Starch is widely used in food and non-food industries because of its unique characteristics. However, native starch shows some weaknesses that restrict its applications. Recently, some studies have demonstrated the benefits of using protein to overcome these limitations. Therefore, the aim of the present study was to investigate the effect of mung bean protein isolate (MBPI)  on the physicochemical, pasting, and thermal properties of native corn starch (NCS), as a novel starch–protein composite. Higher swelling power (SP), water absorbance capacity (WAC), and solubility values of NCS were observed with increasing MBPI concentration. Additionally, by the addition of MBPI, the rapid visco analyzer (RVA) showed a reduction in pasting temperature (77.98 to 76.53 °C), final viscosity (5762 to 4875 cP), and setback (3063 to 2400 cP), while the peak viscosity (4691 to 5648 cP) and breakdown (1992 to 3173 cP) increased. The thermal properties of NCS/MBPI gels investigated by differential scanning calorimetry (DSC) showed higher onset, peak, and conclusion temperatures , but lower gelatinization enthalpy (10.85 to 8.79 J/g) by increasing MBPI concentration. Fourier transform infrared spectroscopy (FT-IR) indicated that the addition of MBPI decreased the amount of hydrogen bonds within starch. Furthermore, after three cycles of freeze-thaw shocks, the syneresis of NCS-MBPI composites decreased from 38.18 to 22.01%. These results indicated that the MBPI could improve the physicochemical properties of NCS, especially its syneresis and retrogradation characteristics. Starch is a semi-crystalline high-molecular biopolymer that mainly consists of linear amylose and branched amylopectin chains . It is oProteins can improve the structural, textural, thermal, and pasting properties of food products through multiple interactions with other biopolymers, especially starch ,16. MoreLegumes are dicotyledonous seeds, rich in carbohydrates (65 to 72%), proteins 18 to 32%), and dietary fibers (10 to 20%). Generally, they have remarkably higher protein content compared to cereal seeds, and are considered as important sources of proteins, essential amino acids, and bioactive peptides for large parts of the world’s population ,18,23. T to 32%, Vigna radiata L.) is a plant species from the legume (Fabaceae or Leguminosae) family, which is cultivated in almost 8.5% of the global legume area. Mung bean seed is a good source of micro- and macro-nutrients, such as carbohydrates, minerals, vitamins, dietary fibers, as well as anti-bacterial, anti-fungal, and bioactive components [Mung bean  and hydrophobic amino acids  are exposed, which could form electrostatic or hydrogen bonds with other components in the system [The hydration properties of starch–protein composite depend on the characteristics of its components, their ratio, and their interactions . MBPI ise system ,35. Highe system . Additioe system  reportede system ,37. Howee system . Besidese system ,31.p < 0.05). However, it should be mentioned that the MBPI is soluble in water, and these solubility values do not represent the actual amylose leaching of corn starch granules [The solubility of NCS with and without the MBPI was shown in granules .p < 0.05). The PT, SB viscosity, and FV of composites decreased  by increasing the MBPI concentration, while the PV and BD viscosity of the composites increased (4691 ± 17 to 5648 ± 13 cP and 1992 ± 20 to 3173 ± 19 cP). These results are similar to those of previous studies [The pasting characteristics of NCS/MBPI gels are shown in  studies ,38.PT is the minimum temperature in which the viscosity starts to increase because of heating, which negatively correlates with the water binding capacity (WBC) of the system . The decThe SB viscosity reflects the reaggregation of the swollen starch granules after cooling of the starch paste and indicates the short-term (amylose) retrogradation tendency . The viso, Tp, and Tc) of composite gels increased with increasing the MBPI concentrations , while the gelatinization enthalpy (ΔH) decreased significantly (p < 0.05) from 10.85 ± 0.23 to 8.79 ± 0.15 J/g. The same results have been reported for other starch–protein composites [o, Tp, and Tc) are related to the internal crystalline structure of starch and its heat stability, and the ΔH reflects the starch crystallinity and the energy needed for double helical dissociation [The thermal parameters of the NCS/MBPI samples are shown in mposites . However−1 in the FT-IR spectra of MBPI corresponded to the C=O stretching, which may be related to the amide I region. Additionally, the peaks at wavenumbers of 1500 to 1580 cm−1 (amide II) corresponded to the C–H stretching and N–H bending vibrations, and 1200 to 1400 cm−1 (amide III) corresponded to the complex mix of α-helix and β-helix. These regions represent the secondary and tertiary structures of proteins [−1 and 2938 cm−1 might correspond to the overlapping of O–H and N–H stretching vibrations and C–H stretching vibrations, respectively [−1, which were related to the O–H stretching, C–H group, water adsorption, angular twisting of CH2, and stretching of the glucose ring, respectively [−1 corresponded to –COOH [−1 and 1075 cm−1 were related to the C=O group and CH2–OCH2 stretching vibrations [−1 was reduced to a wavenumber of 3308 cm−1 after adding 8% MBPI, which indicated the weaker strength of hydrogen bonds and lower retrogradation [−1 and 1525 to 1775 cm−1 became wider and redshifted with higher concentrations of MBPI, which were related to forming the intermolecular hydrogen bonds between MBPI and the amorphous region of the starch molecule [FT-IR spectrometry was used to evaluate the feasibility of the interactions between starch and protein. Spectra of NCS and MBPI powders and the NCS/MBPI gels are presented in proteins . Moreoveectively . NCS shoectively ,10. The molecule . AdditioThe ability of starch to overcome physical changes during temperature shocks is estimated using freeze-thaw stability . SyneresThis study showed that the MBPI could interact with the leached amylose by forming intermolecular hydrogen bonds during the gelatinization, which could disturb the amylose–amylose interaction and retard its rearrangement and retrogradation. Additionally, the presence of MBPI could enhance the WBC of the system and decrease the syneresis, especially at higher concentrations. Overall, these results suggest that the addition of MBPI to NCS could significantly improve the physicochemical and functional properties of NCS, which could prolong the shelf-life of starchy food products by retarding their short-term retrogradation and reducing the syneresis.Mung bean seeds were purchased from a local market in Shiraz, Iran. Native corn starch was obtained from the Behinazma company . All chemical materials were purchased from Sigma Chemical Co.  and were of analytical grade.w/v), and the pH of the dispersion was adjusted to 9 by NaOH. Then, it was stirred for 1 h using a magnetic stirrer at 25 °C and centrifuged for 6 min at 3000× g. After that, the pH of the supernatant was adjusted to pH 4.5 with HCl according to precipitate the protein and then centrifuged at 3000× g for 6 min and then collected. The protein curd was washed three-times with distilled water and neutralized by NaOH before freeze-drying. Finally, the protein content of MBPI was determined using the micro-Kjeldahl technique [The protein isolates were prepared using the alkaline extraction precipitation process described by El-Adawy  with somechnique , which wMBPI was mixed with NCS at different concentrations  using a mixer .g. The supernatant was poured into a pre-weighted Petri dish and dried overnight at 55 °C. The WAC, solubility, and SP of the samples were determined by the following Equations (1)–(3):The SP, WAC, and solubility values of the blend samples see  were detPasting properties were determined using the RVA . For this experiment, 25 mL of distilled water and 3.5 g of NCS-MBPI mixture were poured into an aluminum canister and stirred manually by the plastic paddle of the instrument for 60 s to assure the sample was fully dispersed in distilled water. The slurry was subjected to a programmed 13.0 min heating and cooling cycle, where the sample was held at 50 °C for 1 min, heated to 95 °C at a rate of 5 °C/min, held at 95 °C for 2.7 min, cooled down to 50 °C at a rate of 5 °C/min, and finally held at 50 °C for 2 min. The stirring speed of the paddle for the first 10 s was 960 rpm, and for the rest of the experiment was 160 rpm; then, the pasting parameters, including pasting temperature (PT), peak viscosity (PV), final viscosity (FV), setback (SB), and breakdown (BD) were calculated, and the viscosity was measured in centipoise (cP).o, Tp, and Tc) and gelatinization enthalpy (ΔH).The thermal properties of the blend samples were examined according to the method of Zheng et al. , using a−1 with a resolution of 4 cm−1.The pasted samples from the RVA analysis  were freg. These freeze-thaw cycles were repeated for three cycles, and the syneresis was determined after each cycle by the following Equation (4):The freeze-thaw properties of the blend samples were evaluated by the method of Hoover and Senanayake  with a mThe analysis of variance (ANOVA) and Duncan’s multiple range test were used to study the differences between the mean values at a 5% significance level, using SPSS software, version 26 . All experiments were performed in triplicate.


