Our results strongly contradict the widespread assumption that adenylation and condensation (C) domains coevolve and significantly challenge the attributed role of C domains as stringent selectivity filter during NRP synthesis. Moreover, they teach valuable lessons on the choice of natural exchange units in the evolution of NRPS diversity, which may guide future engineering approaches.Nonribosomal peptides (NRP) are crucial molecular mediators in microbial ecology and provide indispensable drugs. Nevertheless, the evolution of the flexible biosynthetic machineries that correlates with the stunning structural diversity of NRPs is poorly understood. Here, we show that recombination is a key driver in the evolution of bacterial NRP synthetase (NRPS) genes across distant bacterial phyla, which has guided structural diversification in a plethora of NRP families by extensive mixing and matching of biosynthesis genes. The systematic dissection of a large number of individual recombination events did not only unveil a striking plurality in the nature and origin of the exchange units but allowed the deduction of overarching principles that enable the efficient exchange of adenylation (A) domain substrates while keeping the functionality of the dynamic multienzyme complexes. In the majority of cases, recombination events have targeted variable portions of the A Nonribosomal peptides (NRPs) are one of the most diverse and widespread classes of natural products. They are of tremendous importance in microbial ecology as virulence factors and toxins among others such as the siderophore mycobactin. In human health, they serve as life-saving drugs that greatly contribute to human welfare as showcased by the antibiotic vancomycin or the immunosuppressant cyclosporine A . Biochemcore) and a much smaller “sub” domain (Asub), thiolation (T) domains as the amino acid carrier, condensation (C) domains for peptide bond formation, and sometimes additional modifying domains, such as epimerization (E) or methyltransferase (MT) domains  are customized for the synthesis of restricted sets of related compounds . These e domains . Althoug domains , multisp domains , or by s domains , NRPSs a domains .mcyB, which is responsible for the incorporation of the amino acid at position 2, has been shown to be a recombination hotspot . Segments with low π values (near 0) correlate with high homology of sequences, whereas segments with high π values (near 1) correlate with high divergence of sequences, which could be caused by recombination. Putative recombination events have further been validated by using Recombination Detection Program version 4 (RDP4) . We starcore subdomain. These results allowed us to develop an universal evolutionary model for NRPS machineries that is in perfect agreement with recent structural insights in the catalytic cycle of NRPS  replace the conventional T and C domains (TC and LCL) leading to the exchange of TC–LCL didomains with TE–E–DCL tridomains DR) together with the neighboring conserved LPxP motif  but, in strong contrast to their A domain counterparts, do not cluster according to their assumed substrate specificity , ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains, which are responsible for substrate selection and the degree of reduction of the carbon chain. In contrast to that, incongruities have not been observed for ketosynthase (KS) domain sequences, which encode the domains responsible for condensation reactions in polyketide biosynthesis. Phylogenetic trees further suggested that these incongruities result from recombinational replacements within and between biosynthetic gene clusters of S. avermitilis and putative sites for homologous recombination were discovered in the interdomain regions of PKS modules as well as within domains  and then fragmented using pyfasta tool  ensuring with a sliding window of 50aa (-k flag) with 25aa overlap (-o flag) between windows. The resulting windows for each protein sequence were then subjected to BlastP  with its respective documentation at https://mwa-secmet-bgc.readthedocs.io/en/latest/index.html . Proteins have been drawn to scale by using IBS (Module boundaries have been inferred by using the PKS/NRPS Analysis Website of the University of Maryland . Boundaro BlastP . The tabo BlastP . The winsing IBS . Structusing IBS . For detAll code and software used in this study are described and/or are available in the Materials and Methods section.Molecular Biology and Evolution online.msab015_Supplementary_DataClick here for additional data file.


A best evidence topic has been constructed using a described protocol. The three-part question addressed was: In patients undergoing laparoscopic appendicectomy is Hem-o-lok clip safer than Endoloop ligature for closure of appendiceal stump?The search has been devised and 6 studies were deemed to be suitable to answer the question. The outcome assessed was the safety and cost effectiveness of Hem-o-lok clip (Polymer ligation) versus Endoloop ligature for appendiceal stump closure during laparoscopic appendicectomy. We concluded that Hem-o-lok clip is a safe and feasible tool for appendiceal stump closure. It's also a cost-effective way and could be a cheaper option compared to other measures. A BET p2In performing laparoscopic appendicectomy, which technique is the best for appendiceal stump closure? Is it Endoloop ligature or Hem-o-lok clip? Are there any downsides for using either one of them? Therefore, we decided to conduct a systematic review to look for an evidence-based answer to these questions.3In [patients undergoing laparoscopic appendicectomy] is [Hem-o-lok clip] [Polymer clip] safer than [Endoloop ligature] for [closure of appendiceal stump]?4The search was conducted as following:Embase 1974 to 2021 and MEDLINE® 1946 to November 2021 using the OVID interface.[Laparoscopic appendicectomy OR appendicectomy] AND [ Hem-o-lok clip OR Polymer clip OR Polymeric clip] AND [closure of appendiceal stump] AND [Intraoperative complications OR Postoperative complications OR Leak OR abscess].The search was limited to English language and human studies.5250 articles were found. Out of these 6 deemed to be suitable and met the criteria of our search after removing the duplicate and excluding the irrelevant articles. We only included articles which compared these two methods for appendiceal stump closure and excluded studies which compared other methods.1Conference abstracts2Studies not comparing both techniques3Absence of full-text articles4Low evidence papersExclusion criteria:6.8Four randomized clinical trials and two retrospective studies proved that Hem-o-lock is safe and feasible tool for appendiceal stump closure. It's also a cost-effective way and could be a cheaper option compared to other measures.Not applicable.None.SA: devised the idea of the study, conducted literature search and wrote the paper.TS: assisted in literature search and collecting the data.MO: assisted in literature search and writing the paper.RI: assisted in literature search editing and writing the paper.1.Name of the registry: Not applicable2.Unique Identifying number or registration ID: Not applicable3.Hyperlink to your specific registration (must be publicly accessible and will be checked):Sabry Abounozha (SA), Tamer Saafan (TS), Munzir Obaid (MO), Rashid Ibrahim (RI).Not Applicable.None.


Millo corvo variety) bread. As a result, baking always resulted in a general decrease in TPC, even at 150 °C. In contrast, TAC only decreased when temperatures as high as 180 °C were applied. Some relevant individual phenolics were preserved during the whole process as long as 150 °C was used. In particular, the content of the major anthocyanin, namely, cyanidin-3-O-glucoside, hardly decreased from the raw flour to the final bread. The loss of antioxidant activity of Millo corvo raw flour during bread baking was avoided by heating at 150 °C. These results demonstrate the appropriate temperature to bake Millo corvo corn bread without losing the antioxidant characteristics and health-promoting properties of the starting black corn.Black corn is known for its health-promoting properties, which are due to its high content of bioactive phytonutrients. However, the high temperatures required during the processing of bakery products usually trigger thermal degradation, and therefore, the loss of all labile bioactive compounds. In the present study, we evaluated the effect of baking temperature on the phenolic content  and antioxidant activity in black corn ( Corn, together with rice, is one of the most cultivated cereals in the world, and since it is gluten-free, it is also suitable for consumption by those with celiac disease. There are several varieties of corn; however, in recent years, black corn has become particularly popular. From a marker standpoint, black corn is a natural colorant with a plant origin that can be very interesting as an alternative to artificial additives. More importantly, black corn possesses excellent nutritional properties due to its high content of relevant phytonutrients with functional roles in human health .O-glucoside is the most abundant [In this respect, black corn contains some phenolics, carotenoids and particularly high content of anthocyanins . Summingabundant . Beneficabundant .(Millo corvo variety). To this end, the phenolic composition and antioxidant properties of the final bread obtained were compared with those of the starting Millo corvo flour to select the heating conditions that enabled the flour antioxidant content to be preserved. To date, no bibliographic report can be found in the literature about bakery products baked using Millo corvo corn.Corn-based foods require a thermal process prior to consumption. In this context, the inadequate selection of baking time and temperature may cause breakdown problems, such as cracking and checking , affectiO-glucoside standard was provided by Extrasynthase .Ultrapure water was obtained from a purification system . Both formic acid and MeOH (HPLC grade) were obtained from VWR Inc. . 