Artocarpus heterophyllus) seed flour. The drying processes of jackfruit seeds were performed at 50, 60 and 70 °C in order to choose the optimal temperature for obtaining the flour based on drying time, polyphenol content and antioxidant capacity. The experimental values of the moisture ratio during jackfruit seed drying at different temperatures were obtained using Page’s equation to establish the drying time for the required moisture between 5 and 7% in the flour. The temperature of 60 °C was considered adequate for obtaining good flour and for performing its characterization. The chemical composition, total dietary fiber, functional properties and antioxidant capacity were then examined in the flour. The seed flour contains carbohydrates (73.87 g/100 g), dietary fiber (31 g/100 g), protein (14 g/100 g) and lipids (1 g/100 g). The lipid profile showed that the flour contained monounsaturated (4 g/100 g) and polyunsaturated (46 g/100 g) fatty acids. Sucrose, glucose, and fructose were found to be the predominant soluble sugars, and non-digestible oligosaccharides like 1-kestose were also found. The total polyphenol content was 2.42 mg of gallic acid/g of the sample; furthermore, the antioxidant capacity obtained by ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) was 901.45 µmol Trolox/100 g and 1607.87 µmol Trolox/100 g, respectively. The obtained flour exhibited good functional properties, such as water and oil absorption capacity, swelling power and emulsifier capacity. Additionally, this flour had a protective and preventive effect which is associated with the potential prebiotic activity in Lactobacillus casei and Bifidobacterium longum. These results demonstrate that jackfruit seed flour has good nutritional value and antioxidant and prebiotic activity, as well as potential protective effects and functional properties, making it an attractive food or ingredient in developing innovative functional products.The general aim of this study was to evaluate physicochemical properties, prebiotic activity and anticancer potential of jackfruit ( Artocarpus heterophyllus) is highly produced and consumed in the southeast of Asia and Brazil [The jackfruit  from 15 jackfruits. The weight of each seeds was obtained, and the seeds were then cleaned and stored in resealable bags at −20 °C until later use and analysis.Because of the variability that exists in seed drying to obtain the flour, drying kinetics studies were performed to control the drying process. Seed samples were stored overnight at 4 °C before starting the experiments. At the start of each experiment, the seeds were cut into eight pieces, including the shell or aryl, with dimensions of 10 × 7 × 5 mm. About 50 g of these samples were spread in a square tray in a single layer. Drying experiments were performed using three replicates at the temperatures of 50, 60 and 70 °C in a forced air oven  with an air velocity of 1 m/s in all the experiments. The weight reduction of the samples during the drying process was recorded every 30 min with an electronic balance . Drying was stopped when consecutive weight measurements gave constant values. Average data are reported in drying curves. When the drying was complete, the samples were ground separately in a food processor (NutriBullet Rx) and then placed in Tyler sieves in order to obtain flour with a particle size of ≤250 µm. The flour was stored in airtight, amber bottles at room temperature until use.The best temperature of drying was selected based on average drying time, polyphenol content and antioxidant capacity.X) was transformed to a dimensionless form referred to as the moisture ratio (X*) and was then related to the time for deriving the drying curves. The drying curves were then analyzed with Page’s model given by the following equation:X (t), X0 and Xe are the moisture content at any time, at t = 0 and t = ∞ respectively; k is the drying constant (min−1), which is a phenomenological property representative of mass and heat transport phenomena; t is the drying time (min); and n is the dimensionless parameter of the Page equation. The goodness of fit was estimated by the coefficient of determination (r2). Moisture content  equipped with a universal attenuated total reflection (ATR) detector was used. The spectrum of the sample and its duplicate were recorded in transmittance mode with a 4 cmperature .r = 0.99).Carbohydrates characterization was performed using the methodology reported by . 20 μL oThe lipids were extracted using the Soxhlet extraction method with analytical grade ether as the reflux solvent for 4 h. At the end of the extraction, the oil was recovered from the mixture by distillation on a rotary evaporator. Sodium sulfate was added to remove moisture from the extract which was stored at 4 °C until use.Fatty acid methyl esters (FAME) were prepared according to the AOAC 969.33 method. A methanol solution was prepared with NaOH (0.1 g) in methanol (5 mL), and the mixture was heated to dissolve the sodium hydroxide. The extract was placed in a test tube (900 mg) after, 0.8 mL of the methanol solution was added and the mixture subjected to a microwave cycle method using a Discover System Model 908005  with the following conditions: 100 °C, 150 W, 290 Psi, with a running time of 3 min and a warm-up time of 10 min, with high agitation. Subsequently, 1 mL of boron trifluoride in methanol was added, and a microwave cycle was applied to the same conditions. Finally, 1 mL of trimethylpentane was added, and again