15.02 in triplicate. Total dietary fiber (TDF) of dried and defatted samples was determined according to AACC method 32-05.01 and AOAC Method 985.29 (if fat content is >10%) in duplicate using Total Dietary Fiber Assay Kit . The results are expressed se mean value in grams per dry weight of cookie.p < 0.05.The data recorded were subjected to one-way analysis of variance (ANOVA) to establish that there were no significant differences between two fillings of printer syringe with the same dough mixture. Two-way ANOVA was used to identify the effects of flour and fat type on sample parameters. ANOVA for repeated measures was applied on weight, line width, and height of 3D-printed shapes to evaluate the repeatability of printing. ANOVA, Tukey post hoc test, Pearson correlation test, and principal component analysis (PCA) were run with a Statistica 12 . Differences were considered statistically significant when p < 0.001)  . The flo< 0.001) . This is . The flp < 0.001) for mixtures B than for mixtures A, probably because of water contained in the butter.Comparing the values of the consistency and apparent viscosities of all mixtures, mixtures 2 had the highest values. These mixtures contained rye flour, which has gluten, while flours in other doughs are gluten-free. Rye pentosans, proteins, and starch have an important function in water binding . Rye flop < 0.001), regardless on the flour and fat type. The first samples of all six mixtures had the biggest weight compared with each subsequent printed form  between mixtures did not significantly differ, which is also shown as the total sum of all printed samples from the syringe at ted form b. The weThe percentage of mixture inserted into the printer syringe converted to final printed product is shown as dough utilization rate a. The hiThe weight of all shapes was reduced during baking and cooling resulting in baking loss , probabl*, a*, b*, C, and H°. The expected significant difference (p < 0.05) of all color parameters was evident between mixtures 1 and 2, as well as 1 and 3 due to the carob flour added in mixtures 2 and 3. The lightness L* and redness a* were dependent on flour choice, where mixture 1 was the lightest, showing the lowest intensity of red color. The color parameters b*, C, and H° were significantly influenced by the interaction of flour and fat (p < 0.05), being the most intensive in sample 1A (oat with olive oil). Comparing the color of mixtures 1A and 1B .The biggest challenges of 3D food printing are the precision and accuracy of printing, its repeatability, and stability of 3D shapes. The printing quality and the stability of 3D shapes can be achieved not only by controlling the physicochemical, rheological, structural, and mechanical properties of the material  but alsop > 0.05). The cookies from mixture 1 and 2 were higher when butter was used, but cookies from mixture 3 were higher when containing olive oil but the differences were small. The line width was positively correlated with height (r = 0.96) and inversely correlated with dough consistency (r = −0.77). Patterns of mixtures 2 and 3 were less thick and less spilled than patterns of mixtures 1, which indicates that carob flour, i.e., higher fiber content, increases the stability of mixtures.Total height, line height, and width  were sigp = 0.09). All doughs with olive oil had c-values that deviate less than 1 compared to those with butter  depending on both flour and fat type. Minor changes were observed in mixtures 2 and 3 with olive oil, which might be related to their rheological parameters, i.e., higher values of consistency coefficient and apparent viscosity. The increase in viscosity results in better shape retention during 3D food printing [p > 0.05) in average weight between samples, significant (p < 0.001) differences were established in the weight of cookie samples along the printing time. The differences in weight, line width, and height at different printing times are the reasons for printing inaccuracy.The average shape accuracy (97.5 ± 0.9%) was not significantly influenced with the flour or fat choice. Still, printing . The shap < 0.001). Cookies from mixture 1 contained in average 2.02 ± 0.05 g of fiber per 100 of cookie dry matter. On the other hand, cookies from mixtures 2 and 3 were high in fiber containing in average 10.28 ± 0.28 and 8.31 ± 0.30 g of fiber per 100 of cookie dry matter, respectively. This confirmed the great potential of carob flour for the enrichment of cookies with fiber [Whole grains contain insoluble and soluble dietary fiber with multiple health-promoting effects . As expeth fiber . Besidesry fiber . The carry fiber . Carob gry fiber .*, b*, C, and H°), and line width with the dough consistency, redness a*, c-value, and TDF (*, but highest L*, b*, C, H°, line width, and PCA extracted five factors with the average eigenvalue of 3.20, so only first two components above the average value with eigenvalues of 10.50 and 3.34, accounting for 86.5% of the total variance, were considered . The fir and TDF b. The seThe aim of this study was to determine the physical properties of 3D-printed whole grain cookies depending on flour and fat choice as well as on dough rheology. The results showed that the type of flour and the type of fat strongly affect dough rheology and printing accuracy. Mixtures containing more fiber have higher consistency coefficient, which is related with more narrow width of printed line and less spread of the printed layer resulting in better printing repeatability and accuracy. During 3D printing, each subsequent 3D cookie is printed at a lower extrusion rate, has a lower weight and more narrow width than the previous one, and is printed more accurately. The printing speed and extrusion rate are strongly related with the height and width of the printed dough layers. By modifying the cookie recipe and controlling the rheological properties of the dough, a cookie dough with higher nutritional value can be 3D printed with high accuracy and repeatability without the use of additives. Future studies should investigate the role of different sweeteners on the quality of 3D-printed cookies and test their acceptance by consumers.


