OPTC) inhibits the collagen-induced promotion of bioactivities of human retinal vascular endothelial cells (hRVECs). The present in vivo study aimed to further investigate the regulatory role of opticin in vitreous collagen-mediated retinal neovascularization and to elucidate its regulatory mechanisms with regard to integrin α2-I domain–GXXGER complex formation and RhoA/ROCK1 signal change. The regulatory role of Mg2+ on integrin α2-I domain–GXXGER complex formation in the above process was also investigated.It was previously demonstrated that opticin (OPTC-overexpressing plasmids were intravitreally injected to assess the antiangiogenesis effect of opticin. The regulatory role of opticin in integrin α2-I domain–GXXGER complex formation in vivo was analyzed by mass spectrometry. The mRNA and protein expression of RhoA/ROCK1 were examined. The concentration of Mg2+ as an activator of the integrin α2-I domain–GXXGER complex was measured. Solid-phase binding assays were performed to investigate the interference of opticin in integrin α2 collagen binding and the regulatory role of Mg2+ in that process.The zebrafish model of hypoxia-induced retinopathy was established, and OPTC-overexpressing plasmid injection reduced retinal neovascularization in the zebrafish model of hypoxia-induced retinopathy. Mass spectrometry revealed that opticin could inhibit integrin α2-I domain–GXXGER complex formation. The Mg2+ concentration was also decreased by opticin, which was another indication of the complex activation. Injection of OPTC-overexpressing plasmids inhibited mRNA and the protein expression of RhoA/ROCK1 in the zebrafish model of hypoxia-induced retinopathy. The solid-phase binding assay revealed that opticin could block integrin α2–collagen I binding in the presence of Mg2+.Opticin and 2+-modulated integrin α2-I domain–collagen complex formation and suppressing the downstream RhoA/ ROCK1 signaling pathway.Opticin exerts its antiangiogenesis effect by interfering in the Mg As mentioned above, the interaction of Mg2+ and MIDAS exposes the α7 helix at the C-terminal of the integrin α2-I domain, which opens up the collagen binding site and allows further binding to glutamic acid residues in the collagen sequence GXXGER. Thus, it has been hypothesized that opticin may regulate binding of the integrin α2-I domain to collagen by interfering with Mg2+, which will further interfere with the collagen-stimulating angiogenesis. Because RhoA/ROCK1 is activated by integrin signals, a conduit for the transmission of signals into cells is provided.OPTC in the formation of the integrin α2-I domain–GXXGER complex and the signal activities of RhoA/ROCK1.Integrins are a large family of α and β heterodimeric receptors that mediate cell–cell and cell–extracellular matrix (ECM) interactions.The Tg(flk:GFP) zebrafish used in this study were obtained from the Zebrafish Model Animal Facility at the Institute of Clinical and Translational Research of Sun Yat-Sen University. Reagents used for the hypoxia experiment were purchased from Sigma-Aldrich . Retinal neovascularization was visualized using a Zeiss LSM 710 confocal microscope  and quantified using AngioTool64 (v0.6a) software  as described before.OPTC-N1) for overexpression of the OPTC gene were used for the ocular transfection assay. The zebrafish OPTC gene sequence was obtained from GenBank. Plasmids were purchased from HedgehogBio .Plasmids  were purchased from Bioworld Technology . Reagents used for quantitative real-time PCR were purchased from Takara Bio . The primers were synthesized by Sangon Biotech .Q Exactive Plus  was used for the mass spectrometry analysis. All materials used for mass spectrometry were obtained from Thermo Fisher Scientific. A Synergy H1 microplate reader  was used for measurement of the MgRegents used for the solid-phase binding assays were purchased from Biodragon Immunotechnologies . Recombinant human opticin  and recombinant human integrin α2  were used for the solid-phase binding assays. Biotinylated collagens were purchased from KYDBio Technology .,2 concentration was gradually reduced until final 10% air saturation over the course of 48 to 72 hours. Zebrafish were exposed to this hypoxic environment for various lengths of time (12 days at most).The zebrafish facility and all experimental procedures were approved by the Animal Ethical Committee of Sun Yat-sen University. The design of the hypoxia chamber and protocol for hypoxia exposure of adult zebrafish have been described previously.OPTC (0.5 mg/mL) or OPTC-overexpressing plasmids (250 mM) for 10 to 15 seconds under a dissecting microscope, and the right eyes were injected with an equivalent volume of PBS. They fully recovered from anesthesia in fish-tank water for 5 to 10 seconds, and the recovery rate was 100%.Before experimental operations, all zebrafish were anesthetized with 0.02% tricaine. Zebrafish were deeply anesthetized with anesthetic solution prepared in fish-tank water and placed on a moist surface under a dissecting microscope. The left eyes of the zebrafish were injected with 0.6 µL of recombinant human ,Seven days after the injection, the zebrafish were sacrificed by a lethal dose of tricaine, followed by decapitation. The retina was carefully cut four times into four equal quadrants from the edge toward the center using sharp microsurgery forceps. Retinal neovascularization was visualized under the Zeiss LSM 710 confocal microscope and quantified using AngioTool64 (v0.6a) software as described before.All samples containing the protein were collected in each group, and their purity was validated with SDS-PAGE (180 V and 120 mA for 40 minutes) after denaturation at 75°C in the presence of lithium dodecyl sulfate buffer. Gels were stained with SimplyBlue stain for the visualization of protein bands. Strong bands corresponding to the target proteins including collagen and integrin α2 on SDS-PAGE were cut and digested by trypsin. The peptides were desalted by StageTips, vacuum dried, dissolved in 1% formic acid, and loaded on a nanoflow HPLC system coupled to the Q Exactive Plus mass spectrometer equipped with a nano-electrospray ionization source. Data-dependent acquisition was enabled, and higher energy collisional dissociation of the top five ions was performed in each cycle from the survey scan over the mass-to-charge (m/z) range of 350 to 1500 with dynamic exclusion time of 10 seconds. There were three replicates per sample. Spectra were searched with PEAKS software in the SwissProt database, with 10-ppm peptide mass tolerance and 0.02-Da fragment tolerance.2+ concentration in tissues.Retinal tissue homogenate was centrifuged for 2 minutes at 4°C. The absorbance was measured at 510 nm using a microplate reader and was used to determine the MgQuantitative RT-PCR was performed as described previously. The primer sequences used for the relative expression of target genes including integrin α2, RhoA, ROCK1, and MYPT1 were obtained from PrimerBank.The protein expression of integrin α2, RhoA, ROCK1, MYPT1 and p-MYPT1 was detected by western blotting as described previously. Immunostaining of whole-mounted retinas and frozen sections for the detection of protein expressions of integrin α2, RhoA and p-MYPT1 was performed as described by Hu S et al.To mimic integrin α2 collagen binding (through the formation of the integrin α2-I domain–GXXGER complex), solid-phase binding assays were performed as described by Le Goff et al.2+ solution at different concentrations (0 to 2.5 mM) was added to the wells. Residual protein absorption sites were blocked with BSA for 1 hour. Biotinylated collagen I (0.5 μg/mL) was added to the wells, which were incubated for 3 hours. 3,3′,5,5′-Tetramethylbenzidine substrate was added to detect the absorbance.A 96-well plate was coated with integrin α2 (5 μg/mL) in TBS buffer for 1 hour. Mg2+, and biotinylated collagen I (0.5 μg/mL) was pre-incubated with OPTC (100–500 nM) for 2 hours.Following the above procedure, wells were coated with integrin α2 in TBS buffer containing 2-mM Mgt-test and t-test. One-way ANOVA was used for multiple comparisons among different groups. The pairwise comparison between groups was performed using Dunnett's test and the Student–Newman–Keuls (SNK) and q test. The test level was α = 0.05, with P < 0.05 being considered significant. SPSS Statistics 25.0  was used for statistical analysis.The data were obtained from at least three independent assays and are expressed as mean ± SD. The results were compared between two groups by paired P < 0.05, paired t-test)  A–1C.P < 0.05, paired t-test; not significant).To investigate whether opticin regulates angiogenesis under pathological conditions, an intravitreal opticin injection experiment was performed. First, we assessed retinal angiogenesis under normoxia condition and found that there were no differences after opticin injection for 7 days compared with normoxia controls A. Next, OPTC overexpressing plasmids labeled by mCherry. Inclusion of the entire OPTC coding sequence was built in the pmCherry-myc-zOPTC-N1 plasmid .To further investigate the endogenous role of opticin in regulating hypoxia-induced angiogenesis, we performed an in vivo transfection procedure utilizing  plasmid A. Empty 3T cells B, S1C. Tplasmids D, S1E waplasmids AI, 3AII.OPTC overexpression group had significantly decreased vascularization areas, branches, and sprouts in retina compared with the hypoxia group and the empty plasmid injection group . Similarly, subanalysis of different vascularity areas showed that the branches, sprouts, and vascularization areas in the OPTC overexpression group were significantly decreased in high vascularity areas, whereas only sprouts and vascularization areas were significantly decreased in low vascularity areas compared with the hypoxia group and the empty plasmid injection group . These results suggest that overexpression of OPTC could suppress retinal neovascularization in zebrafish.It was found that the on group B . Also, our results showed that opticin had no effect on activating integrin α2-I domain expression under the normoxia condition . There were significant differences in samples in the comparisons among the