Nephrotic syndrome is the most common kidney disease in children worldwide. Our aim was to critically appraise the quality of recent Clinical Practice Guidelines (CPGs) for idiopathic steroid-sensitive nephrotic syndrome (SSNS) in children in addition to summarize and compare their recommendations.Systematic review of CPGs. We identified clinical questions and eligibility criteria and searched and screened for CPGs using bibliographic and CPG databases. Each included CPG was assessed by four independent appraisers using the Appraisal of Guidelines for REsearch & Evaluation II (AGREE-II) instrument. We summarized the recommendations in a comparison practical table.Our search retrieved 282 citations, of which three CPGs were eligible and appraised: Kidney Disease: Improving Global Outcomes (KDIGO) 2012, Japan Society for Pediatric Nephrology (JSPN) 2014, and American Academy of Pediatrics (AAP) 2009. Among these, the overall assessment of two evidence-based CPGs scored > 70% (KDIGO and JSPN), which was consistent with their higher scores in the six domains of the AGREE II Instrument. In domain 3 (rigor of development), KDIGO, JSPN, and AAP scored 84%, 74%, and 41%, respectively. In domain 5 (applicability), they scored 22%, 16%, and 19%, respectively, and in domain 6 , they scored 94%, 65%, and 88%, respectively.The methodological quality of the KDIGO CPG was superior, followed by JSPN and AAP CPGs with the relevant recommendations for use in practice.CRD42020197511.The protocol was registered in the Center for Open Science (OSF) DOI: 10.17605/OSF.IO/6QTMD and in the International prospective register of systematic reviews PROSPERO 2020 The online version contains supplementary material available at 10.1186/s13643-021-01666-w. Two external international experts in nephrology were invited as international collaborators to contribute to this CPG project.We systematically searched MEDLINE and EMBASE databases for relevant guidelines using the Ovid platform and hand-searched EBSCO DynaMed Plus (USA), ECRI Guidelines Trust, Guidelines International Network, International Guideline Library, National Institute for Health and Care Excellence (UK), Scottish Intercollegiate Guidelines Network (UK), and Australian National Health and Medical Research Council . Moreover, we searched databases of national and international societies specializing in fields related to our health topic of SSNS, including the Japanese Society of Pediatric Nephrology (JSPN), KDIGO, International Society of Nephrology, American Society of Pediatric Nephrology, National Kidney Foundation, American Academy of Pediatrics (AAP), and Scottish Paediatric Renal and Urology Network. The search terms used included combinations of subject headings and keywords with various synonyms for idiopathic SSNS, nephrotic syndrome, nephrology, pediatrics, pediatric medicine, child health, treatment, management, pharmacology, practice guidelines, CPGs, healthcare quality, patient safety, evidence-based medicine, AGREE II instrument, quality assessment, critical appraisal, and evidence-based pediatrics  evidence-based with a clear record of their development methods; (2) English or Arabic language; (3) original source CPGs (de novo development); (4) national or international scope; and (5) published by an organization or group authorship and accessible from a CPG database or peer-reviewed journal. Only the most current version of each source CPG was appraised.The exclusion criteria were CPGs that were published earlier than 2009, not in the English or Arabic language, adapted from other CPGs, presented as consensus or expert-based statements, or had a single author.The two CPG methodologists (YA and RA) conducted a capacity building workshop for the review team through hands-on sessions in the concepts of evidence-based medicine and evidence-based CPG standards using the AGREE II instrument tool in 2 days. During the workshop, participants refined the research questions of interest to adapt a CPG to local practice . Afterward, each reviewer scored his/her assigned CPGs. All four reviewers critically appraised each CPG. All appraisers reviewed the full CPG documents, including any updates with any relevant supplementary information or links to online web pages related to the CPG methods or CPG implementation tools. For each item, AGREE appraisers were instructed to record the justifications for their scores in the “Comment” section .www.agreetrust.org) consisted of 23 items organized into six domains: scope and purpose, stakeholder involvement, rigor of development, clarity of presentation, applicability, and editorial independence [The AGREE II instrument  were resolved by discussion with the appraisal group. The online My AGREE PLUS automatically calculated the standardized AGREE domain scores or ratings (%). We agreed upon a cutoff point of 70% for each AGREE standardized domain score or rating. After the appraisal, more weight was emphasized on the scores of domains 3 and 5 to facilitate the filtration and final evaluation of the reporting quality of included CPGs. Similar cutoff values were reported –23. In aFor each AGREE II domain, we calculated standardized scores ranging from 0 to 100% using the methods suggested by the AGREE II instrument. The key recommendations of the eligible CPGs were summarized in a comparative tabular format. The quality of CPGs was classified based upon the rating of domain 3 (rigour of development) where a high-quality CPG will receive a standardized domain rating of more than or equal to 70%, moderate quality CPG (40–69%), and low quality (less than 40%).We retrieved a total of 282 records. After screening titles and abstracts, eight were included for full-text assessment, and only three were eligible for the review as illustrated in the PRISMA  flowcharTable The AGREE II standardized domain ratings are summarized in Table The AGREE II standardized score for domain 1 ranged from 65 to 100%. The scores of two CPGs were > 70% in domain 1 (KDIGO = 100% and AAP = 75%).The AGREE II standardized domain scores for domain 2 ranged from 60 to 86%. The score of a single CPG was > 70% in domain 2 (JSPN = 86%).The AGREE II standardized scores for domain 3 ranged from 41 to 84%. The score of two CPGs were > 70% in domain 3 (KDIGO = 84% and JSPN = 74%). They both reported utilizing the Grading of Recommendations, Assessment, Development and Evaluations method. Moreover, the KDIGO CPG reported its adherence to two sets of CPG standards, namely the Conference on Guideline Standardization Checklist for Reporting CPGs and the Institute of Medicine Standards for Systematic Reviews and Guidelines.The AGREE II standardized scores for domain 4 ranged from 78 to 100%. The scores of all three CPGs were > 70% in domain 4 .The AGREE II standardized scores for domain 5 ranged from 16 to 22%. None of the included CPGs scored > 70%.The AGREE II standardized scores for domain 6 ranged from 65 to 94%. The scores of two CPGs were > 70% in domain 6 .The AGREE II standardized domain scores for the first overall assessment ranged from 58 to 75%. Two CPGs scored > 70% (KDIGO and JSPN), which was consistent with the high scores in the six AGREE II domains.The second  assessment  revealed a consensus between the reviewers on recommending the use of two CPGs.n = 12), among them were six Cochrane SRs [n = 5) including four Cochrane SRs [n = 4) with one Cochrane SR [All included CPGs cited SRs in their reference list. The largest number of SR citations was observed in the JPNS CPG  tool, which addresses the clinical credibility of the CPG recommendations . The selOther limitations, apart from those imposed by the AGREE II, include the following: (i) only English or Arabic language CPGs may have resulted in the exclusion of relevant CPGs intended for use in non-English-speaking and non-Arabic healthcare settings; (ii) this review mainly focused on CPGs for management of SSNS in children and did not evaluate other subcategories of nephrotic syndrome in children as it was out of the scope of this study.The adaptation of CPGs has been identified as a valid alternative to de novo development, which is a resource-extensive process . EvidencThis critical appraisal highlights the importance of quality assessment of CPGs by clinicians to ensure the transparency and strength of the CPG development process according to international CPG standards and support the best practice for children with SNSS. We recommend incorporating the AGREE II appraisal of CPGs in the capacity building of pediatricians and nephrologists.The methodological quality of the KDIGO CPG was superior, followed by JSPN and AAP CPGs. Recommendations including the case definition, genetic testing, diet therapy, treatment of an initial episode of SSNS with steroids, treatment of relapsing SSNS with steroids, steroid therapy in FRNS and SDNS in children, treatment of FRNS and SDNS with steroid-sparing agents, renal biopsy, and vaccination in children with SSNS.Additional file 1. Search strategy.Additional file 2. PICAR statement.Additional file 3. PRISMA checklist.Additional file 4. Evidence-Base Classifications.


