The purpose of the work was to inactivate PPO activity to extend the shelf life of graham flour and at the same time preserve all the of its high-quality properties. The influence of supercritical CO2 (scCO2) treatment on PPO activity in graham flour was investigated. First, graham flour was exposed to scCO2 conditions, then the proteins were extracted, and in the last step the concentration of total proteins and the specific activity of the PPO enzyme were determined by spectrophotometric assay. PPO activity decreased with an increase in treatment pressure. Furthermore, the flour quality characteristics that meet all needs for wheat end-use products after scCO2 treatment have been preserved. No major changes in the structure of the granulate or shape of the flour particles were observed. A slightly reduced value of the moisture content in scCO2-treated graham flour also implies an extension of the shelf life.Graham flour is a form of whole wheat flour made by grinding the endosperm and is thus also the most nutritious. Generally, the enzyme polyphenol oxidase (PPO) catalyzes two different reactions in the presence of molecular oxygen: the hydroxylation of monophenols to ortho-diphenol and the oxidation of  This browning causes the deterioration of food, causing large economic losses. Moreover, a recent study from Zhai et al. also discovers the importance of PPO enzyme in flour onto time-dependent discoloration and darkening of wheat (Triticum aestivum L.) products. Based on the results obtained, it was established that PPO activity in flour is the main cause of undesirable darkening of wheat end-use products [Graham flour is similar to whole wheat flour, as it contains the germ, endosperm, and bran of the wheat berry. Products vary in granulation and percentage content of the whole kernel, and the size of the bran particles affects the baking quality of the mix . Moreoveproducts . Enzymatproducts ,4, and hproducts .o-diphenol to o-quinone [PPO is a dicopper-containing enzyme involved in oxidation of the polyphenols . General-quinone ,7,8. The2 used for inactivation of PPO activity in graham flour is suitable as it does not adversely affect the quality of the flour. Furthermore, the latest study from Guo et al. presented the achievement of high inactivation of the PPO enzyme using the superheated steam treatment procedure in which a very high temperature of up to 190 °C was used [2 technology, it is much more suitable as throughout the whole procedure. The temperature was only 35 °C, which is an additional parameter, which proves that with the scCO2 technology the properties of flour remain the same as those of untreated flour.Enzymatic browning has also been implicated in the discoloration of flour, an issue that has already attracted the attention of some researchers. A heat treatment procedure is often applied for enzyme inactivation with the use of a microwave oven operated at 900 W for 80 s, as the influence of high temperatures above 50 °C could cause several biological, chemical, and physical modifications leading to sensory, nutritional, and textural changes, affecting both the quality and biocomponents of the flour , where dwas used . If we c2 has already been shown to be suitable for enzyme inactivation in our previous research, due to its specific characteristics. Dense carbon dioxide in the supercritical state is nontoxic and non-flammable [2 was used for inactivation of PPO in graham flour in order to prolong the shelf life of the flour and prevent the browning process, which occurs in the presence of the enzyme PPO. For this reason, a specific non-thermal technology with scCO2 was used for the inactivation of enzyme PPO in graham flour. High-pressure carbon dioxide (HPCO2) has previously been reported as a promising non-thermal technology for the stabilization of fresh products. During such treatment, food is in contact with pressurized CO2 [2 are above the critical temperature and critical pressure  [2 is an environmentally friendly solvent that is ideal for food processing [2 pressure is generally within 40 and 300 bar, rarely exceeding 500 bar. The temperature is generally much lower than that seen with conventional treatments, between 20 °C and 50 °C, low enough to represent a non-thermal treatment [2 treatment [2 [The application of scCOlammable , and thuized CO2 . Moreove3.8 bar) ,16. Furtocessing ,18. Typireatment . It has reatment , HP CO2 reatment ,23,24,25reatment , ultrasoreatment , and tretment [2 . Yadav etment [2 . Nonethe2 on graham flour is presented in the current study. The article describes inactivation of the enzyme PPO in graham flour, where the activity of PPO was affected by scCO2 treatment under different conditions. Moreover, the structural properties of untreated and scCO2-treated graham flour were characterized using scanning electron microscopy (SEM), a particle size analyzer and moisture content analyzer.For the first time, the detailed influence of a sustainable process using scCOThe effects of flour pre-storage on the protein concentration of the flour were determined by different methods of pre-preparation. More specifically, glass beads were added to the graham flour before the extraction process, where a slightly better extraction was expected since glass beads can provide efficient disruption, homogenization, and high yields of extracted flour proteins . ProteinPre-storing flour at 4 °C positively affected the thermal stability of the enzyme, resulting in a higher protein concentration after protein extraction from graham flour. The use of glass beads also enhanced the disruption of the protein matrix and facilitated better access to protein molecules , which wBased on these results, the optimal method with the highest protein concentration is a process where glass beads have been added to the flour, and therefore we decided to use this method for extraction of proteins from graham flour in further tests.2 on protein concentration was evaluated after graham flour exposure to scCO2 at different conditions, with the results presented in 2-treated graham flour. As can be seen from the results, the protein concentration in graham flour decreased after scCO2 exposure. Additionally, with increasing pressure it is observed that the protein concentration was reduced by a maximum of 20% after scCO2 treatment for 24 h and 300 bar, confirming that scCO2 induced protein loss in graham flour. The protein loss in scCO2-treated graham flour in this study is mainly ascribed to leakage of proteins into the media. A similar effect was also described in Liao et al. (2010) [2 treatment of E. coli cells by extending the exposure time.The protein concentration after protein extraction from graham flour was determined by the Bradford method. The effect of scCO. (2010) , where t2-treated graham flour was determined, as described in 2 on the residual activity of the PPO enzyme in graham flour at different pressures and with different treatment times at 35 °C is presented in 2 treatment of graham flour at 300 bar was 87%. Moreover, after 24 h of treatment at 200 bar there was an 18% decrease in PPO activity, and after scCO2 treatment at 300 bar the residual activity of PPO decreased significantly to 65% of the initial value of the untreated flour. Similar to the results for protein concentration, at a higher pressure a greater inactivation of the PPO in graham flour was achieved. As the residual activity of PPO in graham flour was lower at higher pressure (300 bar), it is obvious that a pressure of 300 bar for scCO2 treatment is more suitable for the inactivation of PPO.After determination of the protein concentration in supernatant, obtained by extraction after centrifugation, the PPO activity of untreated and scCOThe effects of pressure in a supercritical fluid environment are complex and can be divided into three categories. First, the direct effects of pressure on the conformation of enzyme molecules (stability); second, the effects on intrinsic enzyme kinetics; and third, the effects on the physical properties of supercritical fluids, which can then indirectly modulate enzyme activity, specificity, and stability .2 pressure and temperature. This earlier study describes inactivation of PPO from Agaricus bisporus, where the residual activity of PPO after scCO2 treatment at 300 bar and 45 °C was 60%. With prolongation of the time (30 min), higher pressure (650 bar), and higher temperature (50 °C), the lowest PPO residual activity of 12% was obtained [2 on the PPO enzyme in graham flour. Furthermore, in another study, Marszałek et al. (2017) described the inactivation of PPO in beetroot juice, which showed that the rate of decrease in PPO activity after scCO2 treatment was very low, only a few percent, although a high pressure of 600 bar and high temperature were used [These observations coincide with the work of Marszałek et al. (2019), which explains the sensitivity of the PPO enzyme to scCOobtained . Howeverere used .2, under these conditions had no deteriorating effects on its nutritional value [Our study was focused on lower pressure conditions compared to those in earlier works due to possible changes in flour quality   found that the polyphenol content in potato correlated with PPO activity during storage [The results of total phenolic content in untreated graham flour compared to scCO storage , which i2 on graham flour quality, characterization of flour particles was performed by SEM and particle size analyses. In addition, the moisture content for both untreated and scCO2-treated graham flour was examined.In order to determine the influence of scCO2-treated graham flour were determined. SEM micrographs of the untreated and scCO2-treated graham flour at the same magnification level are shown in 2-treated graham flour were within the size range 16 to 83 μm. Moreover, it can be observed that larger clusters of particles disintegrated after scCO2 treatment, which is consistent with the particle size measurement results, as presented in the 2 does not adversely affect the quality of the flour, nor does it not significantly change the structure. This provides further support for the idea that scCO2 treatment is appropriate for preprocessing of graham flour, which could be further used in the bakery industry.The mean particle size and shape of the untreated and scCO2-treated graham flour was conducted to determine if scCO2 affects flour quality. 