hypoxia, opticin injection, and OPTC overexpression groups . The above findings suggest that activation of the integrin α2-I domain was reduced by opticin. Also, the best unique amino acid sequences of GXXGER on collagen were confirmed by MALDI-TOF, and relative abundance values were detected . The content of GXXGER sequences in samples was significantly decreased with increasing concentrations of opticin .To investigate the regulatory role of opticin in the formation of the integrin α2-I domain–collagen complex in vivo, the expression of activated forms of the integrin α2-I domain and GXXGER peptides was measured using mass spectrometry to monitor the expression of amino acid sequences. Comparison of the closed and open forms of integrin α2-I domain structures showed that the αC helix provided a steric barrier in the closed conformation for attainment of the metal–ligand bond. The open conformation was seen in the presence of ligands, whereas the closed conformation was seen in the absence of ligands. Residues corresponding to the ligand were Gly284–Arg288 (GYLNR). Therefore, the peptide FGIAVLGYLNR covering the GYLNR motif was identified as the open form of the integrin α2-I domain in the samples. The best unique amino acid sequences of GYLNR were confirmed by matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) in the samples A. Comparn groups B , solid-phase assays were performed using purified recombinant proteins. Collagen I was biotinylated as a label. Ligands including integrin α2 and opticin binding to biotinylated collagen I were detected based on the absorbance detected using horseradish peroxidase (HRP)-conjugated streptavidin. The absorbance changes were characterized as a marker for the binding affinity of proteins. Inhibition experiments were performed using a fixed concentration of collagen I and a fixed concentration of Mgt manner A. Inhibinner . The results indicate that integrin α2 bound to collagen I with high affinity in the presence of Mg2+.To further define the role of Mged group A . The findings demonstrate that opticin negatively regulates Mg2+ during pathological angiogenesis in vivo.Considering the paramount importance of Mgplasmids C , which may be regulated by the RhoA/ROCK1 signaling pathway, we explored the status of the RhoA/ROCK1 signaling pathway in vivo in response to opticin/RhoA and ROCK1, two downstream target genes of the RhoA/ROCK1 signaling pathway, as well as integrin α2. On the other hand, the expressions of these genes was downregulated by opticin injection and OPTC overexpression . The mRNA expression of MYPT1 was not changed due to the phosphorylation of MYPT1 at the protein level . These results suggest that opticin negatively regulated activation of the RhoA/ROCK1 signaling pathway following formation of the integrin α2-I domain–collagen complex in zebrafish retina. Similarly, the protein expression of the integrin α2 and RhoA/ROCK1 signaling pathway showed the same trend. Hypoxia induced significantly increased expression of integrin α2, RhoA, and ROCK1 at the protein level. The expression of p-MYPT1, a marker for ROCK1 activation in the intracellular domain, was also downregulated by opticin at the protein level .To clarify the regulatory role of opticin in the RhoA/ROCK1 signaling pathway following the formation of the integrin α2-I domain–collagen complex, zebrafish retinas were isolated, and the expression of target genes was detected at both transcriptional and protein levels. As shown in in level B–7C (P <OPTC overexpressing plasmid injection significantly inhibited the protein expressions of integrin α2, RhoA, and p-MYPT1 (To confirm that the above-mentioned signal changes in zebrafish retina, immunofluorescence staining was performed in cells. The expression levels of target proteins in cells of retinal sections were detected in each group using immunofluorescence staining . Specifi p-MYPT1 D–7I. To ,,OPTC can inhibit collagen-induced promotion of bioactivities of hRVECs.,It is widely accepted that the vitreous body generally resists angiogenesis, and many of the known ECM molecules play central roles in maintaining the angiogenesis balance in the vitreous body.OPTC gene overexpression from the endogenous level. Our study is in line with previous studies reporting that OPTC-deletion mice exhibited increased preretinal neovascularization in oxygen-induced retinopathy models.Next, our results suggest that opticin could inhibit hypoxia-induced retinal neovascularization. Moreover, the reduction in pathological angiogenesis due to opticin is further supported by the observations of impaired angiogenic response to 2+ in zebrafish retina was downregulated by opticin treatment. On the one hand, it was further confirmed in a previous in vitro study that opticin inhibits collagen-induced promotion of bioactivities of hRVECs via integrins. On the other hand, a new specific interference mode and target binding site were found to underlie the antiangiogenic mechanism of opticin, extending previous research by Le Goff et al.Some previous work has attempted to illustrate how opticin inhibits interactions between collagen and integrin in Matrigel, mimicking the collagen environment, but there have been no direct investigations of the underlying mechanism of the antiangiogenic activity of opticin in actual animal models.2+ levels in the ECM.2+ was downregulated by opticin treatment, thus adding another dimension to our understanding of the intricate activation of the integrin α2-I domain–collagen complex. There is a possibility that opticin decreases the concentration of Mg2+ and then downregulates activation of the integrin α2-I domain–collagen complex. It also seems reasonable to speculate that opticin–collagen binding could trigger large conformational changes in the complex, thus making the MIDAS more inaccessible for the Mg2+ and ligand. Neither of the two hypotheses can be excluded, but they both stress the importance of the formation of integrin α2-I domain–collagen complex. These possibilities were further verified by the solid-binding results indicating that integrin α2–collagen binding affinity was facilitated by Mg2+ in a dose-dependent manner. It was also demonstrated that opticin specifically inhibited integrin α2–collagen binding affinity, consistent with the in vivo study results. It is worth noting that solid-binding assays can be used to investigate the activation state of the complex in the presence of Mg2+, which may add another dimension to research on the role of the integrin α2–collagen binding complex. Thus, this study warrants further investigation of the relationship between Mg2+ concentration and opticin and further conformational changes of the complex in pathological conditions.It has been previously reported that integrin α2–collagen binding through MIDAS is dependent on MgThe results of this study demonstrate that formation of the integrin α2–collagen complex is indispensable for the antiangiogenesis effect of opticin, and regulation of the RhoA/ROCK1 signaling pathway also played an essential role. Although the significance of the RhoA/ROCK1 signaling pathway has been well recognized in preretinal neovascularization diseases,OPTC gene prevents hypoxia-induced neovascularization, suggest that opticin-specific inhibitors can be developed as promising therapeutic targets for the treatment of diseases associated with abnormal angiogenesis such as retinal vascular disorders and other proliferative vascular diseases.In summary, the research results provide compelling evidence that opticin is an important regulator of pathological angiogenesis by modulating the formation of the integrin α2-I domain–collagen complex and further suppressing the RhoA/ROCK1 signaling pathway. These results, coupled with the finding that pharmacological overexpression of the


Sorghum bicolor) flour (SF). Endogenous lipids and/or proteins were removed from different portions of SF to obtain defatted (DF), deproteinized (DP), and defatted and deproteinized (DF-DP) flours. Bioactive constituents , antioxidant activities , starch, amylose, starch hydrolysis index (HI), estimated glycemic index (eGI), and pasting properties of treated and control (untreated) flours were determined. The control flour (SF) had significantly higher (p < 0.05) levels of all the bioactive constituents and antioxidant activity tested than the DF, DP, and DF-DP flours, while the DF-DP flour had the least levels of bioactive constituents and antioxidant activity. In contrast, the starch, amylose, HI, and eGI were consistently in the order of DF-DP > DF > DP > control flour (p < 0.05). The control flour had the highest (p < 0.05) peak viscosity, and the least peak time and pasting temperature, while the DF flour had the highest final viscosity. Therefore, endogenous lipids and proteins contribute to the antioxidant, starch digestibility, and pasting properties of sorghum flour.This study evaluated the effect of endogenous lipids and proteins on the antioxidants, starch digestibility, and pasting properties of sorghum ( Sorghum bicolor (L.) Moench] is a major cereal for millions of poor people in semi-arid tropical regions of Africa, Asia, and Latin America (Aceda (thick porridge) and Hulu-mur  (Sorghum [ America . It serv America . Sorghumverages)  in diffeverages) . The floverages) .In addition to its rich nutritive value, including proteins, starch, B-complex vitamins and minerals in the endosperm, as well as lipids and some fat-soluble vitamins in the germ , whole sin vitro starch digestibility and estimated glycemic index of kodo millet flour increased significantly after the removal of protein and/or lipid, especially after the removal of lipid. On the other hand, the addition of the protein was found to inhibit the catalytic activity of amylase against starch granules  of red sorghum grains was procured from the grains market in Minna, Nigeria. The sample was sorted and ground into flour. The sorghum flour (SF) was packed hermetically in an opaque plastic container, and stored at 4°C during analysis. Analytical grades of all the chemicals and reagents were used in all the experiments.Endogenous lipids and/or proteins were