The flow stress characteristics of the alloy were analyzed according to the true stress–strain curve. The constitutive equation was established to describe the change of deformation temperature and flow stress with strain rate. The thermal deformation activation energy Q was equal to 551.7 kJ/mol. The constitutive equation was −1 and temperature range of 920–980 °C, and the deformation mechanism was dynamic recovery. In the β region deformation, the power dissipation rate reached 48% in the range of 0.01–0.1 s−1 and temperature range of 1010–1040 °C, and the deformation mechanism involved dynamic recovery and dynamic recrystallization.The hot deformation behaviors of a new Ti-6Al-2Nb-2Zr-0.4B titanium alloy in the strain rate range 0.01–10.0 s Titanium alloys are important structural materials in ship and aerospace due to high-temperature resistance, the high specific strength, weldability, corrosion resistance, and other excellent characteristics ,2,3,4. IIn this study, the deformation behavior of Ti-6Al-2Nb-2Zr-0.4B alloy was studied by hot compressive test. The influences of strain rate and heating temperature on flow stress were also evaluated. The Arrhenius type thermal deformation constitutive equation was accomplished through the experimental data. The energy dissipation and plastic instability diagrams of the alloy were obtained, and the thermal working diagram was obtained by superposing the two diagrams. The deformation law of the alloy at high temperatures was analyzed and the suitable processing technology was obtained, providing a theoretical basis for the hot processing technology formulation.β) was approximately 1002 °C. Additionally, ultimate tensile strength Rm and the yield strength Rp0.2 were 838 MPa and 745 MPa, respectively. The Ti-6Al-2Nb-2Zr-0.4B alloy studied in this paper was taken from a 60 mm diameter bar, which was hot-forged in the α + β range, and then a stress relief annealing was performed. The chemical composition indicated in The compression specimens with a diameter of 8 mm and a height of 12 mm were prepared by using Ti-6Al-2Nb-2Zr-0.4B alloy bars, as shown in −1) using the Gleeble-3800 thermal simulator, and the compression ratio was 60%. The specimen was heated by electric frequency induction at a rate of 3 °C/s. The deformation temperature was held at the given temperature for 3 min.The compression tests were carried out at different temperatures  and four different strain rates . Bulging was observed in the specimen after deformation; thus, the specimen is close to a unidirectional stress state.During the compression process, a thermocouple welded in the middle of the sample was used to measure the temperature in real time. The temperature control accuracy is ±1.0 °C. The experimental data was collected and calculated using a testing machine equipped with an automatic microcomputer processing system, and finally outputs containing the load-stroke and flow stress–strain data were obtained in the form of a table. The specimen morphologies before and after deformation are shown in L is the displacement, L0 is the sample initial length, F is the load, and S0 is the sample sectional area.Assuming that the volume of the Ti-6Al-2Nb-2Zr-0.4B alloy remains constant during compression deformation, the load and displacement data were obtained using a computer. The true stress–strain curve of specimens during compression deformation can be obtained through Equations (1) and (2).β phase with 12 slip systems increased, the content of α phase with 3 slip systems decreased with an increase in temperature, and the slip system increased. The greater the possibility of metal slip, the lower the flow stress. Additionally, the steady–state flow characteristics of the alloy appeared under all deformation conditions during compression deformation, that is, when the true compression strain exceeded a constant value under certain strain rate conditions and deformation temperatures, the change of flow stress decreased and tended to a stable value by increasing the strain variable. At this point, the hardening and softening mechanisms reached dynamic equilibrium.−1 and various deformation temperatures. At the same degree of deformation, the flow stress was strongly dependent on the temperature and it decreased by increasing the deformation temperature, but it was not a simple linear relationship. For example, the deformation resistance increased sharply under 940 °C. This is mainly because the atomic thermal vibration amplitude increased by increasing the temperature, the binding force between the atoms decreased, and the critical shear stress that causes slippage in the crystal decreased. Thus, the obstacles to the dislocation movement of materials and the sliding between crystalline surfaces were reduced. Simultaneously, the softening mechanisms easily occur by increasing the temperature, such as dynamic recovery and dynamic recrystallization, which reduces the dislocation density and counteracts the work hardening effect, reducing the flow stress of the alloy [he alloy .To accurately reflect the relationship between strain rate, deformation temperature, and flow stress in the process of thermal deformation, the Arrhenius type constitutive equation of Ti-6Al-2Nb-2Zr-0.4B alloy was determined in this paper. The equation can be expressed in three different manners .At low-stress levels:At high-stress levels:T is the absolute temperature (K); Q is the deformation activation energy (J/mol); R is the universal gas constant (8.314 J/mol·K); A1, A2, and A are the material constants; n and n1 are the stress exponents. Moreover, Using hyperbolic sine functions for all stress levels: = n2/n1 ,17,18.T is a constant, it can be obtained from Equations (3) and (4) as follow: When 1 value was 6.95, the The relation diagrams of The material needs to overcome a specific potential barrier to complete the thermal deformation process. The potential barrier is the activation energy during the hot deformation, and its magnitude reflects the difficulty of dislocation initiation, recovery, and recrystallization during the hot deformation of the alloy.Q for deformation can be obtained as:From Equation (5), the activation energy T were approximately linear, indicating that the hyperbolic sinusoidal Arrhenius equation satisfactorily describes the thermal deformation behavior of Ti-6Al-2Nb-2Zr-0.4B alloy. The slope of the fitting line in Q of 551,706 J/mol (the standard deviation is 45.2 J/mol), which is much larger than the activation energy of pure α titanium (204 kJ/mol) and pure β titanium (161 kJ/mol) [The  kJ/mol) . The acttitanium 4 kJ/mol  kJ/mol) ,22. From kJ/mol) ,24.T in high-temperature experiments, which can be expressed using the parameter Z [Zener and Hollomon proposed a “temperature compensated strain rate” to verify the relationship between strain rate and deformation temperature ameter Z ,26:(9)Z Z can be obtained as:From Equation (9), the ln Z was determined, as illustrated in A3 was e54.41 s−1 and n equalled 2.35. The uncertainties of n and ln A3 can be seen in the table in −1 was obtained:The relation graph of J and the energy dissipated by plastic deformation is represented by G. Among them, the process of plastic instability and fracture is related to G, while the microstructure evolution is related to J [P absorbed by the fragments in the process of plastic flow can be expressed as [−1) and J and G is given by [J and plastic deformation G, respectively.According to the dynamic material model, the fragment subjected to deformation is a nonlinear energy dissipation unit. The energy input by external forces to the fragments contributes mainly two aspects: (1) plastic deformation, most of this is converted to heat and a small amount is stored in materials and (2) microstructure evolution, as recovery, recrystallization, and phase transition. The energy dissipated by microstructure evolution is denoted by ted to J . The eneessed as ,28:(12)Pgiven by ,30:(13)mJ can be given by the following equation [−1) and σ is flow stress (MPa).Under certain deformation strain and temperature, equation :(14)J=mmm = 1. The nonlinear dissipation element and ideal linear dissipation element are normalized to produce a dimensionless parameter, which is expressed as [For an ideal linear dissipative unit, essed as :(15)η =Jη reflects the mechanism of microstructure change within a specific range of strain rate and temperature. The variation of η with temperature and strain rate generates the power dissipation diagram of different regions. For material processing, dynamic recrystallization, dynamic recovery, and superplasticity are safe thermal deformation mechanisms, while the formation of wedge-shaped cracks and voids is dangerous and should be avoided. Therefore, the power dissipation rate η plays a very important role in selecting the best processing parameters in hot processing.The parameter The criterion of material rheological instability was proposed by Kumar based on Zeigler’s principle of maximum entropy production rate :(16)ξ(ε˙When Equation (16) is satisfied, an unstable rheological process occurs. The parameter m parameter, the η rate due to the microstructure change, and the ξ and η, which are used as functions of the strain rate and deformation temperature, respectively. The final thermal processing maps are generated by the superposition of the instability maps and power dissipation maps [According to the above principles and methods, the ion maps .When the true strain ε is 0.5, the relationship between The curve between log in logε˙ :(17)logσm at the corresponding points can be determined by substituting the values of η rate of the alloy at different strain rates and deformation temperatures is get by substituting the values of m in Equation (15). The power dissipation diagram under the whole experimental condition can be obtained by the interpolation method, as shown in The values of m for various conditions in Equation (19) and the alloy instability map can be obtained using the interpolation method [The values of n method , as showη rate value and the shaded parts represent the instability areas in η rate value is larger. In (α + β) two-phase region, the power dissipation rate reaches 53% in the range of 0.01–0.05 s−1 and temperature range of 920–980 °C, and the deformation mechanism is dynamic recovery. In β single-phase region, the power dissipation rate reaches 48% in the range of 0.01–0.1 s−1 and temperature range of 1010–1040 °C, and the deformation mechanism is also dynamic recovery. The results show that these two regions are the best deformation parameters of Ti-6Al-2Nb-2Zr-0.4B alloy under laboratory conditions. The instability zone is mainly in the temperature range 890–940 °C and strain rate range 0.1–10.0 s−1. When the deformation temperature is below 940 °C, the deformation resistance is high and it is not suitable for low-temperature forging. When the strain rate is equal or greater than 0.1 s−1, it is easy to crack due to the B element influence and it is not suitable for high strain rate forging, which is consistent with the forging experimental results.The processing maps of the alloy at ε = 0.5 can be obtained by superposing the contour maps of The true stress–strain curves were similar under all conditions. The flow stress reached the peak fast (hardening) by increasing the strain followed by a gradual decrease (softening), exhibiting a typical dynamic recrystallization curve. When the true compressive strain exceeded a certain value, the change in flow stress decreased and tended to a stable value by increasing the strain variable.The thermal deformation activation energy Q was 551.7 kJ/mol, and the constitutive equation was −1 and temperature range of 920–980 °C at the (α + β) two-phase region, and the energy dissipation rate was 53%. The other zone was in the range of 0.01–0.1 s−1 and temperature range of 1010–1040 °C at the β single-phase region, and the energy dissipation rate was 48%.The suitable hot-working zone of the alloy was in the range of 0.01–0.05 s


The goal of this study is to evaluate the primary stability of a cementless augment-and-modular-cage system with and without the addition of cranial straps in a standardized in vitro setting. As the surrogate parameter for the evaluation of primary stability, the measurement of relative motion between the implant components themselves and the bone will be used. Acetabular revision components with a trabecular titanium augment in combination with a large fourth-generation composite left hemipelvis were assembled. These constructs were divided into two groups with (S) and without cranial straps (nS). A total of 1000 cycles was applied at each of three load levels. Relative movements (RM) between the components were measured. Load levels display a significant effect on the amount of RM at all interfaces except between shell/augment. The group assignment appears to have an effect on RM due to significantly differing means at all interfaces. Between bone/shell RM increased as load increased. NS displayed significantly more RM than S. Between shell/augment RM remained constant as load increased. Between shell/cup S showed more RM than nS while both groups’ RM increased with load. We conclude a significant increase of primary stability between the shell and the bone through the addition of cranial straps. Relative motion between components (shell/cup) increases through the addition of cranial straps. A clinical impact of this finding is uncertain and requires further investigation. Finally, the cementless fixation of the augment against the rim-portion of the shell appears stable and compares favorably to prior investigation of different fixation techniques. The number of primary hip arthroplasties is rising all over the developed world . Due to While structural bulk allografts for the weight-bearing area of the acetabular rim present limited integration and consecutive resorption, leading to failure of fixation in the long term, modern macro-porous metal augments present a promising alternative ,5,6,7. TTherefore, the goal of this study is to evaluate the primary stability of two different designs of a cementless augment-and-modular-cage system with and without the addition of cranial straps in a standardized in vitro setting. As the surrogate parameter for the evaluation of primary stability, the measurement of relative motion between the implant components at all interfaces and the bone (composite hemipelvis model) will be used. The Results will be compared to current literature.