2 treated graham flour even indicates an extension of the shelf life of the flour. On the other hand, the moisture content was not so reduced to affect further flour processing in the manufacture of bakery products in terms of its mechanical, thermal, and rheological properties [Moisture content affects the processibility, shelf life, usability, and quality of the final graham flour product. Moisture content determination therefore plays a key role in ensuring quality with regard to applications in the food industry. Consequently, an analysis of the moisture content in both untreated and scCOoperties . It is aoperties .2-treated graham flour changed slightly, and it became lighter due to the reduced moisture content. As a result, certain aggregates disintegrated, which is reflected in the reduced value of the particle size distribution. In short, the scCO2 medium successfully inactivated the enzymes and at the same time did not weaken the key properties of the flour that are crucial in the production of bakery products.The properties of scCOl-3,4-dihydroxyphenylalanine, l-ascorbic acid, and ethylenediaminetetraacetic acid were supplied from Sigma . All other chemicals used in the laboratory were of analytical grade.Graham flour was kindly donated by the bakery Hlebček d.o.o. . Analyses were conducted using carbon dioxide (99.5% purity) produced by Messer, Ruše. Ethanol, phosphoric acid, and Coomassie-Brilliant Blue G250 were supplied from Merck , while bovine serum albumin (BSA), sodium acetate, potassium phosphate, 2 under the described conditions. The process of exposure was followed by protein extraction, where the resulting clear supernatant was used for spectrophotometric measurements of PPO activity and protein determination in graham flour.An overview of the experimental procedure in 2 in a 60 mL high-pressure batch reactor at 35 °C to the desired pressure (200 bar and 300 bar) for a defined time, with the scCO2 system shown in 2 was added to the reactor until the desired pressure was obtained. Afterwards, the reactor was quickly depressurized (Δp/Δt = 0.37 ± 0.08 bar/min). Furthermore, a sample of flour was taken out of the reactor and left at room temperature for some time to ensure that CO2 was released out of the sample. The enzyme activity of the treated samples of graham flour was compared with that of the untreated graham flour.Graham flour was exposed to scCO2 to reach its supercritical state. All experiments were done in triplicate and the error bar represents the percentage error (±3%) in each set of readings.It is known that exposure of enzymes to higher temperatures leads to their inactivation . As the Proteins were extracted from graham flour (5 g) with 0.1 M acetate buffer solution (pH 5.3) by shaking for 90 min at room temperature. To optimize the process of protein extraction from graham flour and obtain the best possible yield, various parameters, which are specifically listed in Protein concentration was determined according to the Bradford method using BSA as the standard protein , where ac (mg/mL) in the samples was calculated using the standard curve of BSA from Equation (1),A595 nm is the sample absorbance measured at 595 nm, and k is the slope of the calibration curve, according to Bradford [The absorbance was measured in the supernatant after centrifugation using a colorimetric method and a UV-spectrophotometer with the wavelength set at 595 nm . Each sample was assayed twice and three individual absorbance measurements per extracted sample were recorded. Moreover, the protein concentration Bradford .tert-butylcatechol, and chlorogenic acid [l-3,4-dihydroxyphenylalanine, 0.072 mM ascorbic acid, 0.0022 mM ethylenediaminetetraacetic acid, and 50–100 units of PPO. One unit of enzyme activity was defined as the amount of enzyme causing a 0.001 change in the absorbency per minute at pH 6.5 at 25 °C in 3 mL reaction mix.PPO activity can be monitored by oxygen consumption or spectrophotometrically using a variety of substrates such as pyrogallol, pryocatechol, 4-methylcatechol, 3,4-dihydroxyphenylacetic acid, 4-nic acid . In the A265 (nm/min) per unit of polyphenol oxidase in a 3.0 mL reaction mixture at pH 6.5 at 25 °C and 0.1 represents the volume (in milliliters) of enzyme used in the assay.Finally, the activity of PPO enzyme was calculated by using Equation (2), following Behzadi et al. ,(2)U=Un2-treated flour. The percentage of residual activity in the graham flour was calculated, using Equation (3) from Hojnik Podrepšek et al. [The residual activity of PPO (%) was defined as the ratio between the enzyme activity of the untreated flour and the enzyme activity of scCOk et al. (3)Resid2CO3 (75 g/L), was added to 0.5 mL of extract (supernatant after protein extraction), and the resulting mixture was thoroughly agitated. The sample was incubated for 5 min at 50 °C. 0.5 mL of distilled water was taken for the control sample. After cooling the samples, the absorbance was measured at 760 nm using a UV/VIS spectrophotometer . Phenol content was determined using gallic acid (GA) as standard and expressed as mg GA/g flour material. The analyses were performed in triplicate, where an average deviation was calculated.The total phenols were determined using the protocol proposed by Škerget et al. (2005) using the Folin-Ciocalteu method . Folin-C2 on graham flour quality, characterization of flour particles was performed by particle size determination and SEM analysis, with the moisture content of untreated and scCO2-treated graham flour also determined.To determine the influence of scCO2-treated graham flour was measured using a laser diffraction particle size analyzer , using a dry analysis method within the selected size range from 0.3 to 300 µm. The sample was placed into the sample hopper and then aspirated into the apparatus. The distribution was reported as the arithmetic mean (µm), where three replicates were performed within a standard deviation of ±5 µm.The particle size distribution of untreated and scCO2-treated graham flour, with the resulting SEM images used to study the changes in size and shape of the particles after scCO2-treatment.The size and morphology of the graham flour particles were analyzed by SEM . The flour particle morphology and microstructural changes were observed on untreated and scCOMoisture content was measured using an HB43-S  halogen moisture analyzer. This performs measurements based on the thermogravimetric principle, i.e., the moisture is determined from the weight loss of a sample dried by heating to 130 °C, within a standard deviation of ±0.2% and with duplicated measurements.2 on the protein concentration and inactivation of enzyme PPO in graham flour after scCO2 exposure. The effects of pressure as a process parameter on protein concentration and PPO activity in graham flour were studied. The results clearly showed significant changes in protein concentration induced by scCO2, which fell by 20% after scCO2 treatment at 300 bar for 24 h, and could be ascribed to the leakage of proteins into the media. These results also indicate that significant changes in enzyme activity caused by scCO2 may be associated with the conformation of enzyme molecules, as evidenced by a 35% decrease in enzyme activity of PPO in graham flour after scCO2 exposure at 300 bar for 24 h. At the same time, the results showed that the quality of the flour remained almost the same or even improved. The current study is important as the technology of scCO2 was recognized as appropriate because it does not adversely affect the quality of the flour, which was proved with SEM analysis, where starch grains of graham flour after scCO2 treatment were not damaged and therefore, scCO2-treated graham flour is still suitable for use in the bakery industry for wheat end-use products. In addition, the correlation of PPO activity and total phenolic content of untreated and with scCO2-treated graham flour was examined. This study provided an effective method of scCO2 technology, which was found to be a suitable technology to inactivate the PPO enzyme, which might be a crucial factor to induce the chemical reactions resulting in undesirable darkening of flour end-use products and can thereby extend the shelf life of the graham flour.The aim of this study was to investigate the effects of scCO


The assembly of specific neuronal circuits relies on the expression of complementary molecular programs in presynaptic and postsynaptic neurons. In the cerebral cortex, the tyrosine kinase receptor ErbB4 is critical for the wiring of specific populations of GABAergic interneurons, in which it paradoxically regulates both the formation of inhibitory synapses as well as the development of excitatory synapses received by these cells. Here, we found that Nrg1 and Nrg3, two members of the neuregulin family of trophic factors, regulate the inhibitory outputs and excitatory inputs of interneurons in the mouse cerebral cortex, respectively. The differential role of Nrg1 and Nrg3 in this process is not due to their receptor-binding EGF-like domain, but rather to their distinctive subcellular localization within pyramidal cells. Our study reveals a novel strategy for the assembly of cortical circuits that involves the differential subcellular sorting of family-related synaptic proteins. The function of the cerebral cortex relies on dynamic interactions between excitatory and inhibitory neurons. These interactions are governed by precise synaptic connectivity patterns, which are laid down during development and subsequently sculpted by ongoing activity to support neural processes such as learning. What are the molecular and cellular rules that underlie the development of specific connectivity motifs? Neurons use cellular and subcellular strategies for the assembly of specific synapses . CellulaThe molecular mechanisms underlying subcellular synaptic specificity likely rely on the sorting of specific mRNAs and/or proteins into distinct subcellular compartments. For instance, the localization of certain mRNAs into the somatodendritic or axonal compartments of neurons depends on alternative 3′UTR sequences . In addiThe tyrosine kinase receptor ErbB4 is involved in the wiring of specific microcircuits in the cerebral cortex . ErbB4 iNeuregulins comprise a family of growth factors that activate receptor tyrosine kinases of the ErbB family . Type IINrg1 and Nrg3 across cortical layers 2–6 during postnatal development and into adulthood  mice, in which a tamoxifen-inducible version of Cre recombinase is under the control of the endogenous regulatory sequences of the pyramidal cell-specific Neurod6 locus  day 30  day 30 . TherefoNrg1 and Nrg3 in the development of inhibitory synapses onto pyramidal cells. We investigated the two types of inhibitory synapses that are known to be altered in the absence of ErbB4 from interneurons: synapses made by Cholecystokinin (CCK+) basket cells onto the soma and synapses made by chandelier cells on the axon initial segment (AIS) (Ai9 (tdTomato) in our breeding scheme  . We perfnt (AIS) . Of notent (AIS) . To ideng scheme . We obsecontrols . In contet cells . These rNrg1 received significantly fewer GAD67+ boutons  or is generally required for GABAergic synaptogenesis, we analyzed the number of PV+ basket cell synapses contacting pyramidal cells lacking specific neuregulins. We used Synaptotagmin-2 (Syt2) to specifically identify the presynaptic compartment of these synapses . We founcontrols . These rsynapses .