We aimed to compare the detection rates of prostate cancer (PCa) and clinically significant prostate cancer(csPCa) by biparametric (bp-) and multiparameter magnetic resonance imaging (mpMRI).A total of 699 patients who underwent transperineal prostate biopsy in the Department of Urology, the Second Affiliated Hospital of Nantong University from January 2018 to December 2021 were retrospectively reviewed. Multivariate analysis was used to explore the influencing factors associated with the detection rates of PCa and csPCa. According to MRI examination before biopsy, the patients were divided into bpMRI group and mpMRI group. The detection rates of PCa and csPCa by bpMRI and mpMRI were compared. Furthermore, stratified analysis was performed for patients in these two groups to compare the detection rates of PCa and csPCa at different tPSA intervals, different prostate volume (PV) intervals and different PI-RADS V2 scores.P > 0.05), with no statistical significance. In the tPSA 10–20 ng/ml interval, the detection rates of PCa  and csPCa  by mpMRI were significantly higher than those by bpMRI, while in other tPSA interval , different PVs  and different PI-RADS V2 scores , the detection rates of PCa and csPCa were comparable between the two groups.A total of 571 patients were finally analyzed in this study after exclusion, and the overall detection rate of PCa was 54.5%. Multivariate analysis showed that patient age, tPSA level, prostate volume and PI-RADS V2 score were independent risk factors affecting the detection rates of PCa and csPCa. The detection rates of PCa and csPCa by bpMRI and mpMRI were comparable (51.3% vs. 57.9%, 44.0% vs. 48.0%, both For detecting PCa and csPCa, bpMRI and mpMRI had similar diagnostic efficacies, whereas mpMRI detected more PCa and csPCa in the tPSA interval of 10–20 ng/ml. Prostate cancer (PCa) is the second most common male malignant cancer worldwide, and its death rate ranks sixth . NowadayThe latest Prostate Image Reporting and Data System (PI-RADS) proposed that the DCE sequence (dynamic contrast enhancement) in mpMRI was with limited efficacy for diagnosing PCa sometimes . Only whIn the present study, we analyzed the clinical data of 699 patients who underwent transperineal prostate biopsy in our center. The detection rates of PCa and csPCa by bpMRI and mpMRI were compared at different tPSA intervals, different prostate volumes and different PI-RADS V2 scores.This is a retrospective study approved by the institutional review board and written informed consent was obtained from all patients. From January 2018 to December 2021, a total of 699 patients suspicious of prostate cancer  underwent transperineal prostate biopsy in our hospital. Patients with a previous prostate biopsy history or a prior diagnosis of prostate cancer were excluded.In this study, all patients underwent transperineal prostate biopsy. They were placed in the lithotomy position, routinely disinfected, and draped with a sterile hole towel. Then 1% lidocaine was used for subcutaneous local infiltration anesthesia of the puncture site in the perineal region. The rectal ultrasound probe was placed in the rectum, and infiltration anesthesia deep to the extraprostatic capsule was done at the puncture site under direct ultrasound guidance. After that, combined cognitive MRI-targeted biopsy and systematic biopsy were performed using 18G puncture needle . Cognitive MRI-targeted biopsy was performed with 2 cores per targeted lesion, followed by 12-core systematic biopsy.The primary outcome was to evaluate the detection rates of PCa and csPCa by bpMRI and mpMRI based on pathological results of prostate biopsies. The secondary outcome was to analyze the detection rates of PCa and csPCa by bpMRI and mpMRI stratified by tPSA level, PV (prostate volume), and PI-RADS score. In different tPSA intervals , the detection rates of PCa and csPCa by bpMRI and mpMRI were compared. Prostate volume was calculated according to magnetic resonance imaging measurements , and the detection rates of PCa and csPCa by bpMRI and mpMRI were compared in different PV intervals . Also, the detection rates of PCa and csPCa by bpMRI and mpMRI were compared stratified by PI-RADS V2 scores .All biopsy samples were reviewed by the same genitourinary pathologist (>15 years of experience). For each prostate cancer-positive biopsy core, the location, Gleason score (GS) based on the International Society of Urological Pathology 2005 consensus , and perP < 0.05 was considered statistically significant.In this study, SPSS 23.0 (IBM) software was used for statistical analysis, and patient characteristics were reported using descriptive statistical methods. Continuous variables such as age, PSA level, PSA density, and prostate volume were compared using the t-test. All continuous variables were expressed in the form of mean ± standard deviation, and the chi-square test was applied for categorical variables, P > 0.05). The overall detection rates of PCa and csPCa were comparable between the bpMRI group and mpMRI group , with no statistical significance.Between January 2018 and December 2021, a total of 699 patients underwent transperineal prostate biopsies in our hospital. Of these 699 patients, 128 were excluded for various reasons , such asP < 0.05), regardless of MRI patterns (P > 0.05) (P = 0.0109) and csPCa  by mpMRI were significantly higher than those by bpMRI, while in other tPSA intervals , the detection rates of PCa and csPCa by bpMRI and mpMRI were comparable , with no statistical significance .The results of multivariate analysis showed that patients' age, tPSA level, PV, and PI-RADS V2 score were independent risk factors for PCa and csPCa detection  . Based o > 0.05)  showed tP > 0.05), with no statistical significance. In addition, we also found that the detection rates of PCa and csPCa by bpMRI and mpMRI were comparable at different PI-RADS V2 scores  , with no statistical significance  and csPCa  by mpMRI were significantly higher than those by bpMRI; in the other tPSA intervals, the detection rates of the two MRI modalities were comparable, with no statistical significance. Our study indicates that when patients' tPSA values are in the 10–20 ng/ml interval, they should undergo mpMRI examination which may improve the detection rates of PCa and csPCa. While in other tPSA intervals, they can only undergo bpMRI examinations for detecting PCa and csPCa instead of mpMRI examinations. We speculate that this difference may be due to the fact that prostate cancer lesions do not perform significantly on bpMRI images in the tPSA 10–20 ng/ml interval, while the addition of DCE sequences can improve the sensitivity of interpretation of suspicious lesions. However, it needs to be further studied.Since the diagnostic performance of the bpMRI method is not inferior to that of the mpMRI, the application of the bpMRI method requires high image quality and reader expertise . In thisAs we all know, the tPSA 4–10 ng/ml is a gray area for prostate cancer determination. When patients' tPSA values is in the gray area, it is often necessary to refer to fPSA and other PSA-related derived indicators such as f/tPSA, PSAD and PSAV (PSA rate) [20]. We supposed mpMRI had higher diagnostic efficiency of PCa and csPCa in the tPSA gray area compared to bpMRI, while further analysis revealed that mpMRI was not superior to bpMRI in this tPSA interval. In addition, in other tPSA intervals , different PVs , and different PI-RADS V2 scores , the detection rates of PCa and csPCa were comparable between the two groups, and the difference was not statistically significant.Our study has some limitations . This waDespite these limitations, our findings validate bpMRI as an alternative to mpMRI for detecting PCa and csPCa in clinical practice. Besides, as a more rapid and simple modality, bpMRI is feasible in our center.The overall detection rates of PCa and csPCa by bpMRI and mpMRI were comparable, but mpMRI detected more PCa and csPCa in the tPSA interval of 10–20 ng/ml. In other tPSA intervals, bpMRI could be an alternative to mpMRI for detecting PCa and csPCa, regardless of different prostate volumes and PI-RADS scores.


Solanum lycocarpum starch bio-based edible films incorporated with a phenolic extract from jaboticaba peel were investigated. Aiming to determine the effect of the polymers and the phenolic extract on the properties of the films, a three-factor simplex-lattice design was employed, and the formulation optimization was based on the produced films’ antioxidant potential. The optimized formulation of the starch-PEJP film showed a reddish-pink color with no cracks or bubbles and 91% antioxidant activity against DPPH radical. The optimized starch-PEJP film showed good transparency properties and a potent UV-blocking action, presenting color variation as a function of the pH values. The optimized film was also considerably resistant and highly flexible, showing a water vapor permeability of 3.28 × 10−6 g m−1 h−1 Pa−1. The microbial permeation test and antimicrobial evaluation demonstrated that the optimized starch-PEJP film avoided microbial contamination and was potent in reducing the growth of Escherichia coli, Staphylococcus aureus, and Salmonella spp. In summary, the active starch-PEJP film showed great potential as an environmentally friendly and halochromic material, presenting antioxidant and antimicrobial properties and high UV-protecting activity.In this study, the antioxidant, antimicrobial, mechanical, optical, and barrier attributes of  The current world demand for improvement in food quality and safety and the concomitant increased concern about the increase in plastic waste production has attracted interest towards the development of eco-friendly packaging presenting low environmental charge in their whole life cycle ,2. RecenSolanum lycocarpum fruit is an excellent source for starch obtention. S. lycocarpum is a common and abundant plant in the Brazilian Cerrado that presents a high resistance to hydric and climatic stress, surviving and fructifying along the year [Amongst the nonconventional starch sources, the year . Its fruCurrently, starch films have been enriched with antioxidant, antimicrobial, and color change indicators to improve the active properties of these films, monitor the freshness, and prolong the shelf-life of perishable products ,4,14. AdPlinia cauliflora) is a Brazilian fruit with a purple peel that is considered a residue of the processing of wine and juice industries [Jaboticaba  as an active component. The film composition was optimized using a three-factor simplex-lattice mixture design. The optimized formulation was characterized by its morphological, physical, optical, mechanical, antioxidant, barrier, pH responsivity, and antimicrobial properties.Citric acid , glycerol , nitric acid , ethanol , methanol , and hydrochloric acid  were purchased from Dinâmica Contemporânea Ltd.a. . Sodium chloride , anhydrous calcium chloride , and sodium hydroxide  were purchased from Neon . Potassium bromide , 2,2-diphenyl-1-picrylhydrazyl (ref. D9132), nylon membrane (ref. WHA7402002), qualitative filter paper (ref. WHA1001325), and cyanidin-3-glycoside chloride (ref. PHL89616) were purchased from Sigma Aldrich Chemical Co. . All other chemicals used were of analytical grade, obtained from accredited companies, and were used as received.S. lycocarpum unripe fruits were collected in