a microwave cycle was applied. The sample obtained had two phases, and the organic phase was used for the injection into the chromatograph.The FAME were analyzed on a 6890N gas chromatograph of an Agilent Technologies model 5973 system, equipped with a ZB-WAX column  and an FID detector. The carrier gas was helium. The temperature of the column was initially 100 °C (for 5 min) and was gradually increased to 240 °C for 4 °C/min. This temperature was maintained for 20 min. The injector temperature was 240 °C, and 1 μL of the organic phase was injected twice. The identification was performed by comparing the retention times of the FAME standards, which consist of a mixture of 37 fatty acid methyl ester.The total polyphenol content was evaluated in the flour obtained from the seeds. For extraction, the sample was allowed to stand for 24 h in the methanol (80%) at 4 °C. The solution was filtered for evaluating the total phenolic content following the Folin-Ciocalteau reagent assay .The antioxidant capacity was measured using two methods. The first was 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical assay following the protocol from Santos-Zea , and theg for 60 min and 3077× g for 25 min, respectively, the measurements were performed at room temperature (25 °C). The supernatant was decanted, and the water and oil absorption were calculated as a percentage [One g of sample was dispersed in 10 mL of deionized water or in 6 mL of soybean oil in a centrifuge tube. After stirring, the sample for water and oil absorption was centrifuged at 4280× rcentage .g for 1 min. The emulsion obtained was centrifuged at 1731× g for 60 min. the measurements were performed at room temperature (25 °C). The emulsifying capacity was calculated as a percentage of the height of emulsified layer/the height of the whole layer in the tube) × 100 [One g of flour was suspended in 20 mL of deionized water, and then 7 mL of soybean oil was added to it. The mixture was emulsified with a T 25 digital ULTRA-TURRAX  at 192× W1) were placed in a tube with 20 mL of distilled water and weighed (W2). The sample was heated in a water bath at 40, 50, 60 and 70 °C for 30 min, then a centrifugation was performed at 2292× g for 10 min. The supernatant was separated and used to determine the solubility, and the tube and its contents were weighed (W3). For solubility, 10 mL of each supernatant (Vs) were taken and placed in a crucible at constant weight (W4). The crucible with the sample was dried in a forced air oven at 105 °C for 12 h [W5) of the sample, these properties were calculated with the following equations:The 500 mg of flour  was used, which is equipped with a parallel stainless steel plates (θ = 60 mm) geometry. The temperature was increased from 30 to 80 °C. A gap of 0.5 mm between the plates was used. The concentrations evaluated were 1, 3 and 5 g/100 mL, and the amount of sample used was 1.5 mL. The viscosity Pa·s was determined as a function of the increase in the shear rate (γ) from 10 to 600 sThe prebiotic extraction was carried out according to a published procedure  with somLactobacillus casei and Bifidubacterium longum was evaluated using a MRS culture medium  with the same composition but replacing the carbohydrates source. Different sources of carbohydrates such as Agave salmiana fructans, commercial inulin and whole wheat flour were used because they are already recognized as prebiotics and we consider it important to compare them with the prebiotic effect of jackfruit flour. The growth of these bacterial was also evaluated in commercial MRS media and MRS without carbohydrates source and were used as control. All carbohydrates source including jackfruit flour were evaluated to 20 g/L of concentration. The broth MRS for B. longum were supplemented with 0.05 g/L of L-cysteine. L. casei was inoculated was 0.06% (v/v) corresponding to approximately 1 × 108 CFU/mL in all culture media (25 mL) and incubated at 37 °C/24 h. The concentration of each inoculum was verified through plate count with MRS agar using the Miles and Misra methods [The prebiotic effect of flour and prebiotic extracted was performed using the methodology  with som methods  the bact2 culture flasks and grown in Dulbecco’s modified Eagle medium  supplemented with 10 % of fetal bovine serum (FBS) (Gibco) and antibiotics . The cells were cultured in a humidified incubator with 5 % CO2 and 95 % air atmosphere at 37 °C. When the cells grew to 80 to 90 % confluence, they were washed with PBS and were trypsinized with 0.5% trypsin-EDTA (Gibco). In addition, the cells were used in subsequent experiments.The human colon epithelial cell lines  CRL1831,  HT29 and  SW480 were transferred to 25-cm5/well with PBS. The PBS was replaced with DMEM supplemented only for the control for the treatments, PBS was replaced with DMEM-supplemented with jackfruit flour at a concentration of 50 µg/mL, 100 µg/mL, 500 µg/mL and 1 mg/mL. The cells were incubated in a humidified incubator with 5% CO2 at 37 °C for 24 h.The HT29, and SW480 cells were seeded in 96-well plates at a density of 1 × 10Viability was assessed with a thiazolyl blue tetrazolium bromide (MTT) assay at 5 mg/mL, at 24, 48 and 72 h, measuring absorbance with a spectrophotometer at a wavelength of 570 nm.g at 4 °C for 5 min. The cell-free supernatants from all the treatments and controls were collected in new sterile tubes. The activities of superoxide dismutase (SOD) and catalase (CAT) enzymes were measured using a colorimetric assay kit