2,2-diphenyl-2-picrylhydrazil (DPPH) and sodium carbonate standards were supplied by Sigma-Aldrich . An antioxidant capacity lipid-soluble (ACL) kit was acquired from Sigma-Aldrich . Foulin–Ciocalteu reagent was obtained from Merck . Chlorogenic, caffeic and ferulic acids, as well as quercetin and quercertin-3-glucoside standards, were purchased from Sigma-Aldrich . Cyanidin-3-Zea mays L., Millo corvo variety) and conventional white corn produced and supplied by Asociación Cultural Meiro  were used. Flour samples were prepared via dekerneling corn by hand and then grinding the whole grain using a traditional stone corn mill. Flour samples were immediately stored at −14 °C until their use. The preparation of bread was accomplished as follows: dough from flour was carried out by mixing 35 g of raw flour with 10% of water. The mixture was then optimally hand kneaded to a smooth dough. The breadmaking process was performed in a scale-down method by adding 4.6 g of fresh yeast to 35 g of the dough previously prepared. After that, it was kneaded by hand up to a homogenous mixture, which was finally baked in an oven at 150 °C for 60 min. Two batches were run for each sample.For the experiments, black corn  to the sample. The resulting mixture was homogenised with an Ultraturrax homogeniser  for 10 min and then centrifuged at 2500 rpm at 15 °C for another 10 min. The solvent was removed and an additional 6 mL of methanol/acetone/water (35/35/30) was added to the extract, which was re-extracted. Then, the extract was taken to dryness using a rotary evaporator. Finally, the dry extract was stored at −14 °C until analysis. The extractions were carried out in triplicate and both the −1) were added to a 0.1 mL volume of the extract (20 mg mL−1). The final mixture was made up to 25 mL with distilled water. After 1 h, the absorbance was measured at 750 nm against a blank . The results were expressed as milligrams of gallic acid equivalents (GAE) per 100 g of sample. All the analyses were carried out in triplicate.The equipment used for TPC measurements was a Beckman Coulter DU-800 spectrophotometer . The method applied was based on the oxidation of the hydroxyl groups of phenols in basic media by the Folin–Ciocalteu reagent, as described in the literature . In brieMillo corvo corn flour and bread samples were diluted with 0.025 M potassium chloride buffer solutions at pH 1 and with 0.4 M sodium acetate buffer at pH 4.5. A 400-700 nm sweep was carried out using a spectrophotometer . TAC was expressed as milligrams of cyanidin-3-O-glucoside equivalents per 100 g of sample based on a molar extinction coefficient of 26,900 L cm−1 and a molecular weight of 449.4 g/L.Total anthocyanin was quantified using the pH differential method reported elsewhere . Millo cTotal absorbance was measured using the equation:−1. This solution was further diluted to final concentrations of 15.6, 62.5, 125, 250 and 500 µg mL−1 before being transferred to a 96-well microtiter plate. Before adding DPPH*, each extraction solution was used as a blank. Each well contained 50 µL aliquot of the sample and 150 µL of DPPH* (400 µmol L−1). Each mixture was incubated at 37 °C for 30 min and then the absorbance at 517 nm was monitored. The absorbance value obtained was compared with that of the DPPH* solution measured immediately after being prepared, which was used as a reference. The absorbance decrease obtained indicated the percentage inhibition of the DPPH* by each dilution level of the samples. A plot of percentage inhibition versus concentration was made and the IC50 values were calculated using linear regression analysis. The experiments were performed in duplicateAntioxidant activity was determined using a DPPH* assay  with sli® device  and it was conducted via an ACL protocol, which allows for the antioxidant capacity of the lipid-soluble components to be measured [−1). A 20 μL volume of this solution was then mixed with ACL reagent and the mixture was placed into the Photochem device. Results were calculated on the basis of standard curves into nanogram Trolox equivalents per microlitre of sample (ng µL−1).The antioxidant capacity was also determined using a PCL assay. This method was applied using a Photochemmeasured . The stumeasured . For the18) column  at a flow rate of 1 mL min−1. To protect the column, an Altima 5 µm C18 pre-column  was used. Both the pre-column and column operated at 20 °C. The elution program was the same as previously optimised [O-glucoside at 520 nm. Stock solutions of the standard compounds were prepared in 70% (v/v) methanol to a final concentration of 1 mg mL−1. In addition, calibration curves of the standards were established for six data points and each standard dilution was run in triplicate. The extracts were also reconstituted in 700 mL L−1 methanol and also run three times at a concentration of 20 mg mL−1 for the quantification of polyphenols.The content of individual phenolics was determined using HPLC. The equipment used for the measurements was an Alliance Separation Module 2695 chromatograph  with an automatic injector and a photodiode array detector 996 . The separation of the target compounds was accomplished on an ODS reverse phase (Cptimised . Blanks −1 weight ± SD). Data from raw flour, heated flour and bread were included in the statistical analysis. Comparisons of means were made by using Fisher’s protected LSD. Differences were considered significant for p < 0.05.An analysis of variance for the TPC, TAC, DPPH and PCL activities and individual phenolic content was carried out using the one-way analysis of variance (ANOVA) method. The results are presented as the average of all values obtained and standard deviation . PCL is a relatively new method for measuring antioxidant capacity. It is based on the optical excitation UV sensitiser that causes free radical generation, which is partially eliminated by naturally occurring antioxidants in the sample [p < 0.05) differences between the two flours were measured. Millo corvo corn flour exhibited significantly (p < 0.05) higher TPC and antioxidant activity from both the DPPH and PCL methods. Different TPC data were reported according to the corn cultivar [Initially, raw e sample . The rescultivar ,16. HoweMillo Corvo corn flour as an ingredient.In a view of these results, the use of white corn flour to bake bread was discarded. The bread study was only carried out using Millo corvo flour was performed by reproducing the baking processing at 150, 180 and 200 °C.The effect of temperature on TPC, TAC, DPPH scavenging activity and individual phenolics in raw p < 0.05) with heating. In particular, TPC dropped from 305.3 mg GAE 100 g−1 in the unheated flour samples to 119.6, 105.6 and 102.3 mg GAE 100 g−1 after heating at 150 °C, 180 °C and 200 °C, respectively. Interestingly, all the temperatures tested provided similar results.As can be seen, the TPC values decreased significantly (p > 0.05) decrease from 381 mg cyanidin-3-O-glucoside 100 g−1 in the unheated sample to 27 mg cyanidin-3-O-glucoside 100 g−1 after heating at 180 °C was measured. The apparent changes observed after heating at 150 °C and 200 °C were not considered since they were not statistically significant.Regarding TAC, values also diminished with heating. In particular, a significant  was not significantly (p > 0.05) affected after heating at 150 °C . However, the application of 180 °C and 200 °C during the baking process did result in a significant (p < 0.05) increase in DPPH activity, particularly at 200 °C .Concerning the DPPH scavenging activity, a similar trend was observed see . The IC5−1) in Millo corvo flour baked at different temperatures  for 1 h. From trans-ferulic acid decreased significantly (p < 0.05) as a result of heating. This decrease suggests a direct correlation between the individual phenolics here determined and those contributing the most to the TPC  differences were found among the temperatures tested. Hence, the individual phenolic content would be similar whatever the temperature applied.As seen in with TAC , the heaO-glucoside, decreased significantly with heating, the increase of the DPPH activity  from 291.1 mg GAE 100 g−1 in raw flour to 48.1 mg GAE 100 g−1 in bread. In contrast, TAC increased significantly (p < 0.05) from 381.2 mg g−1 in raw flour to 507.0 mg g−1 in bread, and the antioxidant activity in terms of DPPH was not significantly affected by heating, obtaining statistically insignificant values for raw flour and bread.From Millo corvo raw flour (p < 0.05) increase in Millo corvo bread as compared with the unheated raw flour  baked at 150 °C for 1 h. In the same way, chlorogenic and caffeic acids, together with quercetin, kept decreasing after baking, whereas ferulic acid was not affected by heating. However, the breadmaking process did minimise the effect of the temperature on the contents of quercetin-3-glucoside and cyanidin-3-O-glucoside. As seen in O-glucoside content obtained in bread with respect to raw flour was in accordance with the higher TAC above mentioned  and the final bread (i.e., IC50 value of 875.3 µg mL−1, Millo corvo bread. The value obtained was 916.19 ng eq Trolox µL−1, which was statistically similar to that of Millo corvo raw flour (i.e., 734.3 ng equivalents Trolox µL−1, Millo Corvo flour in the final bread baked from it in the baking conditions applied here was confirmed.Finally, the statistical comparison between the free radical scavenging activity in terms of the DPPH of raw flour (i.e., ICMillo corvo corn bread. This is why our results cannot be contrasted with the literature. However, the influence of baking on anthocyanins in colored-grain wheat bread was recently studied [To our knowledge, there is no bibliographic report on the antioxidant properties of  studied ,23. ThesMillo corvo corn bread possessed the same phenolic content and antioxidant activity as the starting flour as long as 150 °C was applied during the baking process. This indicates that Millo corvo is a suitable corn variety for bread production due to the remaining antioxidant content after baking. Our objective now is to extend these results to the study of other colored compounds and additional bakery products. In particular, the consideration of carotenoids and additional anthocyanins and the study of new parameters, such as the baking time and the addition of citric acid, are scheduled.The present study demonstrated that


Hollow noodles, also known as Kongxin noodles in China, are traditionally hand‐made noodles produced by spontaneous fermentation. It is easy to cook, nutrient‐rich, and delicious. However, it is difficult to realize industrial production by spontaneous fermentation due to its complexity. More recently, new techniques have emerged for producing such noodles industrially using commercial yeasts. However, there are no reports on how to choose the raw materials for making fermented hollow noodles. Therefore, the suitability of eleven local varieties of wheat flour was determined by evaluating their physicochemical, rheological properties, and pasting properties. Flour and dough properties of wheat flour were also correlated with the quality characteristics of hollow noodles. The correlation coefficient data indicated that the color score was negatively correlated with ash content and positively correlated with starch content. Different from ordinary dried noodles, a negative correlation was observed between cooking time (CT) and protein content. Water absorption (NWA) of hollow noodles was negatively affected by extensograph properties. Water absorption of flour (FWA) and extensibility (E) were found to be highly correlated to hollow rate (Hol‐R), indicating that these two indexes could predict the fermentation status of hollow noodles. Results showed that wheat flours with higher swelling index of glutenin (SIG), FWA, E, and pasting temperature (PT) had better dough fermentation power and stability and thus were beneficial to the production of high‐quality hollow noodles. This study provides a simple method for the industrial production of hollow noodles and provides a basis for the selection of raw materials for their production. Water absorption of flour (FWA) and extensibility (E) were found to be highly correlated to hollow rate (Hol‐R), indicating that these two indexes could predict the fermentation status of hollow noodles. Results showed that hollow noodles with stable fermentation and high quality demanded wheat flours with high swelling index of glutenin (SIG), FWA, E, and pasting temperature (PT). Nowadays, consumers pay more attention to nutritional and healthy noodle products and require the noodles with ready‐to‐eat convenience and superior flavor and taste. Fermentation can convert wheat flour dough into inviting, toothsome and digestible products, and this technology is mainly used in the production of bread and steamed bread. However, in order to improve the nutrition, functional, sensory, and technological characteristics of noodle products, fermentation step as an innovative strategy was applied to pasta making  to improve the peculiar flavor, texture properties, and nutritional value of fermented noodles or pasta , a large number of microholes appear, which confers the hollow noodles with pleasant fermented flavor and good rehydration. Nevertheless, the manufacturing procedures of hand‐made hollow noodles are various, complicated (up to 20 steps), and time‐consuming (up to 70 hr). The fermentation process