removed from different portions (500 g) of SF to obtain defatted (DF), deproteinized (DP), and defatted and deproteinized (DF-DP) flours, according to the method described by Annor et al.  with sliExtraction of flour samples for bioactive constituents and antioxidant assays was carried out as described by Engida et al. . Two graTotal phenolics level of the flour extracts was determined as per Folin–Ciocalteu method described by Singleton et al.  and expr*+ scavenging assay was carried out as described by Re et al.  and K2S2O8 (2.45 mM) were mixed and incubated in the dark at room temperature for 16 h. Afterward, the absorbance of the reagent was adjusted to 0.7 ± 0.02 at 734 nm using 95% ethanol. For the assay, a mixture of 2.0 ml of the ABTS+* reagent and 0.2 ml of each extract was incubated at room temperature for 15 min. Afterward, the absorbance was read at 734 nm in a UV-Vis spectrophotometer. ABTS+* scavenging ability of the extracts was calculated using Trolox calibration curve, and expressed as Trolox equivalent antioxidant capacity in micromole per gram sample dry weight (TEAC μmol/g DW).ABTSe et al. . To prep* scavenging assay was conducted as described by Cervato et al. , was calculated.DPPHo et al. , using a2O. Afterwards, 1 ml of 0.1% ferric chloride was added to each tube and the absorbance was read at 700 nm. Reducing power of the extracts was calculated using gallic acid calibration curve, and expressed as gallic acid equivalent in milligram per gram sample dry weight (GAE mg/g DW).Reducing power assay was carried out as described by Oyaizu . A mixtu(Fe2+) chelating assay was conducted as reported by Puntel et al. (4 (500 μM) was incubated at room temperature for 5 min. Afterwards, 13 μl of 0.25% 1,10-phenanthroline was added, and the absorbance was read at 510 nm in a UV-Vis spectrophotometer. Fe2+ chelating capacity of the extracts was calculated and expressed as half-maximal scavenging concentration (SC50) in mg/mL.Iron (II) l et al. , using a2SO4 were mixed in a test tube, and allowed to cool to room temperature, after which the absorbance was read at 490 nm. Starch content of sample was calculated using a D-glucose calibration curve.Starch content of the flour samples was quantified as described by Onitilo et al.  with min2 in 2% KI), and 9.2 ml of distilled water. The mixture was incubated for 20 min at room temperature, following which the absorbance was read at 620 nm in a UV-Vis spectrophotometer. Amylose content of the samples was computed using amylose standard.Amylose content of the flour samples was determined as described by Juliano et al. . A mixtuAmylopectin level of the samples was calculated as per the formula reported by Juan et al.  as folloin vitro starch digestibility rate and hydrolysis index (HI) of the flour samples were determined as per the method of Goni et al.  and AUC (ref) are the areas under the hydrolysis curves of the flour sample and reference/standard carbohydrate (50 g glucose), respectively.The estimated glycemic index (eGI) of samples was calculated using the following formula: eGI = 39.71 + 0.549HI; where GI = Glycemic index (%) and HI = Hydrolysis index (%).The pasting properties of the flour samples were analyzed using a Rapid Visco Analyzer (RVA)  . The RVAp < 0.05. The 17th version of Statistical Package for Social Science (SPSS) software was used for data analysis.Results of three independent determinations were expressed as mean ± SD. The mean values were subjected to one-way ANOVA, and the mean values of different treatments were compared using Duncan's multiple range test at p < 0.05), such that the control > DF > DP > DF-DP flour. The total phenolics, tannins, total flavonoids, saponins, and anthocyanins contents of the flours ranged from 1.54 ± 0.06 to 7.48 ± 0.51 mg GAE/g; 1.69 ± 0.08 to 8.23 ± 0.62 mg TAE/g; from 0.97 ± 0.04 to 4.69 ± 0.04 mg QE/g; from 0.77 ± 0.03 to 3.74 ± 0.04 mg DE/g; from 1.93 ± 0.06 to 9.35 ± 0.60 CGE mg/g, respectively, in DF-DP and control flours.The levels of bioactive constituents of the control, defatted (DF), deproteinized (DP), and defatted and deproteinized (DF-DP) sorghum flour samples are presented in The total phenolics (7.48 ± 0.51 mg GAE/g), tannins (8.23 ± 0.62 mg TAE/g), and total flavonoids (4.69 ± 0.04 mg QE/g) contents of the control SF were lower than the levels of these bioactive constituents  previously reported for whole sorghum flour . The low*+ and DPPH* scavenging, Fe2+ chelation, and reducing power assays) are presented in p < 0.05) due to the removal of lipids and/or proteins, whereas the ABTS*+ scavenging ability and reducing power were in the order of control flour > DF > DP > DF-DP flour. The DPPH* and Fe2+ chelation SC50 values had a reverse order of DF-DP > DP > DF > control flour. Taken together, these trends indicate that the control flour, containing the endogenous lipids and proteins, had the strongest antioxidant activity, while the DF-DP flour had the weakest antioxidant activity. This notwithstanding, ascorbic acid (a reference antioxidant compound) had stronger DPPH* scavenging and Fe2+ chelation abilities than the control flour, as indicated by its lower DPPH* and Fe2+ chelation SC50 values . However, the DPPH* scavenging ability of the control flour (SC50: 8 ± 0.98 μg/ml) was stronger than the DPPH* scavenging ability earlier reported (SC50: 12.04 ± 0.85 μg/ml) for S. bicolor, as a lower SC50 value represents a stronger scavenging activity (*+ scavenging activity of the control flour in this study (7.52 ± 0.45 mmol TEAC/g) is much higher than the range of ABTS*+ scavenging activity  that Awika et al. -scavenging activity of the flour is indicative that it could help prevent and/or ameliorate oxidative stress when ingested as food. It is important to recall that cellular oxidative stress sets in when the oxidant burden of the cell outweighs its antioxidant defense system. Oxidative stress, in turn, precipitates some non-communicable diseases such as gout, obesity, and diabetes mellitus , which can attack biomolecules, such as and lipid, DNA, and protein. Thus, the ability of the control sorghum flour to chelate Feolecules . In addiolecules .p < 0.05) higher in the DF-DP flour, followed by the DF, DP, and control flours. In contrast, the control flour had the highest (p < 0.05) amylopectin level followed by DP, DF, and DF-DP flours. The amylose/amylopectin ratios of the DF and DF-DP flours were comparable (p > 0.05), but both were significantly higher than those of the control and DP flours. There were significant (p < 0.05) and consistent reductions in the HI and eGI of the flour as a result of defatting, deproteinization, and defatting and deproteinization, such that DF-DP > DF > DP > control flour.Starch, comprising amylose and amylopectin and morphologically occurring as water-insoluble semi-crystalline granules, has a wide range of applications in food products due to its peculiar thickening, gelling, and stabilizing qualities . In bothp < 0.05) HI and eGI, relative to the DF, DP, and DF-DP flours  to the starch granules (in vitro starch digestibility of the control sorghum flour due to the presence of endogenous lipids and proteins suggests that the endogenous lipids and proteins could lower the glycemic index of the sorghum flour.Contrary to the expectation that a lower level of amylose in the control flour would translate to a higher rate of its starch hydrolysis (digestibility) by α-amylase, thereby releasing more monosaccharides (such as glucose and fructose), with a concomitant higher GI , the HI P flours . This buP flours , 47 of tP flours . Similargranules , 11. Takp < 0.05) were observed in the HI and eGI of the treated flours, such that the DF-DP flour had the highest HI and eGI, followed by the DF flour, and lastly the DP flour. This indicates that the removal of the endogenous lipids enhanced the in vitro digestibility of the sorghum starch more than the removal of endogenous proteins. However, this trend is different from the trend observed by Ye et al.  on the pasting profiles of the flour. The control flour had the highest peak and breakdown viscosities , and the lowest peak time and pasting temperature . DF flour had the highest final and setback viscosities , while DP flour had the lowest final and setback viscosities . The result further revealed that the DF-DP flour had the highest trough viscosity (128.04 ± 4.65 RVU) and peak time (9.00 ± 0.01 min). In addition, the pasting temperature of the DF-DP (89.25 ± 1.77°C) and DP (90.50 ± 0.07°C) flours were comparable (p > 0.05), but these were both significantly higher than those of the DF (83.63 ± 0.53°C) and control (80.83 ± 0.04°C) flours. The lower peak time and pasting temperature of the control flour, relative to the treated flours, could be directly attributed to the removal of the endogenous lipids and proteins from the treated flours. As earlier suggested by Ye et al.  of the sorghum flour. Contrarily, the starch, amylose, and in vitro starch digestibility (HI and eGI) of the flour increased as a result of the removal of endogenous lipids and/or proteins. The increase in the in vitro starch digestibility of sorghum flour upon the removal of endogenous lipids and proteins suggests that the presence of endogenous lipids and proteins may lower the glycemic index of sorghum flour. The control flour had the highest peak and breakdown viscosities, while the DF flour had the highest final viscosity. Therefore, endogenous lipids and proteins contribute to the antioxidant, starch digestibility, and pasting properties of sorghum flour.The removal of endogenous lipids and/or proteins led to a decrease in the levels of all the bioactive constituents  and antioxidant activity (ABTSThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.EI conceptualized the study, supervised the analyses, wrote the original draft, and edited the final draft. AA contributed in conceptualizing the study, analyzed the samples, and collated the data. TA contributed in the methodology, samples and data analysis, and writing of the original draft. All authors contributed to the article and approved the submitted version.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.