-expressing axonal terminals contacting PV+ interneurons. We found that conditional deletion of Nrg1 from pyramidal cells does not alter the density of excitatory synapses these neurons make onto PV+ interneurons  and excitatory (outputs) synapse formation independently in pyramidal cells. Nrg1 mediates inhibitory synapse formation onto pyramidal cells, whereas Nrg3 controls excitatory synapse formation onto interneurons.Nrg1 and Nrg3 conditional mutant mice, we performed gain-of-function experiments by electroporating plasmids encoding Nrg1 or Nrg3 into pyramidal cell progenitors in embryonic day (E) 14.5 mouse embryos. We used Type III Neuregulin 1 (hereafter referred to as Nrg1), because it is the most abundant Nrg1 isoform in the cerebral cortex during synaptogenesis  localized to the soma, whereas Nrg3 carrying the EGF-like domain of Nrg1 (Nrg3EGF:Nrg1) localized to the neuropil  increased the density of CCK+ presynaptic boutons innervating the soma of GFP+ pyramidal cells, as visualized by GAD65/CB1R colocalization, in similar quantities than wild-type Nrg1 (EGF:Nrg1), which is efficiently targeted to the axonal compartment, did not result in significant changes in CCK+ bouton density  significantly augmented the density of axonal excitatory synapses that electroporated pyramidal cells made onto PV+ interneurons, at comparable levels than wild-type Nrg3 (EGF:Nrg3), which is targeted to the perisomatic region, did not change excitatory synapse densities in axon terminals  of these proteins might be important for their localization and therefore differential physiological roles . To testype form . We alsoCt:Nrg1; . Remarkay inputs . These rCt:Nrg3) does not increase the number of CB1R+/GAD65+ presynaptic boutons contacting electroporated pyramidal cells (Ct:Nrg1), which is abnormally retained in the soma of pyramidal cells  lose the specific subcellular distribution found in wild-type Nrg1 and Nrg3 KanNeurod6) (CreNeurod6 (tm1(cre)KanNeurod6) (floxedNrg1 (tm3CbmNrg1) (floxedNrg3 (tm1a(KOMP)MbpNrg3) (tdTRCL (tm9(CAG-tdTomato)HzeGt(ROSA)26Sor) (The mouse lines RT2)Kan) , Neurod6cre)Kan) , Nrg1flo1tm3Cbm) , Nrg3floOMP)Mbp) , and RCLato)Hze)  were maiCreERT2Neurod6 , CreNeurod6 , floxedNrg1 , floxedNrg3 , and tdTRCL .The following primer sequences were used for routine genotyping: Nrg1  and Nrg3 . The neuregulin sequences lacked the 5’- and 3’-untranslated regions (UTR) and were preceded by the Kozak consensus sequence. The different neuregulin inserts were cloned into an expression vector plasmid containing the synapsin promoter (pSyn) using the restriction enzymes NotI/EcoRI. These plasmids contained an additional pSyn promoter followed by a green fluorescent protein (GFP) as a reporter to label the electroporated cells  epitope tag upstream of the EGF-like domain (exon 2). It has been previously reported that this tag insertion site in neuregulin loci does not alter their function . The HA EGF:Nrg3. Second, the EGF-like domain of Nrg3 (amino acids 278 to 346) was replaced by the EGF-like domain of Nrg1 (amino acids 223 to 286); this chimeric protein was named Nrg3EGF:Nrg1.For EGF-like domain swapping experiments, the chimeric neuregulins were generated by replacing the EGF-like domain of a neuregulin member by the corresponding domain from its homologous gene. First, the EGF-like domain of Nrg1  was replaced by the EGF-like domain of Nrg3 ; this chimeric protein was named Nrg1Ct:Nrg3. Second, the C-terminal domain of Nrg3 (amino acids 384–713) was replaced by the C-terminal domain of Nrg1 (amino acids 326–700); this chimeric protein was named Nrg3Ct:Nrg1.For C-terminal domain swapping experiments, the chimeric neuregulins were generated by replacing the C-terminal domain of a neuregulin member by the corresponding domain from its homologous gene. First, the C-terminal domain of Nrg1  was replaced by the C-terminal domain of Nrg3 ; this chimeric protein was named Nrg1ΔCt. Second, the C-terminal domain of Nrg3 (amino acids 389–713) was deleted and a stop codon was inserted after amino acid 388 ; this truncated protein was named Nrg3ΔCt.For C-terminal domain deletion experiments, the truncated neuregulins were generated by deleting the C-terminal domain of each neuregulin and inserting a stop codon downstream of the transmembrane domain of the protein. First, the C-terminal domain of Nrg1 (amino acids 329–700) was deleted and a stop codon was inserted after amino acid 328 ; this truncated protein was named Nrg1For in utero electroporation (IUE), neuregulin expression plasmids were used at a concentration of 1 μg/μl. DNA solution was mixed with Fast Green  and 1–2 μl of the solution was injected into the lateral ventricle of E14.5 embryos.https://blast.ncbi.nlm.nih.gov/Blast.cgi). Amino acid sequences were compared for exons 2–3 of both neuregulins—corresponding to the EGF-like domains—, and exons 6–9 and exons 6–10 of Nrg1 and Nrg3, respectively—corresponding to the C-terminal domains.To estimate the similarity between homologous protein domains of Nrg1 and Nrg3, we used the online-based Basic Local Alignment Search Tool (BLAST) from NCBI (In utero electroporation (IUE) was performed as described before . CD-1 eLife. Your revised article has been evaluated by John Huguenard (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Subcellular sorting of neuregulins controls the assembly of excitatory-inhibitory cortical circuits" for further consideration by Overall, the three reviewers agree that the revisions have improved the manuscript substantially and regard almost all critical issues resolved. However, there is one important point that remains to be addressed:It should be tested experimentally whether ErbB4 and Nrg1/Nrg3 are co-localized at the synapses studied, and it should be quantitatively assessed what proportion of synapses contain both, the receptor and ligand. Such colocalization is implied throughout the paper, but no corresponding data are shown, nor do previously published studies adequately resolve this issue. At this point, it would be acceptable to address this issue for the wild-type scenario. Anti-ErbB4 antibodies suitable for this purpose are available.The reviewers regard this issue to be essential because the corresponding data will have a major impact on the overall interpretation of the study.If this information can absolutely not be provided with acceptable effort, the claim that Nrg1/Nrg3 functions are mediated by direct and persistent interactions with the ErbB4 receptor at the synapses of interest is not sufficiently supported. Accordingly, the text would have to be partially rewritten to reflect this issue – e.g. by merely proposing that ErbB4 mediates synaptic functions . Essential revisions1) Evidence that the distribution of overexpressed HA-tagged full-length Nrgs recapitulates the distribution of endogenous Nrgs is needed to support the conclusions of the study. If immunohistochemistry is challenging, the authors could consider a molecular replacement strategy by expressing HA-tagged Nrg1 and Nrg3 in the respective Nrg conditional knockout mice.We thank the reviewers for this important consideration. We agree that our overexpression experiments of HA-tagged neuregulins are insufficient to provide conclusive evidence regarding the endogenous localization of Nrg1 and Nrg3 in the neocortex. Following the reviewers’ advice, we have performed heat-induced antigen retrieval-based immunohistochemistry using antibodies against Nrg1 and Nrg3 to study the endogenous localization of these proteins in the neocortex. The results of these experiments reveal the presence of endogenous Nrg1 and Nrg3 clusters in the same subcellular localisations than we previously observed the HA-tagged versions of Nrg1 and Nrg3 in pyramidal cells , which demonstrates that the distribution of HA-tagged Nrgs recapitulates the distribution of endogenous Nrgs.Nrg1 conditional mutant mice to demonstrate the specificity of the antibodies. The results of these experiments are shown in Figure 3—figure supplement 1.We quantified the density of Nrg1-labelled synaptic puncta in CA1 stratum pyramidale  and stratum radiatum (as a negative targeting control). We used Nrg3 conditional mutant mice to demonstrate the specificity of the antibodies. The results of these experiments are shown in Figure 3—figure supplement 2.We quantified the density of Nrg3-labelled clusters in both the CA1 region of the hippocampus and superficial layers of the cerebral cortex. We used 2) Related to this issue, many data  relate to analyses of chimeric and deletion constructs of Nrg. It needs to be shown that these constructs traffic normally to the cell surface. It is possible that altered protein folding and reduced surface trafficking could result in the distribution patterns shown for the chimeric and truncated Nrg constructs, e.g. due to accumulation in endoplasmic reticulum present in somata and dendrites. Further, specific inhibitory or excitatory synaptic targeting of these constructs needs to be assessed.Following the reviewers’ suggestions, we have examined the synaptic targeting of all the chimeric and truncated Nrg constructs. We have quantified the percentage of colocalisation for all constructs with inhibitory and excitatory synaptic markers. The results of these analyses have strengthened our conclusions about the specific synaptic trafficking of these constructs.We quantified the percentage of co-localization of EGF-like domain chimeric constructs with GAD65+ puncta targeting GFP+ somas in superficial cortical layers. The results of these analyses are shown in Figure 4—figure supplement 1.+/GFP+ puncta innervating PV+ interneurons in superficial cortical layers. The results of these analyses are shown in Figure 4— figure supplement 2.We quantified the percentage of co-localization of EGF-like domain chimeric constructs with VGluT1We quantified the percentage of co-localization of C-terminal domain chimeric constructs with GAD65+ puncta targeting GFP+ somas in superficial cortical layers. The results of these analyses are shown in Figure 5—figure supplement 2.+/GFP+ puncta innervating PV+ interneurons in superficial cortical layers. In addition, we quantified the intensity of HA+ puncta. The results of these analyses are shown in Figure 5—figure supplement 3.We quantified the percentage of co-localization of C-terminal domain chimeric constructs with VGluT1We quantified the percentage of co-localization of C-terminal domain truncated constructs with GAD65+ puncta targeting GFP+ somas in superficial cortical layers. The results of these analyses are shown in Figure 6—figure supplement 1.+/GFP+ puncta innervating PV+ interneurons in superficial cortical layers. The results of these analyses are shown in Figure 6— figure supplement 2.We quantified the percentage of co-localization of C-terminal domain truncated constructs with VGluT13) It is unclear whether the overexpressed HA-Nrg1 that is observed in clusters on GFP+ pyramidal neurons is produced by these neurons or provided in trans. This should be clarified by quantifying perisomatic HA-Nrg1 on neighboring GFP+ and GFP- somata.The possibility that HA-Nrg1 is provided in trans is very unlikely because pyramidal cells very rarely synapse on the soma of other pyramidal cells . We have nevertheless quantified HA-tagged Nrg1 and HA-tagged Nrg3 clusters in electroporated GFP+ pyramidal cell somas as well as in NeuN+/GFP- neuronal somas. The results of these analyses demonstrate that HA-Nrg1 is only present postsynaptically to inhibitory terminals contacting the soma of electroporated pyramidal cells. These results also confirmed that Nrg3 is never found in that location.We have quantified the percentage of co-localization of HA-tagged Nrg1 and HA-tagged Nrg3 with inhibitory boutons targeting GFP+ and NeuN+/GFP- neuronal somas in superficial cortical layers. The results of these analyses are shown in Figure 3—figure supplement 3.4) It appears to be assumed throughout the study that ErbB4 is located pre- or post-synaptically in the respective synapse analyzed (related to axonic vs. somatic Nrg), but this is nowhere shown. Some experiments that address the mechanism of how Nrg1/Nrg3 facilitates synapse formation should be presented. Is this dependent on a long-term (persisting) Nrg1/ErbB4 or Nrg3/ErbB4 interaction? In other words, is Nrg1-GFP located at the majority of the perisomatic synaptic sites