is also vulnerable to the weather. Therefore, the production of hand‐made hollow noodles is limited and it is difficult to carry out large‐scale industrialization.More recently, new techniques have emerged for producing hollow noodles industrially using commercial yeasts. Yihai Kelly Group (China) has launched an industrial fermentation type hollow noodles which have been applied for a patent   and farinographic properties  of wheat flour were highly related with white salted noodle quality Lindsay, . Much reIn this work, the characteristics of 11 brands of wheat flour used for hollow noodles making were evaluated by physicochemical, rheological, and paste properties; then, a simple and industrializable production process is adopted for hollow noodles making and the correlation between characteristics of wheat flour and quality of hollow noodles were investigated. The objective was to provide a theoretical basis for the establishment of the raw material quality standard for making hollow noodles.22.1Eleven different kinds of commercial wheat flours were obtained from the market including Xiangxue high gluten wheat flour (Xiangxue HG) (Shenyang Xiangxue Flour Co. Ltd), Xiangmanyuan high‐quality wheat flour (Xiangmanyuan) (Yihai Kerry Co. Ltd), Xuejian multi‐purpose wheat flour (Xuejian MP) , Sifeng fine wheat flour (Sifeng) (Xinxiang Sifeng Powder Industry Co. Ltd), Xuejian wheat core flour (Xuejian WC) , Sany original wheat flour (Sany) (Zhengzhou Tiandiren Flour Industry Co. Ltd), Golden Arowana multi‐purpose wheat flour (Golden Arowana) (Yihai Kerry Co. Ltd), Wudeli high gluten wheat flour (Wudeli) (Wudeli Group Xinxiang Flour Co. Ltd), Jinyuan refined wheat flour (Jinyuan) (Henan Jinyuan Grain and Oil Co. Ltd), Fengzheng low gluten wheat flour (Fengzheng LG) (Weifang Fengzheng Flour Co. Ltd), Fengzheng high gluten wheat flour (Fengzheng HG) (Weifang Fengzheng Flour Co. Ltd). Active dry yeast (Angel Yeast Co. Ltd) was purchased from a local supermarket. All other reagents used in this experiment were of analytical grade.2.2*, a*, and b* values, was performed in terms of the method of Kaur et al. , the resistance to extension , the dough extensibility , and R/E value at 45 min.Farinograph properties of flour were analyzed by the AACC method 54–22 (2000) using a 300 g Brabender Farinograph equipment (Brabender GmbH & Co KG). Extensograph properties of flour were analyzed by the AACC method 54–10 (2000) using Brabender Farinograph and Extensograph (Brabender GmbH & Co KG). The data were provided with the energy value . Flour  and 25 ml distilled water were added to an aluminum RVA sample canister and then run a heating and cooling cycle program: a heating step, from 50 to 95℃ at 12℃/min (after an equilibration time of 1 min at 50℃), a holding phase at 95℃ for 5 min, a cooling step from 95 to 50℃ at 12℃/min, and a holding phase at 50℃ for 2 min.2.5The preparation process of hollow noodles in this study was not as complicated as traditional method and was more suitable for industrial production. The ingredients used in the preparation of the hollow noodles were 200 g wheat flour, 60 g distilled water, and 2 g yeasts. First, the yeasts and distilled water were mixed in a beaker and then preserved heat at 38℃ in a water bath for 10 min. Dough was formed using a mixer  which was operated at low speed for 3 min and medium speed for next 4 min. After mixing, the prepared dough was placed in a plastic bag and rest for 15 min in a thermostated container at 25℃. The noodle dough was then passed through a laboratory JMTD 168/140 noodle machine  at the gap of 3 mm for 4 passes. Each pass included folding the dough sheet to double the thickness. After being doubled folded, the roll gap was adjusted to 2.0 mm for another pass. Following that, five more passes were made to reduce the gap progressively to 1 mm. Finally, the obtained sheet was cut into rectangles of dimensions of 25 cm ×0.2 cm ×0.1 cm. The resultant noodle strands were hung on rods and placed in an incubator at 38℃ and 85% RH for 2 hr (a fermentation step), then reduced relative humidity, and dried in incubator in two stages. In the first stage, the temperature was kept at 38℃ and relative humidity was at 65% for 10 hr. In the second stage, the temperature was kept at 25℃ for 4 hr and the relative humidity was at 65%. Finally, the moisture content of dried hollow noodles was at 12%–13%. The dried hollow noodles were cut into the length of 10 cm and stored in sealed plastic bags for the next studies.Hollow noodles were prepared on two different days , and two bags of hollow noodles were prepared for each flour type.2.6Cooking properties were determined by the method of Liu et al. . Texture2.7The fermentation state of hollow noodles was determined by hollow rate, and not all noodles were fermented into hollow ones. Satisfactory fermentation states were generally along with high hollow rates. Fifty noodles were randomly selected from each type of wheat flour and the number of hollow noodles (L) was counted. The hollow rate of hollow noodles was expressed as: % hollow rate of hollow noodles =L/50 × 100.2.8Ten trained food engineering students participated in the sensory evaluation of the hollow noodles, including color, firmness, viscoelasticity, smoothness, and flavor. A 9‐point scale (1 = dislike extremely and 9 = like extremely) (ISO 11136:2014) was used for this evaluation. During sensory evaluation, each sample was placed in the same plate coded with a digit number, and drinking water was provided to clean mouths during tasting.2.9r) between different properties and analysis of variance (ANOVA) between different wheat flours by Duncan's test was conducted using software 19.0 (SPSS Inc.). Significance was defined at p < .05.The data reported in all tables are averages of at least three replications. Pearson correlation (33.1p > .05). The protein content, wet gluten content (WG), and gluten index (GI) were in the range from 8.35% to 12.48%, 21.89% to 33.13%, and 52.38% to 95.26%, respectively. GI indicates the information about quantity and quality of wet gluten, which is strongly influenced by environmental factors , indicate the swelling and crystal collapse of starch granules during heating and cooling process in a system . Overcooked noodles are undesirable for too soft and sticky taste. In contrast, a raw flour taste and hard texture are found for undercooked noodles during biting and, hence, cooking time is a key indicator for texture quality of noodles . The hardness (HD), adhesiveness (AD), springiness (SP), and cohesiveness (CO) varied from 4,231.15 to 5,204.14 g, 91.67 to 191.64 g, 0.84 to 0.93, and 0.68 to 0.73, respectively. Hollow noodles made from Xiangxue HG flour exhibited excellent textural properties as evident from the highest HD, AD, and SP values and higher CO value, while Sany flour had lower AD, the lowest SP, and lower CO values. A high degree of gluten network formation would completely encircled starch granules and formed a compact internal structure of noodle strands, which largely contribute to these results  and tensile distance (Ten‐D). The results of Ten‐F and Ten‐D ranged from 10.66 to 14.77 g and 56.30 to 82.78 mm, respectively . The color scores ranged from 5.76 to 8.28, of which Xiangxue HG and Sifeng flours exhibited the highest and Xuejian MP flour showed the lowest. Although there was no significant differences among these samples (p > .05), the scores of firmness, viscoelasticity, and smoothness varied from 6.45 to 7.50, 7.00 to 8.00, and 5.10 to 6.00, respectively. The pleasant degree and uniqueness of the fermented flavor of hollow noodles were determined by the flavor scores. According to table The results of sensory characteristics were reported in the form of scores in table 3.5r = −.632, p < .05). The ash content of flour showed a negative correlation to the color score of cooked hollow noodles , indicating that the flour with higher ash content could darken the brightness and color of noodles. A similar relationship between ash content and noodle color had been reported . Brighter or whiter appearance might be attributed to more light reflected from more starch granules. A slight negative correlation was observed between protein content and CT , which were not in agreement with the results reported by Kaur et al.  while negatively related to the AD of cooked hollow noodles . In addition, the SIG was positively correlated to the viscoelasticity of hollow noodles . This might be due to glutenin, which was linked by interchain disulfide bonds, determined dough strength and elasticity  and the color of cooked hollow noodles .The correlation coefficients between physicochemical properties of different wheat flour and the quality of hollow noodles are presented in Table r et al. , 2016. Ir et al. . In the  Wieser, . The valr = .638, .647, .614, and .615, respectively, p < .05). The DDT exhibited a positive correlation with the firmness and smoothness , but showed a negative relationship with Ten‐D . Furthermore, the DST was highly positively correlated with the firmness of cooked hollow noodles  while was negatively related to the NWA of hollow noodles . Similar results were obtained by previous researches . The A value was negatively correlated to the NFA and CO of hollow noodles . The R related negatively to the NWA , and the E was positively correlated to the Ten‐D and Hol‐R . It is demonstrated that the dough with excellent extensibility could provide hollow noodles with better extensibility and fermentation status. The R/E showed negative correlation with the NWA  and Ten‐D . Similar results were obtained by Yue et al. .The correlation coefficients between farinographic properties of different wheat flours and the quality of hollow noodles were also presented in Table e et al.  who founr = −.627, p < .05). The PT was positively correlated to the SP , while negatively related to the viscoelasticity . Although the correlation coefficient was not significant, the PV related positively to the CT  but negatively to the Hol‐R and fermentation flavor . The TV and FV showed similar relationships with CT, Hol‐R, and flavor. Therefore, higher peak, through and final viscosity were not beneficial for the fermentation of hollow noodles.As shown in Table 4In this study, based on a set of commercial samples, the quality of hollow noodles was associated with physicochemical characteristics, rheological properties including farinograph and extensograph and pasting properties of wheat flours. The CT of hollow noodles was negatively related to protein content while positively related to pasting properties of flour. Extensographic properties had a considerable effect on NWA. The FWA and E were found to be highly correlated to the Hol‐R. A significant positive correlation was observed between SIG and viscoelasticity, PT, and SP. Furthermore, farinographic properties substantially affected the firmness of sensory evaluation. Thus, flour with higher SIG, FWA, E, and PT resulted in hollow noodles with better fermented flavor and better textural quality. So far, there are few studies on the hollow noodles, including formula, quality changes during storage, quality formation mechanism, and further investigations need to be conducted.Xiaoqing Xiong: Software ; Validation ; Visualization ; Writing‐original draft (lead); Writing‐review & editing (lead). Chong Liu: Conceptualization ; Resources (lead); Supervision (lead); Validation ; Visualization ; Writing‐review & editing (supporting). Mengkun Song: Writing‐review & editing (supporting). Xueling Zheng: Formal analysis ; Funding acquisition (lead); Methodology .This study does not involve any human or animal testing. The authors have declared no conflicts of interest for this article.