® Comfort, Peter Brehm GmbH, Weisendorf, Germany) in combination with a large fourth-generation composite left hemipelvis . Of these constructs (implant and hemipelvis), 12 were prepared in the manner listed below and consecutively divided into two groups  and 6 without cranial straps (nS)). Sample size estimation was carried out using G*Power 3.1 Software , [d = 2.5 and α = 0.05, with power (1-β) = 0.8; we based the chosen values on the well-standardized methods of this experiment and laboratory experience, as well as similar literature [We constructed an experimental setup utilizing an acetabular revision component with a trabecular titanium augment (MRS-Titanermany),  using a terature ,15.In order to comply with a frequent clinical setting in cases of acetabular revision arthroplasty, the common morphology of an ADC IIa/Paprosky IIb defect was chosen ,16. The The cementless augment-and-modular-cage construct with cranial straps (S) and without cranial straps (nS) was implanted in a standardized fashion by an experienced orthopedic surgeon in accordance with the manufacturer’s instructions and the preceding digital planning. All shells were size 56 mm left combined with a 14 mm A-Augment and either no cranial straps or two cranial straps (45 mm) with 2 screws holes each extending over the superior aspect of the acetabulum to the iliac bone. The A-Augment was pre-operatively modified by the addition of a metal rod which would later carry marker points during the biomechanical analysis. This step was necessary due to the coverage of the augment by the cage. Therefore, in order to enable a valid measurement of relative motion, the metal rod with marker points was used as an extension of the augment. To compare to a clinical setting, a press-fit implantation prior to the insertion of screws was attempted and achieved in all cases. The press-fit of the implant in combination with an uncontained acetabular defect, however it does not offer the same stability as an undisturbed acetabular rim would.The porous metal augment was attached to the titanium shell and fixated through a singular screw (6 × 12 mm) with 8 Nm torque prior to implantation. After initial press-fit of the cementless augment-and-modular-cage construct, the following screws were added depending on the assigned group (S/nS): S: cranial strap with 4x flathead spongiosa screw 6 × 25 mm, 6 × 30 mm, 6 × 30 mm, 6 × 40 mm; rim with 1 flathead spongiosa screw 6 × 40 mm; dome with 3 flathead spongiosa screw 6 × 25 mm, 6 × 50 mm, 6 × 60 mm; nS: rim with 1 flathead spongiosa screw 6 × 25 mm; dome with 3 screw 6 × 25 mm, 6 × 50 mm, 6 × 60 mm.Finally, in preparation for the biomechanical analysis, all hemipelvis models with the implanted acetabular component were secured by submerging the posterior ilium into a containment device filled with a liquid two-component casting resin block. In a separate step, the symphysis was also secured into a two-component casting resin block with the addition of a stainless-steel ball on the undersurface. In the biomechanical analysis, this ball is in contact with a metal plate only fixating the symphysis in one plane and therefore allowing multiplanar movement and rotation in order to mimic a more physiological fixation ,18.To enable discrimination and recording of relative movement between the components during loading, the implant-hemipelvis-construct was outfitted with optical markers  along the surface of each component planned to be measured. While markers at the implant shell and composite bone were easily attachable, the augment was mostly covered by the rim and cranial straps. Therefore, the implant manufacturer assembled a specialized augment containing a metal rod as an extension in order to attach a satisfactory number of optical markers .To record relative movement between the components, an optical measuring system  applying 3D point triangulation detected the optical marker positions in the defined coordinate system. The 3D micromovements in x-, y-, and z-axes were consecutively measured simultaneously between the different interfaces . The dependent variable was the relative movement between the components (measured in μm). The recorded maximal movements of the component augment, shell, bone and cup were calculated using the formula The biomechanical simulation was carried out by using a material testing machine . The implant-hemipelvis-construct set into the material testing machine is displayed in The vector of the load application coincided with the direction of the greatest load occurring during normal gait ,20. TermAccording to a publication by Bergmann et al., the maximum load during normal walking relates with 233% to the individual’s body weight at 31° of rotation around the x-axis and 5° around the z-axis relative to the described acetabular component system .The experimental body weight was set at 80 kg for each testing (1.8 kN at 100% load). To warrant a proper force closure between the force plate and the implant-hemipelvis-construct, a constant force of 0.2 kN was applied prior to testing. A total of 1000 cycles were applied in a sinusoidal waveform at 1 Hz at each of three load levels: 3% to 30% load ; then 5% to 50% load ; and, finally, at 10% to 100% load (1.8 kN peak load). Relative movements between the components were measured at cycles 1 to 50, 51 to 200, 201 to 500, 501 to 800, and 801 to 995 (average and variance).The statistical evaluation was performed using SPSS Statistics Version 22 . To test for primary stability, the surrogate parameter of relative movement between the components during different loads (measured in µm) was applied. For each interface a separate analysis was carried out . Initial descriptive statistical analysis was applied utilizing the arithmetic mean, range and standard deviation. Normal distribution was assessed through the Kolmogorov–Smirnov and Shapiro–Wilk Test.t-test was applied to test for differences between component design (S/nS) according to load when homogeneity of variance could be established. In cases where homogeneity of variance could not be shown, Welch Test was utilized.A repeated-measures analysis of variance (ANOVA) was calculated for the transformed relative movement . To validate the ANOVA Mauchly’s sphericity test was used. In cases where sphericity could not be verified the Greenhouse–Geisser correction was applied. As covariates we defined the application of cranial straps , force , and their interaction. The unpaired p-value was set at ≤0.05. Hemipelvis ID was chosen as a random intercept, and post hoc comparisons between S and nS, and S/nS were performed using differences of ‘least-squares’ means . The levt-test/Welch Test. Means of maximal relative movement between the different components with and without cranial straps (nS/s) is displayed in p < 0.001), bone and augment  = 89.52, p < 0.001), and shell and cup  = 65.58, p < 0.001). In the interface between shell and augment, no significant effect of load levels on the amount of relative motion could be assessed  = 3.52, p = 0.054).Load levels display a significant effect on the amount of relative motion between bone and shell  = 233,585, p < 0.001), bone and augment  = 279.101, p < 0.001), shell and cup  = 56.686, p < 0.001), and shell and augment  = 36.357, p < 0.001).The group assignment (S/nS) appears to have an effect on relative motion due to significantly differing means in the comparison of bone and shell  = 219.333, p < 0.001; 50%: t(8) = 7.18, p < 0.001; 100%: t = 18.61, p < 0.001).At the interface between bone and shell, relative movement increased as load level increased for the nS-group while remaining constant throughout for S-group . The nS-p < 0.001; 50%: t(8) = −10.87, p < 0.001; 100%: t(8) = −4.65, p < 0.003).At the interface between bone and augment, relative movement increased as load level increased for the nS-group while remaining constant throughout for S-group . The S- p = 0.065; 50%: t(8) = −3.56, p < 0.008; 100%: t(8) = −17.32, p < 0.001).At the interface between shell and cup, relative movement increased as load level increased for both groups. The S-group started with a higher amount of relative movement when compared to the nS-group. Relative movement was significantly different between groups for the 50% and 100% load level, while the difference at the 30% load level was not significant (30%: t(8) = −2.14, p = 0.003; 50%: t(8) = −6.11, p < 0.001; 100%: t(8) = −3.51, p < 0.009).At the interface between shell and augment, relative movement remained constant as load level increased for both groups . The S-gThe aim of this publication is to assess the impact of the addition of cranial straps to a cementless augment-and-modular-cage construct on the amount of relative motion between the different components within the system and between implant and bone. Relative movement is an important in vitro measurement in order to assess primary stability and consecutively determine the potential of osseointegration. The conducted in vitro testing brought many significant results to light. In order to properly interpret these findings, the measurement of relative movement needs to be related to a clinical setting. Prior in-vivo animal and human autopsy studies established cut-off values indicating that successful osseointegration occurs at ~40 µm, while relative movement of 150 µm appears to result only in fibrous attachment ,24. AlthBesides, proper osseointegration relative movement also affects the implant system as a whole. One of the most relevant reasons for failure of an implant is particle debris with consecutive osteolysis and loosening. Increased relative movement has been associated with increased wear of the implant surfaces resulting in particle debris ,26. Due While the usefulness of porous metal augments in cases of severe acetabular bone defects has been established, there remains controversy around the best fixation method between augment and shell or cup. In our findings the form-fitting fixation of the augment against the rim-portion of the shell through a single screw proved stable with a constant amount of relative movement across all load levels for both groups. Even though there was a significant difference between relative movements at all load levels to the disadvantage of the S-group, the small difference is not estimated to present a relevant clinical consequence (nS: 9.52 ± 0.46 µm vs S: 10.83 ± 0.70 µm at 100% load) . The groThe initial design of straps or flanges surfaced in reconstruction rings/cages which would bridge an acetabular bone defect in order for the added bone graft below to be protected until it would eventually consolidate. Due to vast improvements in implant design, modern revision systems offer a variety of different modes of fixation such as cranial or dorsal straps/flanges, iliac pegs, caudal hooks, and iliac lag screws. While there are plenty publications assessing the clinical use of implant systems  which utilize cranial straps, to the knowledge of the authors, no studies exist which objectively quantify the potential gain in stability achieved in a clinical or an in vitro setting. The main objective of this publication is to evaluate if cranial straps provide additional primary stability. To answer this question, the contact surfaces of the implant to the bone are of major concern. In the interface between shell and bone, the addition of cranial straps provided a lower amount of relative movement which remained constant across all load levels. In comparison, in the absence of cranial straps, relative movement started out higher and even increased as load levels increased. Therefore, in addition to the initial design intend, cranial straps seem to provide additional stability to the shell. Especially at the 100% load level, relative movement between the groups were significantly different  . In the Finally, the analysis of the interface between augment and bone provided another interesting insight. Even though relative movement between components within the S-group remained constant over all load levels, it started out significantly higher when compared to the nS-group. The nS-group, starting out significantly lower, displayed an increase across ascending load levels and finally almost aligned with the S-group at the 100% load level . Due to Our methods present several limitations. We chose to utilize composite pelvic models instead of cadaveric bone in this in vitro setup in order to decrease inter-specimen variability and improve availability and ease of use. In addition, the application of a consistent acetabular defect in cadaveric bone with varying anatomical setup and bone density is considered challenging. Even though our choice enabled a reproducible in vitro experiment, correlation with the clinical situation might be limited due to different biomechanical properties such as strain distribution and overall stiffness in a fourth-generation composite model . The impact of the addition of cranial straps was only assessed for a specific augment-and-modular-cage revision system. Therefore, a generally valid statement for the many available systems utilizing cranial straps cannot be obtained as of now. We strongly encourage the application of the presented methods to other implant systems and even different set ups of the same augment-and-modular-cage revision systems by other groups. The applied defect morphology (ADC IIa/Paprosky IIb) was chosen due to a wide incidence in the daily reality of hip revision arthroplasty ,16. DiffWe hope our study inspires future research into the subject. Especially in vivo studies of different fixation techniques between the components within a modular system and the impact of different configuration of that system should be evaluated.Our study evaluated the gain in primary stability through the addition of cranial straps to an augment-and-modular-cage revision system. We utilized a reproducible in vitro setup with composite hemi-pelvis models and measured relative movement between individual components in addition to relative movement between components and the bone as a surrogate parameter for primary stability.We conclude a significant increase of primary stability between the shell and the bone through the addition of cranial straps. Relative motion between components (shell/cup) increases through the addition of cranial straps. A clinical impact of this finding is uncertain and requires further investigation. Finally, the cementless fixation of the augment against the rim-portion of the shell appears stable and compares favorably to prior investigation of different fixation techniques.