Post-translational modifications (PTMs) of Cyclin-dependent kinase 5 (CDK5) have emerged as important regulatory mechanisms that modulate cancer development in patients. Though CDK5 is an atypical member of the cyclin-dependent kinase family, its aberrant expression links to cell proliferation, DNA damage response, apoptosis, migration and angiogenesis in cancer. Current studies suggested that, new PTMs on CDK5, including S-nitrosylation, sumoylation, and acetylation, serve as molecular switches to control the kinase activity of CDK5 in the cell. However, a majority of these modifications and their biological significance in cancer remain uncharacterized. In this review, we discussed the role of PTMs on CDK5-mediated signaling cascade, and their possible mechanisms of action in malignant tumors, as well as the challenges and future perspectives in this field. On the basis of the newly identified regulatory signaling pathways of CDK5 related to PTMs, researchers have investigated the cancer therapeutic potential of chemical compounds, small-molecule inhibitors, and competitive peptides by targeting CDK5 and its PTMs. Results of these preclinical studies demonstrated that targeting PTMs of CDK5 yields promising antitumor effects and that clinical translation of these therapeutic strategies is warranted. CDK5 is an atypical member of cyclin-dependent kinases (CDKs) located in chromosome 7q36, which shares large proportion of amino acid sequence identity with other CDK members , 2. Its . The N-lobe mainly contains 5 β-sheets, while C lobe includes 4 α-helices. The PSSALRE helix and the DGF motif in T-loop forming a stereo-structure in the surface of CDK5 are critical for activator binding, such as p35 or p25 [. In addition, the ATP-binding domain located on the surface of CDK5 can receive ATP for activation by regulating the crossing of DFG motif to PPD motif; during this progress, hinge region can form hydrogen bonds with the ATP cleft. The functions of these domains can be controlled by various PTMs. For example, in ATP-binding domain, phosphorylation of Thr14 and Tyr15 controlled by the dual specificity kinase Wee1 and Myt1 can influence CDK5 activity [. It is clear that PTMs occurred on the different domains play important roles in switching the molecular function of CDK5.Similar to other CDK members, CDK5 structurally contains N-lobe, C-lobe, ATP binding domain, activators binding domain, hinge region, PSSALRE helix and T-loop Fig. a. The N-25 [Fig. b. In addactivity , whereasactivity . S-nitro. Interestingly, Minegishi et al. swapped cognate p10 regions between p35 and p39, and found that p39 showed a slower degradation rate than p35 [CDK5 was recognized to be activated by non-cyclin proteins p35 and p39. p35 is the first regulatory subunit found to bind with CDK5 and control its activity. It is a short half-life protein that can be rapidly degraded through proteasomes –13. WhenFig. . Interesthan p35 . As a unthan p35 . On the than p35 , however. A recent study showed that high expression of CDK5 and p35 was observed in late stage of triple-negative breast cancer and correlated with the poor clinical outcome [Increasing studies showed that CDK5 plays an important role in cancer progress. Abnormal expression of CDK5 and its activators are positively associated with multiple tumorigenesis, as summarized in Fig.  outcome . In glio outcome . Mechani outcome . In cont outcome . Interes outcome , and thuThe allele frequencies of mutations on CDK5 are low in various cancer samples, but some mutations located in key domains of CDK5 likely influence its structure and PTMs, which contribute to tumorigenesis. As summarized in Table . These examples partially explain the co-occurrence of CDK5 and p35 frequently observed in cancer, more upstream regulators should be further investigated.Hyperactivation and overexpression of CDK5, as well as its activators p35 and p39, are frequently observed in colon cancer , breast Fig. . These e. Inhibition of CDK5 kinase activity causes dephosphorylation and perinuclear accumulation of p35 or p39 [2+ homeostasis. Deletion of CDK5 triggers mitochondrial permeability transition pore (mPTP) opening and mitochondrial Ca2+ transfer from the endoplasmic reticulum (ER) to the mitochondria [The cellular localization of CDK5 is closely related to its biological functions, and determined by its PTMs or the activity of p35 and p39. By forming complexes with p35 or p39, CDK5 can attach on cellular membrane or shuttle between cytoplasm and nucleus to exert its molecular functions. N-terminal p10 region of p39 and p35 containing localization motifs undergoes myristoylation that determines its membrane association, while removal of myristoylation on p39 or p35 can induce the nuclear localization of CDK5 . InteresFig. . Inhibit5 or p39 . It is e5 or p39 . Anotherchondria . More PTCIP1 protein degradation [Cell cycle disorder and abnormal proliferation are two hallmarks of cancer cells. Major cell cycle regulators, such as CDKs, have been widely studied for their role in promoting cancer cell proliferation . In addiradation . In non-radation .. In glioblastoma (GBM), CDK5 can amplify EGFR signaling by phosphorylating CRMP2 (collapsin response mediator protein 2) at Ser522, sustaining the pro-proliferation effect on tumor cells [INK4b and p21CIP/WAF1 to promote cell proliferation . It phosphorylates Rb at Ser807/Ser811 to release transcription factor E2F for initiating cell cycle [As a proline-directed kinase, CDK5-mediated phosphorylation signaling cascade exhibits chief effect on multiple cancer progression Fig. , Table 2or cells . Retinobor cells . In meduion Fig. a. It pholl cycle . In addill cycle . Recent ll cycle .Fig. 3P, such as ATM (ataxia-telangiectasia mutant) and Ape1 (apurinic/apyrimidine endonuclease 1) [).During tumor progression, a decrease of damaged surveillance mechanism and an enhancement of genome instability are necessary for cancer cells to achieve uncontrolled growth and the adaptability associated with aggressive tumors . DeregulFig. a, such alease 1) , 54. Thelease 1) . DNA damMany traditional chemotherapeutic agents are DNA damage stimulators, for instant, ionizing radiation and topoisomerase inhibitors can enhance the activity of CDK5 in cancer cells. CDK5 is able to induce the phosphorylation of ATM at Ser729, to activate DNA damage repair to promote cancer proliferation . In addiThe dual role of CDK5 in apoptosis was found in different diseases, depending on the occurrence of PTMs at specific proteins/sites. Hsu et al. found that CDK5/p35 complex can induce the Ser81 phosphorylation of AR (androgen receptor) to inhibit cell apoptosis and promote cell proliferation in prostate cancer . Recent G12D promotes the generation of p25 that further enhances the activity of CDK5 in pancreatic cancer, resulting in the activation of Ral (Ras-Like) pathway and morphological change conducive to cell migration [. Increasing evidences proved that CDK5 is involved in the regulation of the microtubule complex that is necessary for cell movement. For example, the Ser202 phosphorylation of microtubule-associated protein Tau [. Jin et al. found that CDK5 regulates Thr261 phosphorylation in EZH2 (Enhancer of zeste homolog 2), a component of the polycomb repressive complex 2 (PRC2). The phosphorylated EZH2 can bind to FBW7 (F-box and WD repeat domain-containing 7) for degradation to prevant pancreatic cell migration and invasion [. Collectively, CDK5-mediated phosphorylation cascade is critical for tumor metastasis, suggesting that targeting CDK5 is a promising strategy for preventing cancer spread.Metastasis is a key feature in cancer progression, and the activity of CDK5 was found to be associated with invasive phenotype, such as cytoskeleton remodeling . A studyigration , 60, 61.igration  . In U87 glioblastoma and Lewis lung cancer, inhibition of CDK5 can reduce VEGF expression to inhibit angiogenesis [Angiogenesis is the physiological process of growing new blood vessels from existing blood vessels, which provides nutrition and oxygen for tumor growth. Julia Herzog et al. showed that CDK5 is the main regulator of angiogenesis in hepatocellular carcinoma (HCC). CDK5 can directly phosphorylate HIF1α (hypoxia inducible factor 1α) at Ser687 to enhance the expression of its target genes VEGFA, VEGFR1 and EphrinA1, which are essential for the formation of novel blood vessels in tumors   can produce potent anti-tumor immunity . Recent With the advanced mass spectrometric technology developed, new PTMs on CDK5 including glycosylation, phosphorylation, ubiquitin, sumoylation and acetylation were identified to play important roles in tumorigenesis –77. Thes2+ ion coordination sphere, as well as the G-loop to shift away from the ATP binding site [CDK5 phosphorylation and CDK5-mediated phosphorylation events were reported to play critical roles in degenerative neurological diseases and cancer . Phosphoing site . Phosphoing site .For CDK5-mediated phosphorylation events, its substrates are involved in a variety of biological processes regulation and are closely related to the occurrence and development of cancer. For example, the activity of CDK5 during neuronal development can be realized by phosphorylation of p35 at Thr138 position , 80. RecS-nitrosylation refers to the addition of NO group to a cysteine residue of certain proteins, which plays diverse regulatory roles in multiple physiological processes. Its role in cancer development was widely investigated in recent years. In breast cancer, S-nitrosylation of Ras induces MAPK-dependent phosphorylation and activates ETS-1 (Erythroblastosis virus transcription factor-1), a critical mediator of nitric oxide, resulting in an aggressive breast cancer phenotype . S-nitro. On the contrary, zhang et al. proved that the mutation of Cys83 S-nitrosylation on CDK5 restores its kinase activity and enhances dendritic growth and branching [In CDK5, Cys83 and Cys157 can be S-nitrosylated, which leads to overactivation of CDK5 and subsequently contributes to mitochondrial dysfunction, synaptic damage and neuronal cell death. A study showed that the interaction between neuronal NOS1 (nitric oxide synthase) and CDK5 contributes to the formation of SNO-CDK5 . In turnFig. . On the ranching . NO actsranching . ConsideSumoylation is involved in cell biological processes such as cell cycle regulation, senescence, and apoptosis , 89. Rec. However, the biological function of K56 of CDK5 remains to be studied [K33 and K56 are two acetylation sites of CDK5 that were firstly identified by Juhyung Lee et al. in 2014 . They fo studied , 96. Int studied . RegardiHyperactivation of CDKs was frequently recorded in most primary tumors, and thus were deemed as useful targets for clinical cancer therapy. Since CDK activity relies on ATP, most of the current CDKs inhibitors are based on targeting ATP binding pocket. For example, flavopiridol, the first pan-CDK inhibitor used in human clinical trials, can bury in the ATP-binding pocket of CDKs, and inhibits their activity at nanomolar dosage . SeveralConsidering the oncogenic role of CDK5 in cancer, targeting the ATP binding of CDK5 and CDK5-p35 interaction is currently the two major strategies to suppress CDK5 activity, which has been used in preclinical trials of drugs for several types of tumors Table  114–116116. UndeAnother class of CDK5 inhibitors can inhibit the CDK5 activity by abolishing the interaction of CDK5/p35 complex. Roscovetine  was reported to inhibit the activity of CDK5 by competing for the binding site of p35 . TamoxifCDK5 is considered to be an atypical member of the CDKs, and its abnormal expression is involved in a variety of diseases, such as neurodegenerative diseases and cancer. During cancer progression, CDK5 serves as an oncogene to promote cell proliferation, migration and invasion. Currently, emerging types of PTM on CDK5 were identified, and were found to be involved in a wide range of cellular processes in cancer. ATP competitive inhibitors and drugs by targeting CDK5-p35 complexes are currently the major inhibitory tools to reduce CDK5 activity, however, their low specificity/stability and high toxicity remain handicap for clinical application. Increasing clinical studies have found that the CDK5 activity inhibitors are not specific, since they inhibit the activity of other kinases as well Table . In this