Bran is the main by-product of wheat milling and the part of the grain with the highest Zn content. We investigated the effects of the particle sizes  and addition level (5–20%) of Zn-biofortified bran on the quality of flour and Chinese steamed bread. It was studied to determine if the Zn content of steamed bread could be enhanced without deleterious effects on quality. Dough pasting properties, such as peak viscosity, trough viscosity, final viscosity, breakdown, and setback, decreased significantly as the bran addition level was increased from 5 to 20% but did not significantly differ as a result of different bran particle sizes. Bran incorporation significantly increased hardness, gumminess, chewiness, and adhesiveness, whereas the springiness, cohesiveness, and specific volume of steamed bread decreased with the increase in bran addition. The optimal sensory score of steamed bread samples in the control and Zn fertilizer groups were obtained under 5% bran addition resulting in comparable flavor, and texture relative to control. Meanwhile, the Zn content of the steamed bread in the Zn fertilizer group was 40.2 mg/kg, which was 55.8% higher than that in the control group. Results indicated that adding the appropriate particle size and amount of bran would be an effective and practical way to solve the problem of the insufficient Zn content of steamed bread. Zn is one of the most essential trace elements that are closely related to human health. It is a component of more than 100 enzymes in the human body and a participant in the synthesis of nucleic acids and protein. Zn deficiency seriously affects the secretion of enzymes and hormones, thus endangering human health. More than 50% of the world's agricultural soils are deficient in available Zn . Every yWheat is one of the major food crops that is considered to be an important candidate for Zn biofortification. In China, the Zn content of wheat grain grown in major wheat-producing areas is <30 mg/kg , which iThe processing of wheat for consumption always includes two main steps: grinding wheat grain into flour and food preparation. During these processes, bran, which has the highest Zn content, is often discarded, resulting in the large loss of Zn from wheat grains. Therefore, bran can be applied in the processing of steamed bread to improve the content of Zn and increase the utilization rate of Zn in wheat grains. Wheat grain is composed of three components: the bran, embryo, and endosperm. The endosperm is the main ingredient of flour, and the bran is the main by-product of flour processing. Studies have shown that the content of metal elements, such as Zn, in wheat grain embryos, aleurone layers, and seed coats is drastically higher than that in the endosperm . The Zn 4·7H2O  containing 0.01% (v/v) Tween 20 as a surfactant. All the fertilization treatments were performed after sunset. Each plant plot had an area of 50 m2 (5 m × 10 m) with three replications. The line spacing was 0.25 m, and the plant spacing was 0.02 m.The winter wheat cultivar used for the study was “Shannong 29,” which was bred by Shandong Agricultural University. The field experiment was conducted at the Shandong Agricultural University Research Farm . This area has a warm and semihumid continental monsoon climate, an annual average rainfall of 680 mm, and an annual mean temperature of 12.8°C. During the growing season, Zn fertilizer was sprayed at the jointing, flowering, pre-grouting, and late filling stages for the Zn biofortification of wheat. The foliar treatments were as follows: (1) foliar spraying of water as a control and (2) spraying of ZnSOAfter harvesting, part of the samples was milled into flour by using a MM400 Hybrid Ball Mill  for the determination of the Zn concentration of whole grains. The other wheat samples were milled into three core powders , three hide powders , and bran by using a MLU-202 Automatic Laboratory Mill  in accordance with the AACC-approved method 26-21A .The bran collected from the mill was sifted into coarse bran (particle size of 2–2.5 mm) by using a JJSD sieve shaker (Shanghai Jiading Ltd. equipped different sized screens) with differently sized screens. Whole wheat bran was ground by using a fine grinder  into fine-ground bran. The bran samples were then divided into three equal portions. Two were subsequently ground into medium and fine particles by using a Perten 3100 laboratory mill  equipped with meshes of different sizes. Coarse bran (D50 = 375.4 ± 12.3 μm) was superfinely ground into medium (D50 = 122.3 ± 7.1 μm) and fine (D50 = 60.5 ± 4.2 μm) bran. The bran samples with different particle sizes were added to the flour at 0, 5, 10, 15, and 20% levels (on a 14% moisture basis). In this work, whole flour was the flour that contained whole grain. Standard flour was a blend of the 1M, 2M, 3M, 1B, 2B, and 3B fractions and was similar to commercially available flour. Coarse flour was a mixture of standard flour and coarse bran. Medium flour was a mixture of standard flour and medium bran. Fine flour was a mixture of standard flour and fine bran.3-H2O2  in a microwave digester reaction system . Then, the Zn content of the digested solution was determined through inductively coupled plasma optical emission spectroscopy . After use, all the instruments used in the experiment were soaked in 20% nitric acid overnight, then rinsed with deionized water and dried. The moisture content, ash content, and falling number of the flour samples were determined in accordance with the Chinese National Standards GB/T 5497-1985, GB/T 5009.4-2003, and GB/T 10361-89, respectively. Protein content on a dry weight basis was determined through the Kjeldahl method in accordance with the International Approved Method 46-12  in accordance with the AACC International Approved Method 76-21 . The tes*, a*, and b* values. L* represents brightness and is a measure of black to white (0–100). Large L* values are indicative of high whiteness and brightness. a* stands for the red–green phase. A positive a* value indicates redness, whereas a negative a* value reflects greenness. b* stands for the yellow–blue phase. If b* is positive, then the sample is yellow. If b* is negative, then the sample is blue.A Minolta Chroma Meter  was used to determine the whiteness of wheat flour and obtain LThe gluten content and index were determined by using a Glutomatic 2200 system  in accordance with the AACC International Approved Method 38-12.02 . A totalSteamed bread was prepared with 200 g of flour or flour blended with bran, 2 g of yeast, and water (85% of Mixolab water absorption). Before dough mixing, the yeast was dissolved in water (35°C). Then, the yeast solution and flour were poured into a mixing machine  and mixed for 5 min. The mixed dough was sheeted 15 times on a tablet pressure machine to remove air bubbles and split into 100 g portions. Each chunk was rolled with heights of approximately 6 cm by hand and then fermented  for 45 min in a cabinet. After fermentation, the buns were steamed for 20 min in an electric steamer and cooled for 5 min after turning off the fire.The steamed bread samples were cooled to room temperature for approximately 1 h before the measurement. The volume of steamed bread was determined by using the rapeseed displacement method. The specific volume was calculated from the volume-to-weight ratio of the steamed bread. GB/T21118-2007 with a few modifications was adopted for the sensory quality analysis of steamed bread. Specific volume, color, surface structure, appearance, internal structure, elasticity, toughness, stickiness, and scent were scored by six trained evaluators. The highest scores for each item were 20, 10, 10, 10, 15, 10, 10, 10, and 5, which provided a total score of 100 (the specific score was accurate to 0.1). The eating quality of the cooked steamed bread was subjectively evaluated by 16  trained panelists according to Chinese Standard Method GB/T 35991-2018.After sensory evaluation, the steamed bread was subjected to textural profile analysis (TPA) by using a TA XTPlus Texture Analyze apparatus  equipped with a P/35 R probe. Before the test, the steamed bread was cooled to room temperature and then cut into 10 mm-thick slices. The two center slices were taken for TPA. The test parameters were as follows: pretest speed of 1.0 mm/s, test speed of 1.0 mm/s, post-test speed of 1.0 mm/s, compression ratio of 50%, trigger force of 5 g, interval time of 5 s, and data collection rate of 200 pps.p < 0.05) was used to compare significant differences among the samples.All data were collected at least in triplicate. The statistical analysis of the results was carried out with SPSS software , and Duncan's test (p < 0.05), which followed the order of 3M > 2M > 1M. By contrast, no significant differences were found among the Zn concentrations of the three hide powder flours. As can be seen from p < 0.05). Meanwhile, the Zn content of the Zn fertilizer flour had increased by 58.7% compared with that of the control flour. These results showed that the foliar spraying of Zn fertilizer is an effective way for bioaccumulation (p > 0.05) between the CK and Zn groups, indicating that the processing of bran into different particle sizes would not affect Zn content. These results showed that Zn fertilization effectively increased the Zn concentrations of whole wheat grain relative to the control treatment, and bran was the most Zn-rich part of wheat grains.The effects of spraying Zn fertilizer on the Zn content of whole grain and different grain components are summarized in mulation . Figure p < 0.05). These results could be attributed to the following: The grinding of small grains required more time and consumed more energy than that of large grains. The heat generated by the processing equipment in this process may be an important reason for water loss. In addition, the whiteness of flour decreased significantly with the increase in bran addition (p < 0.05). Whiteness is the major sensory indicator, which is actively correlated with consumers' acceptance. Bran was brownish yellow, and the flour was milky white as usual. The color of the blends gradually deepened and darkened as the amount of bran added to the flour was increased. As can be seen from * and b* values increased and the L* value decreased obviously. Dose-dependent lower L* value indicated bran incorporation darkened the appearance of flour  and was positively correlated with bran addition level. Generally, wheat bran contains more than 15% high-quality proteins , indicating that the addition of bran could affect the formation of gluten in the dough. The possible reasons are as follows: on the one hand, although the protein content increased due to the presence of more bran, the bran protein was mainly dominated by globulin and albumin, and the gluten quality was poorer than that of endosperm protein; on the other hand, the presence of bran had an adverse effect on the gluten network, making it difficult for the gluten to form a strong network structure  likely because the addition of bran destroyed the network structure formed by gluten and reduced the quality of the dough. This result was consistent with the changes of gluten content and gluten index. The adverse effect of bran addition on gluten network formation could be explained by its role as a filler suspended in the dough . Bran could act as a filler in the flour, wherein it reduced the proportion of starch and competed with starch granules for moisture, thus reducing the minimum viscosity values of the bran-containing pastes (p < 0.05). The size of the bran had no noticeable effect on the minimum and final viscosities of the flour. The setback value is related to the tendency for paste retrogradation. Starch retrogradation occurs in the cooling stage, wherein the amorphous