according to the manufacturer’s protocol: SOD (EC 1.15.11) Assay kit item no. 19160, CAT (EC 1.11.1.6) Assay kit item no. CAT100 (Sigma-Aldrich), using a plate reader.The CRL1831, HT-29, SW480 colon epithelial cells stored in PBS (100 µL) were homogenized adding proteinase k .All analyses were carried out in triplicate, and the data are given as the mean ± standard deviation. An analysis of variance (ANOVA) and Fisher’ least significant difference (LSD) test were performed at a level of The individual weight of each seed was between 6 and 8 g, and these seeds represent 8.5% of the total weight (0.56 ± 0.3 kg) of jackfruit, which is an attractive percentage for use in flour manufacturing. This value was similar to that reported by  who indip < 0.05) were observed between samples dried at 50, 60 and 70 °C to reach the equilibrium moisture. In 2, n and k (min−1) calculated with Page’s equation are given in 2 values higher than 0.99 was observed. Therefore, Page’s equation is a good model for describing the drying of jackfruit seeds and can be used to calculate the required or desired moisture content of samples at 50, 60 and 70 °C. These results suggest that using any of these temperatures is not required long term to obtain a stable product with a moisture content of less than 10% of the flour. For example, from this figure the time required to obtain a flour with 6.5% of moisture at 60 °C was 8.5 h.The experimental values of the moisture ratio during the drying of jackfruit seeds at different temperatures are shown in The chemical composition values of flour are presented in A high content of total dietary fiber (TDF) was found in the flour (31.5 g/100 g), which suggests that this flour is an ingredient with a high fiber content. The TDF of this flour has not been reported by other authors; only crude fiber was reported (3.19 and 3.72 g/100 g) by , respect−1, which reflects the presence of carbohydrates. This value is in the range of 1189–953 cm−1 reported by [−1 and 1533 cm−1 have been related to amide group characteristics of proteins [−1 the bands correspond to double and/or triple bonds and aromatic amino acids [−1 corresponds to OH stretching vibration, which has been associated with moisture content [orted by . The valno acids , which s content . No signAccording to the HPLC analysis, five types of sugars were identified and quantified in jackfruit seeds flour . The maip > 0.05) in polyphenol content. It was observed, however, that the polyphenol content decreases when the temperature increases. For this reason and for the process time, we considered 60 °C as the average temperature adequate for obtaining the flour. Information on polyphenol content in flour obtained from jackfruit seeds is scarce. The TPC has only been reported in jackfruit seeds  [The total polyphenol content (TPC) and antioxidant activity were determined in the three flours obtained at different temperatures. In extract) , which iextract) , howeverextract) , where 1extract)  also shoextract)  thereforThe antioxidant capacity of ingredients derived from vegetables has been a relevant field in the last years. The antioxidant capacity was determined by DPPH radical and FRAP. DPPH radical is a compound that is capable of generating free radicals and has been widely used to evaluate the ability to capture free radicals in compounds with antioxidant activity. The values of DPPH radical ranged from 1579 to 1617 µmol Trolox/100 g are shown in 3+) to the ferrous (Fe2+) because of their reductive power [p < 0.05) reduced in the flours obtained at 60 and 70 °C, which is correlated with the trend observed in the polyphenol content. The FRAP and DPPH radical scavenging activity assays, however, determine antioxidant capacities through a different mechanism; therefore, these cannot be correlated [The FRAP is a method used to evaluate the ferric reducing capacity by electron transfer. Antioxidant compounds cause the reduction of the ferric  of 34.83 ± 0.001 g/100 mL was obtained in this flour, which is higher than that reported for chia flour . The higp = 0.069) were observed between the evaluated temperatures. A low solubility value may be related to only a slight degradation of starch and leads to having few soluble molecules in the flour [With respect to the water solubility capacity (WSC) and swelling power, he flour . This behe flour . These ahe flour ,8. p = 0.112) were observed, with the values ranging from 4.06 to 5.15%. The increase of swelling power caused by temperature can be explained because more water is retained when the temperature increases, and then the flour begins to swell and volume augments [The swelling power was also directly proportional to the temperature used, and no significant difference (augments . Similaraugments . It was augments . Swellinaugments .The emulsifying capacity (EC) is a molecule’s ability to act as an agent that facilitates the solubilization or dispersion of two immiscible liquids  and it ip < 0.05), the viscosity value to 5% was 0.056 ± 0.0002 Pa·s, while for the concentrations of 1% and 3% the maximum viscosity values reached were 0.0013 ± 0.00001 and 0.011 ± 0.00025 Pa·s, respectively. The increases of viscosity values in dispersions are associated with the higher fiber content, and some components of fiber, such as β-glucans, pectins, hemicelluloses, and cellulose, can retain more water to form a high viscosity suspensions [The dynamic viscosity results of the three flour concentrations are shown in pensions . The