High strength and ductility, often mutually exclusive properties of a structural material, are also responsible for damage tolerance. At low temperatures, due to high surface energy, single element metallic nanowires such as Ag usually transform into a more preferred phase via nucleation and propagation of partial dislocation through the nanowire, enabling superplasticity. In high entropy alloy (HEA) CoNiCrFeMn nanowires, the motion of the partial dislocation is hindered by the friction due to difference in the lattice parameter of the constituent atoms which is responsible for the hardening and lowering the ductility. In this study, we have examined the temperature-dependent superplasticity of single component Ag and multicomponent CoNiCrFeMn HEA nanowires using molecular dynamics simulations. The results demonstrate that Ag nanowires exhibit apparent temperature-dependent superplasticity at cryogenic temperature due to (110) to (100) cross-section reorientation behavior. Interestingly, HEA nanowires can perform exceptional strength-ductility trade-offs at cryogenic temperatures. Even at high temperatures, HEA nanowires can still maintain good flow stress and ductility prior to failure. Mechanical properties of HEA nanowires are better than Ag nanowires due to synergistic interactions of deformation twinning, FCC-HCP phase transformation, and the special reorientation of the cross-section. Further examination reveals that simultaneous activation of twining induced plasticity and transformation induced plasticity are responsible for the plasticity at different stages and temperatures. These findings could be very useful for designing nanowires at different temperatures with high stability and superior mechanical properties in the semiconductor industry. Nanomaterials are new age materials exhibiting exceptional mechanical, electronic, and optical properties due to their unique structure and high surface-area to volume ratio . Among nHigh entropy alloys (HEAs)  have gaiGali et al.  reportedStrain hardening in conventional metals is generally described with Taylor’s relation where an increase in flow stress is related to the increased dislocation densities . Deng etNowadays, HEA nanoparticles are synthesized successfully and are able to exhibit better catalytic performances . The sucAn embedded atom method (EAM) potential developed by Williams and Mishin  is used To describe the interaction among the atoms of CoNiCrFeMn HEA alloy NWs, a second nearest neighbor modified embedded atom method (2NN MEAM) potential developed by Choi et al.  is used To investigate the temperature dependent deformation mechanism during the tensile deformation of CoNiCrFeMn HEA and Ag NWs, we have conducted a series of atomistic simulations at 77, 300, and 1000 K. Molecular dynamics simulations as implemented in a large-scale atomistic/molecular massively parallel simulator (LAMMPS)  are perfBefore we begin the simulation, all the nanowires are first relaxed using conjugate gradient minimization. Subsequently, all the nanowires are equilibrated at their respective temperatures and zero pressure using Langevin thermostat and Berendsen barostat, respectively, in the z-direction. Subsequently, a microcanonical or NVE  ensemble is used to relax the nanowire for 50 ps. The simulation box remains periodic in all three dimensions and the time step for the integration is taken as 1.0 fs. After equilibration, a constant strain rate of  theorem . The ten theorem  is used.On the other hand, in CoNiCrFeMn HEA NW, stress increases monotonically with strain at 77 K, as shown in At 300 K, stress in CoNiCrFeMn HEA NW reaches up to 5.5 GPa at around 5% strain, as shown in At 1000 K, owing to the thermal effect raising the nucleation frequency, the yielding occurs earlier with a value of only half of that at low temperature. Unlike the SS curve of Ag at 1000 K where the flow stress and ductility decay quickly to fulfil the failure, the flow stress of HEA NW can still maintain at a certain degree as good as the flow stress at the low temperature. The good refractory property of the HEA NW in this work is attributed to the same synergistic mechanism as the aforementioned one. The difference is that the phase transformation induced plasticity seems to be more dominant at high temperatures so that the fraction of the HCP phase is much more than the low-temperature cases. We also find that the increasing fraction of the HCP phase is associated with the unlock behavior of the Lomer Cottrell locks. Due to the interaction of leading and trailing partial dislocations, Lomer Cottrell locks also form at 1000 K similar to the cases at low temperature. However, the Lomer Cottrell locks do not become impediments to the following dislocation motion but can be unlocked due to transformation into the HCP phase enabled by accumulation of stacking faults at the Lomer Cottrell locks. With this unique mechanism, the stacking faults at different orientations gradually quit the NW, and eventually, the stacking faults at the same orientation are left behind, as can be seen in Work hardening is often seen in metallic systems, leading to better ductility. Lomer Cottrell locks enable a complex dislocation network and provide a strong resistance to the propagation of moving dislocations and are inherently responsible for the work hardening behavior. In our simulations at low temperature where dislocation density is high as shown in The tensile deformation in CoNiCrFeMn HEA NWs is primarily proceeded by nucleation and glide of Strengthening of the nanowires is dependent on the nature of dislocation interaction at different temperatures. On two different (111) and On further tensile deformation, the interaction of partial dislocations results in the creation of a/6[011] stair-rod dislocation as following:Since the resultant a/6[011] is not on the active slip planes as shown in The superplastic behavior of Ag NW and CoNiCrFeMn HEA NW at cryogenic temperature is shown in the To explore the underlying mechanism of superplasticity in the CoNiCrFeMn NWs, the contributions of dislocations on plastic strain at different temperatures are also shown in Surface dislocation nucleation has been identified as a dominant deformation mechanism in NWs contrast to bulk materials. Twinning and dislocation slip are the two competitive mechanisms of deformation in FCC metallic NWs. The former is responsible for large plasticity while the latter results in limited plasticity. The competition between these two mechanisms is dependent on loading direction and material properties such as stacking fault energy. The energy barrier for the twinning is proportional to the change in surface energy. Thus, the transition of deformation modes is dependent on the change in surface energy associated with stacking fault energy. Stacking fault energy (SFE) is an intrinsic parameter which often depends upon composition, alloying elements, and temperature. Materials may exhibit martensite formations at low SFEs, deformation twinning at intermediate SFEs, and dislocation glide at high SFEs. Here, we explore the role of stacking fault energy using generalized stacking fault energy curves of Ag eference . We furteference  to estimeference .(4)δusfThe relative barrier height (ernstein .(5)τa=1 studies ,55. Even1It is found that both Ag NWs and HEA NWs show unique superplastic behavior during tensile deformation at 77 K. In HEA NWs, dominant deformation mechanism is TWIP, aided by limited FCC-HCP martensitic transformation during the structural re-orientation, while Ag NW deforms via twinning induced surface re-orientation.2At 300 K, during tensile deformation, the motion of partial dislocations is hindered by stacking fault networks resulting in formation of dislocation junctions or Lomer Cottrell locks in HEA nanowire. We speculate that variations in the local chemical composition in HEA nanowires facilitate the formation of Lomer Cottrell locks, which further promotes formation of nucleation sites for the HCP phase transformation. At a later stage of deformation, FCC-HCP martensitic transformation causes unlocking or annihilation of Lomer Cottrell locks. In contrast to deformation at 77 K, martensitic transformation induced plasticity overtakes the twinning induced plasticity in HEA nanowire, while Ag nanowire deforms mainly by dislocation slip with a little contribution from twinning.3At 1000 K, FCC-HCP martensitic transformation dominates in HEA NWs, which results in the TRIP effect enabling additional ductility and refractory properties. Similar to 300 K, dislocation junctions or Lomer Cottrell locks are unlocked and annihilated at a later stage of deformation. On the other hand, Ag nanowires fail by dislocation slip.4Increasing the deformation temperature of HEA nanowires leads to a decrease in the dislocation density, which is also responsible for shifting the deformation mechanism from TWIP to TRIP at elevated temperatures. With increasing temperature, martensitic transformation dominates as an independent mechanism and results in the annihilation or removal of Lomer Cottrell locks at the later stages of deformation. Thus, the synergistic sequence of plastic deformation mechanisms in HEA NWs at different stages of strain are the source of its excellent combination of strength, ductility, and toughness or damage tolerance which are in better agreement with the existing experimental and simulation studies.In the present work, tensile deformation of Ag NWs and multicomponent HEA CoNiCrFeMn NWs was carried out using molecular dynamics simulations at different temperatures. Based on this study, the following conclusions can be made:


Human death domain superfamily proteins (DDSPs) play important roles in many signaling pathways involved in cell death and inflammation. Disruption or constitutive activation of these DDSP interactions due to inherited gene mutations is closely related to immunodeficiency and/or autoinflammatory diseases; however, responsible gene mutations have not been found in phenotypical diagnosis of these diseases. In this study, we comprehensively investigated the interactions of death-fold domains to explore the signaling network mediated by human DDSPs. We obtained 116 domains of DDSPs and conducted a domain–domain interaction assay of 13,924 reactions in duplicate using amplified luminescent proximity homogeneous assay. The data were mostly consistent with previously reported interactions. We also found new possible interactions, including an interaction between the caspase recruitment domain (CARD) of CARD10 and the tandem CARD–CARD domain of NOD2, which was confirmed by reciprocal co-immunoprecipitation. This study enables prediction of the interaction network of human DDSPs, sheds light on pathogenic mechanisms, and will facilitate identification of drug targets for treatment of immunodeficiency and autoinflammatory diseases. The human death domain (DD) superfamily is one of the largest and most studied domain superfamilies. It comprises four subfamilies called the DD subfamily, the death effector domain (DED) subfamily, the caspase recruitment domain (CARD) subfamily, and the pyrin domain (PYD) subfamily , 2. DeatGenetic mutations in DFD-containing proteins often cause various immunodeficiency and autoinflammatory diseases . For exaAlthough much evidence have been accumulated, no mutations of responsible genes for these diseases have been identified, even in phenotypically diagnosed cases . This prIn this study, we focused on domain–domain interactions rather than on full-length protein–protein interactions. As shown in the diagram in Fig. We synthesized FLAG- and biotin-tagged recombinant DDs, DEDs, CARDs, and PYDs using the wheat germ cell-free synthesis system and investigated domain–domain interactions using the amplified luminescence proximity homogeneous assay (ALPHA) to elucidate the interaction network of DDSPs.cDNA clones encoding human DDSP domains . Amplified DNA fragments were subcloned into the pEU-E01-GW-FLAG and pEU-E01-GW-bls vectors using Gibson Assembly seamless cloning. After subcloning, pEU expression plasmids were arranged in a 96-well format and stored as glycerol stocks. The glycerol stocks were diluted with TE buffer and used as templates of PCR. Template DNA fragments for transcription were PCR-amplified using the SPu-2 primer (5′-CAGTAAGCCAGATGCTACAC-3′) and AODA2306 primer (5′-AGCGTCAGACCCCGTAGAAA-3′).The tagged recombinant human DDSP domains were synthesized using a wheat germ cell-free synthesis system . TranscrCell-free synthesized DDSP domains were diluted 20-fold, injected into a 96-well MaxiSorp plate , and incubated overnight at 4 °C. After washing with Tris-buffered saline containing 0.1% Tween 20 (TBST), the plate was blocked with TBST containing 5% skimmed milk for 1 h at room temperature. Next, the plate was incubated with an anti-DYKDDDDK tag monoclonal antibody  or anti-biotin antibody  diluted 1:2000 and 1:1000, respectively, in TBST containing 5% skimmed milk for 1 h at room temperature. Thereafter, the plate was washed three times with TBST and incubated with a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody  diluted in TBST containing 5% skimmed milk for 1 h at room temperature. Finally, 50 µL of tetramethylbenzidine liquid substrate (Sigma-Aldrich) was injected into the plate and incubated for 15–30 min at room temperature. The reaction was terminated by injecting the same volume of 1 M HCl. Absorbance at 450 nm was measured using a SpectraMAX M3 plate reader .http://www.visualdatatools.com/DataGraph/).All ALPHA reactions were conducted in an AlphaPlate-384 microplate . All proteins and reagents were diluted in reaction buffer . Twenty microliters of solution containing 0.4 μL of a biotin-tagged domain in reaction buffer was dispensed into the reaction plate  using a Viaflo automated multichannel pipette and the Viaflo Assist system . Next, 0.4 µL of FLAG-tagged domain was transferred to the reaction plate  using the Janus automated dispensing workstation (PerkinElmer) and Nanohead, a 384-well micro-syringe head (PerkinElmer). This procedure allowed 192 combinations of FLAG- and biotin-tagged domains to be mixed together in duplicate per assay plate. Then, 9.6 μL of detection mixture containing 0.02 μL of an anti-DYKDDDDK tag monoclonal antibody, 0.06 μL of streptavidin-conjugated AlphaScreen donor beads, and 0.06 μL of protein A-conjugated AlphaScreen acceptor beads in reaction buffer was added to each well of the reaction plate using a FlexDrop dropper (PerkinElmer). The detailed dispensing scheme and well layout are shown in Supplementary Fig. 2, mixed with 500 μL of 2× HEPES buffer , and added to each 10 mL well containing HEK293T cells. A total of 1 × 106 HEK293T cells were transfected with 1 μg of each of the following expression plasmid sets: pcDNA3-AIM2-PYD-AGIA and pcDNA3-NLRP9-PYD-FLAG, pcDNA3-AIM2-PYD-AGIA and pcDNA3-ASC-CARD-FLAG, pcDNA3-NLRC4-CARD-AGIA and pcDNA3-ASC-CARD-FLAG, and pcDNA3-NOD2-CARD1-CARD2-AGIA and pcDNA3-CARD10-CARD-FLAG. Transfected cells were lysed in 1000 μL of NP-40 buffer  supplemented with a cOmplete Mini Protease Inhibitor Cocktail tablet (Roche Diagnostics). Cell lysates were centrifuged. Supernatants were mixed with an anti-AGIA tag monoclonal antibody (in-house made)  were conducted in duplicate in the ALPHA assay.3 RLU, among which 236 pairs had signals higher than 2 × 103 RLU and 95 pairs had signals higher than 5 × 103 RLU  in two repeats. The red dashed lines represent the fold change threshold (±1). A total of 98.74% of the points were located between the two red dashed lines. This shows that more than 98% of the reactions were reproducible, demonstrating that this assay was highly reproducible and reliable.We assessed the reproducibility of the assay between two repeats using an MA plot Fig. . Each po3 RLU. Meanwhile, among the 13,340 nonself-interacting pairs located on either side of the diagonal, the interactions of 761 (5.7%) were strong with ALPHA signals higher than 1 × 103 RLU. Comparison of the percentages of positive results between self-interacting (32.8%) and nonself-interacting (5.7%) pairs implies that self-interacting pairs, which interact via homotypic binding, tend to interact more readily than nonself-interacting pairs.Human DDSP domains function in various signaling pathways involved in cell death and inflammation by oligomerizing with each other, which promotes interactions between proteins . In this3 RLU irrespective of the tags used (FLAG × biotin and biotin × FLAG) were extracted and listed in Table We focused on the red points of nonself-interacting pairs symmetrically distributed on both sides of the diagonal in the heat map , generating large multi-subunit structures comprising only one type of protein . HoweverThis study shows many novel possible interactions Tables  and 2. TThe one-sided interactions listed in Table CARD10  domain-containing protein 3 (CARMA3)) functions as a scaffold and is involved in NF-κB activation in response to various types of upstream innate immune signaling or modulates the interactions of deubiquitinating enzymes such as A20 and CYLD . SeveralAlthough the results of this study were highly reproducible, there are several limitations. First, all the recombinant DDSP domains synthesized and used in this study were fused with tags. The type and position of the tag may affect the ALPHA results. Depending on the structure of the domain, the fused tag may be concealed inside the mature domain and inaccessible to the detection antibody or streptavidin. Furthermore, when a domain interacts with partners via amino acid residues close to the terminus, the tag may interfere with the interaction, leading to false ALPHA results. This may explain one of the cause of known pairs of interactors that do not have a high enough ALPHA signal (Supplementary Table In conclusion, we believe that our comprehensive investigation of DDSPs will be helpful for the field of DDSP-related diseases, especially immunodeficiency and autoinflammatory diseases. This work may facilitate future research aiming to identify pharmaceutical targets for drug discovery and to elucidate the pathogenesis of these diseases.Supplementary Figure S1Supplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4


Lycosa sp.) use to close their burrows, serve to maintain favorable conditions of temperature and humidity within them. We removed trapdoors from burrow entrances and monitored changes in temperature and relative humidity in their bottoms, where the spiders reside during the day. We also followed the behavioral responses of these spiders to trapdoor removal at different times of the day and in different seasons and monitored temperature and relative humidity in artificial burrows during summer mornings and at midday. At noon, air temperature at the bottom of open artificial burrows increased by less than 1 °C more than in covered ones, and total humidity remained constant, even though air temperature at the soil surface reached 55 °C when the burrow temperature was 35 °C. The relatively small increase in air temperature in uncovered burrows at midday was probably due to the penetration of direct solar radiation. Thus, it is evident that the presence of a trapdoor has a negligible effect on the microclimate at the bottom of these spiders’ burrows and its roles are more likely predator avoidance and prevention of flooding and stones and sand from falling in.We investigated whether trapdoors, which small Negev Desert wolf spiders ("L. hyraculus") burrows in the Negev Desert serve to maintain favorable environmental conditions within the burrow by removing trapdoors and monitoring the ensuing temperature and relative humidity regime within them. We also monitored the behavioral responses of “L. hyraculus” to trapdoor removal at different times of the day and in different seasons. “L. hyraculus” often spun silk mesh in their burrow entrances in response to trapdoor removal during the day, possibly to deter diurnal predators. The frequency of web-spinning peaked on summer mornings, but spiders began spinning webs sooner after trapdoor removal later in the day. In addition, we monitored temperature and relative humidity in artificial burrows in the summer during the morning and at midday. At noon, air temperature (Ta) at the bottom of open burrows increased by <1 °C more than in covered burrows, but water vapor pressure in burrows did not change. The relatively small increase in Ta in uncovered burrows at midday can probably be ascribed to the penetration of direct solar radiation. Thus, air temperature and humidity at the bottom of the burrow are apparently decoupled from airflow at the surface.Burrows are animal-built structures that can buffer their occupants against the vagaries of the weather and provide protection from predators. We investigated whether the trapdoors of wolf spider (Lycosa sp.; temporary working name  Animal-built structures vary widely in shape and size and have evolved through natural selection on their occupants . Burrows2, CO2, NH3, etc.) due to limited ventilation and thus affect its normal physiological functions. This is, however, not necessarily the case, and several ventilation mechanisms have been reported in the literature for relatively large bi-entrance [The physical isolation of an animal in its burrow could potentially result in the depletion or accumulation of respiratory or other gases  seal their burrows during estivation [Aliatypus  trapdoor spiders that inhabit arid areas in western North America were found to seal their burrows more often than their conspecifics that live in more humid conditions [It has been proposed that forced ventilation of the lower parts of their burrows may be detrimental to ectothermic animals, as implied from the fact that some scorpions build burrows in a manner that reduces gas exchange ,13, and tivation , presumanditions ,16,17.None of the above-mentioned studies, however, are supported by direct measurements of the relevant microclimatic variables in the field. Our aim was therefore to quantitatively assess the effects of the presence or absence of a trapdoor on the environmental conditions at the bottom of burrows of a common, but undescribed, lycosid spider species. To this end, we made detailed measurements in a set of artificial burrows, built in the field to resemble natural ones as closely as possible; we also made observations of spider behavior in their natural burrows after selectively removing trapdoors at different times of the day.Ta) and relative humidity (RH) in the burrows and that their removal would affect environmental conditions at the bottom of the burrow and therefore induce spider responses depending on the prevailing environmental conditions at the surface. Specifically, we predicted that warmer and drier surface conditions would elicit faster responses to trapdoor removal.Our working hypotheses were that the presence of trapdoors would have measurable effects on air temperature (Hammada scoparia (Pomel) Iljin.Field site—Our research site was a 3-hectare plot on the Sede Zin plateau in the Negev Desert highlands, less than 50 m from the north-east corner of the Sede Boqer Campus of Ben-Gurion University of the Negev at Midreshet Ben-Gurion, Israel , where the Jacob Blaustein Institutes for Desert Research (BIDR) meteorology station is located. The station provides hourly-averaged values of standard environmental variables. The loess soil in the study area is sparsely populated by shrubs, dominated by Lycosa hyraculus”  that we have used below. All the spiders studied were adults and we did not differentiate among sexes.Study species—The many groups of spiders that build burrows include wolf spiders (reviewed in ). We stuL. hyraculus” generally remain in their burrows with the trapdoor closed. At night, they actively hunt on the soil surface within the vicinity of their burrows, typically less than 1 m away, and the trapdoors are left open . Like other lycosids, “L. hyraculus” is a generalist predator and we observed them capturing or holding moths, beetles, termites, scorpions, smaller spiders, and other arthropods.During the day, “Lycosa hyraculus” co-occurs with Lycosa olivieri Simon, 1876, also a widely distributed species, but the two differ in their coloration and the construction of their burrow entrances. Lycosa hyraculus covers and camouflages its burrow entrance, usually with a trapdoor made from a piece of soil crust. L. olivieri, in contrast, builds a grass or soil turret around its burrow entrance. The presence or absence of a turret was found to be a reliable species-specific indicator in other burrowing wolf spiders [L. hyraculus” but