Cristianópolis-GO, Brazil (17°11′58″ S and 48°42′12″ W). The isolation of the starch from the fruits was performed in a sequential procedure to obtain insoluble (IS) and soluble starch (SS), following the methodologies described by Pascoal et al. [S. lycocarpum fruits were washed, dehulled, and the seeds were removed. The pulp (20 g) was mixed with 0.8% citric acid solution (100 mL) and milled in a blender  for 5 min. The resultant dispersion was filtered through a sieve of 500 μm, and the solid residue was washed twice with 50 mL of water. The filtrate was left to decant for 12 h at 4 °C, and the IS was separated by centrifugation at 4 °C and 4000× g for 10 min .The l et al.  and Rodrl et al. . Firstly−1) to pH 1.0. The dispersion was incubated at 85 °C (±5 °C) for 30 min, under stirring. Then, the mixture was cooled at room temperature and filtered using a nylon membrane. The starch was isolated by adding 200 mL of cold ethanol to the dispersion. The system was incubated at 4 °C for 12 h, and the precipitated starch was separated by centrifugation at 4 °C and 4000× g for 10 min . The isolated starches (IS and SS) were dried at room temperature (25 ± 2 °C) and stored in airtight vials.The filtration residue was sequentially dispersed in 100 mL of water acidified with nitric acid (2 mol L−1 HCl to pH 4.0) for 2 min, then macerated for 9 h at 4 °C. After maceration, the dispersion was filtered using qualitative filter paper. The filtrate was stored in amber vials at 4 °C. The phenolic [−1). The absorbance of the diluted sample (A) was calculated according to Equation (1):520 is the sample absorption at 520 nm and A700 is the sample absorption at 700 nm .PEJP was prepared according to Montes et al. , using aphenolic  and anthphenolic  were det−1 cm−1), and MP is the molar pathway (1 cm).The total anthocyanin content of the sample was calculated using Equation (2):The starch-based films were produced as described by Prates and Ascheri , varying−2). After autoclaving, the solution was cooled at room temperature (25 °C), and the required content of PEJP was added to the system. Glycerol (0.5% (w/v)) was added to all formulations as a plasticizing agent. The filmogenic dispersion was homogenized under magnetic stirring for 10 min, gently poured on acrylic molds, and left to dry at room temperature until complete solvent evaporation. The dried films were detached from the molds and stored in plastic vials for further analysis.For the film production, SS or IS was firstly dispersed in distilled water and autoclaved  at 121 °C for 20 min  solution (0.15 mmol L−1 in 80% v/v methanol) and incubated for 60 min at room temperature (25 °C) under orbital stirring. The decrease in the absorbance of the resulting solution was monitored at 520 nm in a UV-Visible spectrophotometer . All measurements were performed in triplicates. The antioxidant activity was calculated as a percentage of DPPH radical scavenging using Equation (3):s is the absorbance of the standard DPPH solution at 520 nm and Aa is the absorbance of the mixture reaction containing the tested samples at 520 nm.The antioxidant activity of the starch-PEJP films was determined using an adapted methodology from Brand-Williams et al. . SamplesThe surface and cross-sectional morphologies of the optimized starch-PEJP film were visualized using a JEOL JSM-IT300 scanning electron microscope . The samples were mounted on an aluminum stub using carbon-coated double-sided adhesive tape, sputter-coated with a layer of gold under vacuum , and examined using an accelerating voltage of 5 kV and a magnification varying from 500 to 2000 times. SEM experiments and analyses were performed in the Laboratório Multiusuário de Microscopia de Alta Resolução (LabMic) at the Federal University of Goias, GO, Brazil.−1 (resolution of 2 cm−1), with 16 scans recorded [FTIR spectra of the isolated starches and the optimized starch-PEJP film were acquired on a PerkinElmer FTIR spectrometer . For the test, samples were milled and mixed with potassium bromide (KBr), and the pellets were analyzed in the range of 4000–400 cmrecorded .−1. The thickness of the films was determined using a digital micrometer  at 10 random positions in the sample.The mechanical properties such as tensile strength, elongation at break, and Young’s Modulus of the optimized starch-PEJP films were measured according to ASTM D882-02 standard , using ae, Barueri, SP, Brazil). The analysis was conducted under a nitrogen atmosphere at a heating rate of 10 °C min−1. Samples of approximately 10 mg of the starch-PEJP film were cut as small pieces, placed into an aluminum pan, and heated from 30 °C to 500 °C. The TGA/DTG analysis was performed in the Laboratório de Métodos de Extração e Separação (LAMES) at the Federal University of Goias, GO, Brazil.Thermal properties of the optimized starch-PEJP films were accessed by Thermogravimetric Analysis (TGA) using a TGA/DSC equipment .The light barrier properties of the starch-PEJP film against visible and ultraviolet light were determined by measuring the sample absorbance at a wavelength between 200 and 800 nm using a UV-Visible spectrophotometer . Samples with uniform thickness were cut into 1 × 4 cm strips and placed in the spectrophotometer test cell, and the spectrum was recorded using air as a standard reference. For the opacity determination , the abs600 is the percentage of transmittance at 600 nm, and X is the film thickness (mm).For the transparency tests , the tra−1 HCl solution for achieve pH = 2.0 and 0.1 mmol·L−1 HCL solution for pH 4.0. Milli-Q water was used as the standard for neutral pH (pH = 7.0) and 0.1 mmol·L−1 NaOH solution was the standard for alkaline pH (pH = 9.0).To evaluate the halochromic potential of the starch-PEJP film, 25 μL of different pH solutions were dropped into the film’s surface, and the color changes were registered after 15 min, using a digital camera (Samsung NX 3000). In the tests using acid pH it was used a 10 mmol·L2 were dried at 105 °C for 24 h and weighed. The dried films were immersed in 30 mL of distilled water and stirred at room temperature for 24 h. The flasks containing the film were filtered using a qualitative filter paper , the residues were dried at 105 °C for 24 h, and their final weight was determined. The water solubility was expressed as percentage.To evaluate the water solubility of the starch-PEJP film, it was used an adapted methodology from Rodrigues et al. . Starch-2) were placed on the top of glass cups, filled with 25 g of anhydrous calcium chloride (0% RH), and the edges were covered with parafilm® M to guarantee sealing. After this, each glass cup was placed in a desiccator maintained at 75% RH with a sodium-chloride-saturated solution  and stored at room temperature (25 °C ± 2 °C). The permeability of the starch-PEJP films was determined by linear regression of the constant mass transfer region between weight gain (g) and time (h) which was correlated with the exposed area to determine WVP, as described in Equation (5):−1), X is the average film thickness (m), A is the area of the exposed film surface (m2), and Δp is the water vapor pressure difference between the two sides of the film (1753.55 Pa). WVP was measured for four replicated samples.The water vapor permeability (WVP) of the starch-PEJP films was measured gravimetrically according to the methodology described by Ghasemlou et al. . The sta2 (test area of 1.4 cm2), placed on the top of flasks containing 2 mL of nutrient Broth , and covered the edges with parafilm® M to guarantee sealing. The flasks were left in an open environment at room temperature (25 °C) for 10 days. Open flasks were used as a positive control, and flasks closed with a silicon stopper were used as a negative control. The occurrence of turbidity of the nutrient broth in any flask was recorded as microbial contamination.The capacity to avoid microbial infiltration was tested according to the methodology described by Rodrigues et al. . The sta8 CFU mL−1) of different microorganisms . Then, starch-PEJP films were cut into 4.0 × 1.0 cm strips and laid onto the inoculated plate’s surface. Next, the plate was incubated at 37 °C for 24 h. Antimicrobial efficiency was determined in four replicates by evaluating halo formation around the films and observing microbial growth density.The antimicrobial activity of the starch-PEJP films was evaluated using the agar diffusion method described by Agarwal et al. , with sop > 0.05) by ANOVA. The effect size of each variable and interaction on the antioxidant activity of the produced film was represented using a Pareto chart. The Pareto chart aims to identify the vital few variables that strongly contribute to the response. Pareto rules say that 10% to 20% of the tested elements will determine 60% to 80% of the response. These variables are essential items of concern and must be carefully considered. The desirability function was performed, and the optimum formulation was set to maximal antioxidant activity.The three-factor simplex-lattice design results were analyzed by regression analysis coupled to response surface methodology (RSM) using the software Statistica 10.0 . All measurements were performed at least in triplicate. Results were expressed as mean ± standard deviation (X ± SD). The model was simplified by dropping terms that were not statistically significant (−1 of PEJP and an anthocyanin concentration of 103.2 (± 1.2) μg mL−1. SS and IS were evaluated regarding their antioxidant activity, and results showed 25.2% of DPPH scavenging activity for IS and 78.2% for SS.Before the production of the starch-PEJP films, PEJP was characterized regarding the content of phenolic compounds and anthocyanin, and the results evidenced a phenolic content of 110.4 (± 0.2) μg GAE mLS. lycocarpum starches (IS and SS) and PEJP to produce a film with maximum antioxidant activity, 10 formulations were predicted by a three-factor simplex-lattice design. The starch-PEJP films generally presented a regular and uniform appearance without cracks or bubbles. The results of the antioxidant activity of the prepared starch-PEJP films are shown in To determine the best proportion of As can be observed in However, not only the content of PEJP contributed to the antioxidant potential of the starch-PEJP films, but also the content of SS and the combined effect of PEJP/SS improved the scavenging potential of the films against DPPH radical . The abiThe optimization of the formulation to achieve the maximum antioxidant activity was performed within the composition constraint using the desirability function. The optimal starch-PEJP composition predicted by the model included 1% IS, 0.87% SS and 375 μL of PEJP, with a projected antioxidant activity of 90.83% (d = 0.99). The experimental validation evidenced that the starch-PEJP film presented an antioxidant activity of 91.01% (±0.5), with a thickness of 81.8 μm (±0.6). In this optimized formulation, the starch-PEJP film showed a reddish-pink color, a regular and uniform appearance, and no cracks or bubbles .