and is ErbB4 detected there? Similarly, at how many synapses in the axonal initial segment is Nrg1-GFP/ErbB4 co-localization detected? Alternatively, a persistent Nrg1/ErbB4 or Nrg3/ErbB4 localization at the synapse might not be needed, just short-term interactions could be sufficient to elicit ErbB4 signalling.Nrg1 and Nrg3 conditional mutants (present study) together recapitulate those previously reported for ErbB4 conditional mutants , strongly suggests that presynaptic ErbB4-postsynaptic Nrg1 signalling is involved in the formation of perisomatic inhibitory synapses, whereas presynaptic Nrg3-postsynaptic ErbB4 signalling mediates the formation of excitatory synapses contacting PV+ interneurons. Although we agree that the study of the dynamic interactions between ErbB4 and Nrgs is of great interest, we think that the experiments required to asses them fall outside the scope of the current study.We agree on the interest of understanding the trans-synaptic signalling pathway mediated by neuregulins and ErbB4 receptor. We and others have previously shown the specific subcellular localisation of ErbB4 in different populations of cortical interneurons. In particular, we used electron microscopy to reveal the localisation of ErbB4 at excitatory postsynaptic densities targeting the dendrites of interneurons and at inhibitory presynaptic boutons targeting the axonal initial segment and perisomatic compartment of pyramidal cells . In the present study, we have found that the subcellular localisation of Nrg1 is specifically restricted to the perisomatic compartment of pyramidal cells, whereas the subcellular localisation of Nrg3 is greatly enriched in the axon terminals of pyramidal cells innervating PV+ interneurons. Moreover, the fact that the synaptic deficits observed in The first paragraph in the Discussion summarises the genetic evidence supporting the idea that Nrg1 and Nrg3 interact with ErbB4 for the formation of the synapses described in this study.5) In Figure 3, the localization of HA-Nrg1 to the edges of somata is visible but specific colocalization with GAD65 or especially gephyrin is not clear. Localization of HA-Nrg1 to inhibitory postsynaptic sites needs to be quantified as it is an important part of the conclusions.We do not understand what the reviewer refers to in this comment. HA-Nrg1 is expressed post-synaptically in the pyramidal cell soma, therefore it is opposed to GAD65+ presynaptic boutons and gephyrin puncta should be embedded in the HANrg1 clusters, as shown in Figure 3 and Figure 3—figure supplement 3. To strengthen this point, we have now quantified the co-localisation of HA-tagged Nrg1 and HA-tagged Nrg3 clusters with presynaptic GAD65+ boutons targeting the somas of electroporated GFP+ pyramidal cells.We quantified the percentage of GAD65+ boutons that are in close apposition to postsynaptic clusters expressing HA-tagged Nrg1 or HA-tagged Nrg3 in electroporated GFP+ pyramidal cells. The results of these analyses are shown in Figure 3—figure supplement 3. In this figure, we added HA staining in a colour-inverted image in low and high-magnification to more clearly illustrate the localisation of HA-tagged Nrg1 at postsynaptic locations within the pyramidal cells.6) All main conclusions depend on colocalization analyses of immunofluorescence signals and expressed markers in single confocal optical sections, with no other complementary approaches, such as electron microscopy or electrophysiology. There is a concern with this approach, as to whether individual synapses belonging to specific presynaptic and postsynaptic cells can be sufficiently resolved given the limits of standard confocal imaging. In the images shown, there is no clear correspondence between GAD65 and gephyrin, nor between VGlut1 and PSD95. This is a particular concern for excitatory synapses, as antigen retrieval methods are typically used to visualize PSD95, but do not appear to have been used here, and some of the VGlut1 puncta counted do not appear to fit entirely within the relevant tdT or GFP filled axons. Further, there is a concern that individual excitatory synapses can be resolved given their high density in cortical neuropil without using super-resolution or expansion microscopy approaches, which are now available. Currently, the best way to address this issue could be for the authors to present in an extended view figure large field images of PSD95 and VGlut1, and of GAD65 and gephyrin, showing a one-to-one correspondence of presynaptic and postsynaptic antigens and demonstrating ability to resolve all individual synapses in a field.Nrg1 and Nrg3 mutants compared to their respective controls). This suggests that, even if we may not identify all synapses using confocal microscopy, we do not have artefactual biases in our analysis because we identify the same proportion of synapses in control and experimental groups. Notwithstanding these new analyses, we would like to mention that we have done this type of synaptic analyses for more than 10 years in the context of different projects, and we have repeatedly shown that our histological data is always equivalent to electrophysiological synaptic analyses performed on the same mice .We are aware of the limitations of standard confocal imaging to resolve synaptic puncta. As suggested, we now provide large field-of-view images of the distinct synaptic markers used throughout our study for both loss- and gain-of-function experiments. In addition, we have quantified the proportion of clusters co-labelled with synaptic markers that are used for co-localisation synaptic analysis. Our analyses reveal that, independently of the experimental condition, the proportion in colocalization for both inhibitory or excitatory synaptic markers is 50-70% and, more importantly, is identical for each comparison  The targeting of Nrg3 to excitatory presynaptic sites on axons and presynaptic function has already been reported quite thoroughly in Muller et al., 2018. This information, as well as information on Nrg1 isoforms and their trafficking and function should be presented in the Introduction . In essence, much of the more general introductory background is not essential, but specific background on Nrgs needs to be added.Following the reviewers’ suggestion, we have included these studies in the Introduction. The requested citations are now included in the Introduction.8) In loss-of-function experiments, a conditional Nrg1 allele was used that affects all Nrg1 isoforms, whereas in the gain-of-function experiments a type III Nrg1 cDNA is used. This should be clearly stated and discussed.We thank the reviewers for this comment. A sentence has been added in the Results and Discussion clarifying this point.Overall, the three reviewers agree that the revisions have improved the manuscript substantially and regard almost all critical issues resolved. However, there is one important point that remains to be addressed:It should be tested experimentally whether ErbB4 and Nrg1/Nrg3 are co-localized at the synapses studied, and it should be quantitatively assessed what proportion of synapses contain both, the receptor and ligand. Such colocalization is implied throughout the paper, but no corresponding data are shown, nor do previously published studies adequately resolve this issue. At this point, it would be acceptable to address this issue for the wild-type scenario. Anti-ErbB4 antibodies suitable for this purpose are available.The reviewers regard this issue to be essential because the corresponding data will have a major impact on the overall interpretation of the study.If this information can absolutely not be provided with acceptable effort, the claim that Nrg1/Nrg3 functions are mediated by direct and persistent interactions with the ErbB4 receptor at the synapses of interest is not sufficiently supported. Accordingly, the text would have to be partially rewritten to reflect this issue – e.g. by merely proposing that ErbB4 mediates synaptic functions .+ cells on the soma and by chandelier cells on the axon initial segment of pyramidal cells is to perform double labeling immunohistochemistry for Nrg1 and ErbB4 at the electron microscopy level. We hope that the editors understand that performing this additional experiment is a major effort beyond the expected in a second revision at eLife. However, since we agree with the reviewers’ criticism that our experiments do not demonstrate that the function of Nrg1 and Nrg3 in synapse formation is indeed mediated by ErbB4, we have modified the text to avoid giving the impression that this is the case. In brief, we have avoided any reference to Nrg1/ErbB4 or Nrg3/ErbB4 binding or signaling in the Abstract and Results sections, and we have edited the Discussion to reflect the reasons supporting the view that ErbB4 is the most likely receptor mediating the function of Nrg1 and Nrg3 in cortical interneurons. We think the revisions introduced in the text directly address the point raised by the reviewers.We were happy to learn that the three reviewers consider that our revisions have improved the manuscript substantially and that there is only one important point that remains to be addressed. Following your request, we have performed immunohistochemical experiments to assess, at the light microscopy level, whether ErbB4 co-localises with Nrg1 and Nrg3 at the synapses studied. We found that most ErbB4+ synaptic clusters found in PV+ interneurons also contain presynaptic Nrg3 , which reinforces the view that the role of Nrg3 in the formation of excitatory synapses contacting PV+ interneurons is mediated by ErbB4. We attempted to do a similar experiment for Nrg1, however we could not detect ErbB4 clusters at the inhibitory boutons at light microscopy, precluding the assessment of Nrg1-ErbB4 co-localisation. This result is not surprising since we have previously found using electron microscopy that the levels of ErbB4 at inhibitory synaptic terminals are two or three times lower than those found in postsynaptic densities being contacted by excitatory synapses . At this point, the only way to demonstrate that Nrg1 and ErbB4 co-localise at inhibitory synapses made by CCK