Membrane transporters are integral membrane proteins that mediate the passage of solutes across lipid bilayers. These proteins undergo conformational transitions between outward- and inward-facing states, which lead to alternating access of the substrate-binding site to the aqueous environment on either side of the membrane. Dozens of different transporter families have evolved, providing a wide variety of structural solutions to achieve alternating access. A sub-set of structurally diverse transporters operate by mechanisms that are collectively named ‘elevator-type’. These transporters have one common characteristic: they contain a distinct protein domain that slides across the membrane as a rigid body, and in doing so it ‘drags” the transported substrate along. Analysis of the global conformational changes that take place in membrane transporters using elevator-type mechanisms reveals that elevator-type movements can be achieved in more than one way. Molecular dynamics simulations and experimental data help to understand how lipid bilayer properties may affect elevator movements and vice versa. Structural studies of membrane transporters from diverse protein families have revealed that alternating access may by achieved in many ways (reviewed recently ). The soThe elevator-type transport mechanism offers an alternative solution to achieve alternating access . ProteinPh [The first elevator-type mechanism was described in 2009 for the aspartate transporter GltPh , a membePh . In recePh . In thisPh .z-direction if the membrane plane is defined as the xy plane. In many cases, the domain movement is more complex than a simple translation, and the total distance over which the substrate is displaced is larger than the vertical distance , and the human neutral amino acid exchanger ASCT2 (Alanine Serine Cysteine Transporter 2)  family of solute transporters is structurally well-characterized with 39 available structures of four different family members: the prokaryotic sodium-dependent aspartate transporters Gltoccluded , inward-occluded  and inwaoccluded . We willLike all members of the SLC1 family, neutral amino acid transporter ASCT2 is a homotrimer. Each monomer consists of 8 transmembrane segments (TMs) that form a scaffold domain  and a transport domain . The transport domain additionally contains two helical hairpins (HP1 and HP2). In the outward-facing states the substrate-binding site is close to the extracellular side of the membrane, and the only difference between open and closed conformations is the position of HP2, which works as a gate to provide access to the binding site from the extracellular aqueous environment  Figure . When thTk [Ph [The fixed barrier elevator mechanism with one gate is likely conserved among the SLC1 family, as evidenced by recent single particle cryo-EM structures of GltTk , and molTk [Ph . Fixed bTk [Ph ) have chTk [Ph ,33 and CTk [Ph  indicateATP-binding Cassette (ABC) transporters do not use elevator-type mechanisms of transport, with the exception of the non-canonical subfamily of ECF (energy-coupling factor) transporters. ECF transporters are involved in uptake of vitamins or other micronutrients (reviewed in ). Two suThe concentrative nucleoside transporter CNT (a member of the SLC28 family) is a homotrimer , with ea+/succinate transporter VcINDY [+/citrate transporter SeCitS [It is possible that the location of the binding site between two domains in CNT necessitates the use of two gates, whereas an occluded binding site within the transport domain, as found in SLC1 transporters, may allow the use of a single gate. Most of the transporters with proposed elevator-like transport mechanisms have substrate-binding sites positioned at the interface of two domains . Transpor VcINDY  , and bir SeCitS  of the Sr SeCitS , but addThe bile acid transporter ASBT, and structurally related sodium-proton antiporters have 10 and 13 transmembrane helices respectively, with a transport domain  consisting of TM3–5, TM8–10 in ASBT , and a scaffold domain . Despite the movement of the substrate-binding site across the membrane during sliding of the transport domain relative to the scaffold , ASBT does not have a fixed barrier . Thus, tASBT is exceptional among elevator-type transporters because it is a monomeric protein. Another monomeric transporter, for which an elevator mechanism has been postulated, is CcdA . CcdA isPh show different extents of membrane deformation depending on the position of the transport domain [Ph in the outward-facing state induce very little local membrane curvature [It has been noticed that the TMs of the scaffold domains of many elevator-type transporters are shorter than those in transport domains, and often highly tilted . As a cot domain  , than in liposomes composed of Phosphatidylcholine (PC) [In vivo, allosteric modulation by lipid molecules has been observed in Xenopus oocytes expressing EAAT4 that displayed increased glutamate-induced currents when arachidonic acid was added [In only very few cases have the effects of the lipid environment been studied experimentally. In Gltine (PC) . This efine (PC) , but againe (PC) . In vivoas added . The preas added .The above examples show that lipids may affect protein function directly via interactions with amino acid residues, which could accelerate or slow down transport domain movements or stabilize the scaffold domain in the membrane. In addition, colligative bilayer properties are likely to affect the functioning of elevator-type transporters, because the lipid–protein interface must rearrange substantially during transport. Finally, also the domain structure of the proteins may affect the bilayer morphology, and consequently elevator dynamics.Importance of the field. Since the first description of an elevator-type transport mechanism for GltPh over a decade ago [cade ago , a varieA summary of the current thinking. In elevator-type transport mechanisms, one protein domain brings the substrate-binding site from one side of the membrane to the other by sliding through the lipid bilayer. The extent of the elevator movement, ranging from 21 Å in GltTk to 7.5 Å in ASBT, and number of gating elements (one or two) vary between different proteins  willulations .


Scarabaeus galenus digs a burrow that it provisions by making repeated trips to a nearby dung pile. Even more remarkable is that these beetles return home moving backwards, with a pellet of dung between their hind legs. Here, we explore the strategy that S. galenus uses to find its way home. We find that, like many other insects, they use path integration to calculate the direction and distance to their home. If they fail to locate their burrow, the beetles initiate a distinct looping search behaviour that starts with a characteristic sharp turn, we have called a ‘turning point’. When homing beetles are passively displaced or transferred to an unfamiliar environment, they initiate a search at a point very close to the location of their fictive burrow—that is, a spot at the same relative distance and direction from the pick-up point as the original burrow. Unlike other insects, S. galenus do not appear to supplement estimates of the burrow location with landmark information. Thus, S. galenus represents a rare case of a consistently backward-homing animal that does not use landmarks to augment its path integration strategy.Unusual amongst dung beetles,  Scarabaeus galenus , with their navigational capacity being the focus of intense research for over a century. Ant species that inhabit relatively featureless environments, such as the salt pans of northern Africa, find their way home by computing a straight-line trajectory that ends at the position of their nest (Wehner Locating a tiny (~ 2 cm diameter) and visually inconspicuous burrow entrance from the ground-level perspective of a foraging beetle is no simple task, particularly considering that t Wehner . They dot Wehner , 2007. At Wehner .Ant species that inhabit more cluttered environments, like the pine forests of Greece or the vegetated deserts and bushlands of Australia, in addition to path integration, use landmarks or the skyline panorama to find their way back home  and fed with fresh cow dung. To avoid pseudo-replication, individual beetles were marked with a number on their thorax using correction fluid.All experiments were carried out with the diurnal dung beetle S. galenus  was displaced 50 cm in one of four directions: laterally  or translationally . In this set of experiments, as well as in all experiments described below, each beetle was allowed to make at least 6 foraging runs before they were manipulated to ensure that they were accurately navigating between their burrow and the feeder. Before releasing