−2 assigned to the stretching of -OH groups, a peak around 2940–2910 cm−1 related to the vibration of -CH bonds from glucose, and a peak in the region of 1647 cm−1 assigned to scissors vibrations of hydroxyl groups from hydration water. [−1, ascribed to the C=O stretching of carboxylic acid, which is absent in the FTIR spectrum of IS. However, the formation of saccharic acid in the anomeric regions of the starch molecule did not impair the backbone structure of the starch molecule, with typical peaks being observed in the region of 1200–800 cm−1 from both starches, SS and IS. This region is referred to as a fingerprint region for polysaccharides, presenting bands related to the stretching vibrations of C-O-C and C-O-H from glycosidic linkages [FTIR analysis was used to characterize the SS and IS and evaluate the interactions between the polymers and PEJP in the optimized formulation of the starch-PEJP film. The FTIR spectra of SS and IS a showed n water. ,35. The linkages ,36.−1 observed in the native starches (−1) in the produced film  indicates the extensive degradation of the sample, with a weight loss of 48.9% being observed . In thisThe color changing of starch-PEJP film at different pH values is shown in At pH 4.0, the optimized starch-PEJP film did not present color alteration, and at pH 6.0, it was possible to observe a slight brownish tone starting to appear. This color change can be ascribed to the conjugation of water molecules to the heterocycle structure of the anthocyanin molecules and the increase in pH, which leads to a neutralization of the positive charges and the formation of the colorless carbinol pseudo-base ,54. WhenIn recent years, there has been growing interest in developing intelligent food packaging to reduce food waste and enhance consumer safety. The possibility of developing a smart coating of high sensitivity and safety that provides direct information without damaging the package integrity just by optical/visual color changes is of great interest ,44. TherAiming to evaluate the ability of the optimized starch-PEJP film to avoid microbial contamination and permeation, the microbial penetration was assessed, and the results are shown in Considering the flasks covered with the starch-PEJP film, the results evidenced that the coating could prevent the penetration of microorganisms into the medium, being observed no visible microbial growth, and the maintenance of a transparent medium . This reStaphylococcus aureus (Escherichia coli (Salmonella spp. (Together with avoiding microbial permeation, the inhibition of microbial growth is a desired feature for antimicrobial food packaging and coating materials . To detes aureus a rather hia coli b and Sallla spp. c. This aThe tolerance against antimicrobial molecules is strongly correlated with the structural differences between these two types of bacteria. It is possible that the absence of a lipid membrane in the cell wall of the Gram-positive bacteria could facilitate PEJP to enter and interfere with cellular metabolism. Nevertheless, the more complex nature of the cell wall of the Gram-negative strains, with a thick outer lipopolysaccharide membrane, makes difficult for the PEJP to pass through the outer layer and cause alterations in their cytoplasm ,57.Moreover, this partial inhibition can be ascribed to the concentration of PEJP used in the film production, which could be in insufficient quantity to completely inhibit the microbial load present in the plate. However, considering that it is possible to increase the content of PEJP by increasing the volume of film-forming solution used in the casting procedure, it is possible to infer that the starch-PEJP film is promising as a bioactive material containing natural antimicrobial compounds that avoid microbial penetration and diminish food microbial spoilage.We obtained a halochromic starch-PEJP film with efficient antioxidant and antimicrobial activity in this study. The simplex-lattice mixture design was effective in obtaining mathematical models to predict response values. Consequently, it was a helpful tool to optimize the starch-PEJP formulation to the maximum antioxidant activity. The optimized formulation showed a reddish-pink to greenish color as a function of the pH, suggesting this film is an effective smart material to be used as a natural indicator for the quality of foodstuffs or even be used in the real-time monitoring of food freshness. Future studies are required to explore those applications.The optimized starch-PEJP film also showed high extensibility with low stiffness and good properties of transparency and opacity. Furthermore, the starch-PEJP film demonstrated strong antimicrobial effectiveness, being an efficient barrier against microbial permeation and can reduce the growth of Gram-positive and Gram-negative bacteria. Therefore, the starch-PEJP film has potential applications in the food industry as bioactive coverage with active functionality as a natural indicator with potent antioxidant and antimicrobial properties. Additionally, the results of this study could be used as a guideline for producing environmentally friendly smart active films for the packaging of foods by simultaneously studying various variables and their effects on the material properties and bioactivities.


Estudaram prospectivamente 231 pacientes com AC estabelecida  e os compararam com o mesmo número de indivíduos saudáveis. Alguns pacientes do grupo AC foram avaliados no contexto de acidente vascular cerebral agudo. As características clínicas basais foram bem equilibradas entre os dois grupos. Como esperado, os pacientes com AC moderada a grave  apresentaram níveis mais elevados de marcadores inflamatórios, placas carotídeas mais instáveis e níveis mais elevados de colesterol LDL em comparação com indivíduos saudáveis e de gravidade leve a moderada. Os pacientes com AC apresentaram níveis mais baixos de PM20D1 em comparação com indivíduos saudáveis. Também, os pacientes com placas instáveis e AC mais grave apresentaram níveis significativamente mais baixos. Este biomarcador também está negativamente correlacionado com marcadores inflamatórios, mas não com perfis lipídicos. Nenhuma diferença foi encontrada de acordo com o índice de massa corporal. Este biomarcador apresentou boa acurácia discriminativa pela análise da curva ROC para AC  e AC grave . Infelizmente, apenas relatam dados de regressão logística binária para as variáveis estudadas. Por esse motivo, não é possível confirmar com os dados disponíveis se este novo biomarcador é um preditor independente de desfecho e se tem valor prognóstico agregado em relação aos parâmetros clássicos. Além disso, os autores observaram que pacientes com níveis mais altos de PM20D1 apresentaram níveis mais baixos de colesterol LDL. No entanto, ao contrário do relato dos autores, não foi observada correlação significativa . Os autores não explicaram esse achado; se colocarmos em contexto, a relação entre eles está agora em questão. Por esse motivo, ainda existem importantes questões não respondidas sobre a interação entre os dois.O artigo publicado por Huang et al.No geral, este estudo lançou alguma luz inicial sobre o possível papel dessa via na AC. No entanto, várias questões permanecem sem resposta, e também carece de informações adicionais sobre a aplicabilidade clínica do método, principalmente na prática diária, já que esse parâmetro ainda não está pronto para uso clínico. 1 Furthermore, in the analysis of the Global Burden of Diseases 2019 report, cardiovascular risk factors represent 75% of all cardiovascular burden, particularly hypertension, with an important impact on mortality.1 This is also relevant in Portuguese-speaking countries.2 Carotid intima-media thickness has long been recognized as a surrogate marker for coronary artery disease and has a relevant prognostic impact.3 For that reason, it is a useful tool in cardiovascular risk stratification.Cardiovascular diseases are the leading cause of death worldwide, representing around 30% of all deaths, particularly in developed countries, including cardiovascular and cerebral-vascular diseases.4 PM20D1 and NAAA activity regulation in blood plasma is still largely unknown.4 However, what is already known is that PM20D1 circulates in tight association with both low- and high-density lipoproteins that are powerful co-activators of PM20D1 activity in vitro and NAAA biosynthesis in vivo.4 Serum albumin is also a physiologic NAAA carrier that separates NAAA away from their sites of production, conferring resistance to hydrolytic degradation and establishing an equilibrium between thermogenic “free” versus inactive “bound” fractions.4 It has been hypothesized that lipoprotein particles are probably the main extracellular sites of NAAA biosynthesis, and this supports the concept that a lipoprotein-albumin network regulates the activity of circulating thermogenic lipid family.4N-acyl amino acids (NAAA) are a family of cold-inducible circulating lipids that stimulate thermogenesis, and their biosynthesis in brown adipocytes is mediated by a secreted enzyme called Peptidase M20 domain containing 1 (PM20D1).6 There is also an association with polycystic ovarian syndrome.7 In humans, Increased serum levels of PM20D1 and its catalytic products (NAAA) are also associated with obesity-related glucose dysregulation, insulin resistance and metabolic syndrome and can be potentially used as clinical biomarkers for diagnosing and monitoring these disorders.8Abnormalities in the PM20D1 gene have been associated with several diseases, particularly neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease.9 in the present issue of Arquivos Brasileiros de Cardiologia studied the role of PM20D1 in carotid atherosclerosis (CA). They prospectively studied 231 patients with established CA (assessed by carotid ultrasound) and compared them with the same number of healthy individuals. Some patients in the CA group were assessed in the context of acute stroke. Baseline clinical characteristics were well balanced between both groups. As expected, patients with moderate to severe CA (including those with stroke) had higher levels of inflammatory markers, more unstable carotid plaques and higher LDL cholesterol levels compared with mild to moderate severity and healthy individuals. Patients with CA had lower levels of PM20D1 compared to healthy individuals. Also, patients with unstable plaques and more severe CA had significantly lower levels. This biomarker is also negatively correlated with inflammatory markers but not lipid profiles. No difference was found according to body mass index. This biomarker showed good discriminative accuracy by ROC curve analysis for CA (cut-off 5.4 ng/mL) and severe CA (3.99 ng/mL). Unfortunately, they only report binary logistic regression data for the studied variables. For that reason, it is not possible to confirm with the available data whether this new biomarker is an independent predictor of outcome and if it has added prognostic value compared to the classical parameters. Also, the authors observed that patients with higher levels of PM20D1 had lower LDL cholesterol levels. However, contrary to the authors’ report, no significant correlation was observed . The authors did not explain this finding; their relationship is now in question if we put it into context. For that reason, there are still important unanswered questions regarding the interaction between both players.The paper published by Huang et al.Overall, this study shed some initial light on the possible role of this pathway for CA. However, multiple questions remain unanswered, and it also lacks additional information on the clinical applicability of the method, particularly in daily practice, because this parameter is not yet ready for its clinical use.