Textural measurement depicted that hardness, chewiness, gumminess, springiness, and cohesiveness increased with the substitution of amaranth flour. Rheological parameters like complex viscosity, loss modulus, and storage modulus were also observed in all dough samples. Bread samples with 5%, 10%, and 15% of AF showed lower yellowness (b∗) and higher lightness (L∗) and redness (a∗) values for crust colour while lower L∗ and higher a∗ and b∗ values for crumb colour. The bread prepared by replacing 5% and 10% of AF is nutritionally as well as sensorially acceptable.In this study, the chemical composition, colour analysis, and antioxidant properties of flour and bread were analysed. We also examined the rheological properties of dough and proximate, colour, textural, and organoleptic properties of amaranth wheat bread. Wheat flour was replaced by amaranth flour (AF) at 0-15% levels . AF supplementation increased the moisture (31.06 to 33.24%), ash (0.92 to 1.51%), protein (12.17 to 13.11%), fat (2.16 to 2.77%), and crude fibre content (1.11 to 1.72%) of the bread while the nitrogen-free extract and alkali water retention capacity decreased from 52.58 to 47.65% and 136.00 to 112.02%, respectively. The antioxidant activity evaluated by DPPH, FRAP, and total phenolic content was reduced with increased levels of AF. A significant impact on the physical properties like the weight of bread (increased from 474.00 to 489.30 g), height (went down from 80.00 to 74.33 cm), loaf volume (decreased from 1580.00 to 1518.30 cm Amaranth is a well-known pseudocereal because of its nutraceutical properties. Consumption of this grain helps in improving kidney complaints, constipation, and anaemia. Medicinally, its extracted water is used to treat pain in limbs, tumours, and wounds (Grubbens and Denton 1998). They also have anticancer and antioxidant properties . AmarantTherefore, the aim of our study was to examine the antioxidant activity and chemical composition of flour, rheological properties of dough, and physical, textural, colour, and sensory properties of amaranth wheat bread.Amaranth seeds were purchased from a local market in Jammu, India. The grains were cleaned manually to remove dust and other contaminants. Wheat flour, sugar, salt, vegetable oil, gluten, improver, and yeast were purchased from a local market in Srinagar, India. All the ingredients were stored separately in an airtight storage box at room temperature (25°C) for further analysis. All the chemicals used in this study were of Analytical Reagent (AR) grade.μm sieve to obtain flour of uniform size.Amaranth flour was prepared using a laboratory-grade grinder . The seeds were ground to a fine powder and passed through a 177 The total phenolic content of amaranth flour was evaluated using the modified method of Singleton & Rossi , DPPH byStandard methods of AOAC  were useThe rheological properties of the sample were measured by using a rheometer . After kneading, 7 g of the dough sample with 30 g of yeast added for formulation of bread was kept in-between the plates having parallel plate geometry with a diameter of 40 mm and a gap of 2 mm. The unwanted dough was removed gently, and after a relaxation period of 35 min, the measurements were taken. An oscillation test is the first step in which dough is not damaged when the frequency is increased from 0.1 to 10 Hz at a strain of 0.2%. The dough was further analysed for the creep recovery test for 280 s at a pressure of 110 Pa. Once pressure was removed, there was again relaxation time of 280 s. Nondestructive forces were used to measure different rheological behaviour. Measurements were taken thrice, and average measurements of all the samples did not exceed 5%; otherwise, all the measurements were repeated.Breads were prepared by mixing wheat flour and amaranth flour at three different levels  labelled as A5, A10, and A15 as presented in (1) Height (mm). To determine the height (H), three slices of bread were taken from the middle of the loaf and placed edge to edge. The height of three slices of bread was measured by a scale in mm. The slices of bread were rotated at an angle of 90° for duplicate reading. This was repeated thrice, and the average height was taken in millimeters.(2) Loaf Volume (cm3) and Specific Volume Index (cm3/g). Loaf volume and specific volume of bread samples were measured by the rapeseed displacement method as described by See et al.  and Feili et al. .e et al.  and Feili et al. .The hardness, cohesiveness, springiness, chewiness, and gumminess of bread were measured using a texture analyser  as per the standard method of AACC . A samplL∗, a∗, and b∗) were cut into cubes of 2 × 2 × 2 cm and placed in a digital colourimeter .The samples used for the colour analysis of the bread crumb and crust , were analysed using one-way analysis of variance. Duncan's multiple range test with significance defined at p ≤ 0.05 was used to compare the means. The statistical analysis was performed using SPSS .Data, with three replications , amaranth flour (AF), and their different mixtures are shown in The protein content increased significantly with the increased levels of incorporation of amaranth flour due to higher protein content in amaranth flour as these are rich in some essential amino acids than whole wheat flour . The fibp ≤ 0.05) with the increase in the percentage level of amaranth flour. Similar decreases in DPPH and FRAP were also observed [The antioxidant activity of wheat flour (WF), amaranth flour (AF), and their different mixtures is shown in observed .L∗) value and an increase in redness and yellowness (a∗ and b∗) values with the increase in the percentage of amaranth flour. Similar observations were recorded by Sanz-Panella et al. [The colour measurements of the composite bread substituted with different levels of amaranth flour are depicted in a et al. .G′), loss modulus (G″), and complex viscosity (G∗) and are shown in G′) was higher than the viscous modulus (G″) as depicted in et al. .The rheological properties were measured in terms of the variation of storage modulus  from 52.58% to 47.65% and 136.00% to 112.02%, respectively. This decrease with the increase in the substitution level of amaranth flour could be attributed to the difference in quantitative distribution of protein fractions and physicochemical attributes of WF and AF [Nitrogen-free extract (NFE) and alkali water retention capacity (AWRC) significantly decreased  are depicted in It is observed that by supplementation of AF in the bread, cohesiveness, gumminess, and chewiness increased due to the increase in hardness that results in crumbling of crumb and is usually associated with the loss of water which in turn is determined by the retrogradation properties of wheat flour .p ≤ 0.05) effect on all parameters and is shown in Sensory evaluation is an important parameter for analysing the quality of amaranth bread to meet the consumer requirements. The results are based on a 9-point hedonic scale to measure sensory characteristics of bread like colour, taste, texture, aroma, appearance, and overall acceptability of product . Bread sThe statistically significant difference was observed in aroma and taste. The mean score for aroma and taste decreases from 7.33 to 6.00 and 8.00 to 7.02, respectively, with the increase in addition of amaranth flour. This might be due to the nutty flavour of composite bread produced at a high temperature which could be objectionable in some bakery products. A similar observation was presented by Ayo . The resL∗, a∗, and b∗ values for crust and crumb colour of bread. Amaranth flour causes significant changes in the crumb and crust colour of bread. In general, when AF concentration was raised, the tristimulus colour values  in both crumb and crust were affected. Lightness and redness of bread crust increased by increasing the amaranth content in bread from 50.16 to 54.21 and 16.20 to 18.45, respectively, due to progressive loss of moisture content and oxidation of fats to produce hydrogen peroxide which oxidizes or bleaches pigment materials and results in increase in opacity ([opacity (.p ≥ 0.05) from 8.00 to 6.66. The decrease in the texture of amaranth-based wheat flour bread is due to the incorporation of amaranth flour that decreases the formation of the gluten network that failed to retain vapours produced, and therefore, the symmetry of bread decreased linearly with the increase in concentration of amaranth. These results are in alignment with Emire and Arega [The results pertaining to the mean score for crust and crumb texture are depicted in nd Arega .T0 (8.00) at 0 days of storage, and the minimum score of aroma was found in T3 (7.00) at the 6th day of storage. This might be due to the nutty flavour of amaranth being pronounced with the increase in the concentration of amaranth flour. The results are in agreement with the findings of Ayo [The sensory score for aroma decreased significantly during storage. The aroma score of amaranth-based bread ranges from 8.00 to 7.00. The maximum mean value score for aroma was found in s of Ayo .T3 [The results pertaining to the score for appearance is depicted in T3 .L∗ value increased while a∗ and b∗ values decreased in bread which is in the ratio of 90 : 10. Amaranth flour substitution resulted in the highest total phenolic content in the control (7.08) of all breads. All in all, using two different flours in the production of amaranth-based breads can lead to better products with higher nutrient content. However, a combination of flours could maintain the dough rheology. From the entire work carried out for the development of amaranth-based wheat flour bread, it can be concluded that the panellist preferred T2 (90 : 10) treatment in terms of organoleptic properties. Thus, the inclusion of amaranth flour in bread could be limited, not only for maintaining the product quality but also for preserving the benefits of nutritional ingredientsAmaranth flour could be used as a replacement for wheat flour in the formulation of bread, enhancing the nutritional value and fibre content of the product. In this study, two different flours were substituted, viz., wheat flour and amaranth flour (up to 15%), to examine the changes in physicochemistry, total phenolic content, colour evaluation, physical parameters, texture profile analysis, and sensory attributes of wheat amaranth bread. Results demonstrated that the bread made with 10% amaranth flour had high levels of moisture content 32.60%, 12.96% protein,1.36% fibre, 2.61% fat, and 1.33% ash as compared to the control. Further,


Evolution of damage and fracture behavior of fiber-reinforced mini ceramic-matrix composites (mini-CMCs) under tensile load are related to internal multiple damage mechanisms, i.e., fragmentation of the brittle matrix, crack defection, and fibers fracture and pullout. In this paper, considering multiple micro internal damage mechanisms and related models, a micromechanical constitutive stress–strain relationship model is developed to predict the nonlinear mechanical behavior of mini-CMCs under tensile load corresponding to different damage domains. Relationships between multiple micro internal damage mechanisms mentioned above and tensile micromechanical multiple damage parameters are established. Experimental tensile nonlinear behavior, internal damage evolution, and micromechanical tensile damage parameters corresponding to different damage domains of two different types of mini-CMCs are predicted. The effects of constitutive properties and damage-related parameters on nonlinear behavior of mini-CMCs are discussed. With the development of thermodynamics, the advancement of component integrated design technology, the weight reduction brought by structural simplification and the comprehensive development of material technology, the thrust-to-weight ratio of aeroengines has gradually increased. However, on the premise of maintaining the engine layout and not changing the conventional metal materials, the improvement of aerodynamic, thermal, component design and structural weight reduction techniques can only increase the ratio between the thrust and weight of the aeroengines to approximately 14. For aeroengines with ratios between the thrust and a weight of approximately 12–15 or higher, more breakthroughs must be made in new materials, new process applications and new structural design, such as polymer-matrix composites (PMCs) or metal-matrix composites (MMCs) for cold section components in aeroengine and ceramic-matrix composites (CMCs) for hot section components in aeroengines. Two main types of CMCs are used in aeroengines, including, C/SiC and SiC/SiC. For aeroengines, the operating temperatures of C/SiC and SiC/SiC are approximately 1650 °C and 1450 °C, respectively. Increasing the operating temperature of SiC fibers can raise the operating temperature of SiC/SiC to 1650 °C. Results show that the application of CMCs in the hot section components of the combustion chamber, turbine, and nozzle can increase the operating temperature of the aeroengine by 300–500 °C, reduce the weight by 50–70% and increase the thrust by 30–100% [In order to safely and confidently implement CMCs in different thermo-structural component applications, one should understand the complexity of designing with such brittle composite systems. The mechanical behavior of minicomposites can be used to analyze the internal complex damage evolution of woven composites ,6,7,8. MThe objective in the present analysis is to build the relationship between tensile nonlinear damage behavior and multiple micro internal damage mechanisms of mini-CMCs. A micromechanical nonlinear stress–strain relationship model is given to predict the tensile stress–strain relationship of mini-CMCs when either partial or complete interfacial debonding occurs. Experimental tensile damage evolution and related multiple microdamage parameters of two different mini-CMCs are predicted. Effects of constitutive properties and damage-related parameters on damage and fracture of mini-CMCs are analyzed.