The mid-1970s and early 1980s have witnessed a burst of interest in fibrinolytic proteases as it became clear, primarily through the pioneering work of Edward Reich and his coworkers (The Rockefeller University) that these enzymes might play an important role in tumor invasion and metastasis . The obsThe first major advances in the structural biology of fibrinolytic enzymes and fibronectin were made by the laboratory of Staffan Magnusson (University of Aarhus). Magnusson and coworkers have determined the primary sequence of plasminogen and have shown that five regions in the non-protease part of this protein show significant sequence homology with two internally homologous structures of prothrombin –10. TheyOur research group was attracted to this field at the end of the 70s with a view of clarifying structure-function aspects of plasminogen, plasminogen activators and fibronectin, focusing on kringles of plasminogen and the three types of internal homology units of fibronectin. We have demonstrated that the kringle 5 domain of human plasminogen carries a benzamidine-binding site , but we During the course of these studies we have become aware of the collaborative efforts of the lab of Richard Laursen (Boston University) and the lab of Miguel Llinás (Carnegie-Mellon University) to study the structure of plasminogen kringles by NMR spectroscopy –21. In aIn the late 1980s the focus of our research has moved to other fields, therefore our work on kringles and our collaboration with Miguel, temporarily, has ended in 1988. We have, however, learned an important lesson from our studies on kringles: the conservation of residues in different, non-orthologous kringles reflects their relative importance for the folding autonomy of kringle fold . In thisThe latter finding has led us to initiate a new round of collaboration with Miguel’s group, this time on fibronectin type II repeats. Initially, the primary goal of this collaboration was to explore whether the distant homology of type II units and kringles is supported by their structural and functional similarities. In the first part of our work, we have studied a type II domain of the collagen binding bovine seminal fluid protein PDC-109, resulting in the first structure of a type II domain –50.Our project on type II domains, however, gained additional interest when it turned out that gelatinases also contain type II domains. These metalloproteases play a key role in matrix remodeling, degradation of basement membranes and contribute significantly to the metastatic potential of tumor cells; they appeared promising targets for tumor therapy. Significantly, the three tandem type II domains of gelatinase A were shown to be responsible for the high affinity of the enzyme for collagen .http://scop.mrc-lmb.cam.ac.uk), kringle modules and fibronectin type II modules represent two families of the kringle-like superfamily.The NMR spectroscopic studies of Miguel on the various type II domains of gelatinase A –58 have Although our collaboration with Miguel Llinás was primarily through exchange of research materials via mail, exchanging ideas via email, we had regular personal contacts at the biannual meetings of the International Society for Fibrinolysis & Proteolysis (that has a kringle image in its logo) or at the various Plasminogen Activator workshops. I enjoyed his company as he had a good sense of humor, appreciated the pleasant aspects of life and had a broad interest in culture, history, music. He was an admirer of Bartók so I am glad that when he visited us in Hungary, I could show him the Béla Bartók Memorial House to get an impression about the life of this genius.With Miguel’s passing, science has lost a dedicated scientist and I have lost one of the best friends I acquainted with during the heydays of Plasminogen Activation research.


Banana is a significant source of nutrients, and it has been found to contain carbohydrates and other nutritional components. The present study was conducted to evaluate the proximate composition, antioxidant composition, and physicochemical properties of flour obtained from two different banana varieties (Musa acuminata cv. Pisang awak and Musa acuminata cv. Red dacca) and to evaluate the proximate composition and antioxidant composition of cookies prepared by incorporating both banana flours. Several sets of cookie samples were prepared separately by incorporating each banana flour where wheat flour and banana flour combinations were 85%-15%, 75%-25%, 70%-30%, and 0-100%. These samples were evaluated for sensory attributes, and two best cookie formulations were selected (70% wheat flour and 30% banana flour) for the analysis. Both Awak and Dacca had obtained similar amount of carbohydrates p < 0.05 while Dacca had recorded a higher amount of moisture, fat, protein, ash, and phytonutrients such as polyphenols, antioxidants, and flavonoids. Dacca flour had obtained higher values for physicochemical properties like water holding capacity (WHC) and oil holding capacity (OHC). L∗, a∗, and b∗ values were evaluated for banana flour incorporated cookies. Lightness and the redness of cookies were prominent while yellowness was not prominent. There was no significant difference in texture parameters but hardness was higher in banana cookies as they contained a higher content of protein and fiber.Banana  Musa acuminata (Musa sp.), a very popular fruit in the world market. It can be eaten as raw or processed as well as flour is used as a functional ingredient in various food products  wit wit20] wμL of NaNO2 (5%), 150.0 μL of AlCl3 (10%), and 1.0 mL of sodium hydroxide (NaOH) (1 M) were pipetted into tubes, respectively. After that, the solution volume was increased up to 5.0 mL by adding distilled water. The content of the mixture was vortexed properly, and the absorbance of each sample was measured at 510 nm using a UV-Vis spectrophotometer. The results were expressed as quercetin equivalent (QE) in milligram per g of the dried sample using a calibration curve of quercetin .The total tlavonoid content was determined by the method as outlined in  with sliL∗ ), a∗ (green (−60) to red (+60)), and b∗ (blue (−60) to yellow (+60)). L∗, a∗, and b∗ color values for cookies were measured by using chromameter (CR-400). Color values were taken as replicate (n = 3) in different areas of the biscuit surface  ex exAwak-DBanana being abundant in antioxidants has contained bioactive compounds such as phenolics, carotenoids, dopamine, dopa, carotenes, norepinephrine, and ascorbic acid (vitamin C) which have the ability to protect the body against oxidative stress.  expresseAwak-Dacca flour, respectively, while significantly different (p < 0.05). Effect of extracting solvent which dissolved the sample has contributed to the behavior of the obtained results of flours. DPPH increases when the concentration of phenolic compounds and their hydroxylation increase. The DPPH ranged from 0.04 to 0.15 mg TE/g in Awak-Dacca cookies. The highest DPPH was obtained in Dacca flour and Dacca cookies. [DPPH ranged from 0.15 to 0.68 mg GE/g in cookies.  stated tAwak and Dacca flours, respectively, and there was a significant difference (p < 0.05). FRAP values of the cookies ranged from 3.39 to 7.32 mg TE/g in Awak and Dacca cookies. Dacca still had significantly higher (p < 0.05) antioxidant activity than Awak. ABTS values of Awak and Dacca flours ranged from 59.65 to 68.06 mg TE/g, and results for Awak and Dacca flour cookies were 37.92-56.80 mg TE/g. Both Awak flour and Awak cookies had recorded the lower values for ABTS. The incorporation of banana flour into wheat flour significantly improved the antioxidant capacity of composite flours compared to wheat flour.FRAP values ranged from 25.35 to 28.75 mg TE/g in d ± 0.01 while the FRAP value of 4P% banana flour : 96% wheat flour-incorporated cookies was recorded as 0.64b ± 0.01. According to (adedayo), the ABTS value of Cavendish banana 5.03 ± 0.08a mmol TEAC/g was recorded and Red dacca was recorded as 4.98 ± 0.11a mmol TEAC/g.According to , the FRAAwak and Dacca flours ranged from 0.20 to 0.27 mg QE/g while Awak cookies and Dacca cookies ranged from 0.45 to 0.68 mg QE/g. Baking contributed to a significant increase (p < 0.05) in TFC of cookies compared with the flours.Banana flour is high in TFC compared to wheat flour, and the main classes of flavonoids detected in bananas are quercetin, myricetin, and kaempferol. According to , flavonob ± 0.05 to 23.19e ± 0.11 and TFC of 4% banana flour : 96% wheat flour-incorporated cookies was 33.74d ± 0.17. Baking contributed to a significant increase in TFC of cookies compared to banana flour. This difference can occur due to the development of meladoins. Meladoins, brown color pigments, are the products of Maillard browning reaction which occur during the baking process. Meladoins are produced as a result of high temperatures due to reactions between amino acids and reducing sugars, and they give different flavors and brown color in baked food. Based on the literature [According to , the resterature , formulaAwak flour-incorporated cookies  and Dacca flour-incorporated cookies . The sensory scores for the sensory attributes of cookie samples varied significantly (p < 0.05).Mean sensory scores for sensory attributes of the cookies produced by wheat-banana flour and banana flour blends at different proportions are shown in T3 (70 : 30) was better accepted by panel members as the best Awak flour-incorporated cookie. However, T3 was compared favorably well with treatments T1, T2, and T0 (control) in texture, appearance, odor, taste, aftertaste, and overall acceptability. Treatment T2 had the highest rating for appearance. The lower ratings for appearance in treatment T3 may be due to the initial dark color in banana flour compared to the wheat flour that is lighter in color. The color or appearance of cookies becomes darker in the baking process as the chemical reactions take place due to higher sugar content. Treatments T1, T2, and T0 (control) had almost similar scores for overall acceptability while sample T3 has recorded the highest.Cookie, treatment T3− (70 : 30) was better accepted by panel members as the best Dacca flour-incorporated cookie from the sensory evaluation results T3− has obtained the higher mean score for all the attributes. Treatment T0− (control) had the lowest scores for all the attributes. Treatments T1− and T2− had obtained almost similar scores. Treatment T3− has recorded the highest score for appearance due to low sugar content (less darkness) [Treatment T3− 70 :  was bettHylocereus undatus) peel flour blends. According to [b ± 1.31 while the value of 15% pitiya peel flour : 75% wheat flour cookies was 4.93ab ± 1.48. The value for overall acceptability of 100% wheat flour cookies was 4.97a ± 1.45, and the value for overall acceptability of 15% pitaya flour : 75% wheat flour cookies was 5.23a ± 1.54, respectively.A