extends towards the cephalothorax in L. olivieri  and between 7 and 21 mm in diameter . Ninety-eight percent of the burrow entrance diameters were less than 17 mm. Diameters and depths were linearly correlated (diameter (mm) = 4.51 + 0.073 × depth (mm); r2 = 0.3228; F = 92.47; p = 0.0001). Burrows are lined with a layer of silk, which may stabilize the soil [L. hyraculus" constructs the trapdoor from materials available in its environment, most often pieces of soil crust, but also small pebbles and snail shells, and attaches it to the burrow entrance with silk strands [L. hyraculus" trapdoors are usually lined with silk.Natural burrow structure: We found that “the soil  and faci strands ,21. The tapeta lucida in our headlamp beams. We located over 200 “L. hyraculus” and their burrows between May 2016 and July 2017, recorded the GPS location  of each and marked it with a flag and identification number. We measured burrow entrance diameters and spider body lengths  using digital calipers . The spiders remained immobile on the soil surface near their burrow entrances in our headlamp beams and we could lay the caliper next to them to measure body length. Depth was manually measured with a thin rod against a steel ruler.Natural burrow survey: At night, we found the spiders visually from reflections of their Artificial burrows: We made artificial burrows of two diameters that we chose to represent the range of natural burrow dimensions  to standardize burrow shape and size. To make a burrow, we first slightly wetted the soil surface to stabilize the soil crust. We then drove a hollow pipe  100 mm into the ground, and slowly twisted it up to extract soil. We fitted the burrows with sensors and immediately covered them with opaque, soil-colored plaster discs , 10–20 mm in diameter and 2–3 mm thick, to mimic natural cover and to prevent the soil from drying out at the bottom of the burrow. The covered burrows were allowed to equilibrate with the surrounding soil at least 24 h before measurements were made.Ta in ten artificial burrows of each size  twice a day (early morning and midday) on six days in June 2017. Each burrow was fitted with a type-T thermocouple  at the bottom of the burrow with the junctions bent upwards to ensure that they did not touch the bottom or sides. The thus measured air temperatures are indicative of the environment in which spiders likely reside on hot days [Ta 10 mm above the soil surface with a thermocouple shaded by a white polyurethane board. All thermocouples were connected to dataloggers , which recorded Ta at 10 s intervals and averaged once per minute.Measurements: (1) Temperature: We measured hot days . We conc(2) Relative humidity: We measured relative humidity (RH) and temperature simultaneously using compact probes , whose overall accuracy was at 25 °C is ±2% RH. We calibrated the probes against a humidity sensor conforming to the Israel Meteorological Service standard  at the BIDR meteorology station for 24 h on 30 May and 8 August 2017.e). We first solved for the saturation water vapor pressure  at the given temperature (using Equations (2.5) and (2.6) of [e, as e = RH × es.Since RH depends on temperature, we examined changes in the water content of the air at the bottom of the burrow by calculating the water vapor pressure ((2.6) of  and calcdrad) is:D is the burrow diameter and α is the solar elevation angle. We calculated the elevation angles for the time of opening and closing of the burrows at midday using the NOAA Solar Position Calculator  (accessed on 1 September 2021). Typical values were 81° and 71° at the beginning and end of the measurement trial (13:00–14:00) during the period of 14–18 June 2017 . Thus, we could determine if and for how long direct radiation reached the bottom of the natural “L. hyraculus" burrows.(3) Estimation of solar radiation penetration: The thermal and hydric conditions in the burrow may be affected by diffuse and direct radiation entering open burrows. Diffuse radiation from the celestial hemisphere that enters the burrow can be neglected because the solid angle subtended by the burrow’s entrance is extremely small. Direct radiation penetration into the burrow depends, however, on the elevation of the sun and the size of the burrow opening. Assuming a straight, vertical burrow, the maximum depth that direct radiation could penetrate the burrow  On each trial day, we randomly chose three burrows and opened them for one hour at 07:00, and then three different burrows were opened for one hour at 13:00, leaving four burrows with trapdoors untouched as controls. Opened burrows were closed after each trial. Trials were repeated on three different days for both large (17 mm diameter) and medium-sized (11 mm) burrows. Trial (2) We monitored RH on the 30 July 2017, only at the bottom of the large (17 mm diameter) burrows due to the size of the sensors. We measured RH and temperature simultaneously in six burrows (three of them randomly uncovered) at 1-minute intervals, for 1 h (13:00–14:00), for at least 15 min before and after uncovering. Each set of trials  was analyzed separately. Opened burrows were closed after each trial.L. hyraculus" responses to trapdoor removal at different times of the day and in different seasons. For each trial, we removed the trapdoor and monitored the response of the spider for up to 24 h.Spider responses to trapdoor removal: We examined changes in “n = 16; July 2017, n = 33) and the winter . For each trial day, we removed half of the trapdoors in the early morning (06:00) and half at midday (12:00). We monitored burrows at 1-hour intervals for 3 h following trapdoor removal and at 3-hour intervals thereafter until sundown. We defined tweb,h as the time in hours from trapdoor removal until when we first saw a complete mesh in the burrow entrance. If a spider partially covered its burrow entrance but never completed it, we assumed that we had disturbed the process during our examination and noted the time of completion rounded up to the next full hour. We also used data collected at the BIDR meteorology station to relate changes in spider responses to soil and air temperatures.Seasonal patterns: We compared spider responses to trapdoor removal during the summer  positioned on a short tripod above and to the north of a burrow entrance to avoid shadows and set the camera to take 100 photographs at 1-minute intervals. The first picture was taken with the burrow covered and a ruler next to the trapdoor. Thereafter, we carefully removed the burrow trapdoor and left the area. We moved the camera to a different burrow every 2 h, starting at 07:00 and ending at 19:00 over 15 days between 6 April and 5 May 2017. Since creating the mesh often involved several bouts of activity, it was difficult to define exactly when the repair behavior ended. Thus, we defined tweb among groups using one-way ANOVA [Data analysis of daily spider activity patterns: For each experiment, we compared mean ay ANOVA . We testt-test for unequal variances, where necessary. In our comparison between seasons, we ran a post-hoc test with a Bonferroni correction for multiple comparisons after chi-squared analysis [We examined differences in the fraction of spiders that spun meshes among groups using chi-square analyses. To determine whether and how this fraction changes over the course of a day, we ran a test of equal proportions followed by a chi-squared test for trends in proportions . We alsoanalysis .No differences between the temperature measured at the bottom of open and closed burrows, narrow or wide, were observed for the morning measurements (data not presented). The noon burrow-bottom temperature measurement of the wider burrows, however, showed a clear pattern.Tt as:We defined ΔT = temperature (°C)t” denotes = measurement timeSuperscripted “Superscripted “0” denotes reference time (five minutes before uncovering)bi” = uncovered burrow i (i = 1 to 3)Subscripted “bck” = covered burrow i (k = 1 to 4)Subscripted “Tt data are presented in Tdiff during the first seven minutes after uncovering can be observed. This rise is followed by a moderate decrease in ΔTt and a sharper drop upon re-covering the burrows 60 min after uncovering. To assess the significance of these observations, we analyzed the data for two critical times: 10 min and 60 min after uncovering. The temperature at the bottom of each burrow was measured five minutes before uncovering; this measurement was used as a reference temperature to reduce the effect of the between-burrow temperature variability in our calculations. We compared the changes in the burrow-bottom air temperature of each burrow to its corresponding reference temperature and analyzed the treatment effect 10 and 60 min after the removal of the doors.Δt-tests for 14/6: mean difference = 0.9042 ± 0.2172 SD, t = 5.45, df = 5, p < 0.001; for 15/6: mean difference = 0.5058 ± 0.1031 SD, t = 6.42, df = 5, p < 0.001; and for 17/6, Welch–Satterthwaite t-test: mean difference = 0.5558 ± 0.1023 SD, t = 5.99, df = 2.1, p < 0.013). Sixty minutes after uncovering and immediately prior to covering the burrows, the mean differences were smaller . Upon trapdoor removal, direct radiation penetrated into the burrow and illuminated the northern portion of the wall, from the surface down to the bottom, but never illuminating the bottom itself , particularly when compared to soil surface temperatures, which reached 55 °C and 39 °C 100 mm above the soil surface.Ta increased as expected after uncovering burrows, water vapor pressure differences between covered and uncovered burrows were not significant (data not presented). The fact that both water vapor pressure and temperature appear to be decoupled from the above-ground air flow indicates that resonant mixing or eddy penetration [Ta in the artificial burrows probably represent the maximum effect of the trapdoor in 10-millimeter deep burrows because natural burrows typically have narrower entrances and because there was no forced ventilation in the bottom of the burrow. Thus, the fact that both water vapor pressure and temperature were not affected by above-ground atmospheric conditions strengthens our conclusion that changes in air temperature at the bottom of the burrow when uncovered were negligible, i.e., the microclimatic conditions at the bottom of the burrows is effectively decoupled from the above-ground airflow.Although RH decreased and etration ,10 are nDirect radiation calculated from the dimensions of 181 natural burrows reached a maximum of 42% of total burrow depth at 13:00 on a typical summer day . In onlyn = 181) and corresponding depths for solar zenith angles at 13:00  and at 14:00  on 24 June 2017. The length of each light grey line represents the depth of solar radiation penetration, and the lower boundary of the dark grey line corresponds to burrow depth. Burrows marked with arrows would receive direct radiation at the bottom of the burrow at 13:00 local time.Burrows are ordered by depth. Penetration depth (light gray) was calculated using measured spider burrows diameters  no visible change or activity; (2) spinning a mesh in the burrow entrance during the day ; or (3) ot noted ,26.The spiders appeared to manipulate the soil crust with their chelicera and by adding silk to fit the burrow entrance, rather than build a new door from soil and silk, as do other spiders . HoweverF3 = 85.5, p < 0.001; Tukey post-hoc p < 0.001 for all comparisons against winter morning group; Seasonal patterns: Spiders responded to trapdoor removal slowly on winter mornings, with six hours passing, on average, before beginning to spin a mesh in the burrow entrance . Response times were significantly longer in the morning , with a decreasing trend over the course of the day. As with the seasonal responses, decreases in response time were associated with increases in temperatures in the upper layer of the soil burrow (~100-milimeter deep). The fraction of spiders that spun meshes also decreased over the course of the day (test for equality of proportions p < 0.001; chi-squared test for trend in proportions, p < 0.001, L. hyraculus” burrows and probably do not affect spider behavior. In addition, the hydric environment was unaffected, and we therefore conclude that under the weather conditions that this study was done, the conditions at the bottom of the burrows were essentially decoupled from the above-ground airflow. The fact that most “L. hyraculus" do not attempt to cover their burrow entrances immediately upon trapdoor removal at midday in the summer lends support to this conclusion. In this light, the trapdoor’s roles are more likely predator and parasite avoidance and prevention of flooding, and debris from falling in.The results obtained from the artificial burrow field trial imply that during the summer months, the absence of trapdoors has a marginal effect on the thermal environment at the bottom of “