structure of amylose and amylopectin begins to recrystallize through hydrophobic interactions and hydrogen bonds. The competition of the bran for water leads to the redistribution of water in the paste system, resulting in a decrease in starch retrogradation  with the increment in the addition level of coarse, medium, and fine bran. Compared with that of the flour without bran, the Zn concentrations of the flour in the Zn fertilizer groups and control groups had enhanced by 5.6–21.7% and 4.35–33.35%, respectively. Obviously, the addition of bran effectively increased the Zn content of flour, and the Zn content of Zn fertilizer flour was significantly higher than that of CK flour (p < 0.05). In addition, the different grain sizes of bran had no significant effect on the Zn content of flour.The Zn content of flour with different addition levels and particle sizes of bran are summarized in p < 0.05). When the amount of bran exceeded 10%, the change in the Zn content of steamed bread in the Zn fertilizer group was no longer obvious. The Zn content of the steamed bread without bran increased by 73% relative to that of the control group. At the 5% bran addition level, the Zn content of the steamed bread in the Zn fertilizer group increased by 55% and that of the control group increased by 29% on average. At the 20% bran addition level, the average Zn content of steamed bread had improved by almost 106% and that of the CK group increased by 131% on average. Therefore, bran addition had an excellent effect on the Zn content of steamed bread. As the bran retains most of the minerals in grains, it seems to favor the use of integral flour for the manufacture of bread and pasta products.The effects of bran addition on the Zn content of steamed bread are shown in As can be seen from p < 0.05). Hardness is usually closely related to product acceptability  of bran dough-based products . The senp < 0.05). However, particle size had no significant effect on pasting properties. The whiteness and L* of the mixed flour gradually decreased with the increment in the particle size and addition amount of bran. The hardness, gumminess, and chewiness of steamed bread showed an upward trend with the increase in bran addition level, whereas springiness and cohesiveness declined. The sensory total scores of steamed bread in the control and Zn fertilizer groups were optimal at the 5% bran addition level. By adding 5% medium bran to wheat flour, the Zn content of the steamed bread in the Zn fertilizer group reached 40.2 mg/kg, whereas that of the steamed bread in the control group was only 25.8 mg/kg. The Zn content of the steamed bran bread was 55.8% higher than that of the control group. This increment indicated that the Zn content of this staple food was enhanced. In conclusion, the results of this study could be useful in the application of wheat bran as an ingredient for enhancing the content Zn of steamed bread. On one hand, we offer a sustainable and low-cost way to provide essential micronutrients (Zn) to people in both developing and developed countries. On the other hand, wheat bran as the main by-product of wheat milling usually has low value but in this way it can be a good source of Zn. The influence of bran on steamed bread structure, nutritional ingredients, and qualities need to be further researched. Meanwhile, further research is needed to minimize the detrimental influence of bran on the quality and sensory acceptance of bran addition products, such as applying physical mode  to modify the functional properties of bran and enhance the consumption of bran dough-based products.As a result of the foliar application of ZnSO4, the Zn content of wheat grains and flour increased by 47.4 and 58.7%, respectively, which met the target levels for Zn biofortification. The addition level and particle size of wheat bran affected the mixing characteristics and pasting properties of flour and thus weakened mixing tolerance. The addition of wheat bran significantly decreased peak viscosity, trough viscosity, final viscosity, and setback value .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


As the environmental pollution issue has recently become significant, environmental regulations in Europe and the United States are being strengthened. Thus, there is a demand for the quality improvement of emission after-treatment systems to satisfy the strengthened environmental regulations. Reducing the amount of welding heat distortion by optimization of the welding order of each part could be a solution for quality improvement since the emission after-treatment system consists of many parts and each assembly is produced by welding individual ones. In this research, a method to derive a welding sequence that effectively minimizes welding deformation was proposed. A two-stage simulation was performed to obtain the optimal welding sequence. In the first stage, the welding sequence was derived by analyzing the number of welding groups in each assembly of a structure. The derived welding sequence was verified by performing a thermal elasto-plastic analysis and comparing it with the experimental results. There are residual stresses and deformation on a welding part due to local heating and cooling that occurs during the welding process. In fusion welding, fusion occurs when a molten part is created locally due to intensive heat input and the surrounding area near the fusion part goes through heating and cooling processes while forming a non-uniform temperature distribution. These residual stresses and deformations cause cracks, shape and dimensional errors when a welded structure is produced. In general, a metal expands and softens at the same time as it is heated, therefore reducing the deformation resistance. At extremely high temperatures, there is almost no resistance, so the stress at high temperatures is not very large. However, significant shrinkage and tensile residual stress are always present in a welding part since the shrinkage stress generated during cooling increases with the temperature drop. This thermal stress creates thermal deformation and the deformation that remains permanently after the welding process is called welding deformation. The welding deformation occurring in a welded structure changes the dimensions of the structure or makes the assembly process after the welding process difficult, so additional after-treatment process such as correction is required.This study focuses on the structure of an emission after-treatment system that can comply with the strengthened environmental regulations. The device can be applied to off-road vehicles such as construction machinery, agricultural machinery, and specialty vehicles and is designed to comply with the Euro 6 emission regulation policy. The Euro 6 for vehicles in Europe, the strictest emission regulation, requires the application of a diesel particle filter (DPF) that filters out particulates in an exhaust gas. The DPF is a type of exhaust gas after-treatment device that physically collects and burns particulate matters in the exhaust gas of a diesel engine. In the United States, the on-board diagnostics (OBD) were enacted for automobile emission control laws in accordance with the regulations for an automobile self-diagnosis device. In Europe and the United States, these regulations have been enacted to reduce the emissions of nitrogen oxides (NOx), carbon monoxide (CO), hydrocarbons (HC), and particulate matter (PM). As the environmental pollution issue has recently become significant, there is a demand for the quality improvement of an emission after-treatment system, which is an assembly of a number of parts such as catalysts and sensor mixers, as well as advanced catalytic technology due to the reinforcement of emission gas regulations such as elevated related regulations, introduction of defect check inspection system, and extension of exhaust gas warranty period, etc. The emission after-treatment system consists of many parts . These parts configure a diesel oxidation catalyst (DOC) assembly, DPF assembly, and outlet assembly and these assemblies configure an emission after-treatment device. Since a catalyst is added into the emission after-treatment device, the catalyst cannot be exchanged later if the assemblies are welded, so the individual assemblies are clamped. Since each assembly is produced by welding individual parts, there is an issue that leakage occurs in a fastening joint during final clamping due to welding heat deformation in each part. To solve this problem takes a lot of time and cost for additional post-processing. Since each assembly consists of 3 to 10 welding parts, accumulated tolerances during welding deteriorates the quality of the assembly. Reducing the amount of welding heat distortion by optimizing the welding order of each part could be a fundamental solution.Numerous numerical methods have been proposed to predict the amount of welding deformation ,2. ThermOn the other hand, the simplified method can shorten the analysis time, but the accuracy is lower than that of thermal elasto-plastic analysis ,17,18,19When optimizing the welding sequence and accurately predicting the final welding deformation, thermal elasto-plastic analysis has a limitation in its application due to the time taken to perform the analysis. Gannon et al.  presenteIn this study, a method to derive a welding sequence that effectively minimizes welding deformation was proposed. The welding sequence was derived by analyzing the number of welding groups in each assembly of a structure. Using the derived welding sequence, thermal elasto-plastic analysis was performed to calculate the amount of welding deformation of an actual structure and it was compared with the experimental results. The proposed method was verified by performing a simulation to derive the sequence of minimizing the welding heat deformation amount of each part for the emission after-treatment system structure and comparing it with a case where the actual optimal process was applied.The muffler structure, the subject of this study, consists of a DOC, DPF, and outlet assembly as shown in The outlet assembly used the same 2.0 mm thickness. For the DPF assembly, a thickness of 2.0 mm and a thickness of 12.0 mm were used. The DOC assembly used two types of welding combination. The first has a thickness of 2.0 mm and a thickness of 1.0 mm. The second has a thickness of 2.0 mm and a thickness of 4.0 mm. All structures are constructed of stainless steel 436.After welding for each assembly, the shape of the bead was measured as shown in SF-436 with a diameter of 1.2 mm was used as a filler material for welding the muffler structure. The welding equipment used in this study was Fronius’ TPS4000, and automatic welding was performed using a six-axis robot. The contact tip to working distance (CTWD) was fixed at 15 mm and the torch angle was fixed at 55°. As a protective gas, 100% argon gas was continuously supplied at 20 L/min. The welding current and welding speed were set to 150 A and 10 mm/s, respectively.In this study, two-stage simulation was performed. In stage 1, the sequence of minimizing the amount of welding deformation for each assembly was derived. In stage 2, based on the sequence derived in stage 1, the thermal elasto-plastic analysis was performed to calculate the amount of welding deformation of a structure. The flow chart to calculate the final welding deformation is shown in Each assembly went through finite element modeling for simulation as shown in The parts are welded to form each assembly structure. In A simulation was performed as follows to identify the effect of each welding part on the welding deformation of a structure. For example, to calculate the effect of Part 6 of the DPF assembly on the welding deformation of a