(1)Domain I, the linear domain.(2)Domain II, the nonlinear domain due to microdamages of matrix fragmentation.(3)Domain III, the secondary linear and final fracture domain due to gradual fibers fracture and pullout.The tensile nonlinear behavior of mini-CMCs is mainly attributed to multiple microdamage mechanisms. Through in situ experimental observations and monitoring on internal damage state ,13, damaη is adopted to describe composite internal damage evolution.In this section, the micromechanical nonlinear constitutive relationship of three damage domains mentioned above is developed and related to the microdamage state of matrix fragmentation and fiber fractures and pullouts inside of mini-CMCs. A micromechanical parameter of interface debonding fraction σ and composite stress–strain εc can be determined by Equation (1) [Ec is the elastic modulus of the undamaged composite.In Domain I, no damages occur in mini-CMCs, and the linear relationship between applied stress tion (1) .(1)εc=σVf and Vm, and fiber radius rf) and damage state . The fiber axial stress distribution can be determined by Equation (2) [τi the interface frictional shear stress, σfo and σmo the axial stress of the fiber and matrix in the interface bonding region, and ρ the shear–lag model parameter. The stochastic model in Equation (3) can be used to determine the fragmentation length of the brittle matrix [lsat is the saturation matrix fragmentation length, σm is the stress carried by the matrix, σR is the characteristic stress for matrix fragmentation, and ζd is the debonding energy of the interface.When multiple microdamage mechanisms occur in mini-CMCs, the micro stress field of the damaged composite is affected by composite constitutive properties  .(2)σflc=lc is equal to the strain of intact fibers, and the nonlinear stress–strain constitutive relationship can be determined by Equation (5).Ef is the elastic modulus of the fiber, η is the ratio between debonding length ld and half of fragmentation length of the brittle matrix lc, αf and αc are the axial thermal expansion coefficient of the fiber and the composite, and ΔT is the temperature difference between testing and fabricated temperature.The composite strain εP the probability of fiber being broken.Upon approaching saturation of fragmentation of the brittle matrix, some fibers gradually fracture and pullout inside of the minicomposite. Considering intact and broken fiber in the different damage regions, the axial stress of the fiber can be determined by Equation (6) .(6)σf(xtion (7) .(7)σVf=Considering fibers fracture inside of mini-CMCs, the nonlinear stress–strain relationship at damage Domain III is acquired by Equation (5).Nonlinear tensile damage and fracture behavior of two different types of mini-CMCs are predicted. The effect of heat treatment at 1300 and 1500 °C on tensile nonlinear damage development behavior of mini-CMCs at room temperature is also analyzed. The material properties of different mini-CMCs are listed in TM SiC/SiC mini-CMC. The predicted results of tensile curves and fragmentation density of brittle matrix evolution curves both agreed well with experimental data. Three main damage domains are obtained from the nonlinear tensile curve, including:(1)σmc = 200 MPa, and the first fragmentation density of the matrix is approximately λmc = 0.02/mm, and the corresponding composite tensile strain is approximately εc = 0.06%.Domain I, the linear domain. The domain starts from the initial loading to the first matrix fragmentation stress (2)σmc = 200 MPa to the saturation matrix fragmentation stress σsat = 560 MPa, and the saturation fragmentation density of the matrix is approximately λsat = 1.76/mm, and the composite tensile strain is approximately εc = 0.34%.Domain II, the nonlinear region due to matrix fragmentation. The domain starts from (3)σsat = 560 MPa to composite tensile strength σuts = 644 MPa.Domain III, the secondary linear and final fracture domain. The domain starts from TM Type S SiC/SiC minicomposite. The predicted results of tensile curves and fragmentation density of brittle matrix evolution curves agree with experimental data. Three main damage domains are obtained from the nonlinear tensile curve, including:(1)σmc = 200 MPa with λmc = 0.05/mm and εc = 0.08%.Domain I, the linear domain. The domain starts from initial loading to (2)σmc = 200 MPa to σsat = 470 MPa with λsat = 1.49/mm and εc = 0.3%.Domain II, the nonlinear region due to matrix fragmentation. The domain starts from (3)σsat = 470 MPa to σuts = 488 MPa.Domain III, the secondary linear and final fracture region. The domain starts from TM ZMI SiC/SiC minicomposite. Predicted results of tensile curves and fragmentation density of brittle matrix evolution curves agree well with experimental data. Three main damage domains are obtained from the nonlinear tensile curve, including:(1)σmc = 150 MPa with λmc = 0.01/mm and εc = 0.05%.Domain I, the linear region. The domain starts from initial loading to (2)σmc = 150 MPa to σsat = 350 MPa with λmc = 1.49/mm and εc = 0.198%.Domain II, the nonlinear region due to matrix fragmentation. The domain starts from (3)σsat = 350 MPa to σuts = 498 MPa.Domain III, the secondary linear and final fracture region. The domain starts from (1)σmc = 35 MPa with λmc = 0.04/mm and εc = 0.013%.Domain I, the linear region. The domain starts from initial loading to (2)σmc = 35 MPa to σsat = 340 MPa with λsat = 1.27/mm and εc = 0.6%.Domain II, the nonlinear region. The domain starts from the (3)σsat = 340 MPa to σuts = 399 MPa.Domain III, the secondary linear and final fracture domain. The domain starts from (1)σmc = 50 MPa with λmc = 0.02/mm and εc = 0.012%.Domain I, the linear region. The domain starts from initial loading to (2)σmc = 50 MPa to σsat = 210 MPa with λsat = 0.28/mm and εc = 0.13%.Domain II, the nonlinear region due to matrix fragmentation. The domain starts from (3)σsat = 210 MPa to σuts = 362 MPa.Domain III, the secondary linear and final fracture region. The domain starts from (1)σmc = 50 MPa with λmc = 0.005/mm and εc = 0.017%.Domain I, the linear region. The domain starts from initial loading to (2)σmc = 50 MPa to σsat = 220 MPa with λsat = 0.58/mm and εc = 0.39%.Domain II, the nonlinear region. The domain starts from (3)σsat = 220 MPa to σuts = 246 MPa.Domain III, the secondary linear and final fracture domain. The domain starts from (1)σmc = 130 MPa.Domain I, the linear region. The domain starts from initial loading to (2)σmc = 130 MPa to σsat = 250 MPa.Domain II, the nonlinear region. The domain starts from (3)σsat = 250 MPa to σuts = 348 MPa. The interface debonding fraction increases to 2ld/lc = 0.78 at tensile strength σuts = 348 MPa.Domain III, the secondary linear and final fracture domain. The domain starts from In this section, the constitutive properties and damage-related parameters on tensile nonlinear stress–strain curves of SiC/SiC mini-CMC are discussed.Vf = 25%, the debonding fraction increases from η (σd = 166 MPa) = 0 to η (σuts = 521 MPa) = 0.33, and the failure strain of the composite is εf (σuts = 521 MPa) = 0.6%. For high fiber volume Vf = 30%, the debonding fraction increases from η (σd = 204 MPa) = 0 to η (σuts = 626 MPa) = 0.32, and the failure strain of the composite is εf = 0.64%. At high fiber volume, the debonding fraction at the same applied stress decreases, and the composite strain at nonlinear Domain II decreases, and the composite tensile strength and failure strain increase.i = 20 MPa, the debonding fraction increases from η (σd = 166 MPa) = 0 to η (σuts = 521 MPa) = 0.5, and the failure strain of the composite is εf = 0.73%. For high interface shear stress τi = 40 MPa, the debonding fraction increases from η (σd = 168 MPa) = 0 to η (σuts = 521 MPa) = 0.25; and the failure strain of the composite is εf = 0.25%. At high interface shear stress, the debonding fraction at the same applied stress decreases, and the composite strain at nonlinear Domain II decreases, and the composite failure strain decreases.ζd = 4 J/m2, the debonding fraction increases from η (σd = 192 MPa) = 0 to η (σuts = 521 MPa) = 0.31, and the failure strain of the composite is εf = 0.59%. For high interface debonding energy ζd = 6 J/m2, the debonding fraction increases from η (σd = 234 MPa) = 0 to η (σuts = 521 MPa) = 0.57, and the failure strain of the composite is εf = 0.57%. At high interface debonding energy, the interface debonding stress increases, the debonding fraction at the same applied stress decreases, and the composite strain during nonlinear Domain II decreases, and the failure strain of the composite decreases.In this paper, the tensile damage and fracture behavior of two different mini-CMCs are investigated. Multiple microdamage mechanisms of matrix fragmentation, fibers failure and pullout are considered in the analysis of tensile nonlinear curves. Experimental tensile nonlinear curves and internal damage evolution of SiC/SiC and C/SiC mini-CMCs are predicted. The effects of material properties and damage-related parameters on tensile damage and fracture at different domains are analyzed.(1)Predicted tensile nonlinear curves and matrix fragmentation density evolution curves agree with experimental data, and the matrix fragmentation approaches saturation before tensile fracture, and the interface partial debonding remains till tensile fracture.(2)Microdamage parameters of first matrix fragmentation stress, saturation matrix fragmentation stress and density, composite tensile strength and failure strain are obtained from tensile stress–strain curves and can be used to characterize tensile nonlinear behavior of mini-CMCs.(3)At higher fiber volume, the debonding fraction at the same applied stress decreases, and the composite strain at nonlinear Domain II decreases, and the composite tensile strength and failure strain increase.(4)At higher interface shear stress and interface debonding energy, the debonding fraction at the same applied stress decreases, and the composite strain at nonlinear Domain II decreases, and the failure strain of the composite decreases.