Toxoplasma gondii for humans. The study described here was designed to search for a novel group of optimal B cell and T cell epitopes from Toxoplasma membrane proteins using genome-wide comprehensive screening.There is presently no effective and safe vaccine for T. gondii were obtained from the UniProt database. The ABCPred and BepiPred servers were employed to predict the linear B cell epitopes. The Immune Epitope Database (IEDB) online service was utilized to forecast T cell epitopes within T. gondii membrane proteins that bind to human leukocyte antigen (HLA) class I (HLA-I) or HLA-II molecules.The amino acid sequences of membrane proteins of T. gondii, a total of 14 linear B cell epitopes embedded in 12 membrane proteins were identified. Eight epitopes for major histocompatibility complex (MHC) class I (MHC-I) molecules and 18 epitopes for MHC-II molecules were ultimately selected, for which world population coverage percentiles were 71.94% and 99.76%, respectively. The top rated combinations of linear B cell epitopes and T cell epitopes covering both BALB/c mice and a majority of the human population were identified for the development of a protective vaccine.From the 314 membrane proteins of T. gondii infection by inducing humoral and cellular immune responses.The ultimate vaccine construct described here, which comprises B cells, MHC-I and MHC-II epitopes, might protect individuals against The online version contains supplementary material and is available at 10.1186/s13071-022-05497-z. Toxoplasma gondii is an intracellular parasite that causes toxoplasmosis in humans and animals [Toxoplasma infection is important for public health. animals , 2. Infe animals . Moreove animals . Thus, pT. gondii than chemotherapy [T. gondii for humans, mainly due to limitations in vaccine development strategies and lack of good candidate vaccine molecules [T. gondii, and there has been no extensive and systematic search of Toxoplasma proteins. Moreover, experimental results derived from mice, the most widely used animal model for T. gondii vaccine development, are inevitably biologically biased due to the genetic differences between mice and humans [Vaccination is a more effective approach for otherapy . Howeverolecules . Most ofd humans .Toxoplasma membrane proteins (including uncharacterized proteins). The steps of the in silico analysis are illustrated in Fig. The present study was designed to unbiasedly search for a novel group of optimal B cell and T cell epitopes. Bioinformatics and immunoinformatic analysis were conducted for a genome-wide range of T. gondii were obtained from the UniProt database (https://www.uniprot.org/uniprot/). ABCPred [Surface proteins and cell membrane proteins were identified as targets. The amino acid sequences of membrane proteins of  ABCPred , and the ABCPred  servers T. gondii membrane proteins that bind to human leukocyte antigen (HLA) class I (HLA-I) or HLA-II molecules [The Immune Epitope Database (IEDB) online service was utilized to forecast T cell epitopes within olecules , 10. Forolecules .H2-Db, H2-Kb and H2-Ld) and two MHC-II alleles (H2-IAd and H2-IEd) from BALB/c mice were employed [BALB/c mice, the laboratory animals most commonly used in vaccine studies, were selected as the targets to screen for major histocompatibility complex (MHC) molecules that bind to epitopes. Three MHC class I (MHC-I) alleles . The global population coverage rate as well as those of 11 major regions of the world were computed. For HLA-I, nine world regions showed more than 50% population coverage, with the highest population coverage in Europe, at 81.48%, and in North America, at 76.79%  with a score > 0.7 480–494 peptides in the vaccine construct, which are experimentally determined epitopes .Additional file 13: Figure S3. Population coverage analysis. (a) Population coverage analysis of the 13 Recommended HLA-I alleles; (b) Population coverage analysis of the 19 Recommended HLA-II allelesAdditional file 14: Figure S4. Selected T cell binding epitopes. (a) CD8+ T cell epitopes and their corresponding MHC-I alleles in mice and human; (b) CD4+ T cell epitopes and their corresponding MHC-II alleles in mice and humanAdditional file 15: Table S1. The initial result of the joint prediction using BepiPred-2.0 and ABCPred.Additional file 16: Table S2 Antigenicity, allergenicity, toxicity, transmembrane localization, signal peptide and hydrophilicity profiling of selected peptides.Additional file 17: Table S3 Alignment of experimentally verified epitopes and selected sequences in the vaccine construct.


Anthocyanins reduce starch digestibility via carbohydrase-inhibitory pathways, but food matrix effects during digestion may also influence its enzymatic function. Understanding anthocyanin-food matrix interactions is significant as the efficiency of carbohydrase inhibition relies on anthocyanin accessibility during digestion. Therefore, we aimed to evaluate the influence of food matrices on black rice anthocyanin accessibility in relation to starch digestibility in common settings of anthocyanin consumption—its co-ingestion with food, and consumption of fortified food. Our findings indicate that black rice anthocyanin extracts (BRAE) had reduced intestinal digestibility of bread to a larger extent for the co-digestion of BRAE with bread (39.3%) (4CO), than BRAE-fortified bread (25.9%) (4FO). Overall anthocyanin accessibility was about 5% greater from the co-digestion with bread than fortified bread across all digestion phases. Differences in anthocyanin accessibility were also noted with changes to gastrointestinal pH and food matrix compositions—with up to 10.1%  and 73.4%  reductions in accessibility with pH changes, and 3.4% greater accessibility in protein matrices than starch matrices. Our findings demonstrate that the modulation of starch digestibility by anthocyanin is a combined result of its accessibility, food matrix composition, and gastrointestinal conditions. The shift in diets to one rich in polyphenols has been advocated as a dietary strategy against the pathogenesis and regression of metabolic disorders ,2. AnthoThe efficacy of carbohydrase inhibition is, however, limited by the availability of free anthocyanins from food matrices under the physiological conditions of digestion. In nutrient metabolism, accessibility is generally referred to as the fraction of nutrients liberated from the food matrix into the gastrointestinal tract and are available for absorption . NutrienLimited studies have investigated the gastrointestinal accessibility of anthocyanins in foods ,9,10 or Materials used for the preparation of bread included bread flour (13% protein), vegetable shortening, cane sugar, salt, and dry active yeast, which were purchased from a local supermarket. BRAE used in the fortification of bread was  obtained from Shaanxi Bolin Biotechnology Co Ltd. .Aspergillus niger (A7095), which were obtained from Merck Pte Ltd. . The D-glucose hexokinase assay kit (K-GLUHK) was obtained from Megazyme Inc. .Materials used for in-vitro experimentation included wheat starch (S5127) and gluten (G5004), porcine pancreatic α-amylase (A3176), porcine pancreatic pepsin (P6887), porcine pancreatin (P7545), and α-amyloglucosidase from The bread was decided upon as a potential delivery vehicle for anthocyanins as bakery products are among the most commonly consumed foods rich in rapidly digestible carbohydrates . Bread sA simulated gastrointestinal digestion was conducted to assess the impact of BRAE fortification on bread digestibility, according to the standardised methods developed by Brodkorb et al. . Additiow/v) to reduce the viscosity of the oral bolus. Simulated salivary fluid (SSF), α-amylase solution (75 U/mL), and 0.3 M CaCl2 (1.5 mM) were mixed with 2 g of homogenised samples. The mixtures were topped up to volume with distilled water and incubated for 2 min at 37 °C in a water bath with agitation. Aliquots of 100 μL were quenched in 900 μL of ethanol after incubation.To simulate oral digestion, the mastication process was first replicated via a brief homogenisation  of breadcrumbs and distilled water (1:4 2 (0.15 mM) were added to the oral digesta and adjusted to pH 3.0. The mixtures were topped up to volume with distilled water and incubated for 2 h at 37 °C in a water bath with agitation. Aliquots of 100 μL at 0, 15, 30, 45, 60, 90, and 120 min were quenched in 900 μL of ethanol.To simulate gastric digestion, simulated gastric fluids (SGF), pepsin solution (2000 U/mL), and 0.3 M CaCl2 (0.6 mM) were added to the gastric digesta and adjusted to pH 7.0. The mixtures were topped up to volume with distilled water and incubated for 4 h at 37 °C in a water bath with agitation. Aliquots of 100 μL at 0, 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, and 240 min were quenched in 900 μL of ethanol.For intestinal digestion, simulated intestinal fluids (SIF), α-amyloglucosidase (1.3 U/mL), pancreatin , and 0.3 M CaClQuenched aliquots were centrifuged for 10 min at 15,000 g. The resulting supernatant was used to quantify the glucose released according to the Megazyme D-glucose hexokinase assay kit (K-GLUHK). Starch hydrolysis data was fitted into the first order equation Equation (1).Using the trapezoid rule, area under curves (AUC) was determined from the fitted hydrolysis plots generated from Equation (1).g at 20 °C for 10 min to quench the mixtures. The supernatants were collected for anthocyanin content determination, as described in To determine the effects of food matrices and digestive phases on the gastrointestinal accessibility of anthocyanins, the anthocyanin content in each digestion phase was quantified for both 4FO and 4CO samples. Aliquots of digesta from the end of each simulated digestion phase  were centrifuged at 8000× w/w) and heated at 100 °C for 5 min. The mixtures were allowed to cool to room temperature, and to allow for starch gelatinization. Next, 2 g of each mixture was mixed with 80 mg of BRAE. Simulated fluids without digestive enzymes, corresponding to the volumes used in To evaluate the effects of interactions between anthocyanins and digestion matrix constituents on anthocyanin accessibility, the anthocyanin content of mixtures comprising BRAE with starch, gluten, or enzymes was quantified. Briefly, different distributions of wheat starch and gluten were mixed with water (1:4 v/v) in methanol using at 200 rpm. After each maceration, the samples were centrifuged for 10 min . The supernatants were combined and filtered through a 11 μm pore-sized filter paper and condensed at 40 °C under vacuum. The condensed liquid extracts were topped up with 0.1% formic acid and stored away from light at −20 °C until further analyses. A ×200 and ×10 dilution of condensed extracts from BRAE and lyophilized 4FO samples were performed, followed by filtration through a 0.22 μM PTFE filter, prior to analysis.The extraction of anthocyanins from BRAE and 4FO was performed according to the methods by Ou, Yu, Zhou, and Liu . BRAE  on a HPLC system  connected with a diode array detector (DAD). The injection volume was 50 μL. The flow rate and oven temperature were maintained at 1 mL/min at 25 °C. A gradient elution process was applied : 0% B for 5 min, 10% B at 20 min, 13% B at 40 min, 20% B at 44 min, 25% B at 50 min, and 100% B at 55 min. Detection of anthocyanins was performed at 520 nm. Identification and quantification of each anthocyanin was based on matching the retention time and peak areas with the external calibration curve of respective standards.To determine the distribution of major anthocyanins in the extracts and supernatants, samples were analyzed using a reverse-phased Cp < 0.05.Experiments were carried out in triplicates, with three repeats conducted for each replicate, and expressed in means with standard error of means, unless stated otherwise. Statistical analyses were conducted using Prism 9 . Differences in means between CON, 4FO, and 4CO were assessed using one-way analysis of variance (ANOVA) with Tukey’s post hoc test, in which differences were considered statistically significant at The total anthocyanin content of BRAE and 4FO was determined to be 240.6 mg/g of extract and 127.3 mg/g of bread in C3G equivalents, respectively. The distribution of major black rice anthocyanin species in BRAE and 4FO were also quantified and presented in k) for intestinal starch hydrolysis were statistically significant in 4CO, but not so in 4FO  in the aglycone [On top of the possible inhibitory pathways of starch digestibility, we further explored other factors determining the extent of anthocyanin accessibility in a gastrointestinal environment. From our results, the gastrointestinal transit of food had an impact on anthocyanin accessibility. By quantifying changes in the release of anthocyanins between adjacent digestion phases, we noted a strong influence of changing pH conditions on the accessibility of anthocyanins during gastrointestinal passage , Table 2aglycone , which maglycone . Incidenaglycone ,30.While recent studies have confirmed the non-covalent interactions of anthocyanins with starch and/or proteins ,23,31, tAssessing the inter-relationship among food matrices, anthocyanin accessibility, and the gastrointestinal digestibility of starch is overtly a complex task, making it inherently difficult to attribute our observations to a singular mechanism. From our findings, it can only be generally concluded that the extent of starch digestibility and nutrient  accessibility is a collective result of concurrent interactions among food matrix constituents, anthocyanins, and gastrointestinal conditions. A simplified visualisation of the roles played by the aforementioned factors in starch digestion and nutrient accessibility is presented in This work investigated the effects of bread matrices on the modulatory effects of BRAE in relation to starch digestibility. Simulated gastrointestinal digestion of bread had not only confirmed the inhibitory effects of BRAE on bread digestibility, but also showed that the inhibition was reduced by the bread matrix. While anthocyanin accessibility was suppressed by the bread matrix and intestinal conditions, the bread matrix also had a protective effect over free anthocyanins. Notably, the observed impact of food matrix compositions on the release of anthocyanins during digestion has suggested the food matrix-specific nature of polyphenol accessibility. Conclusively, our findings highlight that the overall bread digestibility is a result of several non-exclusive interactions among black rice anthocyanins, bread matrices, its dietary constituents, and gastrointestinal conditions functioning in unison.