othL. casei, significant (p < 0.05) differences were observed, commercial inulin and MRS (control positive) showed the highest bacterial growth: 8.79 ± 0.1 and 8.77 ± 0.1 Log CFU/mL, respectively. The flour from jackfruit seeds and A. salmiana fructans showed the lowest growth: 8.02 ± 0.1 and 8.08 ± 0.2, respectively. Jackfruit seed flour and A. salmiana fructans had a positive prebiotic score (higher than 8 Log CFU/mL), which was similar to that reported in other studies [p < 0.05) better fermented than the flour of jackfruit seeds by L. casei, which could be associated with the fact that in the prebiotic extract the non-digestible carbohydrates are better concentrated. B. longum showed a higher growth (9.07 ± 0.4 Log CFU/mL) in a prebiotic extract of jackfruit seed followed by jackfruit seed flour and whole wheat, where the value was the same: 8.92 ± 0.25 Log CFU/mL. No significant differences were observed, however, between them. Nevertheless, the prebiotic extract was significantly different (p < 0.05) with A. salmiana fructans, commercial inulin, the positive control (MRS COM) and the negative control (MRS WCS). The A. salmiana fructans were better (p < 0.05) fermented by B. longum than L. casei, and these results coincide with [A. salmiana fructans on Bifidobacterium spp. and Lactobacillus spp. in vivo. For commercial inulin, however, the same (p > 0.05) behavior was observed in both bacteria. The growth of B. longum was higher in all tested prebiotics than the controls. The differences observed in bacterial growth can be associated with the ability that these bacteria have for fermenting non digestible carbohydrates. According to our results, we can say that B. longum is capable of degrading prebiotic compounds that are more complex than L. casei because jackfruit flour has a variety of nutrients like dietary fiber, polyphenols, resistant starch, and ketose. Additionally, it has been reported that L. casei preferentially degrades fructans with a low degree of polymerization [One of the main characteristics of prebiotics is the stimulation of microorganism probiotics to improve the health of the gut microbiota . For thi studies ,52. The ide with , where trization . These r2), hydroxyl radical (OH+), singlet oxygen, and hydrogen peroxide (H2O2). These radical species are involved in damage cell in such a way that oxidative stresses can lead to carcinogenesis, inflammatory diseases, cellular senescence, and neurodegenerative diseases, among other pathological processes. In the organism, there is an antioxidant protection system formed by enzymes and low molecular weight compounds. Two of the enzymes involved in the protection and consequently the maintenance of the oxidant/antioxidant balance are catalase (CAT) and superoxide dismutase (SOD).When natural compounds such as jackfruit flour are analyzed to evaluate cytotoxicity in normal colon cancer cells, it is expected to find that they have no cytotoxic effect on cellular stability. In the case of CRL1831 cells, all treatments maintained cellular balance. Statistically significant differences were only found when comparing the control (50 μg/mL and 100 μg/mL) against the 1 mg/mL concentration at 72 h, where a slight increase in absorbance can be observed, which means a greater amount of MTT -2,5-diphenyltetrazolium bromide) was transformed by the cell or greater cell viability A. When tThrough the different treatments with jackfruit flour, it was observed that the control epithelial cells  presented a significant difference when 100 μg/mL, 500 μg/mL, and 1 mg/mL of jackfruit flour was added at 24 h, as well as 100 ·g/mL and 1 mg/mL of the jackfruit flour at 48 h, and finally 500 μg/mL of proteins of jackfruit flour at 72 h A. No sigFor the CRL 1831 cells, in the case of SOD, it was found that the highest concentration of jackfruit flour (1 mg/mL) at the maximum of 72 h showed significant differences when compared with the control and even with each treatment A. In theTumorigenesis, as the hallmark of cancer, consists of a dysregulation of cellular proliferation, leading to uncontrolled cell growth. Therefore in this work we tested the effect of jackfruit flour on cell viability in a control colonic epithelial cell line and in two colonic cancer cell lines. Jackfruit flour did not alter cell viability in a controlled colonic epithelial cell line, either by showing cytotoxicity nor by inducing proliferation. It has been reported that artocarpin, a phenolic compound found in the wood of jackfruit decreased cell viability in five different colon adenocarcinoma cell lines  but not in normal colon fibroblast cells (CCD-18Co). It induced apoptosis and autophagy, inducing G1 phase cell cycle arrest by targeting Akt1 and Akt 2 kinase activity, and reduced tumorigenesis in vivo .On the other hand, the flour decreased cell viability in the HT29 cell line, which is a cancerous cell line, and thus showed anticancer effects. Reference  recentlyViability in the SW480 cell line was very high in basal conditions, which is expected since it is a cell line from advanced cancer stages characterized by Broders’ classification as grade 4, and shows multiple mitosis and high ROS production . JackfruDrying curves of jackfruit seeds were satisfactorily described by mathematical models based on Page’s equation and can be used to calculate the required or desired moisture content of samples at 50, 60 and 70 °C. Jackfruit seeds flour obtained at 60 °C is a nutritional food because of the presence of