structure, thermal elasto-plastic analysis was performed on Part 6 to calculate the welding deformation on both joints of the DPF assembly after making an FE model that excludes Part 6 of the DPF assembly.As mentioned earlier, the total number of simulation cases to calculate the effect of welding parts of each assembly on welding deformation is as many as the number of corresponding welding parts since the number of welding parts of outlet assembly, DPF assembly, and DOC assembly are 3, 10, and 8, respectively. In other words, the total number of welding cases of DPF was 10!, but the number of simulations was drastically reduced to 10.To perform the thermal elasto-plastic analysis, the temperature-dependent of material properties of stainless steel 436, i.e., the material of a muffler structure, was used . The che2·K and the convective heat transfer coefficient at the surface in contact with a jig was 200 W/m2·K, and the analysis was performed in an environment similar to the actual welding process [This simulation used Ansys Workbench 2021 R1 to implement a welding process environment similar to the actual environment. The finite element model was meshed using three-dimensional solid elements (SOLID186 and SOLID187 in Ansys). The transient thermal elasto-plastic analysis was performed at the actual welding speed of 10 mm/s and the time step size was set to 2 s. A specimen test was performed to calculate the heat source during welding and the heat source in the final welding part was estimated by measuring the temperature data using a thermocouple. In the heat transfer analysis, the natural convection condition was applied to the surface of a structure exposed to the air. The convective heat transfer coefficient under the natural convection condition was 20 W/m process . In addiAfter simulation on the welding parts of each assembly using these conditions, the welding deformation at the joint was calculated and shown in In By considering the process and simulation results, the welding sequence that can minimize the welding deformation at the clamp joint was derived for each assembly. A thermal elasto-plastic analysis was performed for each assembly using the derived welding sequence. Welding deformation at the joint was calculated using the same welding conditions as in From the thermal elasto-plastic analysis, the welding deformation on the fastening surface of each assembly was found to be less than a maximum 0.41 mm.The welding sequence was derived from Surveyor model DS-4060  3D scanner was used to measure the amount of welding deformation at the joint of a welded assembly. The experimental results and elasto-plastic analysis results for the welding deformation of the final muffler structure are summarized in When comparing the experimental and simulation results, these show that the maximum welding deformation error at the joint is 13.89% or less. The allowable deformation of the clamping surface is under 0.5 mm. This is a value that does not cause leakage problems during the airtightness test after the final assembly of the muffler structure.In the case of a structure with many welding parts, it is impossible to calculate all the parts through experiments and simulations because the number of cases for welding sequence increases exponentially. Therefore, in this study, a method to derive a welding sequence that effectively minimizes welding deformation was proposed. First, to derive the sequence of minimizing the welding deformation in each assembly joint, the effect of welding parts on the welding deformation of a structure was calculated, respectively. Based on the calculated results, the sequence was derived so that the part that has the greatest influence on welding deformation was welded last. Since the rigidity of a structure increases when other parts are welded first, it is advantageous in terms of welding deformation of an entire structure when the parts that significantly affect the welding deformation are welded later.In this study, the welding sequence showing the minimum welding deformation at the joint of each assembly was derived for an assembly in which there are 3, 10, and 8 welding parts, respectively. Based on the results, the amount of welding deformation was calculated using thermal elasto-plastic analysis. To verify the simulation results, an experiment was performed using the same process as the simulation and then the welding deformation was measured. Through this process, it was verified that the proposed analysis method is appropriate, and it is expected to be of great help when determining the welding sequence of structures with many weld lines.


RTK KIT regulates a variety of crucial cellular processes via its cytoplasmic domain (CD), which is composed of the tyrosine kinase domain, crowned by the highly flexible domains—the juxtamembrane region, kinase insertion domain, and C-tail, which are key recruitment regions for downstream signalling proteins. To prepare a structural basis for the characterization of the interactions of KIT with its signalling proteins (KIT INTERACTOME), we generated the 3D model of the full-length CD attached to the transmembrane helix. This generic model of KIT in inactive state was studied by molecular dynamics simulation under conditions mimicking the natural environment of KIT. With the accurate atomistic description of the multidomain KIT dynamics, we explained its intrinsic (intra-domain) and extrinsic (inter-domain) disorder and represented the conformational assemble of KIT through free energy landscapes. Strongly coupled movements within each domain and between distant domains of KIT prove the functional interdependence of these regions, described as allosteric regulation, a phenomenon widely observed in many proteins. We suggested that KIT, in its inactive state, encodes all properties of the active protein and its post-transduction events. Receptor tyrosine kinases (RTKs) are cell surface receptors with a highly selective affinity to numerous ligands—growth factors, cytokines, and hormones. Each RTK acts as a sensor for its specific extracellular ligand, whose binding triggers dimerization of the receptor, activation of its kinase function, and auto-phosphorylation of particular tyrosine residues in the cytoplasmic domain . This meEach RTK, depending on the location of its different domains, is composed of an extracellular domain (ED) and cytoplasmic domain (CD), linked by a single transmembrane helix (TM) A. In turThe ligand-induced stimulation of RTKs promotes conformational changes of the ED, governing its dimerization (except IsulinR) and the signal transmission from the extracellular environment to the intracellular area. The conceptually straightforward mechanisms for the ligand-induced dimerization of RTKs are surprisingly different and encoded primarily by the sequence and structure of the extracellular domain ,5. As thThe numerous studies focused on the regions containing tyrosine residues—JMR, A-loop, KID, and C-terminal—put in evidence their roles in different steps of RTK activation. For instance, in several RTKs, FLT3 , KIT 9]9], and tKIT, one of 58 human RTKs, is activated by the binding of a growth factor, the stem cell factor (SCF), and regulates a variety of critical cellular processes, such as proliferation and differentiation, cell survival and metabolism, cell migration, and cell cycle control ,15. The The human KIT is composed of 976 amino acids, in which residues L525–Y545, K546–K581, W582–S931, and T932-R946 represent the TMD, JMR, TK domain, and C-tail, respectively. In response to SCF binding, KIT activates several signalling pathways, using its rich set of phosphorylation sites localised on different fragments, i.e., JMR, A-loop, KID, C-lobe, and C-tail, which are the docking sites (scaffolds) of numerous proteins. In KIT, eight tyrosine phosphorylation sites have been identified in vivo  , as wellAs expected from the critical role of JMR in the regulation of KIT kinase activity, two tyrosine residues, Y568 and Y570, are involved in the maintenance of the auto-inhibited conformation that blocks the regulatory αC-helix and ATP-binding P-loop, thereby suppressing kinase activity ,22. UponThe A-loop Y823 is likely to play a role of a pseudo-substrate, interacting with D792 in C-loop and, thus, maintaining the inactive conformation of KIT. It was reported that phosphorylation of Y823 is not required for KIT activation ; howeverIt is widely accepted that KID does not influence the kinase activity of KIT , and itsConsequently, the different KIT regions regulate the catalytic process and events that activate and control the signalling cascade. As such, KIT functions are dependent on more than one region, and these regions should be directly or collaterally coupled. The study of interconnections between different, distant, or adjacent KIT regions, at the structural and dynamical levels, may shed light on their cooperativity required for different KIT functions.Such study is pertinent because the 3D model of the nearly complete cytoplasmic domain of KIT, in which the empirically  determined kinase domain, completed by the de novo KID and C-tail, was reported . To studWe suggested that such a level of description of KIT, with the fully reconstructed JMR, KID, and C-tail, will elucidate the structural and dynamical properties of its different functional regions that contain (or not) the phosphotyrosine residues. Such characterisation can more explicitly explain the role of each region in maintaining KIT inactive state and establish the relationships between the regions showing either their cooperation or autonomy.The 3D model of the KIT CD possessing KID and C-tail  was compThe root-mean-square deviations (RMSDs), computed for each conformation, respective to the same initial conformation, display comparable profiles in all cMD trajectories, demonstrating the good reproducibility of the generated data . As showThe secondary structure interpretation of KIT conformations indicates that the folding is generally well-conserved in the TK domain and corresponds perfectly to those in the crystallographic structures 1T45 (inactive state) and 1PKG (active state), while JMR, KID, and C-tail changed their folding during and between each trajectory . The int10-helices (I563-N566 and D572-Q575) into turn or bend, as well as the partial instability of two β-strands, are good arguments to classify JMR as an IDR. Similarly, the alteration of α-, 310-helices, bend, turn, and coil in C-tail proves its disordered nature. A-loop also displays a noticeable transformation of its fold, evidenced as a reversible transition of a turn to a 310-helix in three distinct A-loop segments, and a full unfolding of the antiparallel sheet, as observed in trajectory 2 , KID (CKID), C-tail (CC-tail), and the TK domain (CTKD), are the nodes of a dynamical tetrahedron that reflects the displacement of each region, respective to one another  an unsteady position of the flexible KID, JMR, and C-tail, with respect to the relatively stable TK domain, (ii) the intrinsic properties KID, JMR, and C-tail, connected to their unstable (metastable and transient) folding and/or conformational changes. Each KIT region can be regarded, in a first approximation, as a pseudo-rigid body with its local centre of gravity . Centroids, determined on JMR  suggests that the different positions of KID, derived mainly from its turn round (twist), relative to the TK domain and not from a linear displacement. As the αH1-helix is the most structurally stable element of the disordered KID , it can During the first 0.5 µs of simulations, the distance between the tyrosine residues of αH1- and αE-helix varies slightly around their mean values (mv of 25–27 Å), while the bending angle between the αH1- and αE-helix shows quite