the displaced beetles, their burrows were covered over with sand and the ground carefully brushed to remove potential olfactory cues. The path taken by the beetle when searching for its burrow after the displacement was filmed for 4 min . In an additional test, some beetles were also displaced 50 cm towards their open burrow.These beetles were picked up just before entering their burrows after their 7th visit to the feeder—by carefully placing a white opaque plastic container on top of the beetle as it walked over a sandpaper covered plate placed just outside the burrow. The beetles  were then displaced back towards the former position of the feeder . Here, the plate was carefully placed on the ground and the plastic container removed to release the beetle back where the feeder had been located. Before the beetles were released, their burrows were covered over with sand and the area brushed to remove potential olfactory cues.Beetles were picked up during their 7th visit to the feeder (by carefully placing a white opaque plastic container on top of the feeder) and transferred about 30 m away from the original burrow to a different experimental arena surrounded by a different array of natural landmarks unfamiliar to the beetles. The transfer took approximately 30 s, after which the feeder was carefully placed on the ground and the plastic container removed to release the beetle. The beetle’s subsequent path and search for its burrow was filmed for 4 min. To control for possible effects of the transfer on the beetle’s navigational performance and to generate a performance baseline for this experimental condition, foraging beetles arriving at a feeder at the original experimental site were also covered with the plastic container and transported for 30 m (15 m away and 15 m back) for a duration of ~ 30 s before being placed back on the ground at the exact same position as from where they had been picked up. Before releasing these beetles, their burrows were covered over with sand and the ground carefully brushed to remove potential olfactory cues.https://doi.org/10.1137/141000671), unless stated otherwise.All analysis and data management were done using Julia  SIAM Review, 59: 65–98. The paths taken by beetles in each of the experiments were tracked using a custom-made Matlab (Mathworks Inc.) software . Pixel coordinates representing the beetle’s position were converted to real world coordinates using the Camera Calibration toolbox in Matlab, which also accounted for any distortion in the images caused by the camera optics. The coordinates of the beetle’s trajectory were smoothed using a parametric spline from the Dierickx Fortran library) with a smoothing factor of 500 and an order of 2. The smoothing spline removed some of the erratic movements of the beetle as well as compensated for any human errors accumulated during the tracking phase. Path straightness was calculated as the distance between the ends of the path divided by the length of the path.The turning point  were used to evaluate the effect of displacement on the resulting turning points and centers of search. Generalized Linear Mixed Models (GLMM) were used when the response was non-linear and the same individual beetles were repeatedly tested . In addition, we used a permutation test for comparing variance between nominal groups (displaced versus not displaced beetles) and a simple one sample x and y axis) separately. Apart from the added benefit of simpler statistical methodology, we base this decision on the fact that all the displacements were along only one of these two dimensions . Since there is no reason to believe that there is an interaction between turning points and centers of search, we kept the analysis of those two indicators separated as well.In all comparisons, we chose to test the two spatial dimensions  in Figs. Scarabaeus galenus is frequently found foraging in antelope dung middens or on sparsely distributed piles of pellets in savanna and woodland-savanna areas across South Africa  wherever the substrate seems soft enough for them to dig into, including open sandy areas or grassy plains ). After some time , they will then return to the pile, pick-up individual pellets in their hind legs and carry them backwards towards their burrow . Excavated burrows have been found to contain up to 50 pellets and it is common to see individual beetles performing more than 10 foraging trips, with up to 125 trips having been observed . On its way to the food source, S. galenus walks forward on six legs  and their walking speed increases .Once the burrow is complete, the first outbound foraging trip is often quite tortuous but nonetheless directed in the approximate direction of the previously discovered food source Fig. . This isn = 8)  , with a minor deceleration over the last 50 cm before the burrow , these beetles initiated a systematic search pattern Fig. a, b. Thi 8) Fig. c. The me 8) Fig. d, indicarow Fig. e.Fig. 4Tx = 0.93, y = 0.89), centers of search: ) and highly significant .When collecting a 7th pellet, the beetle and feeder were passively displaced 50 cm either laterally (to the left or right of the feeder) or translationally (towards or away from the burrow). From this new location, the beetles immediately set out with a dung pellet from the release location in a straight line parallel to the original homewards path before initiating a search Fig. . We testS. galenus only followed a home vector, obtained by path integration, as if they had not experienced a displacement.The location of the turning points and centers of searches were, for all conditions, centered around the location of their respective fictive burrow, i.e., the site where the burrow should be if it had been displaced along with the beetle Fig. . This st28 unique permutations) on the variance of the x and y values  of the turning points and centers of search separately (n = 27). Although the variance of the turning points of the displaced groups of beetles (σ2 = 218) was significantly larger than for the unmanipulated (‘none’) control group of beetles along the y axis (P < 0.001), we saw no difference along the x axis, . This suggests that the distance measured  by the beetles was more affected by the passive displacement than their perception of direction . The same analysis showed no significant difference for the centers of search , displacement σ2: , P = ), which supports the suggestion that S. galenus perceives distance less precisely than direction.To test how the precision of the home vector  was affected by passive displacements, we centered all displaced groups to their means and ran an one-tailed permutation test ; n = 10)  and could thus no longer rely on path integration to locate their burrow. Once the container was removed, the beetles immediately set out with their pellet from the release location, but almost immediately initiated a search Fig. a. Again,10) Fig. b, c. Alt10) Fig. a. This fn = 10). The center of the search was located 12 cm ± 21 cm to the right of the burrow and 13 cm ± 24 cm in front of it (n = 10). ; center of search: P, one sample t test, n = 8). Again, these experiments indicate that S. galenus rely on path integration, rather than familiar landmarks, to return to their burrow.In the next set of experiments, 10 beetles were covered by an opaque container while at the feeder and then transported along with the feeder for ~ 30 s to a novel experimental site ~ 30 m away. Here, the feeder and the beetle were placed gently on the ground and the container removed. As soon as they were released, the beetles started to move backwards along a straight path, carrying a pellet in their hind legs, in a direction that corresponded to the direction of the fictive burrow . On covering the appropriate distance approximately, the beetle then initiated a search. Fig. a. The tu0). Fig. b, c. Botn = 9). The center of the search was located 1 ± 11 cm to the left of the burrow and 11 ± 11 cm after it (n = 9). This confirms that S. galenus can still successfully locate its burrow with high precision after having been caught and carried in a closed container.As a control for any disturbances caused by covering, transporting and releasing the beetles, another group of beetles were picked up at the feeder and transported ~ 30 m  for 30 s before being released at the feeder’s original location. Upon release, these ‘control beetles’ carried their pellet back to the location of their now covered burrow, where they initiated a search. The turning point was located 2 ± 7 cm to the left of the burrow and 6 ± 14 cm after it , σ2novel, P; center of search; σ2back, σ2novel, P ). This further strengthens our observation that S. galenus do not use familiar topographical features of the