proteins, monosaccharides, oligosaccharides and polysaccharides, as well as omega-3 and omega-6 fatty acids. This flour also provides bioactive ingredients, such as total dietary fiber, polyphenols and antioxidant properties. Additionally, this flour exhibits good functional properties, such as water and fat absorption capacity, swelling powder and emulsifier capacity. The flour and prebiotic extract of jackfruits seed showed a potential prebiotic effect compared with other prebiotic sources. Moreover, jackfruit flour has a protective and preventive effect in colon cancer cells. Additional research, however, is needed to optimize the application of this flour, to assess its effect as a texture adjuvant in food matrices like meat products, bread, beverages or to develop new food products with functional ingredients that may prevent intestinal diseases.


Z and grain size, which determined the microscopic deformation behaviour. There were found to be two high microscopic strain regions in the α phase: intragranular deformation bands through the most favourable slipping and near the GBs through multiple slipping, which promoted continuous and discontinuous DRX, respectively. With the decrease in parameter Z or grain size, the activated dislocations accommodating GBS were found to no longer pile up inside the grain, but instead travel across the grain interior. Finally, methods to improve the macroscopic plastic formability were proposed for the difficult-to-form titanium alloys experiencing non-superplastic hot deformation.High-efficiency and low-cost hot forming technologies for titanium alloys have been developed for producing complex-shaped, thin-walled tubular components under non-superplastic forming conditions. Under these forming conditions, there exist complex and highly integrated material evolution processes including microscopic heterogeneous deformation, microstructure evolution and damage behaviour. This paper presents an integrated crystal plasticity finite element model of near-α titanium alloys during non-superplastic hot deformation conditions considering grain boundary sliding (GBS), dynamic recrystallisation (DRX), as well as void evolution. The polycrystalline model of a near-α TA15 titanium alloy was established, containing α phase, β phase and grain boundary (GB) regions, in which the GB region was a visualised representation of GBS. The quantitative strength ratio between the GB regions and α phase was calculated according to the Zener–Holloman parameter  Near-α titanium alloys are widely used in the aerospace industry due to their outstanding properties, including their superior specific strength and excellent corrosion resistance . HoweverThe hot microscopic deformation behaviour of titanium alloys can be divided into three aspects: the heterogeneous deformation (stress and strain distribution caused by the non-uniform deformation mechanism), the microstructure evolution  and the damage behaviour (consisting of the void and microcrack evolution). To address the complexity of coupling these three aspects, the crystal plasticity-based simulation framework is an ideal alternative tool for the study of microscopic strain heterogeneity , microstThe common hot deformation mechanisms of titanium alloys include dislocation motion, grain boundary sliding (GBS), twinning and diffusion where multiple mechanisms may coexist. High deformation temperature and high Al content limit the occurrence of twinning , while dThe heterogeneous deformation of grains caused by the non-uniform deformation mechanism determines the microstructure evolution such as texture evolution, dislocation density evolution, dynamic recovery (DRV), dynamic recrystallisation (DRX) and phase transformation. The changes of texture, dislocation density, grain morphology and phase fraction in turn alters the subsequent heterogeneous deformation. The prediction of microstructure evolutions during thermo-mechanical processes through crystal plasticity modelling is still challenging. Li et al. established a 3D coupling model of crystal plasticity and cellular automata (CA), in which the heterogeneous deformation and the non-uniform distribution of the dislocation density were calculated through a crystal plasticity model, and the morphological evolution of DRX was calculated with CA through a semi-probabilistic switch rule. The coupled effect of the heterogeneous deformation, mechanical response and DRX evolution during the isothermal compression in the β-single phase region of a near-α titanium alloy was well-captured . SubsequThe formability of titanium alloys during non-superplastic hot deformation is limited. The damage behaviour is governed by the mechanisms of void nucleation, growth and coalescence, and the voids tend to nucleate on the phase boundaries for the two-phase titanium alloys . The micThis paper aims to develop an integrated crystal plasticity model of near-α titanium alloys during non-superplastic hot deformation, considering the deformation mechanisms (dislocation motion and GBS), microstructure evolution  and void evolution. The coupling effects of microscopic heterogeneous deformation, microstructure evolution and void evolution, as shown in A near-α TA15 titanium alloy rolled sheet with a chemical composition of Ti-6.5Al-2Zr-1Mo-1V (in wt%) and a thickness of 2 mm was utilised in this work. The microstructure of TA15 samples was characterised by EBSD performed on a FEG LEO 1530 SEM with a scan size of 0.3 μm. The inverse pole figure of