similar study has been done to evaluate the sensory attributes of cookies prepared from wheat flour and pitaya , redness (a∗), and yellowness (b∗).L∗, a∗, and b∗ values of Awak and Dacca flour-incorporated biscuits ranged from 59.18 to 60.29, 6.82 to 9.20, and 28.94 to 31.12, respectively. The color changes occurred due to the extent of Maillard reaction as banana contains glucose, fructose, and protein. The least L∗ value was observed in Awak flour-incorporated cookies because caramelization or Maillard reaction caused browning during baking of banana flour at high temperature as Awak contains more sugar. As a∗ values indicate the redness of cookies both banana flour-incorporated cookies had obtained positive a∗ values which were significantly different (p < 0.05). The redness of the crust was higher in Dacca flour-incorporated cookies due to the highest antioxidant and phenolic content. The b∗ color value represents the yellowness of biscuits, and the yellowness of both biscuits was more prominent than redness [L∗, a∗, and b∗ values of 100% wheat flour-incorporated biscuits were recorded as 66.00, 4.06, and 27.53, respectively.The  redness . Accordi redness , L∗, a∗,L∗, a∗, and b∗ values as 51.767c ± 0.945, 5.433b ± 0.473, and 20.467c ± 0.378, respectively, while 100% wheat flour cookies has recorded L∗, a∗, and b∗ values as 65.000d ± 0.529, 4.067a ± 0.058, and 27.533d ± 0.757. L∗, a∗, and b∗ values of cookies have been decreased with the substitution of banana peel flour.According to , 4% banaTextural quality is one of the most important quality attributes for cookies. The texture of the two best banana cookie formulations was tested against the texture of 100% wheat flour cookie which was considered a commercial type cookie. The primary TPA parameters, hardness, springiness, and cohesiveness, and secondary parameters of gumminess and chewiness are shown in p < 0.05) differences in the texture parameters of the cookies. Hardness can be defined as the peak force required to break the cookie, increased as incorporating banana flour. Baking conditions, the type and quantity of ingredients, and protein content and fiber content of the flour influence its hardness and other textural attributes. The mechanical changes attributed to the replacement of wheat flour by banana flour in cookies are related to the increases of fibers, liquid oil, proteins, and reduction in carbohydrates. Cookies are made out of a continuous glass-like sugar structure in which ungelatinized starch granules and fat are embedded in the structure. Due to starch gelatinization recrystallization, protein denaturation has occurred and the structure and texture are developed. Though protein content is higher in cookies, the gluten content is reduced in the dough. And also, in baking, moisture migrates from the wet area to the drier surface and the product is turning in dry.There were no significant (There was no significant difference in texture parameters but hardness is higher in banana cookies as they contained a higher content of protein and fiber. Protein and fiber are more important for texture than the lowering of carbohydrates (starch). Previous studies  also disa ± 1.17 while the hardness of 4% banana peel flour : 96% wheat flour-incorporated cookies was recorded as 3572.83d ± 1.14.According to , hardnesa and 31.01 ± 6.0c,b. It states that increasing of okara flour is affected by the fracture force and the fracture force is increased as the okara flour content is increased. Because the dough structure is compacted by proteins and fiber.According to , 100% whPisang awak and Red dacca) were higher than banana cookies except for protein and fat as they are added in the cookie preparation process. OHC in Red dacca was higher and WHC in Pisang awak was less than Red dacca as they contain more sugars and reduce the hydration properties by limiting the availability of water to hydrate the starch. Cookies enriched with banana flour reduce the postharvest loss of banana while improving the nutritional quality of cookies. Textural attributes of cookies are increased with the incorporation of banana flour instead of using 100% wheat flour in cookies. L∗, a∗, and b∗ values of cookies have been decreased with the substitution of banana flour. Among the three formulations, 70% wheat flour : 30% banana flour cookies achieved the highest value for the overall performance.The results clearly revealed the proximate composition and antioxidant composition of banana flour (


Myxococcus xanthus, c-di-GMP regulates type IV pilus-dependent motility and the starvation-induced developmental program that results in formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP effectors. We confirm that the stand-alone PilZ domain protein PlpA is important for regulation of motility independently of the Frz chemosensory system and that Pkn1, which is composed of a Ser/Thr kinase domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pilus-dependent and gliding motility in a Frz-dependent manner as well as development. The acetyltransferase domain is required and sufficient for function during growth, while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility independently of the Frz system, likely by setting up the polarity of the two motility systems. Our results support a model whereby PlpA, PixA, and PixB act in independent pathways and have distinct functions in regulation of motility.In bacteria, the nucleotide-based second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In IMPORTANCE c-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts in a manner that depends on the Frz chemosensory system to regulate motility via the acetyltransferase domain, while the intact protein and c-di-GMP binding are essential for PixB to support development. In contrast, PixA acts in a Frz-independent manner to regulate motility. Taking our results together with previous observations, we conclude that PilZ domain proteins and c-di-GMP act in multiple independent pathways to regulate motility and development in M. xanthus. Bacteria have evolved different strategies that allow them to sense and subsequently adapt and differentiate in response to changing environmental conditions. One strategy centers on signaling by a variety of nucleotide-based second messengers . The higSignaling by c-di-GMP depends on its regulated synthesis by diguanylate cyclases (DGCs), which contain an enzymatically active GGDEF domain, and degradation by phosphodiesterases (PDEs), which contain either a catalytic EAL or HD-GYP domain , 3. Outp4–9–18–Myxococcus xanthus, a Gram-negative deltaproteobacterium, is a model organism for studying how social behaviors in bacteria can be modulated by environmental cues   –45. Occay system .The characteristic polarized assembly of the two motility machineries at the leading pole is regulated by a set of four proteins that make up the so-called cell polarity module. The key protein in this module is the small GTPase MglA, which in its GTP-bound form localizes at the leading cell pole to stimulate assembly of the Agl/Glt complexes as well as formation of T4P , 47–50. 47–50–50–M. xanthus and at a 10-fold-higher level during development M. xanthu xanthus . Despite in vivo . MXAN_29 in vivo  and contPilZ domain protein in xanthusM. A and B, that both bind c-di-GMP in vitro. PixA is a response regulator with a single PilZ domain and involved in regulation of the cellular reversal frequency independently of the Frz system. PixB is composed of two PilZ domains and an acetyltransferase domain. PixB regulates the reversal frequency in a Frz-dependent manner and is also important for development. The acetyltransferase activity is essential for activity and is likely stimulated by c-di-GMP binding to the PilZ domains during development.Here, we report the identification of two PilZ domain proteins, renamed PixA (MXAN_1087/MXAN_RS05220 [old/new annotation]) and PixB (MXAN_2604/MXAN_RS12590), for M. xanthus genome encodes 24 proteins with a PilZ domain .The ∼110-amino-acid PilZ domain is widely distributed in bacteria and can be found either as a stand-alone domain in PilZ single-domain proteins or in combination with other domains . Genome Z domain  pilA muta control , the WT  complex , 70, didA mutant .mrpC mutant, which lacks a transcription factor important for development and served as a negative control  family. Both proteins as well as the genetic neighborhood of the respective genes are conserved in closely related fruiting Cystobacterineae .PixA is a response regulator of two-component systems with an N-terminal PilZ domain that possesses all necessary residues for c-di-GMP binding ; Fig. S1pixA and the downstream gene supports the idea that pixA is not part of an operon, while a similar analysis of the pixB locus supports the idea that pixB could be in an operon with the two downstream genes . To determine whether pixA and pixB are indeed important for motility and development in the case of pixB, we ectopically expressed the full-length genes under the control of their native promoter (Pnat) from the attB site in the ΔpixA and ΔpixB mutants . Motility assays on 0.5% and 1.5% agar plates revealed that the motility defects of both mutants were fully corrected in the two complementation strains .Frz-independent reversals can be caused by interfering with the correct localization of the proteins of the polarity module. In particular, mutations that cause a more bipolar localization of MglA cause Frz-independent hyperreversals , 50, 74.nchanged . In agreA lack of PlpA also causes a shift in MglA-GTP toward symmetric bipolar localization . Altoget6 and PixB-His6  is involved.We conclude that PixA binds c-di-GMP in vivo, we ectopically expressed FLAG-tagged full-length WT proteins and mutant variants from the Mx8 attB site in the relevant in-frame deletion mutants . Mutation of the phosphorylatable Asp to Glu mimics the phosphorylated form in some response regulators , 76, whig growth . AltogetWT-FLAG as well as truncated variants that lacked either the AT domain (PixBΔAT-FLAG) or the two PilZ domains (PixBΔPilZx2-FLAG) in the ΔpixB mutant. Because the two truncated variants could not be detected by Western blotting when expressed from the native pixB promoter, all variants were expressed from the stronger pilA promoter. The two variants as well as the WT protein accumulated at a higher level than PixBWT-FLAG expressed from the native promoter during growth and development  is involved in this binding. By analyzing truncated variants of PixB or variants with single amino acid substitutions in the sequence motifs for c-di-GMP binding, we found that the AT domain was necessary and sufficient for correct reversals during growth. In contrast, the two PilZ domains alone did not complement the reversal defect in the ΔpixB mutant. Along the same lines, substitutions in the c-di-GMP binding motifs did not interfere with PixB activity