surrounding environment to locate the position of their burrow, but rather use the direction and length of their home vector.To further test how the familiarity of the area affects the variance in the beetles’ ability to locate their burrow, we ran a one-tailed permutation test on the variance of the x and y values of the turning points and centers of search for the beetles transported to a ‘novel’ experimental site and for the beetles transported ‘back’ to the original position of the feeder. This did not reveal any significant difference in performance   Fig. . This sung) Fig.  but, unlS. galenus fail to locate their burrow at the end of their homing path, they initiate a systematic search that is characterised first by a clear deviation from the straight-line path, followed by a series of loops. The location of the turning point that indicates the start of the search pattern lies very close to the location of the real burrow (in the case where the burrow has been covered)  Fig.  or to thed) Fig. . This suIf the beetles are passively displaced from the feeder location , they faithfully return along a bearing that is 180° from the direction they walked to the feeder and then initiate a search at a distance that is almost exactly the same as the distance walked on the outbound journey Fig. . While tS. galenus homes at a nearly constant speed  and can no longer rely on path integration to locate their burrow. If the beetles were using the skyline panorama or landmarks to find their way home, they should nonetheless be able to locate their burrow after this displacement, as ants are able to do  between the dung and their burrow. Perhaps over these relatively short foraging distances, the path integrator of S. galenus does not accumulate enough errors  may not be particularly useful for homing in S. galenus as far as we know, don’t occupy long term, permanent nests. Instead they fly long distances between bouts of feeding, to new habitats, where they would have to learn a new set of landmarks for a brief, once-off, provisioning session, only to move on again once that store has been depleted.A third reason could be that most dung beetles, including S. galenus possibly face if employing such an idiothetic odometer, coupled to its legs, is that while the beetles leave their burrow in a standard six-leg tripod gate, they return on only four legs, holding the pellet of dung tightly between the last pair , or transported under an opaque container (transfer experiments), all displayed an increase in the spread of the length of their home vector, while their direction estimate remained unchanged. This suggests that their estimate of distance may be based on idiothetic measurements, which are easily disturbed by the sudden sideways deflection from the terrain or by induced or self-induced movements during food transport. A likely candidate for this estimator is a step-based odometer, similar to the one found in ants (Wittlinger et al. air Fig. c, e. The


The Domain Interface Method (DIM) is extended in this contribution for the case of mixed fields as encountered in multiphysics problems. The essence of the non-conforming domain decomposition technique consists in a discretization of a fictitious zero-thickness interface as in the original methodology and continuity of the solution fields across the domains is satisfied by incorporating the corresponding Lagrange Multipliers. The multifield DIM inherits the advantages of its irreducible version in the sense that the connections between non-matching meshes, with possible geometrically non-conforming interfaces, is accounted by the automatic Delaunay interface discretization without considering master and slave surfaces or intermediate surface projections as done in many established techniques, e.g. mortar methods. The multifield enhancement identifies the Lagrange multiplier field and incorporates its contribution in the weak variational form accounting for the corresponding consistent stabilization term based on a Nitsche method. This type of constraint enforcement circumvents the appearance of instabilities when the Ladyzhenskaya–Babuška–Brezzi (LBB) condition is not fulfilled by the chosen discretization. The domain decomposition framework is assessed in a large deformation setting for mixed displacement/pressure formulations and coupled thermomechanical problems. The continuity of the mixed field is studied in well selected benchmark problems for both mixed formulations and the objectivity of the response is compared to reference monolithic solutions. Results suggest that the presented strategy shows sufficient potential to be a valuable tool in situations where the evolving physics at particular domains require the use of different spatial discretizations or field interpolations. The growing demand of industrial complex simulations, together with the continuous improvement of computer technology, have stimulated the popularity of algorithms capable of processing large systems of equations. This is the case of algorithms based on domain decomposition techniques which basically stem from the need of decomposing a large discretization into a number of smaller discretizations, e.g. using finite elements (FEs), or from the assembly of independently generated meshes which form part of a whole system. The latter scenario can be found for instance during simulations of an aircraft where both wings and fuselage discretizations are designed independently. In these situations, the type of algorithms capable of gluing non-matching meshes are crucial and represent the main focus in the present contribution.Examples of non-conforming interfaces arising from the assembly of non-matching grids can be found in , 25 wherThe most general scenario at a non-conforming interface mentclass2pt{minimMany Domain Decomposition techniques assume that the interface is geometrically compatible  but moreIn mortar methods, the coupling constraints are enforced in a weak sense, i.e. in an integral or average sense along portions of the interface, and are often referred to as segment-to-segment techniques. In general, a displacement field at the interface, One significant drawback of such classical mortar techniques arises when the coarsest discretization is selected for the reference solution field. In such scenarios, the performance of mortar methods might be less optimal as argued in , 18. MorIn contrast with established mortar methods, the DIM  is basedinf-sup condition) [Lagrange multipliers  are typindition)  in orderndition)  is consindition)  and the ndition) . A main mentclass2pt{minimmentclass2pt{minimThe geometrical aspects of the DIM introduced in  are summFinite strain theory is considered in our developments, although an infinitesimal deformation approach can be recovered by considering small displacements (i.e. compared to the domain dimensions) and infinitesimal displacement gradients. Compact notation is utilized for tensor quantities in this contribution.s and q.Consider the union of two non-overlapping domains k as indicated in step (ii). As discussed in [k does not interfere with the results although An independent FE discretization of the two bodies, leads to a number of ussed in , the magA Delaunay triangulation defining the interface domainTriangular linear elements r method .The corresponding deformation map for each domain D at time As shown in , an exprcf. Fig. .n. Due to the linear interpolation of the incremental displacements in   37) is eloped in , 28. TheIn order to stabilize the pressure field for the case of compressible and nearly compressible materials the projection term in  is addedThe identification of the pressure Lagrange multipliers tions in . The exptions in  concernie gap in  and the b stands for the the discrete nodes corresponding to the Lagrange multipliers interpolation using the domain-wise constant shape functions A Galerkin-based spatial discretization is considered in which the solution field components and their variations at each domain cated in  to 177)\documentments in  to 49)  \documenailed in  and 101) is eTo this end, the solution The work performed at the interface by the Lagrange multipliers The identification of the temperature Lagrange multipliers tions in . The exptions in  concernie gap in  and the The variational statement for the thermal constraint equations reads:equation ). In othAs argued in  and brieentclass1pt{minimassion in  and enfocf. Fig. .L stands for the length of the base-side of k denotes the thermal conductivity coefficient.The stabilization parameter As seen in the monolithic formulation \documentmentclass2pt{minimribed in .The corresponding FE approximations of the thermal variational statements in , 122) a a122) anBoth mechanical  and