the undeformed TA15 sheet is depicted in 3, which contained 50 α grains. The β phase was distributed at the GBs of the equiaxed α grains. The initial grain orientations of the polycrystalline model were extracted from the EBSD data of the undeformed TA15 sheet. Fifty randomly selected initial orientations were distributed into the polycrystalline model randomly, which was able to properly characterise the initial texture of the TA15 sheet  orientation, indicated the occurrence of multiple slipping. In order to quantitatively analyse the contribution frequency of each slip system to strain, the corresponding shear strains in the overall model, intragranular deformation bands and deformation bands near the GBs were extracted and normalised as shown in As for the intragranular deformation band, the contribution of prismatic slipping to the deformation increased, and the effect of basal and pyramidal-1 slipping was suppressed. However, as for the deformation bands near the GBs, an opposite pattern appeared. It can be supposed that, due to the constraint of the GBs on the grain rotation resulting from dislocation slipping, the multiple slipping must be activated to meet the higher requirement of deformation compatibility near the GBs.−1–true strain 0.3, the simulated and experimental results of the recrystallised grains and fractions are shown in The initial microstructure of the TA15 sheet was almost entirely recrystallised. It could not be determined whether the recrystallised grains of the tensile specimen were attributed to the initial recrystallised microstructure or the recrystallised microstructure induced by the tensile deformation. Therefore, the recrystallised grains with a size of more than 5 μm in the tensile sample were defined as the initial recrystallizesed microstructure. Under the tensile deformation condition of 800 °C–0.001 s−1–true strain 0.3) are shown in The activated dislocations gradually accumulated near the GBs and formed a recrystallised nucleation. Therefore, the grain rotation resulting from dislocation slipping had a crucial effect on the GBs misorientation and the orientation of the recrystallised grains. The analysis of the GBs misorientation of the recrystallized grains of the TA15 tensile samples . The close agreement between the experimental and simulated results proves the effectiveness of the crystal plasticity model. The cumulative plastic strain energy of the β phase region was significantly greater than the other regions, and the voids nucleated at the phase boundary first.−1). The voids first nucleated at the phase boundary and subsequently elongated along the tensile direction. Simultaneously, when the region around the voids met the void nucleation criterion, the voids grew. Finally, when the grown voids expanded and meet in the β phase region, microcracks that were 45° to the tensile direction were formed.Generally speaking, the local stress concentration led to void nucleation. However, due to the prominent strength difference and compatibility requirement between the β phase and the GB region, the β phase experienced significantly greater strain as shown in microscopic heterogeneous deformation of the TA15 titanium alloy during non-superplastic hot deformation was determined by the strength difference between the constituent structures. According to the constitutive model parameters shown in The According to the kinetic model of dislocation motion and GBS, the simplified strength expressions of the GB region and the α phase of the TA15 samples could be obtained as shown in Equations (20) and (21). The strength ratio of the GB region and the α phase is shown in Equation (22). Due to the low initial dislocation density of the TA15 sheet, the relative dislocation density of the α phase was set to 1 (According to Equation (22) and the model parameters in −1, only when the average grain size was less than 2 μm could the strength of the GB region be equal to or less than the α phase, as shown by point B in When parameter The simulated results of the microscopic strain distribution of the TA15 tensile samples with a true strain of 0.1 with a different strength relationship are shown in When Atomic diffusion and dislocation motion are regarded as the main accommodation mechanisms of GBS during hot metal forming. Hot in-situ tensile and shear deformation tests in the temperature range from 700–900 °C were performed to study the mechanisms responsible for the superplastic effect in Ti-6Al-4V with the similar equiaxed microstructure to that of Z or grain size, the plastic strain contribution from GBS gradually increased. As for the two-phase titanium alloys, regardless of hot deformation condition, the weaker β phase made a greater contribution to the accommodation of the preferential GBS compared to the stronger α phase and, thus, dislocation activities in the β phase occurred from the early stage of deformation. The higher the volume fraction of the β phase, the more crucial the role on the accommodation mechanisms of GBS  30°, [(4)The dislocation motion accommodating GBS fall into two categories: intragranular dislocation activities and that near the GBs. When the strength of the GB regions is greater than that of the α phase, the activated dislocations pile up inside the grain, which promote the continuous DRX. In turn, the dislocations travel across the grain interior. The average thickness of the accommodating regions near the GBs decreases with the decreasing parameter Z or grain size.