From the results, CaAl4O7 plays a vital role in the slag corrosion resistance of the three refractories. Regarding CA6 refractory, the double pyramid module in CA6 crystal structure was destroyed very quickly, leading to the rapid collapse of its structure to form the denser CaAl4O7 in high amounts. As a result, a reaction layer mainly composed of CaAl4O7 formed, which effectively inhibited the slag corrosion, so CA6 refractory exhibits the most excellent slag corrosion. Meanwhile, the formation of CaAl4O7 can also avoid CA6 particles entering the molten steel to introduce exogenous inclusions. For Al2O3 refractory, the generation of CaAl4O7 is much slower than that of CA6 and CA6-Al2O3 refractory, and the amount generated is also quite small, resulting in its worst slag corrosion among the three crucibles. Therefore, CA6 based refractory has excellent application potential in ladle refining and clean steel smelting. In this study, to clarify the corrosion mechanism of CA During the ladle refining process, the refractory is corroded severely due to the continuous reaction of the refining slag ,2,3. The2O3 system refractories, due to their high density and stable high temperature properties. Many researches have been conducted to investigate the corrosion process between Al2O3 refractories and the refining slag [2O3 refractories is influenced by factors such as temperature and refining slag composition [12O19 generated on the surface of Al2O3 refractories can prevent further corrosion effectively, which is a critical mechanism. After years of development, an understanding of the corrosion resistance of Al2O3 refractories has been profound, but there are few reports on the introduction and control of inclusions. So far, as Al2O3 refractories, the problem of introducing inclusions into molten steel is still unavoidable. With the development of cleanliness steel smelting, the effect of refractories on inclusions has attracted more and more attention. In order to prepare refractory materials with excellent corrosion resistance and less introduction of inclusions, in our previous work [6  base raw material and studied the slag corrosion resistance. After that, some researchers focused on optimizing CA6 structure and tried to dope N3− or Zr4+ into CA6 [6-based refractories not only have excellent slag corrosion resistance but can also reduce the size of inclusions and absorb sulfur in steel. Researches on the preparation and structural improvement of CA6 refractory are consummate. However, its slag corrosion mechanism is not clear, and the effect on the exogenous inclusion is not confirmed, which cannot provide strong support for promotion and application. Therefore, it is essential and meaningful to study the slag corrosion of CA6-based refractory, which has a great significance on refining ladle and even on the whole steelmaking process.At present, the primary materials for the refining ladle are Aling slag ,22,23, aposition ,25,26,27position ,29 a smaous work ,32,33,34into CA6 ,36, tryi6-based refractory (pure CA6 and CA6-Al2O3 composite) and refining slag was investigated and analyzed, and the Al2O3 refractory commonly used in the refining ladle was also used as a comparison. The microstructure and distribution of elements in the corrosion area were characterized, and the corrosion process was deduced and simulated by the thermodynamic software. The results of this work proved that CA6 refractory has a great application prospect in the metallurgical industry due to excellent and distinctive slag corrosion resistance.In this study, the reaction mechanism between two CA6, Al2O3, and CA6-Al2O3 crucibles were prepared with CA6 powder , Al2O3 powder , and the CA6 and Al2O3 powder (mass ratio CA6: Al2O3 = 1:1), respectively. The static crucible method was adopted for the corrosion resistance test. Firstly, the dried powder was pressed under 30 MPa into 80 × 80 × 80 mm3 cubes. Secondly, cylindrical holes with a diameter and height of 40 mm were drilled in the cubes as slag holes. Finally, the crucibles were fired at 1650 °C for 3 h. The properties of the crucibles are shown in The CA2O3 , MgO  and SiO2. The chemical composition of the refining slag is shown in The static crucible method was used to investigate the slag corrosion behavior of the three refractories. Put 70 g refining slag into the crucibles and then heat the crucibles to 1600 °C at a heating rate of 5 °C/min in the box furnace (KSL-1700X-M). After holding for 3h, the furnace stopped working and the crucible was taken out when it cooled to room temperature. The crucibles were cut along the center, and the corrosion area was made into a mosaic sample and then polished. The composition of the refining slag was analyzed by X-ray fluorescence spectrometry . The micro-morphology of the slag-refractory interface was observed by scanning electron microscopy . The elemental distribution at the slag-resistant material interface was analyzed by SEM equipped with Energy Dispersive Spectrometer .During the corrosion test, only the final result can be determined clearly and the intermediate process cannot be observed directly. Therefore, thermodynamic simulation for the slag corrosion process is necessary.In this work, Factsage7.0 was used to simulate the corrosion process of three crucibles. The calculation mechanism is shown in The variable <X> in the Equilib module is used to calculate the inverse crossover interdiffusion, based on which to simulate the corrosion process of the refractory from thermodynamic aspect. When X = 0, the refining slag is all the composition of the system. When x = 1, the refractory is all the composition of the system. At the beginning of the reaction, X was defined as 1. With the decrease of X, more and more slag is involved in the reaction. The oxide data included in the calculation are available in the FToxid module. The calculated temperature was set at a constant 1873 k and the pressure was 1atm.2O3 increased in all three crucibles. It indicates that the CaO and MgO in the slag reacted with or entered into the refractories, and Al2O3 in the refractories diffused into the slag during the corrosion process. The content of the SiO2 in the slag remained almost unchanged, which is due to the large ionic radius of the silicate ion. The composition of the slag variation shows that the three crucibles were corroded by the refining slag in varying degrees.The composition of slag always changed due to the reaction with refractory. 6 crucible after slag corrosion are shown in 6 crucible shows excellent slag resistance. At the slag-crucible interface, the width of the reaction layer is 50~60 μm, which is the thinnest of the three crucibles . The microstructure results of the CA4O7 generated at the slag-refractory interface (reaction layer), and most of the components in the reaction layer are CaAl4O7 (such as the EDS results of area 1). At the experimental temperature, liquid slag penetrated into the refractory through the pores and reacted with refractory to form CaAl4O7, which is the main reaction during the corrosion test. The corrosion of the refractory by the liquid slag was effectively inhibited due to the high viscosity of the CaAl4O7. As the time increased, CaAl4O7 continued to react with CaO in the liquid slag to form CaAl2O4 in the reaction layer. In addition, a very small amount of slag phase of Al2O3-CaO-SiO2-MgO was found in the reaction layer. It can be clearly found that the microstructure of the reaction layer was denser than that of the original bricklayer. One of the reasons is the gaps and pores in the reaction layer were filled by CaAl4O7 and CaAl2O4, which is also an important reason for preventing further corrosion of the refining slag. To reveal the penetration degree of the refining slag, area 2 was selected randomly for EDS analysis. The very limited Mg and Si were detected, indicating that the CaAl4O7 layer effectively prevents the penetration of the refining slag.The EDS results show that CaAl2O3 crucible after the static crucible test. As can be seen from 2O3 generated in the reaction layer and it was formed by the reaction between MgO in the refining slag and Al2O3 in the refractory, which is the main component in the reaction layer. Some researchers [2O3 at the interface has some hindrance to the penetration of slag. Meanwhile, trace amounts of CaAl4O7 were also detected in the reaction layer, but its generation mechanism is different from the CA6 crucible. The Al2O3 in the refractory and CaO in the slag reacted to form CaAl12O19 first, and then the CaAl12O19 continued to react with CaO in the slag to form CaAl4O7 in the reaction layer. Compared with the CA6 crucible, EDS results (area 3) show that the content of Mg in the reaction layer increased obviously and the amount of Ca decreased. It is confirmed again that the main phase was the MgO·Al2O3 and the content of CaAl4O7 was limited in the reaction layer. Meanwhile, it can be noticed that the liquid slag phase was found in the reaction layer, and its content is more than that in the CA6 crucible. In the penetration layer, except for the Al2O3 particles, the CaAl12O19 is also found in this area, proving that the Ca in the refining slag gradually penetrated into the refractory through the pores or cracks and reacted with the refractory. In addition, some of slag phase of Al2O3-CaO-SiO2-MgO was found in the penetration layer, proving that the reaction layer does not have an advantage in preventing slag penetration. The EDS results of area 4 show that the content of Mg and Si in the penetration layer is more than that of CA6 and CA6-Al2O3 crucibles, indicating the Al2O3 crucible has the worst slag corrosion resistance of the three crucibles. The EDS results show MgO·Alearchers ,27 point6-Al2O3 crucible after the slag corrosion. From left to right are the slag layer, reaction layer, penetration layer, and original brick layer, respectively. From 6-Al2O3 crucible, and its width was about 200 μm, which was wider than that of the CA6 crucible and thinner than that of the Al2O3 crucible. The microstructure of the reaction layer was denser compared to the original brick layer. In the reaction layer, CaAl4O7 is found from the EDS results, and it