therIn order to obtain scalabilities in a massive number of processors it is convenient to recast the global systems  or 142)142) in tAfter solving the flexibility problem in  and obtaoblem in  which isIf one or more subdomains sions in  to 149)\documentained in , 20, 22.In this view, exploiting the novel features of the DIM method, i.e. a continuous fictitious discretization of the interface, a new methodology to handle rigid body modes has been proposed in  and adopments in  to 46)  \documenments in  to 114)\documentcf. Fig.  and \docfound in  for the Note that the term t}cθ˙ in  allows fcated in  leading The linearized set of equations in  and 142142 obtai methods ). In thicated in .Assuming a fix domain decomposition and a given Delaunay interface discretization, the iterative scheme for the coupled thermomechanical non-linear DIM framework is summarized in Box Two representative tests are selected to assess the performance of the multifield domain decomposition framework. Attention is focused on the continuity of the hybrid solution field at the interface, the convergence rate upon mesh refinement and the influence of the stabilization parameters as well as the activation of the Lagrange multipliers for the different solution fields at the interface. Finite strain theory and plane strain conditions are considered in all two dimensional examples.F is applied at one end of the membrane while the opposite end is fixed in both directions.The mixed ons cf. , 37). Th. Th\docuThe membrane is considered elastic with Young’s modulus In order to assess the convergence of the proposed methodology, five different discretization scenarios are considered. These concern a monolithic approach and the DIM on the body partitioned in two domains, i.e. mentclass2pt{minimThe vertical displacement of point A cf. Fig.  is monitIt is observed that all analyses provide an analogous convergent behaviour and the differences between the monolithic approach and the domain decomposition strategy are hardly visible beyond the third discretization, i.e. around 200–250 FE. In both compressible and incompressible analyses the DIM with non-conforming interfaces shows a slightly slower convergence with respect to the domain decomposition with conforming interfaces.The influence of the activation of the pressure Lagrange multiplier mentclasspt{minimaIt is observed that the activation of h  presented in  is extenThe extended DIM method for multiphysics problems essentially relies in an explicit discretization of the interface by means of a zero-thickness Delaunay triangulation as in the original methodology. This strategy allows setting a full tied contact in the most demanding scenarios, e.g. between geometrically incompatible interfaces. Continuity of the mixed solution field is satisfied across domains through the incorporation of Lagrange Multipliers. A Nitsche method is adopted in which a stabilization term is added at the constraint equations of the mixed field, i.e. one stabilization term per field. The incorporation of the stabilization term avoids the appearance of zero diagonal terms at the global system and instabilities are avoided if the LBB condition is not fulfilled by the chosen discretization. It is important to remark that, since the penalized term is part of the Euler-Lagrange equations of the variational principle, it will vanish upon mesh refinement .The formulation presented in this manuscript is based on the identification of all Lagrange multipliers to connect the mixed field and their consideration in the weak variational form together with the corresponding consistent stabilization term. This technology is first assessed for a mixed displacement/pressure formulation to tackle incompressible materials in which a Polynomial Pressure Projection method is employed to account for the stability of the resulting systems within each domain. It is demonstrated that the pressure field is transferred with continuity across the non-conforming interfaces only if the Lagrange multiplier concerning the pressure field is taken into account, i.e. the continuity of the displacement field at the interface, enforced by the displacement Lagrange multipliers, does not guarantee the continuity of the pressure field at the interface. In fact, this effect vanishes upon mesh refinement since the stabilization term is no longer needed and vanishes too. It should be highlighted that the sensitivity of the results to the stabilization parameter is, however, very small. For the mixed displacement/pressure formulation, a variation of eight orders of magnitude in the pressure stabilization parameter would cause an insignificant jump in the pressure field.The assessment of the DIM formulation for multiphysics problems is completed with a coupled thermomechanical problem with applications to bulk metal forming processes. In this case, a mixed displacement/pressure/temperature field is considered together with the corresponding Lagrange multipliers at the interface. Continuity of the three fields is obtained across all interfaces and the distribution of the mixed field is in very good agreement with the reference monolithic solutions. The mechanical response resulting from the upsetting of a billet studied in this example is remarkably close to those reported in literature which validates both the adopted constitutive model and domain decomposition technique.An alternative non-intrusive methodology to handle RBMs was introduced in the original contribution presenting the DIM method. This methodology essentially adds an extra stabilization term to the energy functional with contributions of all adjacent interface patches avoiding, in this manner, the calculation of a pseudo-inverse at floating domains. As a result, the band structure of the global system is preserved and, for this reason, possible parallel solution strategies employed in other dual domain decomposition methods can still be employed. In the analyses considering the mixed displacement/pressure problem no singular modes may arise from the pressure field. However, in the thermomechanical problem, singular temperature modes with constant temperature distribution may arise at floating domains. An extra stabilization term analogous to the term restricting the RBMs should be considered but in the present formulation the subdomain matrices with diagonal temperature coefficients can still be inverted due to the introduction of a transitory term which acts in a similar way than the mass matrix in a dynamics problem.The DIM method for mixed fields inherits all advantages outlined for the irreducible formulation, i.e. the interface connections are set up as a result of the connectivity in the automatic Delaunay triangulation and do not rely in user criteria for the master and slave DOFs or intermediate interface projections as it is done in established techniques such as the mortar approach. For this reason, the methodology shows potential to be used in cases where completely different discretizations are needed among domains since different physical phenomena are tacking place therein. The use of the DIM method could be specially advantageous when a particular domain discretization needs to be changed at a certain stage of the problem in order to improve the simulation of the physical process.


Fosfosal is the O‐phosphorylated derivative of salicylic acid, with documented clinical use as a prodrug for the treatment of inflammatory diseases. We recently discovered that fosfosal itself inhibits the protein‐protein interaction domain, the SH2 domain, of the tumor‐related transcription factor STAT5b. Here, we demonstrate that fosfosal is selective for STAT5b over its close homologue STAT5a. This selectivity is mediated by the STAT5b residue Arg566, located in the SH2 domain‐adjacent linker domain. Our data provide further evidence for the role of the STAT linker domain in determining the activity of small molecules against the SH2 domain. We present a refined binding model for fosfosal and STAT5b, which can serve as the basis for the development of fosfosal‐based STAT5b inhibitors. On the edge: Fosfosal, a prodrug for the treatment of inflammatory diseases, inhibits the tumor‐related transcription factor STAT5b with selectivity over the close homologue STAT5a. Comparative analysis between wild‐type and mutant STAT5 proteins revealed that fosfosal binds not only to the protein‐protein interaction domain, the SH2 domain (green), but also to Arg566 in the adjacent linker domain (magenta). Our study reveals that the STAT5b‐selectivity of catechol bisphosphate‐based inhibitors has its origins in the lead structure fosfosal, and provides further evidence for a contribution of the STAT linker domain to the function of the SH2 domain.In summary, we show that the STAT5b inhibitor fosfosal exhibits fivefold selectivity over the highly homologous STAT5a, and that this selectivity is mediated by Arg566 in the STAT5b linker domain. The refined binding mode of fosfosal presented here will facilitate the development of improved fosfosal‐based STAT5b inhibitors, and is consistent with the binding mode of catechol bisphosphate‐based STAT5b inhibitors.