(5)In order to improve the macroscopic plastic formability of difficult-to-form titanium alloys during non-superplastic hot deformation, the control methods of initial microstructure, for example, refining phase/grain size, introducing substantial deformed structures and isothermal hydrogenation treatment can be performed. In addition, fast heating can suppress the grain growth and martensite transformation, and a loading path with controllable variable–parameter Z can suppress damage behaviour by the stimulative DRX behaviour, which all enhance the formability.In this paper, an integrated crystal plasticity finite element model of near-α titanium alloys during non-superplastic hot deformation was developed, considering the deformation mechanism of dislocation motion and grain boundary sliding (GBS), the microstructure evolution of dislocation density, dynamic recovery (DRV), dynamic recrystallisation (DRX) and void evolution. The following conclusions were reached:


Rosmarinus officinalis and Origanum majoricum) were evaluated in multiple human samples. The basal activity was different depending on the type of tissue, the liver, jejunum and adrenal gland being the ones with the highest amount of superoxide. The antimalarial drugs studied showed specific behavior according to the type of human tissue analyzed, with atovaquone and quinidine producing the highest percentage of superoxide formation, and doxycycline the lowest. In conclusion, the analysis of superoxide production evaluated in cell membranes of a collection of human tissues allowed for the characterization of the safety profile of these antimalarial drugs against toxicity mediated by oxidative stress.The production of reactive oxygen species (ROS) increases considerably in situations of cellular stress, inducing lipid peroxidation and multiple alterations in proteins and nucleic acids. However, sensitivity to oxidative damage varies between organs and tissues depending on the triggering process. Certain drugs used in the treatment of diverse diseases such as malaria have side effects similar to those produced by oxidative damage, although no specific study has been conducted. For this purpose, cell membrane microarrays were developed and the superoxide production evoked by the mitochondrial activity was assayed in the presence of specific inhibitors: rotenone, antimycin A and azide. Once the protocol was set up on cell membrane isolated from rat brain areas, the effect of six antimalarial drugs  and two essential oils ( Tomplex I , which iomplex I ,76,77. Tomplex I , althougRosmarinus officinalis and Origanum majoricum) were evaluated using human CMMA in nine human tissues and organs, including multiple body systems such as the digestive, cardiac, renal and immune systems. The superoxide production evoked by NADH dehydrogenase activity was different depeninding on the tissue analysed, being liver and adrenal gland the ones with the highest basal rate, both of them tissues with abundant mitochondria [Once the protocol for determining superoxide production by rat CMMA was establised, the effects of six antimalarial drugs  and two essential oils (chondria . This tichondria  Moreoverchondria . This feAtovaquone, a naphthoquinone structurally analogous to dUQ, acts in mitochondria by inhibiting complex III of the MRC of Plasmodium falciparum causing alterations in membrane potential . Renal aPlasmodium heme detoxification leading to the parasite’s death [Mefloquine increased superoxide formation only in liver and WBC in absence of dUQ, while in the presence of this electron carrier the superoxide production was also incremented in adrenal gland and spleen. During malaria infection, mefloquine has been shown to induce apoptosis mediated by superoxide formation in the parasite as well as destabilization of the mitochondrial membrane potential, activation of caspases and inhibition of ’s death . The cyt’s death .R. officinallis and O. majoricum, evoked an increase in superoxide formation in several of the tissues tested, being especially higher in white blood cells, where the electron carrier dUQ totally inhibits this effect. This blockade, as with tafenoquine, suggests that dUQ competes with the active compounds of the EOs for binding sites at the site of action of NADH dehydrogenase enzymes. The complex composition of EOs makes it difficult to determine whether compounds with antimalarial properties are also responsible for the formation of tissue-specific ROS or whether it is due to other compounds present in the oils. Further studies should therefore be conducted to identify antimalarial compounds that produce fewer reactive oxygen species. In this way, a safer profile could be achieved, reducing the toxicity and side effects produced by superoxides.Doxycycline was shown to promote low superoxide formation in the nine tissues analysed, being only significant in adrenal gland in combination with dUQ. These modest actions observed on digestive tissues contrast with the numerous gastrointestinal adverse effects described for this antibiotic and antimalarial drug , suggestIn conclusion, the analysis of superoxide production determined in human tissue CMMA can provide relevant data on the oxidative stress generated in specific human tissues by antimalarial drugs. Thus, this technology could be a suitable tool to complement toxicity studies by assessing the vulnerability of a human tissue to a compound by quantifying oxidative stress in the preclinical phases of drug discovery.