in regulation of the reversal frequency under the conditions tested. These observations support a model whereby the AT activity is key to the effect of PixB on Frz-dependent reversals during growth, while the two PilZ domains and c-di-GMP binding are not important. How then would the AT domain act to inhibit reversals? Acetyltransferases of the GNAT family can acetylate amino groups of different substrates, including small molecules, peptidoglycan, nucleotide-linked monosaccharides, and proteins; however, the substrate cannot be determined based on protein sequence  or on 1% CTT–1.5% agar plates at 32°C with addition of kanamycin (40 μg ml−1) or oxytetracycline (10 μg ml−1) (Escherichia coli cells were cultivated in LB (−1) or tetracycline (10 μg ml−1). All plasmids were propagated in E. coli Top10 (Invitrogen Life Technologies) unless stated otherwise.All T DK1622 . In-framT DK1622 . All M. μg ml−1) . Escheried in LB  liquid mM. xanthus cells were harvested at 5,000 × g for 5 min and resuspended in 1% CTT to 7 × 109 cells ml−1. From this suspension, 5 μl was spotted on 0.5% agar with addition of 1% CTT to a final concentration of 0.5% for T4P-dependent motility and on 1.5% agar with the addition of 1% CTT to a final concentration of 0.5% for gliding motility. Cells were incubated in the dark at 32°C for 24 h. Colony morphology was imaged using a Leica M205FA stereomicroscope with a Hamamatsu ORCA-flash V2 digital CMOS camera and a Leica DMi8 inverted microscope with a Leica DFC280 camera.Motility assay were done as described elsewhere, with modifications . BrieflyM. xanthus development assays were performed as described previously  1.5% agar plates and under MC7 buffer . Briefly, exponentially growing cells were harvested at 5,000 × g for 5 min and resuspended in MC7 buffer to 7 × 109 cells ml−1. Then, 20 μl of cell suspension was spotted on TPM agar, and 50 μl was added to 350 μl of MC7 buffer in 24-well polystyrene plate . At 24 h and 120 h, fruiting bodies were imaged with a Leica M205FA stereomicroscope with a Hamamatsu ORCA-flash V2 digital CMOS camera and a Leica DMi8 inverted microscope with Leica DFC280 camera. To determine sporulation efficiency, cells at 120 h of development were harvested from one 24-well polystyrene plate . Cells were sonicated for 1 min  to disperse fruiting bodies and then incubated at 55°C for 2 h. Sporulation efficiency was calculated as the number of sonication- and heat-resistant spores formed after 120 h of development, relative to the WT. Spores were counted in a counting chamber .eviously  on solid4, 2 mM CaCl2) and incubated in the dark at room temperature for 30 min. Cell movement was recorded for 10 min at 20-s intervals. For analysis, Metamorph (Molecular Devices) and ImageJ 1.52b  and 1 . The localization patterns were binned from the ω values as follows: unipolar (ω > 0.9), bipolar asymmetric (0.9 > ω > 0.2), and bipolar symmetric (ω < 0.2). Diffuse localization was determined when no polar signal was detected. For time-lapse epifluorescence microscopy, cells were prepared as described and recorded for 15 min, with images captured every 30 s. Data were processed with Metamorph 7.5 and ImageJ 1.52b.Protein concentration was measured using a Bradford assay (Bio-Rad). Rabbit polyclonal anti-PilC   and anti6, PixAR9A-His6, PixB-His6, PixBR121A-His6 and PixBR331A-His6, PixBR121A/R331-His6, and PixB-His6, E. coli Rosetta 2 (DE3)/pLysS (Novagen) was transformed with pSK51, pSK141, pES09, pPK21, pES13, and pPK22, respectively. Cultures were grown in 1 liter of LB with addition of chloramphenicol and kanamycin at 37°C to an OD600 of 0.5 to 0.7. Protein expression was induced by addition of isopropyl-β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM for 3 h at 37°C. Cells were harvested by centrifugation at 3,800 × g for 10 min at 4°C and resuspended in lysis buffer . Next, cells were disrupted with a French press and centrifuged at 48,000 × g and 4°C for 40 min. Cleared cell lysate was filtered with a 0.45-μm sterile filter  and loaded onto a 5-ml HiTrap chelating HP column  preloaded with NiSO4 as described by the manufacturer and pre-equilibrated in wash buffer . The column was washed with 20 column volumes of column wash buffer. Proteins were eluted with elution buffer A  using a linear imidazole gradient from 50 to 500 mM. Fractions containing purified His6-tagged proteins were combined and loaded onto a HiLoad 16/600 Superdex 75 pg  gel filtration column that was equilibrated with buffer . Fractions containing His6-tagged proteins were pooled, frozen in liquid nitrogen, and stored at −80°C.To purify PixA-His32P-labeled c-di-GMP as described elsewhere (32P-labeled c-di-GMP was prepared by incubating 10 μM His6-DgcA with 1 mM GTP/[α-32P]GTP (0.1 μCi μl−1) in reaction buffer  overnight at 30°C. The reaction mixture was then incubated with 5 U of Antarctic phosphatase (New England Biolabs [NEB]) for 1 h at 22°C to hydrolyze unreacted GTP. The reaction was stopped by incubation for 10 min at 95°C. The reaction was centrifuged , and the supernatant was used for the binding assay. 32P-labeled c-di-GMP was mixed with 20 μM protein and incubated for 10 min at RT in binding buffer . Fifteen microliters of this reaction mixture was transferred to a nitrocellulose filter, allowed to dry, and imaged using a STORM 840 scanner (Amersham Biosciences). For competition experiments, 0.4 mM unlabeled c-di-GMP (Biolog) or GTP (Sigma-Aldrich) was added.c-di-GMP binding was determined using a DRaCALA assay with lsewhere , 91. 32PMyxococcales genomes using a reciprocal best BLASTP hit method. Protein domains were identified using Pfam v33.1 (pfam.xfam.org) (https://myhits.isb-sib.ch/cgi-bin/motif_scan) (The KEGG SSDB (Sequence Similarity Database)  was usedfam.org)  and MyHiif_scan) . Multiplif_scan)  was usedif_scan)  (pairwis


Phaseolus vulgaris L.) are an important source of nutrients with beneficial effects on human health. However, they contain lectins, that limit the direct use of flour in food preparations without thermal treatment, and phytic acid, that reduces mineral cation bioavailability. The objectives of this research were: to obtain biofortified snacks and a cream using an untreated common bean flour devoid of active lectins (lec−) and with reduced content of phytic acid (lpa) and to evaluate the sensorial appreciation for these products. The main results of the present work were: the products with the lpa lec− flour did not retain residual hemagglutinating activity due to lectins; they showed higher residual α-amylase inhibitor activity (from 2.2 to 135 times), reduced in vitro predicted glycemic index (about 5 units reduction) and increased iron bioavailability compared to the products with wild type flour; products with common bean flour were less appreciated than the reference ones without this flour, but the presence of an intense umami taste can be a positive attribute. Results confirmed that the use of the lpa lec− flour has important advantages in the preparation of safe and nutritionally improved products, and provide useful information to identify target consumers, such as children and elderly people.Common beans ( Legume seeds (pulses) are an excellent and inexpensive source of proteins, dietary fiber, vitamins, minerals, and bioactive components, and their consumption is associated with multiple health benefits, such as lower risk of incidence of cancers, type II diabetes, or cardiovascular diseases ,2,3,4,5.Snacks occupy a considerable slice of the ready-to-eat food market and are appreciated by consumers of all ages, eaten during work-breaks by adults and during school recreation by children and adolescents. In some cases, snacks have become meal substitutes; the tendency to consume low-value snacks instead of having a complete and balanced meal is frequent and common, mainly among teenagers. This phenomenon has been associated with an increase in the body mass index and poor academic performance of students . The incBecause of their high nutritional value and their positive performance in heat-treated products, legume flours are suitable ingredients for use in baked goods, and bean flour seems to receive a positive response in the formulation of ready-to-eat snacks ,14,15,16Phaseolus vulgaris L.) is an essential source of proteins and many macro and micronutrients. Therefore, its consumption has beneficial effects on human health and related pathologies, such as reducing cardiovascular diseases and diabetes mellitus, preventing different types of cancer, and controlling some metabolic functions [The common bean  exist ,30,31,32lec− ,36) and ec− [lpa ,38). In ec− [lpa . α-AI caec− [lpa  and is wec− [lpa . Moreoveec− [lpa ,43. Throec− [lpa ,36. For ec− [lpa .lpa mutants, affecting the PvMRP1 phytic acid transporter, have been isolated and characterized to date, showing a 75–90% reduction in seed phytic acid content [lpa1 seeds was higher than from wild type beans, showing that the lpa1 beans are biofortified [lpa1 beans could cause adverse gastrointestinal symptoms due to a hard-to-cook (HTC) phenotype concomitant with increased thermal stability of lectins in these lines [lpa1 mutation is present [Two allelic common bean  content ,38. Fromortified . Howeverse lines . As rece present .lpa1 mutant have been used to prepare biscuits [lec−) is safe, as no lectin activity is present in the biscuits. Moreover, it was shown that baking did not fully inactivate α-AI, a trait that may contribute to lowering the predicted glycaemic index (pGI) of the biscuits. When the lpa1 flour was used to prepare biscuits, a 50% reduction of phytic acid content in the final product was observed [Unprocessed common bean flours obtained from the Lady Joy genotype, and the biscuits . Resultsobserved .lpa) with the absence of an active lectin (lec−) while maintaining the accumulation of α-AI, were analyzed. Crackers with two different proportions of bean flour, two different types of biscuits and a cream, with a content of bean flour ranging from 9% (cream) up to 38% (Cracker 2) were produced. Our general purpose is to promote bean consumption by developing biofortified and low antinutrients beans which are well suited for directly use as bean flours. More specifically, in this study, we aimed, on the one hand, to verify how some nutritional characteristics of the bean flours  were influenced by different formulations and/or cooking conditions and, on the other hand, to assess the sensory properties and related acceptance level of bean-based products, according to a trained evaluation panel.In a previous study  the use P. vulgaris L. genotype, belonging to the bean market class Borlotto and an lpa lec− genotype with the same genetic background of Borlotto. This genotype (lpa lec−) was a BC1F5 line derived from a cross between a BC2F2low phytic acid (lpa) line [lec−).Bean flours were obtained from a wild type (wt) pa) line  and the pa) line , charactComposite flours, containing different percentages of the bean flour, wheat flour type 2, whole wheat flour and buckwheat flour were used to prepare biscuits, crackers and a cream. Recipes were developed with the technical assistance of the backers and pastry chefs Mr. Matteo Consolo and Mr. Ferruccio Farioli at ENAIP  Lombardia, Busto Arsizio . Crackers with two different amounts of bean flour, two types of biscuits, and a cream were produced. Formulations are reported in lpa lec− bean genotype or replacing the common bean flour with an equivalent amount of wheat flour type 2 or whole wheat flour (Biscuit 2). Cream was prepared with the lpa lec− bean flour or with rice flour. Crackers and biscuits prepared with the two different common bean flours (wt and lpa lec−) were used for the different analyses, except the sensorial ones. For these last analyses, crackers, biscuits, and cream prepared with the lpa lec− flour were compared to the same product prepared without the common bean flour.Crackers and biscuits were both prepared in three variants, using the flours from the wt or the For cracker preparation the dough was produced by mixing wheat flour type 2, bean flour, with 95% of water. The dough was left to rest to activate the autolysis process. After 30 min, yeast was added and the remaining 5% of water was gradually added to the dough. The dough was divided in pieces of the same weight and each loaf was reduced to a thin sheet. Each sheet was put in a baking tin, previously oiled, brushed with olive oil and sprinkled with salt to taste. Sheets were pierced and cut in regular pieces and then cooked in an oven at 190 °C for approximately 8 min.Shortbread biscuits (Biscuit 1) were prepared by mixing sugar with butter in a planetary mixer until they were completely homogenized. Then the other ingredients were added. The dough resulted was soft and biscuits were formed using a sac a poche. Biscuits were cooked in an oven at 190 °C for 14–18 min.Buckwheat biscuits (Biscuit 2) were prepared by mixing all the ingredients together to produce the dough. Once all the ingredients were completely homogenized, the dough was worked by “cylinder processing” and formed. Before cooking, biscuits were sprinkled with a mixture of sugar or salt and cornmeal. They were cooked in an oven at 190 °C for 14–18 min.For the cream, the ingredients were mixed together and cooked for approximately 15 min at low temperature (65 °C) to pasteurize the egg yolks. The final cream was stored in the fridge at about 4 °C.The chemical composition of food samples was assessed according to AOAC standard methods . SamplesThe evaluation of in vitro starch digestion of food products over time was performed following the multi-enzymatic protocol detailed by Giuberti et al.  and as d4, 15 mM NaCl, pH 7.4). Hemagglutinating activity in the extracts was determined by a serial dilution method using a human type A erythrocyte suspension. Drops of blood were drawn from the donor finger and collected inside 1.5 mL Eppendorf tube: about 150 μL of blood was collected. Erythrocytes were separated from the serum by washing with about 10 volumes (1.5 mL) of PBS. This step was repeated three times. The serum was eliminated, and the erythrocytes were stored at 4 °C in 10 volumes of PBS. At the time of use, erythrocytes were diluted 1:10 with PBS.Bean flours, or defatted food samples, were extracted with 20 volumes of phosphate buffered saline buffer (PBS: 10 mM KHPOFor each sample (PBS samples both from flours and defatted products), serial dilutions in PBS, ranging from 1:2 to 1:256, were assayed. Agglutination was visually determined after 4 h incubation at room temperature.The analysis was based on a protocol that measures the inhibitory activity of the sample against human salivary α-amylase  by the increase of iodine staining after the action of the salivary α-amylase with soluble starch . Briefly3. Both solutions were preliminary subjected to batch treatment with Chelex-100  to eliminate any residual polyvalent metal ion. A quantity of 1.65 g food sample was dissolved in 10 mL water in a 50-mL screw-cap culture tube. The pH of each sample was adjusted to pH 2.0 with 5.0 N HCl. A volume of 0.5 mL of the pepsin solution was added and incubated for 60 min on a shaker at 37 °C. The pH of the sample was then raised to 6 with 1 M NaHCO3 and 2.5 mL of the pancreatin-bile extract mixture was added. Finally, the pH was adjusted to 7 with 2 M NaOH, and the volume was brought to 15 mL with a 120 mM NaCl/5 mM KCl water solution.A quantity of 0.2 g porcine pepsin  was dissolved in 5 mL 0.1 N HCl; 0.05 g pancreatin  and 0.3 g bile extract porcine  were dissolved in 25 mL 0.1 NaHCO2 in collagen-treated 6-well plates  and cultured at 37 °C in Dulbecco’s modified Eagle medium containing 25 mmol/L HEPES, and 10% fetal bovine serum (complete medium) in 5% CO2 atmosphere. On day 13 from cell confluence, the complete medium was removed and replaced with minimum essential medium (MEM) supplemented with 10 mmol/L PIPES, 4 mg/L hydrocortisone, 5 mg/L insulin, 5 µg/L selenium, 34 µg/L triiodothyronine and 20 µg/L epidermal growth factor (enriched MEM), to ensure background levels < 80 µg Fe/L. A Transwell insert ring (Costar Corp.) was then added to each well, in which the upper chamber was formed by fitting the bottom with a dialysis membrane . A volume of 1.5 mL digested sample was added to the upper chamber and incubated for 2 h. Then, inserts were removed, and 1 mL of enriched MEM was added. Cells were incubated for a further 22 h at 37 °C. Then, the monolayer was washed with 140 mM NaCl, 5 mM KCl and 10 mM PIPES pH 7 (rinse solution). The rinse solution was aspirated and 2 mL of a freshly prepared rinse solution containing 5 mM sodium hydrosulfite and 1 mM athophenanthroline disulfonic acid  was added for 10 min. The removal solution was aspirated and the monolayer was washed with rinse solution. Next, 2 mL of deionized water was added and cells were sonicated for 15 min with a benchtop sonicator. Finally, cells were recovered by scraping, collected along with the 2 mL water and stored at −20 °C. A 10-µL sample was used for ferritin measurement using the Human Ferritin ELISA (Enzyme-Linked Immunosorbent Assay) kit (SIGMA-Aldrich). Cell protein was measured using the Bio-Rad DC protein assay kit (Bio-Rad).The in vitro digestion model from  was adopv/v) HNO3 by a microwave digester system  by applying two-step power ramps . After 20 min cooling, the mineralized samples were transferred into polypropylene test tubes and diluted 1:20 with MILLI-Q water (Merck). Iron concentrations were measured by inductively coupled plasma-mass spectrometry .For iron content evaluation, 300 mg samples were digested in Teflon tubes filled with 5 mL of 65%  and temporal dominance of sensations (TDS) ,54, wereSamples were distributed to the judges, coded with three-digit numbers and presented randomly. Mineral water was distributed to the judges to clean their mouths between samples.For DA, each judge was delivered one product at a time and asked to evaluate the attributes  using a The TDS was performed, considering only 5–7 attributes  linked t® . The sensorial profiles were analyzed through ANOVA and post hoc test (Tukey’s HSD) and displayed in a visual plot. For the TDS, the proportion of runs for which each attribute was considered as dominant was calculated for each point in time. These proportions were traced over time using the SAS® TRANSREG procedure and named “TDS curves”.Results were elaborated by statistical analysis using the software SASlpa lec− common bean flours were analyzed for their proximate composition , which was characterized by the highest bean flour content . Biscuits contained the lowest protein content (average 11.3 g/100 g DM), according to the lower percentage of the bean flour in their formulation .Crackers and biscuits prepared with wt or position . ResultsThe lipid content was higher in Biscuit 1 (average 32.4 g/100 g DM) and consistent in Biscuit 2 (average 15.2 g/100 g DM), since, in both cases, butter was one of the main ingredients. In contrast, crackers, which were prepared with no added fats (except for the olive oil used to sprinkle the pan), showed a lower lipid content (average 3.0 g/100 g DM).The carbohydrate fraction appeared variable between baked products. The starch content was higher in Cracker 1 (average 45.4 g/100 g DM), but was lower in Cracker 2 (average 38.3 g/100 g DM), where the bean flour was present in a higher proportion. In contrast, starch content was higher in Biscuit 1 than in Biscuit 2 , despite the fact Biscuit 1 contained a higher percentage of common bean flour. This difference might be explained by the composition of Biscuit 2, in which wheat flour was replaced by whole wheat and buckwheat flours that further contributed to lowering the starch content. On the other hand, these ingredients did not influence the content of total dietary fiber (average 16.7 and 17.9 g/100 g). The total dietary fiber also constituted a significant fraction in crackers and increased with the increasing level of bean flour in the products, being higher in Cracker 2 than in Cracker 1 (20.9 g/100 g DM against 16 g/100 g DM). In crackers, total sugars were negligible, while they were relevant in biscuits for the saccharose fraction  that correlated to the added sugar in the recipes, that was almost three times higher in Biscuit 2 than in Biscuit 1 .Finally, the ash content was higher in crackers (average 6.5 g/100 g DM in Cracker 1 and 7.6 g/100 g DM in Cracker 2), while Biscuit 2 had a slightly higher ash fraction (average 1.8 g/100 g DM) than Biscuit 1 (average 1.4 g/100 g). These differences might have been due to the bean flour present in the products, together with the amount of less refined flours in Biscuit 2.lpa lec− bean flour had higher protein content and lower starch content compared to those made with wt bean flour, in agreement with the composition of the respective flours (27.7 g/100 g vs. 23.1 g/100 g for proteins and 36.0 g/100 g vs. 44.9 g/100 g for starch)  .lpa lec− genotype . To quantify the amount of lectins in the different bean-based products, equal amounts of extracts were compared with equal amounts of extracts from flours of the wt genotype as control, and genotype .lpa lec− genotype) was not able to agglutinate blood erythrocytes at any dilution. On the contrary, wt flour extract (containing PHAs) was able to agglutinate blood erythrocytes after a serial dilution until 1/128 . Biscuit 1 was characterized by limited retention of α-AI activity when made with wt bean flour (20.44%), while biscuits made with the flour from the lpa lec− genotype showed higher inhibitory activity (51.19%). Biscuit 2 was characterized by higher residual α-AI activity compared to Biscuit 1, however it behaved in a similar way as regards the selected genotype for the bean flour; when lpa lec− bean flour was