is quite possibly generated by the reaction between CA6 in the refractory and CaO in the refining slag. Of course, a tiny part of CaAl4O7 may also come from the multistep reaction of Al2O3 in the refractory and CaO in the refining slag. In the CA6 crucible, the CaAl4O7 can inhibit further corrosion of the refining slag. However, the content of CaAl4O7 in the CA6-Al2O3 crucible was lower than that in the CA6 crucible due to the limit of raw materials, which is one of the reasons for the wider reaction layer than that of the CA6 crucible. In the reaction and penetration layer, a similar position to that of the CA6 and Al2O3 crucibles was selected for EDS analysis. The results show the content of Ca in the reaction layer is less than that in the CA6 crucible but more than in the Al2O3 crucible, so it can be deduced that the amount of CaAl4O7 in the reaction layer is between the other two crucibles. In the penetration layer, the amount of Mg and Si was decreased compared with the Al2O3 crucible, indicating the slag corrosion resistance of the CA6-Al2O3 crucible is better than the Al2O3 crucible but worse than the CA6 crucible.6-Al2O3 crucible. From 2O3 crucibles, which was also confirmed by the EDS result of area 6, proving the slag resistance of the CA6-Al2O3 crucible was worse than the CA6 crucible but better than Al2O3 crucible.6 crucible shows the best slag corrosion resistance, followed by the CA6-Al2O3 crucible, and the Al2O3 crucible is the worst. The reaction mechanism of the CA6 and Al2O3 crucible with the refining slag is shown in 6 particles reacted with the refining slag to form CaAl4O7 that can fill the pores or the gaps between the particles. Due to the high viscosity of CaAl4O7, the slag-refractory interface will be denser and has a positive effect on inhibiting the slag corrosion. At the same time, it should be noticed that the probability of CA6 particles entering the molten steel is greatly reduced due to the formation reaction layer, so the introduction of exogenous inclusions from the refractory is greatly reduced. In our recent work, it has been proved that the reaction product of the CA6 refractory and refining slag can decrease the number and size of the inclusions. For the Al2O3 crucible, in addition to reacting with the Al2O3 particles at the interface, the refining slag also reacts with the inside Al2O3 crucible by penetrating into the refractory through the pores and the gaps between the Al2O3 particles, as shown in 2O3 particles gradually dissolve or fall off into the refining slag, causing the more serious corrosion of the crucible, which is shown in 6-Al2O3 crucible, the CaAl4O7 is generated faster and the content is more due to the CA6 raw materials compared with the Al2O3 crucible. Therefore, the slag corrosion resistance of the CA6-Al2O3 crucible is better than that of the Al2O3 crucible.Through the above analysis, it can be found that the CA6 crucible are shown in The results of the thermodynamic simulation of the corrosion process of the CA4O7, Ca2Mg2Al28O46, CaAl12O19, and liquid slag were obtained. With the decrease of X, the content of CaAl4O7, Ca2Mg2Al28O46, and liquid slag increased while the content of CaAl12O19 decreased sharply. When X = 0.76, the content of Ca2Mg2Al28O46 reached a maximum of 40.95% and the content of CaAl12O19 was zero. Ca2Mg2Al28O46 and CaAl4O7 generated by the reaction of Equations (1) and (2):From 2Al16O27 appeared and increased with decreasing X while the content of Ca2Mg2Al28O46 gradually decreased until the content was zero at X = 0.7, indicating that Ca2Mg2Al28O46 gradually transformed into CaMg2Al16O27 and CaAl4O7 following the Equations (3) and (4) respectively:When 0.76 < X < 0.7, a new phase of CaMg2Al16O27 decreased gradually, and the content of CaAl4O7 increased gradually with the decrease of X, which indicated the CaMg2Al16O27 continues to be converted to the CaAl4O7 by the equation (6). When X = 0.6, the content of CaMg2Al16O27 was zero, while the content of CaAl4O7 and spinel reached the maximum content of 49.04% and 8.91%, respectively:When 0.7 < X < 0.6, the spinel phase appeared in the system at X = 0.66 through the reaction of Equation (5). The content of CaMg4O7 decreased sharply and the liquid slag increased rapidly. When X = 0.47, the content of CaAl4O7 was zero. When 0.47 < X < 0.14, two phases of liquid slag and spinel were in the system, and the content of spinel decreased to zero at X = 0.14. When 0.14 < X < 0, the liquid slag was the only phase in the system. When 0.6 < X < 0.47, the content of the CaAl2O3 crucible and CA6-Al2O3 crucible are shown in 2O3 and CA6-Al2O3 crucibles are roughly similar to the CA6 crucible. However, significant differences existed in the beginning stage of the corrosion. For the Al2O3 crucible, when 1 < X < 0.76, the Al2O3 converted to CaAl12O19 gradually by Equation (7), and the Ca2Mg2Al28O46 can also be generated through Equation (8). When X = 0.76, the phase of CaAl4O7 generated in the system and its content reached the maximum of 34.18% when X = 0.48, which is lower than that of the CA6 and CA6-Al2O3 crucible. For the CA6-Al2O3 crucible, the Al2O3 converted to CaAl12O19 first, and then CaAl12O19 started to react with the refining slag to form CaAl4O7 at X = 0.86. And the maximum content of CaAl4O7 is 40.11% when X = 0.54, which is higher than that of the Al2O3 crucible but lower than that of the CA6 crucible: The results of the thermodynamic simulation of the Al4O7 is the critical point in enhancing the slag resistance of the crucible [4O7 of the CA6, Al2O3, and CA6-Al2O3 crucible is 49.04%, 34.18%, and 40.11%, respectively, through the thermodynamic simulation results, which the trend is consistent with the EDS results. The CaAl4O7 was formed at the beginning of the reaction for the CA6 crucible. For the Al2O3 and the CA6-Al2O3 crucible, the CaAl4O7 was formed at X = 0.54 and 0.86, respectively. Therefore, the CaAl4O7 generated fastest and the amount was the most during the corrosion process of the CA6 crucible, which had a great benefit on the slag resistance corrosion.From the analysis of thermodynamic simulation, it can be found that the high melting point phase CaAlcrucible ,38,39,406 crucible can rapidly generate a higher amount of CaAl4O7 by reacting with the refining slag, while the Al2O3 crucible generates the CaAl4O7 through a series of reactions. In this section, the crystal structure of the CA6 and Al2O3 is analyzed, and the reason for the difference in the CaAl4O7 generation rate and quantity between the CA6 and Al2O3 is also explained.The results of the EDS analysis and thermodynamic simulations show that the CA6 and Al2O3. It can be seen that one Al and five O combined to form the double pyramid module in the mirror layer of the CA6 crystal structure, as shown in the area circled red dotted line. The double pyramid module is the active site of CA6 and is unstable during the reaction process. When the refining slag reacts with the crucible, the active site will be destroyed quickly, resulting in the rapid collapse of the CA6 crystal structure to form the denser CaAl4O7. The crystal structure of CaAl4O7 is more stable and can effectively inhibit the further corrosion of slag, which is one of the reasons for the excellent slag corrosion resistance of the CA6 crucible. Al2O3 crystal is an octahedral structure and more stable compared to CA6, so the reaction rate with Ca in the refining slag is very slow, resulting in a low content of CaAl4O7 in the reaction layer and poor corrosion resistance. More importantly, Al2O3 particles with high stability may directly enter the refining slag and molten steel, resulting in the generation of exogenous inclusions, which has a negative impact on the control of inclusions. At this point, the CA6 refractory can well avoid this problem. So, the excellent slag corrosion resistance of the CA6 crucible has a great application prospect in the ladle refining process, especially for the smelting clean steel.6 crucible, Al2O3 crucible, and CA6-Al2O3 crucible) were selected to investigate the corrosion resistance of the refining slag through laboratory experiments and thermodynamic simulations. The following conclusions were obtained.Three crucibles (CA6-Al2O3 crucible. The Al2O3 crucible shows the worst slag corrosion resistance.(1) The three crucibles show different slag corrosion resistance, CA6 crucible has the best corrosion resistance, followed by the CA6 to the raw materials has a positive effect on improving the slag corrosion resistance of the Al2O3 crucible.(2) The addition of CA4O7 is the critical factor for inhibiting the further corrosion of the CA6 and CA6-Al2O3 crucible. The CaAl4O7 was also detected in the Al2O3 crucible, but Al2O3 in the refractory reacts with CaO in the refining slag to produce CaAl12O19 firstly, and then the CaAl12O19 reacted with slag to form CaAl4O7. Therefore, the generation of CaAl4O7 in Al2O3 crucible is slower than that of CA6 crucible and the amount generated is relatively less, which results in a worse slag corrosion resistance of Al2O3 crucible compared to CA6 crucible and CA6-Al2O3 crucible.(3) The generation of high melting point CaAl6 will be destroyed quickly, resulting in the rapid collapse of the CA6 crystal structure to form the denser CaAl4O7, which is an essential reason for the excellent slag corrosion resistance. At the same time, it also avoids CA6 particles entering the molten steel to introduce exogenous inclusions, so CA6 has great application potential in ladle refining and clean steel smelting.(4) When the refining slag reacts with the crucible, the double pyramid module of CA