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Cdk5−/− mouse embryos, we show that absence of Cdk5 causes a significant increase in both mPTP opening and mitochondrial Ca2+ level. Analysis of subcellular fractions of MEFs demonstrates that Cdk5 localizes in the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) and Cdk5 loss in MAMs causes increased ER-mitochondria tethering, a process required for Ca2+ transfer from the ER to the mitochondria. Loss of Cdk5 also causes increased ATP-mediated mitochondrial Ca2+ uptake from the ER. Inhibition of ER Ca2+ release or mitochondrial Ca2+ uptake in Cdk5−/− MEFs prevents mPTP opening, indicating that mPTP opening in Cdk5−/− MEFs is due to increased Ca2+ transfer from the ER to the mitochondria. Altogether, our findings suggest that Cdk5 in MAMs regulates mitochondrial Ca2+ homeostasis that is disturbed upon Cdk5 loss, which leads to mPTP opening.We previously demonstrated that loss of Cdk5 in breast cancer cells promotes ROS-mediated cell death by inducing mitochondrial permeability transition pore (mPTP) opening . However, the molecular mechanism by which Cdk5 loss causes mPTP opening remains to be investigated. Using primary mouse embryonic fibroblasts (MEFs) isolated from  Cyclin dependent kinase 5 (Cdk5) is a small proline-directed serine/threonine kinase that serves various cell functions such as in the regulation of oxidative stress , 2, mito2+, the inner mitochondrial membrane (IMM) contains the mitochondrial calcium uniporter (MCU) complex that regulates mitochondrial calcium ([Ca2+]mt) influx mt concentration [2+]mt homeostasis.Mitochondria are cell organelles that execute a number of key cellular functions, including in energy conversion, intermediate cellular metabolism, cell differentiation, immune response, calcium signaling and homeostasis, and cell death. While the outer mitochondrial membrane (OMM) is highly permeable to Ca) influx . As MCU activity . Mitocholum (ER) , 10. A clum (ER) . MAM reglum (ER) , 13, andlum (ER) , 15, forlum (ER) , 17. MAMlum (ER) , mitofuslum (ER) , and conlum (ER) . Once Cachondria  through chondria . Severalntration –25. The ntration , 27. Thi2+ concentration leads to increased mitochondrial respiratory chain activity and ATP synthesis mt, has been shown to be a critical regulator of mPTP opening [2+]mt in Cdk5−/− MEFs by tracing cytoplasmic Ca2+ level, [Ca2+]cyt, following the addition of the protonophore and oxidative phosphorylation uncoupler, FCCP. FCCP causes depolarization or collapse of the mitochondrial membrane potential, resulting in mPTP opening and release of Ca2+ from the mitochondria [2+ level following FCCP treatment in wt as well as Cdk5−/− MEFs corresponds to [Ca2+]mt. To proceed with [Ca2+]mt measurement, wt and Cdk5−/− MEFs loaded with the cell-permeable intracellular calcium indicator, Fluo-4-AM, were subjected to single cell Ca2+ imaging before and after FCCP treatment. As shown in Fig. 2+]cyt in Cdk5−/− MEFs than in wt, indicating increased [Ca2+]mt in Cdk5−/− MEFs compared with wt. Quantitative analyses revealed a 58% increase (p < 0.05) in peak amplitude  in area under the curve , contains channels for calcium transfer mt, ultrathin sections of wt and Cdk5−/− MEFs were subjected to TEM  in Cdk5−/− MEFs than in wt (67% vs 52 %). Figure Cdk5−/− MEFs is also greater (p < 0.01) in Cdk5−/− MEFs than in wt (40% vs 15%). In addition, as shown in Fig. p < 0.05) in Cdk5−/− MEFs compared with wt (44% vs 25%).ER-mitochondria tethering is essential for the formation of ER-mitochondria contact sites and subsequent Cachondria , 44. To TEM Fig.  as descrchondria , 45, 46,2+ uptake from the ER in wt and Cdk5−/− MEFs. To do so, cells in Ca2+-free buffer and pre-loaded with a mitochondrial Ca2+ probe, Rhod-2 AM [2+ release [2+]mt in Cdk5−/− MEFs than in wt. Quantitative analyses showed a 26% increase (p < 0.05) in peak amplitude . This was unexpected but supports differential physiology and roles of Cdk5 in normal and cancer cells, and calls for more comprehensive investigations on the function of Cdk5 in mitochondrial calcium dynamics in normal and disease conditions.mPTP is a calcium-dependent and CsA-sensitive high-conductance channel. mPTP opening induces permeability of the IMM due to the opening of its pore forming proteins. The components of mPTP and the pore-forming proteins remain unresolved. Nonetheless, several studies implicate physiological roles of mPTP in Caur group  and otheur group , 55, 56 2+ from the ER to the mitochondria upon opening of the IP3-gated channels [2+ transfer between the ER and the mitochondria [2+ in the absence of Cdk5 is expected to contribute to [Ca2+]mt accumulation and subsequent mPTP opening. Nevertheless, our observations indicate that Cdk5 in MAM serves to control ER-mitochondrial tethering, and loss of Cdk5 causes an upsurge in mitochondrial calcium uptake by increasing ER-mitochondrial tethering. While studies are underway to further characterize the specific role of MAM Cdk5 in ER-mitochondria tethering, our finding that inhibition of either ER Ca2+ release with XeC or mitochondrial calcium uptake with RuR blocks the increase in mPTP opening induced by Cdk5 loss suggests that elevated mitochondrial calcium influx underlies the increase in mPTP opening in the absence of Cdk5. Altogether, our studies demonstrate a novel mechanism whereby Cdk5 controls mPTP opening by regulating the formation of ER-mitochondria contact site structures and subsequently, calcium transfer from the ER to the mitochondria and [Ca2+]mt level.MAM, a proteinaceous link between the ER and mitochondria, is important for calcium and metabolite transfer between these organelles. In this study, we show that Cdk5 is enriched in pure MAM fractions, a finding that is consistent with the mass spectrometry analysis by Poston et al., identifying, with high confidence, Cdk5 as one of the MAM proteins in mouse brain . For effchannels , 24, 58.channels , 59, andchannels , 27. Ourchondria , it is nDulbecco’s modified eagle’s medium (DMEM), heat-inactivated fetal bovine serum (FBS), EDTA-Trypsin, antibiotic-antimycotic, Calcein AM, Fluo-4 AM and Rhod-2 AM were from Life Technologies Inc. The protease inhibitor cocktail, Cyclosporine A (CsA), Ruthenium Red (RuR), ATP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and Xestospongin C (XeC) were from Sigma. Antibodies to Cdk5 (C-8), FACL-4 (F-4), VDAC1 (B-6), GRP78 (A-10), PCNA (PC-10) and LDH-A (E-9) were from Santa Cruz Biotech. Sanglifehrin A (SFA) was a gift from Novartis (Switzerland).Cdk5−/− mouse embryos following the protocol described by Durkin et al.  and 4 ml of MRB then centrifuged at 95,000 × g for 30 min at 4 °C. Pure mitochondria (MT) settles at the bottom of the tube while the MAM fraction appears as a white band above the MT fraction. The MAM and MT fractions were each resuspended in 14 ml of MRB buffer and centrifuged at 6300 × g for 10 min at 4 °C. The resulting supernatants (~14 ml) of the MAM fraction, designated as crude MAM (C-MAM), were further subjected to centrifugation at 100,000 × g for 1 hr. Pellets containing pure MAM (P-MAM) or pure MT were each resuspended in 50 µl of MRB buffer.Crude and pure MAMs, and mitochondria were isolated using Percoll density gradient centrifugation as described previously . BrieflyIsolated subcellular fractions (50 µl) were loaded in 12.5% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked in 5% skimmed milk then incubated with the indicated primary antibody (1:1000 dilution) at 4 °C overnight. After washing with Tris-buffered saline (TBS) + Tween-20 (TBST) , membranes were incubated with horseradish peroxidase-conjugated secondary antibody  for 1 h. Immunoreactive bands were detected using ECL reagent (Amersham).2 (1 mM) in Hank’s balanced salt solution (HBSS), and incubated for 15 min at 37 °C under 5% CO2. Cells were then washed with HBSS buffer and images were taken under an Olympus 1 × 71 fluorescence microscope using a ×10 objective with or without the ×1.6 magnification changer.Calcein fluorescence was measured using the Image-IT live mitochondria permeability transition pore assay kit (Thermo Fisher Sci.) as per the manufacturer’s protocol. Briefly, cells seeded on a four-chamber cover glass (Lab-Tek) were loaded with calcein AM (1 µM), MitoTracker Red (200 nM), treated with or without CoCl4) were harvested using Trypsin-EDTA and pre-incubated with CsA (1 µM), SFA (2 µM), XeC (3 µM) or RuR (3 µM) for 1 h then stained as indicated above. Cells were then washed with HBSS, resuspended in ice-cold PBS and analyzed by flow cytometry using a fluorescein isothiocyanate filter (530 nm) for measuring calcein fluorescence.For flow cytometry, cells  were loaded with Fluo-4 AM (5 µM) in HBSS  for 30 min at RT. Cells were washed three times with KRH buffer  then subjected to single cell calcium imaging using a Leica TCS SP8 confocal microscope (×20 objective). Fluorescence signals were quantified as ratio (F/F0) of the fluorescence after addition of FCCP, relative to the basal fluorescence (F0) before stimulation.For cytoplasmic Ca2+ measurements, MEF cells seeded on 3.5 cm glass bottom petri dishes (80% confluency) were loaded with Rhod-2 AM (5 µM) in KRH buffer  for 30 min at RT. Cells washed three times with KRH buffer were subjected to single cell calcium imaging using a Zeiss LSM 510 Meta laser scanning confocal microscope (×20 objective). Fluorescence signals were quantified as ratio (F/F0) of the fluorescence after addition of ATP, relative to the basal fluorescence (F0) before stimulation.For mitochondrial Cat test  was used. Significance was set at p < 0.05.Student’s