different angular positions, from parallel to orthogonal . ObviousTKD∙∙∙CJMR) is a very approximate metric to estimate the position of the long and flexible JMR, composed of multiple segments—the JM-Proximal (JM-P), JM-Buried (JM-B), JM-Switch (JM-S), and JM-Zipper (JM-Z)—functionally specific and, in the 3D structure of KIT, are largely distant [TKD) to the selected residues of each JMR segment—Y547 (JM-P), Q556 (JM-B), and Y570 (JM-S)—except for JM-Z, which is adjacent to the TK domain N-lobe, while maintaining its position , while the two ends of A-loop  changed slightly. Displacement of the β-hairpin, with alternating secondary structures and a wide range of A-loop curvature, reveals heterogeneous conformations of A-loop, indicating its partial disorder.The distance between the centroids of TKD and JMR (C distant . To imprposition . The disThe transmembrane (TM) helix conserves its perfect α-helical structure but displays a change in its orientation in space. To find out if pivot spaces were preferred, the distribution of the bending and kink angles  were obtThe geometry of the multidomain cytoplasmic region of KIT can be described as relative to the structurally conserved TK domain, through the bending angles between the representative fragments, taken as hints. The bending angles show the abundant diversity of the TMD orientation, in respect to the TK domain, which is richer for the N-lobe (TM-helix versus αC-helix) than the C-lobe (TM-helix versus αE-helix) B. As wasFinally, the positions of the selected hints , superimposed on the mean conformation of KIT, show their relative dynamical positions during the cMD simulations, reflecting the generic geometry of KIT .We have suggested that the positions of JMR, KID, and C-tail, observed in KIT conformations, are controlled by non-covalent interactions, primary by H-bonds. This hypothesis is based on the analysis of non-covalent contacts stabilising the crystallographic structure of the kinase domain of the inactive KIT , which eFor our analysis, in each MD conformation of KIT, the H-bonds involved in the formation of regular structures (helix or sheet) and those contributing to the intra-domain framework  were excluded. The remaining H-bond contacts represent an H-bonding pattern of multidomain KIT that displays the interaction between distinct domains . In termBy focusing on the functionally significant regions contributing to the activation/deactivation mechanism of KIT, we found that the extended JMR forms multiple and regular H-bonds, involving its different segments interacting with distinct fragments of kinase domain—the JM-Proximal segment  with the antiparallel sheet of A-loop (H-bond K546∙∙∙N828), JM-Buried segment  with αC-helix (H-bond Y553∙∙∙E640), JM-Zipper  with the αC-helix (H-bond P577∙∙∙S645), and JM-Switch  with C- (H-bond W557∙∙∙H790 and bifurcate H-bond H790∙∙∙Q556∙∙∙R791) and A-loop (H-bond Q556∙∙∙F811). Similarly, the catalytic (C-) and activation (A-) loops are tightly coupled by the salt bridge (D792∙∙∙R815) and H-bonds D792∙∙∙Y823. The position of the P-loop in the N-lobe of the kinase domain is maintained by the H-bond P600∙∙∙K626.This extensive H-bond pattern of KIT performs crucial functions—(i) maintaining the JMR in its auto-inhibited position , (ii) reIn particular, the C-tail localised at proximity to KID interacts simultaneously with charged residues of KID and C-lobe, serving as a bridge between these two KIT domains E. For thThe position of the phosphorylation sites in the KIT structure strongly depends on their guest fragments from their conformational features and relative position in KIT. Thus, the large displacement  of KID from the TK domain is reflected in the expanded distributions of the Cα-atoms position and hydroxyl groups of Y721, Y747, and Y730, located on the highly flexible fragments of the disordered KID, while the OH groups of Y703 in the stable αH1-helix form the narrower cluster .Likewise, the compact distribution of the Y553, Y568, and Y570 locations, viewed by the Cα-atoms and the OH groups, reflect the stable position of JMR-B, JM-Z, and JMR-S, while the wide-ranging distribution of the Y547 location corresponds either to the multiple inherent JMR conformations or ample displacement of JM-P, with respect to the TK domain. The three clusters of the unique phosphotyrosine of A-loop, Y823, apparently reflect three different conformations of A-loop.To characterize the inherent structural and conformational properties of the variable KIT regions, i.e., JMR, KID, A-loop, and C-tail, and estimate the contribution of these properties to the structural and dynamical relationships of KIT, each of these fragments was analysed individually. First, the conformations of each fragment were grouped by ensemble-based clustering  using di10-helix in JM-Z, regularly undergoing reversible folding–unfolding events. The representative conformations of the most populated clusters, 1 and 2, differ only in the position of the JM-P segment containing Y547, which was identified in vitro as a phosphotyrosine [The majority of JMR conformations forms the two most populated clusters, C1 (68%) and C2 (31%), composed of the conformations observed in each cMD trajectory . All JMRtyrosine . Such alThe conformations of the intrinsically disordered KID, previously characterised in , were gr10-helix), and differ mainly by the orientation of the C-terminal residues. All clusters regroup conformations generated over the three independent trajectories, and, apparently, C-tail secondary structures do not influence a cluster separation. Indeed, the small transient helix is only observed in trajectories 1 and 3, while the conformations from trajectory 2 are folded as a random coil  and C2 (39%), and two lowly populated clusters, C3 (4%) and C4 (3%). The representative conformations of C1, C2, and C3 show similar secondary structures, described as an extended random coil with a small transient helix in the middle  is composed of conformations containing a transient β-hairpin , forming a map of well-defined blocks, distorted by A-loop. Both TK lobes correlate negatively with the KID and TM helix and positively with JMR. C-tail correlates positively with C-lobe and negatively with N-lobe. The inter-lobe correlations are not homogeneous, and, apparently, P-loop and αC-helix of N-lobe play a specific role in the motion correlations. The motions of TM helix and JMR are contrariwise.Such correlation patterns can be partially explained by the overall architectural features of the studied KIT, which has a strongly extended shape. Movements of one end (TM helix) are counterbalanced by movements of the opposite end (KID) to provide a stable balance of KIT around its centre of gravity. On the other hand, the highly coupled motion in KIT may reflect the allosteric effect(s) and has the functionally related content—regulation function. In both cases, the cross-correlation pattern indicates the strongly coupled movements of the largely distant domains/regions and reflects the block-segmented movement of the multidomain KIT. The similar pattern, even more pronounced and contrasted, was observed on the cross-correlation matrix , calculaC- to N-ends. The amplitude and direction of motion of the TM helix and KID differ in the three trajectories  the superimposed KIT conformations of the three cMD simulations provide a wide coverage (overlap) of PC subspaces  the conformational density is extended in ubspaces C.Since the RTK KIT contains at least four disordered fragments, showing reversible protein folding–unfolding events and a large conformational diversity, its conformational space can be represented more explicitly as a ‘free energy landscape’ model. Such interpretation of the intrinsically disordered multidomain protein leads to quantitatively significant results, allowing the comparison between different states. The relative Gibbs free energy, ΔG, defined on chosen coordinates called ‘reaction coordinates’ or ‘collective variables’, describes the conformations of a protein between two or more states, measured as the probability of finding the system in those states. Such representations of protein sampling, with the use of reaction coordinates, can be the quintessential model system for barrier crossing events in proteins .For the evaluation of the relative free energy (ΔG) and reconstruction of its landscape, the distant measures—radius of gyration Each The unimodal Gaussian distribution of Nevertheless, these FELs, reconstituted on two different sets of reaction coordinates, showed that the ensemble of KIT conformations can recover the two-state picture characterized by two global minima (wells); each of them is composed of similar conformations, which are very distinct between the two minima . The comThe low approximate population of conformations in the two deepest wells on the free energy landscape of KIT  and deep well completed by similar conformations, while the second well, if it still exists, is considerably reduced and contains 4% conformations . The secSurprisingly, the FEL determined on the JMR showed only one highly populated deep well (47%). By comparing this observation with the ensemble-based clustering of JMR , its impThe free energy landscape of KID differs from that reported in  and was The FEL profile of C-tail shows unique low energy deep minimum, which is complemented by two wells with the higher energy values. As C-tail conformations have similar secondary structures and differ mainly in the orientation of the C-terminus. These intrinsic properties do not contribute significantly to the FEL, which is primarily impacted by the alternate location of the C-tail, with respect to KID and/or to TK domain.Remarkably, the multidomain modular RTK KIT consists of a quasi-stable TK domain, crowned by at least four intrinsically (inherently) disordered (ID) domains/regions—JMR, KID, A-loop, and C-tail. These disordered regions contain functional tyrosine residues and act as a ground platform for the recruitment of signalling proteins. The implication of disordered regions in post-translational modifications is well-known ,47,48. NThis paper focuses on the functionally significant domains in KIT signalling and their disorder, described at two levels, such as the intra-domain intrinsic disorder  and extrinsic disorder (inter-domain), observed at the KIT level.Since KIT ID domains have no single, well-defined 3D structure, they cannot adequately be described as simple statistical coiled or helically folded chains equally populating all MD conformations allowed by their backbone torsion angles. Instead, KIT ID domains contain transient, short- and long-length structures, which display various degrees of compaction and elongations. Therefore, each ID domain of KIT is not homogeneous, but represents a very complex mixture of a broad variety of differently folded conformations, ranging from the partially folded to fully unfolded, which, in turn, are foldable. In KIT, these regions are mainly composed of polar and charged residues, while the portion of hydrophobic residues is reduced, presenting an archetypal sequence composition of the intrinsically disordered proteins ,50.The ID regions of KIT belongs to two types—the very elongated (extended) and poorly folded regions , as well as the globule-like (collapsed) KID, with a high level of the helical structures.The high content of unfolded residues (random coil) in JMR and A-loop implies a potentially high degree of disorder in these regions. However, since these regions, in the inactive state of KIT, are attached to the TK domain by numerous H-bonds, which anchor each end of A-loop and bind the JMR along most of its length, their disorder is limited. Only the short segments of these regions, not stabilized by the H-bonds, show the local