For any food, it is important to know consumption, preference, and the characteristics as quality parameters that are important to consumers of a product. The descriptive methodologies are an important tool to know the quality attributes of the products. Within these methodologies is the flash profile (FP), which is based on the generation of the distinctive attributes of the products without any expensive and time-consuming training sessions. The aim of this research was to study the consumption and preference of flour tortillas by consumers and to develop the descriptive characterization of the tortillas by using the flash profile method. The wheat flour tortillas used were two commercial and two handcrafted samples. Ten experienced panelists participated as the FP panel. The panelists generated 22 descriptors, six for texture, seven for appearance, five for odor, and four for flavor. These descriptors differentiate the samples of the flour tortillas. The panelists’ performance was assessed using the consensus index (Rc = 0.508). The first two dimensions of the Generalized Procrustes Analysis represent 83.78% of the data variability. Flash profile proved to be an easy and rapid technique that allowed the distinctive attributes of flour tortillas to be obtained. In the bakery industry, the flour tortilla segment has increased significantly in North America . The torTherefore, flour tortillas have various end-uses and characteristics. The hot-press procedure is today the most used 90%) in the total production of tortillas. The dough is cut into small balls of the same weight and shape, then they are crushed into discs through heated platens to obtain the tortillas . Flour t0% in theStronger tortilla doughs that absorb a greater volume of water are obtained by the die-cut procedure. This procedure consists of passing the dough into the rollers so sheets can be formed and which are cut into uniform circles. The die-cut process is appropriate for a bigger production capacity. It is more efficient and generates lower-cost products compared with the hot-press process. Nonetheless, the tortillas made with this procedure are not as adhesive, soft as the ones made with the hot-press process, and the flexibility is lost more quickly during storage. Despite these disadvantages, these types of tortillas are used to prepare processed foods, such as enchiladas, burritos, and chimichangas ,8,9.The third procedure used to obtain flour tortillas is the hand-stretch method. The tortillas are generally thinner and firmer than the ones made with hot-press or die-cut processes. Unfortunately, for the production of tortillas by this method, more personnel are required, and the production speed is slower, which is why tortilla manufacturers do not use this method on a large scale ,5. For aFor any of the flour tortilla-making processes, other ingredients, such as antimicrobial and antioxidant agents, among others, can be added to extend the shelf life and to improve the sensory characteristics demanded by consumers ,2,10. Yeverino et al.  determinA common practice for some time in the food area has been to describe the sensory characteristics of products, either to identify the characteristics of the product, identify the result of the change of an ingredient or phase of the process, evaluate the shelf life, match the standard product, development of a new product or as a means of quality control ,13,14. AConventional descriptive methodologies allow obtaining sensory profiles, however, with certain limitations that delay investigations. Some of the limitations are related to the difficulty of measuring perception, training time, and the resources necessary to form and maintain the descriptive panel ,16,17. FAs a way to characterize and quantify sensory differences and similarities among products, conventional descriptive profile methodologies are generally applied. This requires the training of panelists and the rating of the products’ attributes based on a consensual vocabulary ,16,17. TOriginally, FP was developed to be carried out by experienced panelists. The panel could be sensory experts and/or professionals who have developed sensory experiences with the products. For the number of panelists, the original FP method used eight panel members , but latRecent studies have also explored the application of the FP method with consumer panelists. In these studies, 24–50 consumers were considered sufficient ,21,27,28The use of the Flash profile technique is relatively new but has been applied to the analysis of a great variety of products, including dairy, meat, poultry products, snacks, bakery products, jams, juices, marmalades, among others food samples ,25,26. TThe aim of the present study was to investigate the consumption and preference of flour tortillas by consumers and to generate a descriptive characterization for wheat flour tortillas by applying the Flash profile method.Four wheat flour tortilla samples  , Reyna bA four-question survey was applied to 100 consumers in Saltillo, Mexico. The questions included age range, the brand of flour tortilla consumed, kind of flour tortilla preferred, and frequency of consumption. Panelists were included on the basis of 50 women and 50 men, age range between 20 and 45 years, being a flour tortilla consumer, and their willingness to participate in the survey. The answers were counted and expressed as a percentage.Wheat flour tortilla samples were purchased from two local retailers and two commercial brands in Saltillo, Mexico. The two local manufactures of flour tortillas were labeled as “Bomberos” and “Super García”, and the two commercial brands were labeled as “Soriana” and “Tía Rosa”. The total number of samples was four. The diameter and thickness of each brand were as follow: “Bomberos” 5.91 in diameter and 0.08 thick, “Super García” 5.51 in diameter and 0.08 thick, “Soriana” 6.7 in diameter and 0.10 thick, and “Tía Rosa”. The tortillas were given at a temperature ready to eat (45 ± 5 °C).The panel was made up of ten trained panelists  who are habitual consumers of flour tortillas. The panelists were recruited from staff and students of the Agrarian Autonomous Antonio Narro University. Their selection was based on experience in evaluating food products with descriptive sensory analysis and consumption of flour tortillas. All of them had previously participated in discriminative, consumer, and sensory profiling analysis. They were members of the trained panel of the Antonio Narro Autonomous Agrarian University and students of the engineering career in food science and technology. All were familiar with wheat flour tortilla attributes. However, they were not specifically trained in the evaluation of flour tortillas, and they had not evaluated flour tortillas previously. Each panelist generated from 12 to 22 attributes adding a total of 187 sensory descriptors. The attributes of the samples of flour tortillas generated corresponded to appearance, texture, smell, and taste.A survey with 4 questions was applied to 100 consumers, of which 50 were women and 50 were men, to determine the consumption and preference of the flour tortilla. The age range of the surveyed population was 20 to 45 years, 65% of the population was within the age range of 20–25 years, 19% people over 45 years, 9% people between 25 and 35 years old, and 7% people between 25 and 35 years old.By following Montanuci et al. (2015) , the senThe results were analyzed through Generalized Procrustes Analysis (GPA) using XLSTAT  in order to minimized differences between panelists of the sensory analysis. From the data matrices of the profiling assay, GPA calculates a consensus, and the generated plots demonstrate the differences and similarities of the flour tortilla according to the graphic interpretation.Consumers were asked what brands of tortillas they consumed were. The following were considered within the proposed variants: flour tortillas without brand, tortillas made with La Perla flour, and commercial tortillas “Tortillinas”. According to the surveyed population, 52% consumed wheat tortillas without a registered brand, while 23% preferred to make their tortillas, 20% purchased the commercial brand “Tortillinas”, and 5% consumed the brand name La Perla.According to the kind of tortilla that was consumed, 74% of the surveyed population consumed white flour tortillas, 17% whole wheat flour, and 9% both.Regarding the frequency of consumption, the results of the surveyed population, 62% consumed it three times a week, 14% consumed it once a day, 9% every day, 9% occasionally, and 6% consumed flour tortilla more than once a day.The consumption of the flour tortilla is a deeply-rooted phenomenon in the north of the country since it is where most of the wheat is harvested. Tortillas are an extraordinary alternative to bread, which can be consumed alone by making a roll or cut into 4 or 6 pieces to collect food, or they can be used to wrap different stews . Like alThe residual variance for each manufacture after performing GPA is shown in In relation to the residuals per panelist, the results demonstrated that panelists 2, 3, 4, 5, 6, and 9 conferred an exceptional agreement about the attributes that have been used . The resp < 0.0001, taking as the consensus index the percentage of the consensus variance in the total variance (Rc), while in the determination of discrimination, the correlation coefficient was applied to reveal which sensory terms were correlated with the principal components (CP) since the sensory term is important for the characterization of the samples in the sensory space [The consensus index (Rc) was used to determine the consensus of the panelists. The evaluation of the consensus of the panelists was carried out using the permutation test with a ry space ,13,30. Try space  in the ery space  in the ery space  in the sry space  in the sSer  applied The result of the Flash profile analysis represented in two dimensions (59.21% for the first dimension and 24.57% for the second dimension) explained 83.78% of the variability of the data . The conThe panelists characterized Bomberos flour tortilla as flour flavor, roughness, salty odor, brown spots appearance, toasted appearance, and raw appearance. Soriana’s flour tortilla was described as rubberiness, thickness appearance, flour odor, elasticity, greasy odor, greasy flavor, and sweet odor. The Super García was characterized as thin appearance, buttery odor, rollability, greasy appearance, and sponginess. The Tía Rosa flour tortilla was described by homogeneity, softness, sweetness, and salty taste. According to Kobayashi and Benassi , only th® and Flash Profile (69.0%) and Silva et al. [The variability percentage of this study (83.78%) was higher than that obtained by Rason et al.  in the sa et al.  in the cThe majority of the analyzed population preferred white, homemade, and unbranded wheat flour tortillas and the frequency of consumption was three times a week. The Flash profile methodology is a useful tool to characterize flour tortillas. It allows quick access to product positioning and its distinctive attributes. The methodology provides information that differentiates the samples, and as well, it does not require the training stages of a trained panel for conventional descriptive methods, reducing the time and cost of descriptive tests. The panel consensually generated the 22 descriptors with the Flash Profile method for flour tortilla samples. The descriptors obtained are an initial step to detect and help to understand the important sensory attributes for the consumer of flour tortillas, which will allow their measurement and definition by a trained panel with the use of the quantitative descriptive analysis (QDA), providing a complete, detailed and accurate information about this product.