Nowadays, brewers’ spent grains (BSG) is considered the most abundant and low-cost brewing by-products, presenting a great potential as a functional food ingredient. Since BSG is rich in dietary fiber and protein, it can be a raw material of interest in bakery products. However, blending wheat flour with BSG can affect dough rheology and the structural and sensorial properties of products. In this context, BSG flour at different levels  was used to enrich three commercial soft wheat flours, and to develop new formulations for bakery products . As expected, the enrichment caused a significant increase of proteins, dietary fibers, lipids, and ash related to the BSG enrichment level. Significant changes in dough rheological properties  and in the color of the crust and crumbs of bakery products were also observed. At last, the consumer test pointed out that the 5% BSG enrichment showed the higher overall acceptability of proposed bakery products. In recent years, increasing efforts are being directed towards the reuse of agro-industrial by-products, with the goals of reduction of organic-wastes, contribution to the environmental preservation, preservation of bio-resources, production of value-added foods at low cost, production of molecules to reuse in food and pharmaceuticals or cosmetics, and promotion of the technological development. The brewing industry generates huge amounts of by-products. As matter of fact, for every 1000 tons of beer produced, 137 to 173 tons of solid waste is created in the form of spent grain, spent yeast, spent hops, and unwanted material. These by-products are widely used as additives and supplements for feed or disposed of as waste. Contrariwise, they can be a low-cost source of valuable and innovative products , and their reuse can contribute to the EU Zero Waste Policy ,3.In particular, brewers’ spent grain (BSG) resulting after the mashing and filtration stage represents 85% of total by-products . BSG maiKnowledge of the health benefits associated with increased consumption of BSG dietary fiber and natural bioactive compounds can lead to the inclusion of this active ingredient into popular and affordable foods like bakery products. Previous studies have considered the potential of BSG to increase the protein content and fiber content in biscuits, bread, snacks, and pasta ,10,11,12In light of these considerations, the aim of this work was to explore the potential of brewing-derived by-products as sources of functional ingredients. Therefore, new formulations of bakery products  enriched with brewers’ spent grain were developed considering three wheat flours with different physicochemical characteristics. The technological, nutritional and sensory properties of flours, and the consumer acceptance of the new products were assessed.The choice to investigate the effects of BSG on different bakery products  is due to the fact that these cereal-based products are widely consumed in Italy. Some brewers usually sell BSG to bakers or try to reuse them to make pizza or snacks for brewpubs customers. However, it is quite difficult to make standardized products as many factors  influence the result, and they must be carefully considered. Therefore, the general purpose of the study was to provide useful information, through various rheological tests and cooking tests, on the behavior of commercial soft wheat flours enriched with BSG at different levels that can be used to produce different bakery products. The functionality and versatility of flours and enriched flours are associated with the capacity of their storage proteins—gliadins and glutenins—to form gluten. Consequently, each wheat flour can organize its storage proteins into a viscoelastic network, leading to a different class of wheat suitable for different types of products and to deliver certain functional attributes. The technological behavior of flours is also the result of complex interactions between macromolecules that are responsible for dough performances and BSG. Therefore, the dough rheological properties are important in order to determine the dough strength properties useful for predicting bakery quality after BSG enrichment. −4 J), C2 was a medium flour (W < 260 × 10−4 J), and C3 was a strong flour (W < 350 × 10−4 J).Three representative samples  of commercial soft wheat flours , different in centesimal composition and related technological properties, were considered in this study. C1 was a weak flour  < 170 × 10Fresh barley brewers’ spent grain (BSG) was obtained by a craft brewery in Latium, Italy, and about eight hours after the production was oven-dried at 105 °C for 78 h until the moisture content was less than 10%. Then, the dried BSG was ground by a Kenwood milling machine  and sieved to obtain a fine flour with particle sizes from 400 to 600 μm. Flours, fresh compressed yeast, salt, and extra virgin olive oil were bought from a local market.Moisture, proteins, lipids and ashes were determined by the ICC standard methods 110/1, 105/2, 136, 104/1 respectively  using a The dough strength (W), dough tenacity (P), dough extensibility (L), and configuration ratio (P/L) of flours was determined by means of a Chopin Alveograph (Method 54-30 A) .Water absorption (WA) at 14% moisture content, dough stability (DS), and development time (DT) were obtained with a Brabender farinograph, according to Standard Method No. 54-21 .® device, recently proposed by Brabender® GmbH and Co. , as described by Amoriello and Carcea [The gluten viscoelastic behavior of samples was also investigated by means of the GlutoPeakd Carcea . The torBread loaves were produced from 100% wheat flour (control) and from two wheat–BSG flour blends (95–5% and 90–10% composites). The dough was made with 1000 g flour , water (the optimum quantity was determined by means of the Farinograph), 20 g of compressed baker’s yeast, and salt (2% on flour weight). The ingredients were mixed for 10 min in a planetary bread mixer . Once the dough was formed, it was fermented for 30 min in a fermentation cabinet at 30 °C with 85% relative humidity. Then, the dough was scaled into four equal pieces, which were placed in baking tins and proofed for 50 min at 30 °C with 85% relative humidity. At the end, they were baked for 30 min at 220 °C in a convection/steam oven. Baking tests were performed on each blend by using four replicates.Breadsticks were produced from 100% wheat flour (control) and from two wheat–BSG flour blends (95–5% and 90–10% composites). Dough samples were made by mixing 1000 g flour and water as per Farinograph readings, salt at 2% on flour weight, 30 g of compressed baker’s yeast, 70 g extra virgin olive oil, and 10 g sugar. After mixing, the dough was allowed to ferment for 50 min at 30 °C with 80% relative humidity in a fermentation cabinet. Then, the breadsticks were formed into shape (15 cm length and 1 cm width), proofed for a further 15 min, and baked for 35 min at 210 °C in a convection/steam oven. Pizza doughs were produced from 100% wheat flour (control) and from two wheat–BSG flour blends (95–5% and 90–10% composites). 