The deep afterslip revealed that a creeping process from steady-state “secondary” creeping to accelerating “tertiary” creep in the deep of fault. The future seismic hazard deserves further attention and research.On 3 July 2015, the Mw 6.5 Pishan earthquake occurred at the junction of the southwestern margin of the Tarim Basin and the northwestern margin of the Tibetan Plateau. To understand the seismogenic mechanism and the post-seismic deformation behavior, we investigated the characteristics of the post-seismic deformation fields in the seismic area, using 9 Sentinel-1A TOPS synthetic aperture radar (SAR) images acquired from 18 July 2015 to 22 September 2016 with the Small Baseline Subset Interferometric SAR (SBAS-InSAR) technique. Postseismic LOS deformation displayed logarithmic behavior, and the temporal evolution of the post-seismic deformation is consistent with the aftershock sequence. The main driving mechanism of near-field post-seismic displacement was most likely to be afterslip on the fault and the entire creep process consists of three creeping stages. Afterward, we used the steepest descent method to invert the afterslip evolution process and analyzed the relationship between post-seismic afterslip and co-seismic slip. The results witness that 447 days after the mainshock (22 September 2016), the afterslip was concentrated within one principal slip center. It was located 5–25 km along the fault strike, 0–10 km along with the fault dip, with a cumulative peak slip of 0.18 m. The 447 days afterslip seismic moment was approximately 2.65 × 10 W 6.5 earthquake occurred at Pishan in Xinjiang  Catalog, aftershocks of MW 4.5 and MW 4.6 successively struck 2 h after the mainshock. Zhang et al.10 used the digital broadband seismic data recorded by Xinjiang network stations to obtain the aftershock sequence, which extended unilaterally along an NWW direction with a spreading length of approximately 50 km. The mainshock fault inclines towards the SW, showing the characteristics of a shovel thrust fault10. Ainscoe et al.13 used InSAR data and seismic waveforms to solve the fault parameters, to inverse the slip distribution of the co-seismic deformation. It was revealed that the earthquake was induced by a preexisting slope, reverse‐dipping toward the Tibetan Plateau, the range of ∼9–13 km in deep blind fault13. The Pishan earthquake triggered the root faults to produce small swarms, but the previously accumulated tectonic stress was not completely released15. The future seismic hazard deserves further attention.Based on the solved focal mechanism that a thrust rupture triggered the MMW 4.6 and MW 4.5 aftershocks, the study of the co-seismic deformation and fracture mechanism of Pishan MW 6.5 was insufficient to reveal the intrinsic dynamic correlation of mainshock and aftershocks.Since the co-seismic displacements recorded by InSAR include the influence of Therefore, to further probe into the seismogenic condition and post-seismic deformation mechanism, we adopted Sentinel-1A data and employed the Small Baseline Subset InSAR (SBAS-InSAR) technique with the correction of atmospheric error and orbit error to extract the mainshock deformation within 447 days. Then the SDM method was applied to invert the afterslip distribution. The post-seismic deformation transmits pivotal feedback in the temporal evolution of the afterslip. A comparison of time evolution between the afterslip and the aftershocks was conducted to understand the mechanism of aftershock triggering. We comprehensively studied the relationship between afterslip and co-seismic slip to examine regional tectonic movement.W15. A strand of aftershocks following the mainshock primarily focused on the Pishan anticline, which mirrored seismic events evoked by enhancing stress forbye the locked fault portion15.The entire 9 SAR images were gathered from descending Path136 (hereafter referred to as D136). The earliest image acquisition time is 15 days after the earthquake, i.e., 18 July 2015, and the latest image is 22 September 2016. We calculated a time series and referenced it in time to the image acquired 15 days after the mainshock. Figure We selected 9 dates of accumulated LOS deformation within 447 days of the mainshock, as shown in Fig. 17:Three patches  processes over all the upper crust21. Whereas, ductile (plastic) deformation primarily throughout the lower crust, governed by thermally-induced dislocation creep21. The mainshock was a characteristic reverse thrust belt earthquake, not only induced folding of the upper crust but also leading to aftershocks on the Pishan anticlines15. The post-seismic deformation mainly occurred on the fault plane was a manifest of the stress change caused by co-seismic rupture. According to the phenomenon of afterslip and aftershocks, the temporal evolution of both are corresponding, Yang et al.17 believed that aftershocks were possessed by afterslip. Perfettini et al.21 found that post-seismic displacement and the speed of seismic events show a coetaneous decline in the region of aftershocks. For the upper crust, it was considered that the afterslip dominated the elastic deformation, giving rise to displacement change observed on the ground21. The temporal evolution of aftershocks and the displacement feature in the satellite LOS direction was corresponding with previous studies21. Similarly, we conceived that afterslip undertakes a major impact, in the short term, on near-field displacement after the Pishan earthquake.A histogram in Fig. 14. According to the analysis of the afterslip , the afterslip formed one slip centerFig. , which mFig. 17 N m, the moment magnitude of statistical equivalently, for MW 5.03  after the mainshock was approximately 0.5 .03 Fig. ; the aft5.5 Fig. . The rat14 and iteratively searched the model parameters and inverted slip distribution of the constructed model. The same Sentinel-1A data as Wen et al.14 was used for coseismic deformation and coseismic slip inversion analysis technology, to process from ascending and descending LOS data Figure 11. We calculated standard deviation of the slip distribution with 100 perturbed datasets, as shown in Fig. The fault was enlarged to 36 km strike direction, to 40 km down-dip direction. The fault plane was subdivided, along-strike and down-dip separately, to 1 .04 Fig. . The fin15 found several tensile-type ground fissures at the top of the seismic anticline, and all of the strikes were the same as the NW direction and the anticline. It is conceived that the amount of dislocation near the deep source is bound up with ground fissures and is gradually transformed into the fold of the stratum during propagation and expansion along the fault from stress in the transition. The geodetic moment was approximate 6.36 NW 6.5. From MW 6.2 (USGS) to MW 6.5 (CENC) for seismological computations were compared, matched group estimated in Table 17 N⋅m, which accounts for approximately 4.1% of the co-seismic geodetic moment.At a dip depth of 24–27 km, 22–23 km width, the remarkable slip was embodied a distinct feature, to the top magnitude of 0.9 m. At 0–7 km of the upper layer closed to the value of ~ 0.2 m, there has a nearly uniform slip  and the coseismic slip of the fault were superimposed, as shown in Fig. W 6.5 Pishan earthquake on 3 July 2015, which was derived from 9 Sentinel-1A TOPS SAR images from 18 July 2015 to 22 September 2016 using the SBAS-InSAR technique. The evolution process of post-seismic afterslip distribution was inverted by the SDM method. We comprehensively analyzed the relationship between post-seismic slip and co-seismic slip. The main conclusions are as follows:The post-seismic deformation of the Pishan Ms6.5 earthquake was manifested as an uplift in the satellite line-of-sight (LOS) direction in the western portion of the epicenter, which had a significant positive value of up to 25 mm in the satellite LOS direction and a negative value of up to 20 mm in the satellite LOS direction. As of August 3, 2015, 98 aftershocks of Mw At 447 days after the mainshock , the afterslip fields form one slip center. It was located 5–25 km along the fault strike, 0–10 km along with the dip, with a peak magnitude of 0.18 m. The geodetic moment that was based on the afterslip distribution 447 days after the mainshock was approximate 2.65 NThe post-seismic mechanism was a dominant up-dip of the seismogenic portion of a fault zone. The temporal evolution of the deformation feature in the satellite LOS direction and aftershocks supported the scenario that afterslip on the fault plane was the most likely mechanism. The temporal evolution characteristics of afterslip and aftershocks supported the standpoint that aftershocks are dominated primarily by afterslip.We presented a spatial–temporal distribution of post-seismic deformations from the M27. These will be the focus of future studies.Our study helps understand the characteristics of regional fault activity. It will lay a foundation for us that understanding the spatial distribution of fault locking, slip rate deficit and stress accumulation, to better realize mainshock-aftershock activity28. Meanwhile, the results of InSAR observations show that uplift occurred in the western part of the epicenter, and subsidence occurred in the southern part of the epicenter. There are obvious differential deformations on both sides of the fault after the earthquake. The strong earthquake activity near the northwestern margin of the Tibetan Plateau (the West Kunlun orogenic belt) and the Tarim Basin has a certain dynamic relationship with the collision between the Indian Plate and the Eurasian plate, so, close attention should be paid to this region.It has been determined that the Pishan Mw 6.5 earthquake was the result of the N-direction extrusion of the Tarim block in the Tibetan Plateau, and the seismogenic fault was concealed in the Early-Middle Pleistocene14 depicted that the focal depth was mainly distributed in 7–15 km. Nonetheless, the post-seismic afterslip extended to 0–10 km in the shallow part of the northern section, and the slip trend was increased overtime at the deep SSE of the fault  were used for orbital corrections. Elimination of topographic phase effects using 90 m resolution (https://srtm.csi.cgiar.org) Shuttle Radar Topography Mission (SRTM) digital elevation model (DEM) provided by NASA.Thirteen descending Sentinel-1A Terrain Observation with Progressive Scans (TOPS) SAR images (C-band) acquired from D136, from 18 July 2015 to 22 September 2016 covering the earthquake zone, were collected to estimate the LOS deformation time series. The Sentinel-1A Interferometric Wide (IW) imaging mode uses the TOPS method to obtain three sub-bands with a total width of approximately 250 km and resolutions of 5 m × 20 m along with the range and azimuth directions\* MERGEFORMAT34. It combines pairs of images with multi-master image methods, obtains the time series of surface deformation of each set by using the least square estimation method, and obtains the minimum norm least square solution of the surface deformation rate between image sequences by using singular value decomposition.We adopted the Small Baseline Subset InSAR (SBAS-InSAR) technique to process 9 Sentinel-1A TOPS SAR images over the research area. SBAS-InSAR is a method that effectively mitigates decorrelation phenomena by analyzing distributed scatters (DS) with high coherence based on an appropriate combination of interferograms35:Assuming that a set of 36. We obtained interferograms involving data pairs with Doppler centroid differences within the SAR image Doppler bandwidth. Twenty-eight groups of small baseline interferometric pairs were selected, and the parameters of the interferometric pairs are shown in Table We computed a set of multi-look  filtered) differential interferograms characterized by constraints on the maximum temporal and perpendicular baseline37. To estimate the atmospheric disturbances, we select high phase coherence (HPC) pixels on the image grid to estimate the atmospheric phase screen (APS), which can be interpolated on the grid. Both operations (filtering and resampling) can be performed at the same time using kriging interpolation38.Atmospheric components have a strong correlation at short distances38:Estimate the mean value of the estimated atmospheric components in the 38:The phase of each slave image can be modified by these estimated quantities. The new set of phases of the modified slave images will be represented as39. Estimate the orbital error phase of a single interference image pair as:Although the orbital parameter correction has been performed on the Sentinel-1A data using the precision ephemeris orbit data before the data interference processing, there are still residual orbit errors. In this paper, the polynomial optimization method is used to remove the residual orbit errors based on the atmospheric delay error correction40. Assuming that the terrain phase has been removed by DEM, the interference phase on the i-th point target can be expressed as42:The deformation rate was estimated using the least-squares method43:41.The phase of the interference 44:There is a linear relationship between The interference phase model can be further expressed as:42. The high-order polynomial simulates the deformation process;42. We obtain the deformation time series by solving the above interferometric phase model.The To reduce the amount of inversion calculation and improve the signal-to-noise ratio of the data, we first performed a quad-tree downsampling processing on the post-seismic deformation fields for the 8 dates, and the number of deformation points is reduced from 21,927 to 967. Each date of the post-seismic deformation fields had the same number of observation points after downsampling, which is beneficial for comparison in the afterslip inversion. In the case of quadtree downsampling, the sampling in the near-field region was relatively dense, and the sampling in the far-field region was relatively sparse; therefore, the high-resolution information of the deformation field near the epicenter was obtained to the greatest extent.26. In general, the fault parameters of the afterslip inversion can refer to the fault parameters of the co-seismic deformation nonlinear inversion43. The parameters of the fault model were set by referring to the existing co-seismic fault model parameters  is extended to 36 km, and the lower boundary is extended downward (in the direction of the dip) to 40 km along the fault plane. According to the focal mechanism solutions given by USGS, GCMT, and CENC, the range of the fault strike change is set between 95° and 125°, the range of the dip change is set to 18° to 35°, and a search calculation is performed every 1°. After many inversion experiments, the combination of strike and dip angles with minimum root mean square error of fitting residual and the highest modulus correlation coefficient was selected as the optimal value45. When the fault geometry parameters are determined, the slip parameters on the fault plane and the InSAR observed deformation values can be transformed into general linear problems. The expression is:The SDM program developed by Professor Wang Rongjiang is used to constrain the post-earthquake afterslip. The SDM program uses the steepest descent method for inversion calculation, which is an iterative algorithm for constrained least-squares optimizationTo obtain a more detailed fault slip distribution, the fault plane was divided into discrete rectangular elements of 1 km 20. The smoothing factor is generally obtained by plotting the compromise curve between the roughness and the misfit of the test data. The determined optimum smoothing factor was 0.04 using an L-curve plot (Fig. 46 model was used for fault slip inversion, and the model parameters are shown in Table The smoothing constraint was applied to the slip distribution to overcome the instability of the inversion resultslot Fig. . MeanwhiSupplementary Information.