disorder, which is evidenced by the alternating positions of JM-P on JMR and the unfolding of the β-hinge in A-loop. Since the disordered segment of JMR contains the functional tyrosine Y547, this residue is highly dislocated in 3D space over a wide range and, therefore, is accessible for phosphorylation events, even in inactive KIT or its intermediate state. Y823 in A-loop is also irregularly positioned in space, located in at least in three different positions, but its side chain (hydroxyphenyl) is still oriented towards the active site of KIT.We suggest that, upon activation of KIT, induced by SCF binding, JMR displaced from its packed auto-inhibited position will achieve the most extended and flexible conformation, so that its level of disorder will increase, and, therefore, the level of its adaptability required for scaffolding (docking sites) and recruitment of different protein binding partners will also increase.The position of Y936 and orientation of its phosphorylable sidechain is highly conserved in all conformations of the extended C-tail, regardless of the location of its disordered C-end, either near KID or close to the TK domain. It may well be that these results do not fully reflect the role of the C-end; in our model, it was shortened, with respect to the native length.The higher portion of charged and polar residues in KID, compared to JMR and A-loop, is reflected in the enhanced disorder of KID, which involves almost all residues of the domain, except of a single helix (αH1), which shows stable folding (secondary structures) but varies greatly in its orientation, respective to the TK domain. The structure of the αH1-helix is conserved, not only in the KID fused to the tyrosine kinase domain but also in KID simulated as a cleaved polypeptide .10 helices, as well as between a helix and non-regular structure , whose length and type change. Second, as KID helices are connected by flexible linkers of varying length, depending on the order of the helical folding, this promotes many relative orientations of the helices, leading to a large set of very heterogeneous conformations. These two factors, transient folding and high conformational flexibility, are the main items characterizing the intrinsic intra-domain  disorder of KID. Nevertheless, as we have reported [The KID disorder is manifested first as transient folding (secondary structures) and presented as a collection of highly variable helices, permanently converting between α- and 3reported , the intMoreover, this compact globule-like domain is displaced (linear and angular displacement) as a ‘pseudo-rigid’ body, with respect to the TK domain, such representing the inter-domain extrinsic disorder. This movement is also highly heterogeneous, both in space and time, with a variable contribution of the linear and angular components, manifested in the form of small dislocations and large-scale rearrangements. This all-scale movement can involve distinct segments of KID, again showing the highly anisotropic nature of the intrinsic disordered in this domain.The two-level disorder (intrinsic and extrinsic) provides the high conformational variability and adaptability of JMR, KID, and C-tail required for the scaffolding (docking sites) and recruitment of different protein partners of KIT and facilitates the regulation of cellular processes. The overall structure of KIT represents a continuous spectrum of conformations, with a different degree and depth of disorder, as was reported for the other functional proteins ,51, therClassically, multiple binding events in ID KIT, regulated by phosphorylation, can be characterized as binary on/off switches. However, it has been reported that phosphorylation can generate more complex responses , and mulSuch simplified and flattened energy landscape has shown that KIT is extremely sensitive to different environmental changes  that can alter its free energy landscape in different ways, e.g., lowering some energy barriers, while raising certain energetic minima. This explains the conformational plasticity of ID regions, allowing them to evolve faster than protein domains that adopt defined stable structures, and its ability to interact with several different partners and, therefore, fold in different ways.Since ID domains are multiple in RTK KIT, we asked, does the disorder/order of one domain depend on the disorder/order of other remote regions? In other words, we wish to understand whether the folding–unfolding process of the intrinsically disordered domains in inactive KIT is orchestrated or not. Does the allosteric regulation of KIT involve the disordered domains/regions?Highly coupled motions between distant sites of KIT, as evidenced by the cross-correlation maps, suggests its association with the functional dependence of these regions, which is classified as allosteric regulation, the phenomenon largely observed in many proteins ,3,53,54.The mechanism of regulation of the RTK regions directly involved in cell signalling is still a matter that is little studied, due to the absence of structural data characterizing these regions.To the best of our knowledge, we have presented, for the first time, a model of a full-length cytoplasmic region of an RTK KIT attached to a transmembrane helix and its molecular dynamics simulations, under conditions that mimic the natural environment of the KIT. We demonstrated the tight coupling between the KIT remote regions populated by phosphotyrosines , which serves as a scaffold for the recruitment and activation of signalling proteins.The classical molecular models, the Koshland-Nemethy-Filmer  and MonoThe manifestation of allostery in intrinsically disordered proteins (IDP) is one of the most sophisticated phenomena observed in the last decade ,67,68,69The full-length cytoplasmic domain of KIT in the inactive state. The 3D structure of the inactive RTK KIT (K558-R946) was taken at 2 μs of molecular dynamics (MD) simulation, as reported in [https://www.uniprot.org/uniprot/, accessed on 17 June 2021) using Modeller 10.1 [orted in . Structuorted in . Five thler 10.1  and the ler 10.1  and sterler 10.1 .T544-R946), allowed the latter modelling steps.As the generated N-terminal of KIT was not accessible for its linking to the transmembrane domain, the alternative conformations were calculated by the normal mode analysis (NMA) from the R library bio3D . FifteenThe full-length cytoplasmic domain of KIT in the inactive state linked to the transmembrane helix. The 3D model of the transmembrane α-helix (L521-L543), created with the Builder module of PyMOL 1.9 , and last conformation of KITT544-R946, from the 1-ns MD simulation, were used as templates for construction of the full-length cytoplasmic domain of KIT, in the inactive state, with its transmembrane helix (KITL521-R946).L521-R946 were generated with Modeller 10.1 [Five thousand (5000) models of KITler 10.1  using thler 10.1  and sterler 10.1 .The full-length cytoplasmic domain of KIT with transmembrane helix inserted into membrane. A phosphatidylcholine (POPC) lipid bilayer was generated using Charmm-Gui [L521-R946 was oriented manually, so that its transmembrane helix was positioned perpendicularly to the bilayer with polar residues, next to the phospholipids’ polar heads and apolar residues among their tails. Finally, to reduce the number of residues located in the extracellular area, the N-terminal extremity of KIT was reduced to I516-R946 (KITI516-R946).armm-Gui . As a siarmm-Gui , the oriSystem set-up. Each system, KITT544-R946 and KITI516-R946, wrapped in a 23 Å-width leaflet lipid bilayer of POPC (2), were solvated with TIP3P water model in a rectangular box, with the PACKMOL-Memgen [http://ambermd.org/AmberTools.php; accessed on 17 June 2021), using the ff14SB all-atom force field [T544-R946, in the water solution (1), and KITI516-R946, wrapped in a 23 Å-width leaflet lipid bilayer of POPC (2), contained 71,062 atoms in total, with 6415 atoms of protein and 64,647 atoms of water (1), as well as 172,368 atoms in total with 6869 atoms of protein, 42,074 atoms of the lipid membrane, and 123,423 atoms of water (2).L-Memgen  and LEaPce field  for protMinimisation, equilibration and data generation. The systems were equilibrated using the Sander module of AmberTools20. For system (1), 30,000 minimization steps were performed, i.e., (i) 10,000 on the all-atom fixed protein to relax the water, (ii) 10,000 with fixed Cα atoms to allow the relaxation of sidechains, and (iii) 10,000 without any constraints on the system. For (2), the positional constraints of various forces were applied and subsequently decreased at each minimisation/equilibration step to allow a smooth equilibration. The values were for: all protein atoms—10, 10, 2.5, 1, 0.5, and 0.1; the phosphate atom of POPC—2.5, 2.5, 1, 0.5, and 0.1 kcal/mol; the dihedral angle around the double bond of the oleoyl chain of POPC (restricted to 0°) and dihedral angle formed by the glycerol carbon and the oleoyl ester oxygen atoms of POPC (restricted to 120°)—250, 250, 100, 50, and 25 kcal/mol. The system (2) was minimised during 5000 steps (2500 steps of steepest descent then 2500 steps of conjugated gradient).For both systems, the following steps were executed. A 100 ps thermalisation step was performed, where the temperature (atoms velocity) was gradually increased from 0 to 310 K using the Berendsen thermostat . Then, ahttp://ambermd.org/AmberMD.php; accessed on 17 June 2021) using the PMED Cuda module, running of the supercomputer JEAN ZAY at IDRIS . For system (1), a unique short trajectory of 1 ns and, for system (2), three extended trajectories of 2 µ were performed.All equilibration steps and molecular dynamics simulation were carried out with an integration step of 2 fs. Non-bonded interactions were calculated with the particle mesh Ewald summation (PME), with a cut-off of 10 Å, and bonds involving hydrogen atoms were constrained with SHAKE algorithm . The ini(1)The RMSD and RMSF values were calculated for the Cα-atoms, using the initial model (at t = 0 ns) as a reference. The RMSD, RMSF, and cross-correlations were calculated for the Cα-atoms on the initial conformation (t = 0 μs) as a reference.(2)Secondary structural propensities for all residues were calculated using the define secondary structure of proteins (DSSP) method . The sec(3)Clustering analysis was performed on the productive simulation time of each MD trajectory, using an ensemble-based approach . The alg(4)The H-bonds between donor (D) and acceptor (A) atoms N, O, and S were monitored, according to the following geometrical parameters: d(D—A) ≤ 3.6 Å, (5)The principal components analysis (PCA) modes were calculated for the backbone atoms  after least-square fitting on the average conformation calculated on the concatenated data. The eigenvectors were visualized with NMWiz module for VMD .(6)The normal modes and cross-correlation matrices of the average conformation of each replicate were calculated with the R library bio3D , at 310 (7)1 in P-loop), L637−G648 (αC helix), A701−N705 (αH1 in KID), S771−L783 (αE in C-lobe), and V824−K826 (β9 in A-loop). The kink angle of the TMD helix (P524−C537) was calculated according to [Curvature angles of selected secondary structures (helices or β-strand), relative to others or to their initial position (t = 0 μs), were calculated with Equation (1):(8)The radius of gyration (2) from :(2)Rg=∑i(9)The relative Gibbs free energy of the canonical ensemble was computed as a function of two reaction coordinates with Equation (3) :(3)ΔG =−All standard analyses were performed using the CPPTRAJ 4.25.6 program  of Amberhttps://pymol.org/2/, accessed on 14 September 2020). The VMD 1.9.3 program [Visual inspection of the conformations and figure preparation were performed with PyMOL ( program  was used program , which i