used, the residual α-AI activity was more than double that of Biscuit 2 containing wt bean flour (84.17% for lpa lec− and 37.33% for wt). In the case of the cream a moderate retention of α-AI activity was observed (27.28%). This product was made only with the lpa lec− bean flour as, if using wt flour, a high lectin activity would be expected due to the mild cooking conditions  was analyzed for crackers and biscuits using common white bread as reference. Results are reported in Considering the different bean-based products, Cracker 1 had the highest pGI (average 74), which correlates to its high percentage of starch (average 45.4 g/100 g), primarily deriving from wheat flour. In contrast, in Cracker 2, the higher amount of bean flour (38% in Cracker 2 vs. 26% in Cracker 1) contributed to reducing the starch fraction (37.5 vs. 45.4 g/100 g) and to increasing the dietary fiber and the crude protein contents, thus lowering the pGI value.The biscuits had a more complex composition and a similar pGI (average 41.3 for biscuits 1 and 47.7 for biscuits 2). The starch fraction was higher in Biscuit 1 (20.2 g/100 g vs. 16.7 g/100 g), while sugars, consisting almost entirely of saccharose, were more than doubled in Biscuit 2 (30.4 g/100 g vs. 12.0 g/100 g). Saccharose was thus primarily responsible for the higher pGI of Biscuit 2. Biscuit 2 was also characterized by the presence of whole wheat and buckwheat flours that counterbalanced the effect of sugars by contributing to an increased total fiber fraction, which was quite similar to that of Biscuit 1 (17.9 g/100 g vs. 16.6 g/100 g) in which a major contribution derived from bean flour, which also contributed, together with a reduced saccharose content, to the slight reduction of pGI.lpa lec− bean flour, a reduction of about 5 units of the pGI, corresponding to about 7% of the reference (white bread) pGI; was detected .Interestingly, if data were compared considering the contribution of the bean genotype, it clearly appeared that, when products contained the lpa lec− bean and Biscuit 1 flours tended to have a higher ferritin value than their wt counterpart. In addition, ferritin levels in lpa lec− Biscuit 1 were significantly higher compared to cells alone.  between “Neither Like nor Dislike” and “Dislike Slightly”, corresponding, respectively, to 79% and 73% of the score obtained by the Cracker R (5.6) .Regarding the TDS analysis for flavor, in Cracker R (0% bean flour) the flavor of wheat was predominant over the whole tasting; salty showed an important role in the first phase of tasting A. With tBiscuit 1 had a more intense odor and flavor of legumes and wholemeal/bran, as compared to Biscuit 1R. Moreover, Biscuit 1 was perceived as grainier, bitter and with a higher intensity of umami. The control biscuit (Biscuit 1R) was perceived as more consistent, sweeter with a stronger shortcrust pastry flavor . Both biThe TDS of Biscuit 1R A was maiBiscuit 2, as compared to its reference, had a higher mouth coating, while the umami taste was perceived more, as well as the flavor of legumes. The Biscuit 2R showed a higher biscuit odor, consistency and crunchiness; it was perceived to be sweeter than Biscuit 2 and with a more intense flavor of butter and biscuit . A signiThe TDS of biscuit 2R with 0% bean flour A showed Regarding the TDS flavor of Biscuit 2 A, the swRegarding the two cream preparations it was possible to observe several significant differences. In particular, the bean flour Cream had higher odor and flavor of legumes and flavor of almond. Moreover, it was rated as grainier and more sapid  . The CreThe TDS taste of Cream R A showed The nutritional quality of traditional snacks is often considered low due to their high sugar and fat content and their protein deficiency . Howeverlpa lec−) bean genotypes. Common approaches to increase essential mineral bioavailability and to reduce antinutrients are mainly based on a combination of technological processing, such as soaking, dehulling, heating, cooking, fermentation, germination and extrusion [lpa and lec− mutations do not need specific treatments, such as soaking, to reduce phytic acid or heating of the flour to inactivate the lectins, as demonstrated and discussed in this study and in a previous one [If an increase in protein and total dietary fiber and a decrease in starch contents in the bean-based products could be expected, the major element of novelty of the present study relates to the fact that the bean-based products were obtained using flours of biofortified and low antinutrient  than in the wt genotype Biscuit 1 (about 10–12%). In both Biscuits 1 and 2, the lpa genotype retained a more pronounced activity that correlated with higher agglutinating activity of the seed flour. Due to the more efficient heat penetration that allowed complete PHA inactivation, the crackers did not show any residual agglutination activity for all the used common bean genotypes, confirming that proper heat treatment is necessary to obtain a significant reduction or complete inactivation of the toxic protein fraction in PHA-containing genotypes.Lectins, and in particular common bean PHA, may exert a very toxic action on the consumer if not properly heat inactivated, as shown by Petry et al. (2016) who observed unexpected adverse gastrointestinal symptoms in Rwandese women with low iron status participating in a clinical trial aimed at evaluating the biofortification potential of i et al.  demonstri et al. , which dlpa lec− bean flour showed higher activity than that measured in samples made with wt bean flour. This finding was not completely unexpected and confirmed the findings of a previous study in which it was observed that B14 biscuits made with Lady Joy bean flour (the parent used to introgress the lec− and α-AI traits in the lpa lec− genotype) showed higher α-AI activity than that of B14 biscuits made with wt bean flours of Taylor and Billò genotypes [lpa lec− flour was lower than that of the wt, these data, together with the above observations, strongly suggest that the α-AI associated to the lec− trait should be particularly resistant to thermal denaturation and deserves further study to understand the basis for its behavior.In contrast to what was observed for PHA, when the residual α-AI was assayed, all samples containing the enotypes . Since tlpa lec− bean flour showed pGI reductions of about 5 units compared to those of corresponding products made with wt flour. The presence of active α-AI, the activity of which was particularly pronounced in baked products made with lpa lec− bean flour, is most likely the reason for the observed reduced pGI values. Indeed, B14 biscuits made with bean flour devoid of α-AI showed a pGI about 4–6 units higher than that of the same product made with a wt bean flour [lpa lec− bean products here described should be predictive of hypoglycemic functional properties. A first step toward the expansion of the legume-based snacks market to more specific consumers’ sectors is to ensure their sensorial appreciation by target consumers. The incorporation of legumes into various products usually results in “beany” flavor/back notes, which are considered “unpleasant” by some people. Furthermore, fortification of cereal-based products with legume flours affects the texture of the end-product [Legume flours are highly recommended to decrease the glycemic index as they are rich in resistant starch and have a high dietary fiber content. For instance, lupin-based biscuits snacks offered to Type 2 diabetes mellitus (T2DM) patients suggested they may improve both their glycemic control and satiety and a number of clinical trials have been conducted, determining the postprandial glycemic response and the pGI of snacks enriched with legume flours ,68. Theoan flour . In a vean flour , has beean flour . Thus, i-product . In this-product  liking s-product . Moderat-product , accordi-product ,73 low fInterestingly, all the bean-based products demonstrated an intense umami taste. Umami, which is also described as the fifth taste, stimulates salivation, improves hypogeusia by enhancing the gustatory-salivary reflex, enhances appetite and satiety, ameliorates eating disorders, and increases the peristaltic reflex and pellet propulsion through the distal colon . This islec− bean flour drastically reduced the phytic acid content, increased antioxidant capacity and, most interestingly, did not affect α-AI activity [These results, although encouraging, also underlie the need to modify the product formulations  and to improve their technological, functional and sensorial properties. To this end, sourdough fermentation (by lactic acid bacteria) technology looks very promising as it may improve texture and flavor characteristics, as well as nutritional quality. The use of this type of fermentation on activity . Therefoactivity . Furtheractivity .The acceptance of bean-based products by the consumer is important to embrace the possibility of extending their market to a more diverse and wider group of people. Beyond their interesting use in feeding of children, bean-based products can be offered to elderly people, as well as successfully employed as efficient substitutes in diets for people suffering from celiac disease, diabetes and obesity. It is quite common that products characterized by the presence of unusual ingredients, including whole wheat, buckwheat and bean flours, tend to have a lower degree of acceptability  . Howeverlpa lec−), instead of that from a wt bean genotype, further improved the nutritional properties of different products. The lpa lec− bean flour guaranteed the absence of any possible poisoning due to lectins and provided more bioavailable iron, as demonstrated by using a Caco-2 cells model system. All the products made with the lpa lec− bean flour retained higher α-AI activities and had lower pGIs. It would be worthwhile in the future to evaluate the impact of such snacks, particularly those with the lowest pGI and the highest α-AI activity, on the glycemic response, weight gain, and appetite control using an animal model system, thus providing evidence of the health benefits of such products as has been performed for other types of snacks [In the present study, it was demonstrated that the use of a biofortified and lectin free bean flour (f snacks .These products may meet the nutritional needs of children and elderly people well, as they provide more bioavailable iron, are more protein-rich, have lower pGIs, and their umami taste may enhance appetite and satiety, ameliorating eating disorders.Although these results are encouraging, there remains a need to improve the technological, functional and sensorial properties of the products and, most importantly, to evaluate the extent of these improvements in the light of how consumer perception may change with information about the nutritional and functional advantages derived from consuming the bean-based products.