WingMesh. This is an application for simple yet precise automatic modeling of insect wings. Using a series of examples, we showed the performance of our application in practice. We expect WingMesh to be particularly useful in comparative studies, especially where the modeling of a large number of insect wings is required within a short time.Manual modeling of complicated insect wings presents considerable practical challenges. To overcome these challenges, therefore, we developed Distmesh2D, a popular meshing tool, to develop a powerful application for the modeling of geometrically complex objects, such as insect wings. The use of the burning algorithm (BA) in digital image processing (DIP) enabled our method to automatically detect an arbitrary domain and its subdomains in a given image. This algorithm, in combination with the mesh generator Distmesh2D, was used to develop detailed FE models of both planar and out-of-plane  domains containing discontinuities and consisting of numerous subdomains. To easily implement the method, we developed an application using the Matlab App Designer. This application, called WingMesh, was particularly designed and applied for rapid numerical modeling of complicated insect wings but is also applicable for modeling purposes in the earth, engineering, mathematical, and physical sciences.The finite element (FE) method is one of the most widely used numerical techniques for the simulation of the mechanical behavior of engineering and biological objects. Although very efficient, the use of the FE method relies on the development of accurate models of the objects under consideration. The development of detailed FE models of often complex-shaped objects, however, can be a time-consuming and error-prone procedure in practice. Hence, many researchers aim to reach a compromise between the simplicity and accuracy of their developed models. In this study, we adapted  The finite element (FE) method is a numerical technique which is generally used to simulate a physical phenomenon in the virtual world by solving complex boundary value problems ,2. FE soAlthough providing a user with a high degree of flexibility, all available FE packages have a common need: an accurate model. A model is a domain which is subdivided into smaller polygonal or polyhedral meshes, so-called elements . A modelDistmesh2D [f = 0 or by values of a discrete set of points, as explained by the authors, is a time-consuming and error-prone procedure for complex geometries. Due to the use of a mathematical scheme to define the distance function  objects. We specifically designed and used our method to develop models of insect wings. The proposed application, called WingMesh, draws extensively on Persson and Strang’s account in an attempt to offer a simple, but more practical, meshing tool.Here, we aimed to address these challenges and improve the performance of WingMesh, consists of several algorithms that interact with each other. The method requires an input image to identify the boundaries of a given domain as well as subdomains within that. The code Distmesh2D is then employed to mesh the identified domain. Other algorithms were added to the main algorithms to model out-of-plane domains and create a *.inp file, which is the Abaqus input file format [The modeling method presented in this study, called e format . The keyThe burning algorithm (BA) was used to extract the boundary of a domain in a given image ,21. The The function BA can also detect subdomains within a given domain. When the main domain contains any subdomain, the application first finds the white pixels outside the domain. This process eventually results in the detection of the boundary of the domain.To find the boundary of each subdomain, the user should select a pixel inside that subdomain in the input image. By this, the BA finds the pixels located on the boundary of the subdomain using the same method explained earlier. This process continues as long as the user selects a pixel in a new subdomain.The function BA can detect any discontinuity, such as holes, cracks, etc., in a given domain. For this purpose, if any discontinuity exists, the user should select a pixel in each discontinuity in the input image . After tIn a recent study, we developed a method for modeling out-of-plane  domains . Here, wi and j are the index of the row and column of the pixels in the image. This equation recursively updates the color intensity of the pixels in the secondary image and, thereby, the height of those pixels in the model. The number of iterations, which is set by the user, controls the sharpness of corrugations in the developed model. The values of pixels in the secondary input image, which are between 0 and 1, represent the relative heights of corrugations.The recursive Equation (1) is used to smooth the corrugations to avoid any abrupt change in the height of a model at the location of an extremum.Distmesh2D is a mesh generator in Matlab which employs a distance function, d, to describe the geometry of a domain [Distmesh2D to generate triangular meshes. The first line of the code Distmesh2D, the calling syntax, represents inputs and outputs of the Matlab code:fd, the distance function that defines the boundary of the domain.fh, the distance function, which controls the convergence of the size of elements. The size of the elements decreases near fh.h0, the distance between nodes in the initial distribution.bbox, the bounding box in which the domain is located.pfix, defines nodal points, which are set as fixed points while generating elements.a domain . The DelDistmesh2D produces the following outputs:p, gives the coordinate of the nodal points.t, indicates the connection between the nodes.fd and fh for the mesh generator Distmesh2D. In addition to the distance functions fd and fh, Distmesh2D has three other inputs: h0, bbox, and pfix. h0, the distance between initial nodes, can be set to 1, because the minimum distance between two pixels is 1. bbox is equal to the frame size of the imported image (size of the input matrix). The pixels located on the boundaries of the subdomains, extracted by the BA, are defined as fixed points, pfix.Here, coordinates of the nodes on the boundary of a given domain, which are obtained by the BA, are used to define the distance functions Distmesh2D can generate both structured and unstructured elements. However, in this study, we set it to create only unstructured elements, because this type of element fits better with our aim for developing models of geometrically complex structures.WingMesh generates a *.inp file  which contains information regarding the coordinates of the nodal points, their connections, type of elements, sections of the domain, and the material properties of sections. Detailed information about *.inp files can be found in the WingMesh was coded in Matlab 2019a, and Matlab App Designer was employed to develop a GUI. This GUI makes the method easy to implement and eliminates the need to know a programming language. The GUI is available in Example 1: An in-plane domain.inp output file developed by the method is available in Example 2: An in-plane domain consisting of two subdomainsWingMesh was able to detect the border between the subdomains. The subdomains have meshed separately as two sections of a single model. The generated *.inp file is available in Example 3: An in-plane domain with subdomains and a discontinuity.inp file is available in We added a circular hole within one of the two subdomains of the domain given in the previous example e. After Example 4: An irregular-shaped in-plane domain with several discontinuitiesDistmesh2D. However, the use of the DIP technique enables WingMesh to mesh such geometries. .inp file is available in Example 5: A complex-shaped in-plane domain with several subdomains.inp file is available in Example 6: An asymmetric out-of-plane domain with one height maximum and one height minimum.inp file is available in In this example and the next three cases, we used the domain shown in Example 7: An out-of-plane domain with two height maxima.inp file is available in Example 8: An out-of-plane domain with two height maxima and a height minimum.inp file is available in In Example 9: An out-of-plane domain with circumferentially oriented height extrema.inp file is available in In this example, we aimed to test the precision of our method by developing a more complex corrugated domain. In this domain, the corrugation spot is circumferentially oriented, compared with the other domains that had longitudinal corrugations. Example 10: A beetle wingAllomyrina dichotoma (Coleoptera: Scarabaeidae). WingMesh offers several advantages over existing manual modeling techniques using commercial software packages, such as CATIA, SolidWorks, Abaqus etc.:The application is user-friendly and can remarkably reduce the modeling costs.WingMesh is possible by the use of only an image of a given domain.Two-dimensional modeling using Modeling three-dimensional (3D) out-of-plane domains is simple and can be done by the use of one additional image that contains information on corrugated spots.WingMesh can develop meshed models of domains that consist of several subdomains and discontinuities.WingMesh is particularly useful for modeling of a large number of insect wings for comparative investigations.WingMesh is applicable for insect wings that contain a high degree of geometric complexity.Considering the use of computer vision to extract geometric wing features, WingMesh should have only sufficient resolution. This is in contrast to existing tools for extracting morphological features of insect wings using an image, which usually requires high-resolution images at a large size [The input image for rge size ,24.WingMesh has improved the applicability of Distmesh2D, as listed below:Extracting the distance function for complex geometries is a time-consuming and error-prone task, which has been overcome by the use of the computer vision in WingMesh.WingMesh generates a *.inp file as the output, which is a frequently used file format.WingMesh has an improved ability to mesh structures that contain many discontinuities. This ability was poor in Distmesh2D, especially when dealing with domains with more than one discontinuity.In contrast to Distmesh2D, that can mesh domains that have no subdomains, WingMesh is capable of modeling domains with numerous subdomains.Compared with Distmesh2D, WingMesh can model out-of-plane domains.The application presented in this study can be used for modeling a wide range of objects in both science and engineering, where a planar FE model is required. For example, models developed by our application could be used to understand the mechanical behavior of biological structures, such as insect wings, plant leaves, etc. . WingMesh is available on our website: https://wingquest.org/wingmesh/.More information on