1000 g flour , with optimum water quantity as determined by means of the Farinograph, 20 g of compressed baker’s yeast (2% on flour weight), 20 g of salt (2% on flour weight), and 40 g of extra virgin olive oil (4% on flour weight) were mixed in a planetary machine for four minutes. After mixing, the dough was divided into parts to form dough balls, each weighing 250 g. The balls were allowed to ferment for 3 h at 30 °C with 80% relative humidity in a fermentation cabinet, up to double their initial volume. The balls were then spread out to a disk of about 20 cm diameter, pressed to a thickness of 7 mm, left to rest for another 30 min, and baked for 4 min at 300 °C in a convection/steam oven.3 g−1. The results are the average of four samples and were measured after 24 h. The volume of bread loaf was determined using method 10–05.01 , based oL*a*b* scale. The tests took place on day 1, and the results are the average of measurements of four different points per sample.Color measurements were taken on the crust for breadsticks and pizza, and on crumb and crust for bread, using a Chroma Meter CR-200  and (CIE) In order to evaluate the sensory perception in BSG-enriched bread, a consumer test was conducted with forty consumer volunteers. Their age ranged from 16 to 59 years. About 2-cm thickness slices of bread, two breadsticks, and a piece of pizza for each type of sample were served on white plastic plates. After each tasting, water was used for rinsing. Questions about some aspects of the products were asked to the consumers. The sensory characteristics were assessed for taste, appearance, aroma, texture, color, and overall acceptability. The intensity of each attribute was rated on a 7-point Hedonic scale , according to Moskowitz . The scoAll tests were replicated three times, and mean values and standard deviations were calculated. A one-way analysis of variance (ANOVA) employing the Kruskal-Wallis non-parametric test at a significance level of 5% was carried out to determine significant differences in all measured properties. Data were processed using SPSS statistical software .® devices can provide practical information for interpreting the behavior of dough processing and quality.Characterization of rheological properties of dough is effective in predicting the processing behavior. Farinograph, alveograph, and GlutoPeakThe main results for quality characteristics and rheological properties of three commercial flours, BSG, and their mix at different ratios are reported in The moisture content ranged between 13.0 ± 0.2% to 14.0 ± 0.2% for wheat flours  and 8.8 ± 0.2% for BSG. As expected, a significant decrease (about 4%) of moisture content was observed in all flour samples when 10% of BSG was added. Protein content varied between 10.1 ± 0.1 to 12.7 ± 0.4 g/100 g for wheat flours , while it was 16.4 ± 0.2 g/100 g for BSG. A slight increase of protein content was already found at low BSG addition (5%) in all enriched flours samples, although it was not statistically significant for C2 and C3. The ash content was similar ; it was significantly higher for BSG (4.16 ± 0.01 g/100 g). The BSG enrichment at 5 and 10% levels caused a significant ash content increase . The total fat content and total dietary fiber of BSG were 8.2 ± 0.2 g/100 g and 14.6 ± 0.2 g/100 g, respectively, whereas a significantly lower content of these two nutritional parameters was found in wheat flours. Fat, TDF, and protein content significantly increased as the BSG content increased for each blend and wheat flour, as reported by Ktenioudaki et al.  and Stoj−4 J for C1 and 267 ± 10 × 10−4 J for C3; the dough tenacity (P) between 45 ± 6 mm for C1 and 75 ± 5 mm for C2; the dough extensibility (L) between 86 ± 7 mm for C2 and 142 ± 9 mm for C1; and the configuration ratio (P/L) between 0.32 ± 0.03 for C1 and 0.87 ± 0.03 for C2. The addition of BSG caused a significant decrease in the alveographic dough tenacity for the three samples, especially for C3, for which the addition of 10 g/100 g of BSG flour reduced the value by more than 26%. Similar behavior was observed for dough extensibility, although no significant differences were observed between the control and the blend with 5% BSG, except for C1 blends  ranged between 158 ± 16 × 101 blends A. The glRegarding the farinograph parameters of wheat flours, the water absorption values (WA) were distributed between 54.0 ± 0.2% for C2 and 58.7 ± 0.1% for C3; the dough stability (DS) between 2.0 ± 0.6 and 20.00 ± 0.09 min ; and the development time (DT) between 17.3 ± 0.2 and 18.8 ± 0.4 min for C1 and C3, respectively. The Farinograph data demonstrated variation in dough mixing characteristics, but, in general, all the flours behaved quite similarly. The incorporation of BSG increased water absorption and reduced dough development time for all samples. Dough development time and dough stability sharply (except for C3) decreased up to 10% BSG enrichment, indicating dough weakening. However, dough stability showed different behavior among all blends. In fact, DS decreased with fibred addition for C1 and C2, while it increased for C3. These results reflect the discordant trends found in previous studies . The hig® parameters, only those highly correlated with the common technological indexes were chosen for analysis, as showed by Amoriello et al. [® parameters can help to classify different dough viscoelastic traits: high-quality flours generally show short peak maximum time (PMT) and high maximum torque (BEM), whereas weaker flours showed long PMT value and low BEM values. In fact, flours with high protein content and high dough strength form the gluten network in a shorter time than samples with poor quality and need higher energies for gluten aggregation [Of all the GlutoPeako et al. , in orderegation . Our resregation . In generegation .At the same time, the BSG addition, especially at a higher level, caused an increase in the maximum torque (BEM) corresponding to the peak occurring due to gluten aggregation. In fact, the BEM without or with BSG ranged from 60 ± 3 to 71 ± 4 BU for C1, from 46 ± 3 to 58 ± 2 BU for C2, and from 68 ± 3 to 81 ± 3 BU for C3, respectively.The quality characteristics of the bakery products were significantly affected by the BSG addition . Dhingrap < 0.05) as BSG flour increased for C2 and C3 (flours with the highest protein content), whereas no significant differences were observed between C1 control and C1 composites . The decreased specific volume observed in the enriched C2 and C3 flours can be attributed to a reduction of the extensibility and weakening of the gluten network due to dilution and disruption effects on wheat proteins, water holding, and interaction with fibers and non-gluten proteins, which also diminish the gas-retention ability ,27. RegaThe color properties  of bakery products with BSG addition were reported in p < 0.05) in color intensity. The behavior of the three types of flour was similar for all products. As the BSG level increased, the lightness parameter (L*) of pizza crust, breadstick crust, and bread crumbs strongly decreased. On the contrary, a decrease of lightness in the bread crust was not shown with BSG addition, probably due to the cooking treatment that enhances the Maillard reaction on the bread surface, hiding the BSG effect. Redness (a*) was strongly affected by BSG in all bakery products, highlighting significant differences in the a* values of bread crust, pizza crust, bread crumbs, and breadstick crust proportional to the BSG flour. Regarding the yellowness, a low decrease was reported only for the crust and bread crumbs. At last, adding BSG resulted in a low decrease in yellowness only for the crust, more accentuated for the bread crumbs. The higher amount of amino acids in the starch mixture, due to BSG addition, could have favored the Maillard reaction, leading to a decrease of lightness and an increase of redness. Our results are in accordance with previous studies [BSG incorporation caused a significant change  in aroma intensity, crust color, crumb color, saltiness and bitterness, especially for bread with 10% BSG flour (The BSG addition in bread caused a lower consumer acceptability and significant differences (SG flour . In partp < 0.05) on C1 for almost all the parameters  as the BSG content increases, compared to the C1 and C2 samples  in appearance, aroma intensity, crust color, toughness, elasticity (not for C3), sweetness, and bitterness . From a technological point of view, as the flour quality increases, the addition of BSG in the blends caused a decrease in the dough strength, tenacity, extensibility, and development time, while an increase of water absorption was observed. Furthermore, the sensory analysis, although conducted on a small sample of consumers, suggested that the best enrichment with brewers’ spent grain was 5 g/100 g BSG level. Our results can help to better understand the effects on the technological and sensorial performance of the BSG addition on flours of different quality. They can also help guide the choice of flour according to the bakery product  to be obtained.