NOTCH2NLC) gene, which produces a toxic polyglycine-containing protein (uN2CpolyG) in patients with NIID, has been associated with the pathogenesis of ALS. These results suggest that sporadic NIIDs may have more diverse forms. To date, no autopsy cases of NIID patients with an ALS phenotype have been reported. Here, we describe the first autopsy case report of a patient with sporadic NIID who had been clinically diagnosed with ALS. A 65-year-old Japanese man with no family history of neuromuscular disease developed progressive muscle atrophy and weakness in all limbs. The patient was diagnosed with ALS . He had no cognitive dysfunction or neuropathies suggestive of NIID. He required respiratory assistance 48 months after onset. He died of pneumonia at the age of 79 years. Postmortem examinations revealed neuronal loss in the spinal anterior horns and motor cortex. In these affected regions, eosinophilic, round neuronal intranuclear inclusions were evident, which were immunopositive for ubiquitin, p62, and uN2CpolyG. No Bunina bodies or TDP-43-positive inclusions were observed in the brain or spinal cord. Our findings suggest that a small proportion of patients with NIID can manifest a clinical phenotype of ALS. Although skin biopsy is commonly used for the clinical diagnosis of NIID, it may also be useful to identify cases of NIID masquerading as ALS.Amyotrophic lateral sclerosis (ALS) is one of the differential diagnoses of diseases that occur in adulthood and lead to progressive generalized muscle weakness. Neuronal intranuclear inclusion disease (NIID) is a disease in which histopathologically eosinophilic nuclear inclusion bodies are found in various systems. Both familial and sporadic forms of the disease have been reported. Most cases of sporadic NIID are of the dementia type, in which the main symptom is dementia at the first onset. Familial NIID is more diverse, with the main dominant symptoms being muscle weakness (NIID-M), dementia (NIID-D), and parkinsonism (NIID-P). Furthermore, recently, a GGC-repeat expansion in the Notch 2 N-terminal like C ( There are a variety of neuromuscular diseases that cause progressive generalized muscle weakness; of these, amyotrophic lateral sclerosis (ALS) is a disease that presents with both upper and lower motor neuron signs. Clinically, the degree of disturbance of upper and lower motor neurons in ALS varies between cases. The characteristic pathology of sporadic ALS is the loss of upper and lower motor neurons and the presence of Bunina bodies and TAR DNA binding protein 43 (TDP-43)-positive skein-like or round inclusions in lower motor neurons Yoshida, . In addiNeuronal intranuclear inclusion disease (NIID) is a neurodegenerative disease with slow progression and very low incidence. In this disorder, cells of the central nervous system (CNS), peripheral nervous system, and autonomic nervous system, as well those of the visceral organs  and skin biopsy showing intranuclear inclusions in dermal cells can be indicative of NIID  and wasting of both arms, first dorsal interosseous muscles, thenar muscle, and legs. Fasciculations were observed in the tongue and both vastus medialis muscles. Additionally, paresthesia and ataxia of the right upper and lower limbs persisted after thalamic hemorrhage. Deep tendon reflexes were hypoactive and both plantar reflexes were extensors. No sensory impairment was observed in any extremity.Needle electromyography showed active denervation  and chronic neurogenic changes  in his biceps, first dorsal interosseous, vastus lateralis, and tibialis anterior muscles. Motor nerve conduction studies revealed compound muscle action potential reduction in the bilateral median nerves. Sensory nerve conduction study findings were normal .Magnetic resonance imaging (MRI) of the cervical spine revealed mild cervical myelopathy. However, MRI with short T1 inversion recovery showed no intramedullary signal changes, which could not explain the progressive limb weakness.The patient met the El Escorial diagnostic criteria for probable ALS: laboratory-supported ALS with upper motor signs in one region and lower motor signs in three regions, as defined by electromyographic findings. Riluzole was initiated for ALS. The patient developed dysphagia and dyspnea owing to progressive weakness of the respiratory muscles and required artificial respiratory support and gastrostomy at the age of 68 years. Brain DWI at the age of 76 years showed no abnormalities in the cortical medullary junctions and cerebellar paravermis  as seen FMR1 and polyglutamine. The results of other immunostaining reagents used to evaluate the immunoreactivity of nuclear inclusion bodies are listed in At autopsy, the brain weighted 1,330 g before fixation. Macroscopic examinations showed no atrophy of the brain, while the spinal cord was atrophic with thinning and brownish discoloration of the anterior nerve roots. Histopathological evaluations revealed slight loss of neurons in the motor cortex . ModeratThe patient had a clinical course of progressive muscle weakness and met the El Escorial diagnostic criteria for ALS. However, autopsy confirmed the pathology of NIID but not that of ALS.NOTCH2NLC , limb weakness (NIID-M), and parkinsonism (NIID-P)  of 545 patients clinically diagnosed with ALS, which led the authors to suggest that ALS may be a specific phenotype of NIID , 22H02948(KW)] and the Sakurai Memorial Fund for Medical Research (YM).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Several tools have been used to assess muscular stiffness. Myotonometry stands out as an accessible, handheld, and easy to use tool. The purpose of this review was to summarize the psychometric properties and methodological considerations of myotonometry and its applicability in assessing scapular muscles. Myotonometry seems to be a reliable method to assess several muscles stiffness, as trapezius. This method has been demonstrated fair to moderate correlation with passive stiffness measured by shear wave elastography for several muscles, as well as with level of muscle contraction, pinch and muscle strength, Action Research Arm Test score and muscle or subcutaneous thickness. Myotonometry can detect scapular muscles stiffness differences between pre- and post-intervention in painful conditions and, sometimes, between symptomatic and asymptomatic subjects. Muscular stiffness, described as passive or dynamic ,2,3, is Both passive and dynamic stiffness are essential for adequate muscle contraction  and perf(1)elastography  [p < 0.05)] [p < 0.05)]  ; (c) lat< 0.05)] .In turn, from the previously mentioned methods described to assess muscle stiffness, to our knowledge, only ultrasound shear wave elastography has been used to assess myotonometry validity.The validation of myotonometry, particularly of Myoton as an instrument to measure muscular stiffness, by the correlation with this method when comparing muscular stiffness variables ,18,49,56Despite the concurrent validity of myotonometry against elastography is the more frequently adopted approach, the differences between these two methods ,3 shouldMyotonometry reliability was already assessed for several muscles of different body segments ,48,56,63The reliability values range from 0.229 , for UT,As can be seen in Responsiveness is a psychometric property traduce as the ability of an instrument to detect a meaningful change, in a clinical state, over time ,76. RegaSeveral muscles have been assessed with myotonometry ,25,49,56The trapezius is a standout muscle for scapular stabilization that act in a strong relation, mainly, with the major scapular mover—the serratus anterior . In shouPrevious studies regarding myotonometry, had already presented muscular assessment point references for the trapezius portions ,87,88,89The superficial references for serratus anterior assessment with surface electromyography ,94,95,96In its turn, the assessment of muscular stiffness of levator scapulae and pectoralis minor, that was already done with shear wave elastography ,108,109,The relevance of scapular muscle stiffness to the shoulder complex and the possible muscle’s stiffness changes resulting from the scapular position and their influence in muscular length  had led UT stiffness was compared between subjects, or body sides, with and without pain conditions . While tThe trapezius stiffness comparison of pre- and post-intervention moments has already been made for several rehabilitation techniques . Four stMyotonometry assessment of serratus anterior and levator scapulae muscles are needed to validate the purposed assessment points, to define the myotonometry psychometric properties considering these muscles and to increase the knowledge about these muscles’ mechanical properties.The myotonometry psychometric properties should also be researched in subjects with different conditions, such as pain.The use of myotonometry not only at rest condition but also during contraction, could bring new information that could help to standout adaptations in muscle stiffness modulation, given the muscular activity required and variation in the range of motion used in this muscular condition ,113,114.In studies with the intention to infer about intervention effects, the inclusion of follow-up moments could help to understand whether stiffness changes will be kept over time.The summary of the information gathered in this review is presented in The aspects that should be considered in future studies are summarized in The present narrative review has limitations. First, for being a narrative review, the present study could present some possible bias given the absence of predefined hypothesis and protocol-based , the lack of necessity of following guidelines (as the ones purposed by PRISMA) or to the reduced database consulted during search. Although this review presents the guidelines for the correct use of myotonometry, it should be considered that these are specific for one equipment. Moreover, given the equipment limitation regarding the possible interference of subcutaneous fat, it would be important in future studies consider the measurement of subcutaneous fat in the specific measurement point of each muscle of interest as a criteria to use myotonometry.The advantages of myotonometry together with the well-defined guidelines, the mostly high to very high values of reliability, the inferred responsiveness regarding the affected limb of stroke patients and its possible applicability to assess different scapular muscles stiffness, seems to support its use as a non-invasive method in the assessment of muscular mechanical properties as stiffness (N/m), for clinical practice or research. However, caution should be taken given the variable or no correlation with elastography, even if this may be justified by differences in the outcome measured.