We use cysteine-crosslinking and reporter-gene assays to simultaneously and independently probe the signaling state of PhoQ’s sensor and autokinase domains in a set of over 30 mutants. Strikingly, conservative single-site mutations distant from the sensor or catalytic site strongly influence PhoQ’s ligand-sensitivity as well as the magnitude and direction of the signal. Data from 35 mutants are explained by a semi-empirical three-domain model in which the sensor, intervening HAMP, and catalytic domains can adopt kinase-promoting or inhibiting conformations that are in allosteric communication. The catalytic and sensor domains intrinsically favor a constitutively ‘kinase-on’ conformation, while the HAMP domain favors the ‘off’ state; when coupled, they create a bistable system responsive to physiological concentrations of Mg2+. Mutations alter signaling by locally modulating domain intrinsic equilibrium constants and interdomain couplings. Our model suggests signals transmit via interdomain allostery rather than propagation of a single concerted conformational change, explaining the diversity of signaling structural transitions observed in individual HK domains.Transmembrane signaling proteins couple extracytosolic sensors to cytosolic effectors. Here, we examine how binding of Mg Two-component system sensor Histidine Kinases (HKs) are conserved signaling modules in bacteria responsible for sensing a myriad of environmental stimuli and orchestrating transcriptional responses along with their cognate transcription factors  . These sThis question is especially perplexing in light of the various modular architectures of HKs, involving the insertion of one or more signal transduction domains between sensors and the conserved autokinase domain. It is abundantly clear that the same conserved autokinase domain that defines this protein class can be regulated by a myriad of structural inputs, ranging from short alpha-helical dimeric coiled coils, to well-folded tertiary folds such as HAMP, PAS, and GAF domains . Moreove2+, it is hypothesized that in the absence of such cations, the electrostatic repulsion between an acidic patch in the sensor domain and the negatively charged bacterial inner membrane enforces the ‘kinase-on’ conformation of the sensor and results in high-kinase/ low-phosphatase activity in the autokinase domain. In the presence of divalent cations, the electrostatic interaction between the sensor and inner-membrane are bridged resulting in a different ‘kinase-off’ sensor conformation that corresponds to low-kinase/ high-phosphatase autokinase function  signal strength at the limiting low [Mg2+], and (3) the midpoint of the [Mg2+] dependent transition – over the physiologically relevant concentration ranges that E. coli encounters (0.1–10 mM). We further evaluate the intrinsic signaling equilibria of the sensor and autokinase domains by disrupting the allosteric coupling between them using poly-glycine insertions in the signal transduction pathway and show that both domains are highly biased to the ‘kinase-on’ state when uncoupled from each other. The intervening HAMP domain serves as a negative allosteric modulator of both these domains and balances the stability of the ‘kinase-on’ and ‘kinase-off’ states so that they can become responsive to physiological concentrations of Mg2+. With these concepts in mind, we establish, fit and evaluate a semi-empirical 3-domain allosteric coupling model to account for the sensor-autokinase coupling and high/low asymptote and midpoint of transition behaviors of 35 distinct point-mutations and poly-glycine insertions, and highlight the advantages of inserted signal transduction domains in robustly modulating the signaling behavior of HKs.Using this approach, we evaluate the extent to which the sensor’s conformational state couples to and dictates the conformational activity of the autokinase domain for a set of over 30 mutations, representing substitutions throughout the signal transduction pathway from the sensor to the autokinase domain. We show how these mutations can modulate the three basic characteristics of a PhoQ signaling response which need to fit the biological role of the HK (1) signal strength at limiting high , and the low cross-linking sensor state corresponding to low autokinase activity at high [Mg2+] (2+] even though the sensor remains in a high-crosslinking ‘kinase-on’ state (e.g. L254A). Similarly, some mutants retained high kinase-activity in the autokinase despite the fact that crosslinking in the sensor showed WT-like crosslinking in a [Mg2+]-dependent manner . Finally, some mutants produced higher levels of kinase activity at low-Mg2+ than WT PhoQ (e.g. E232A is more active than WT PhoQ at low [Mg2+]). Therefore, mutations along the signal transduction pathway have profound effects in altering or uncoupling sensor-autokinase correlation.Illustrative data in h [Mg2+] . Some mu7) to interrupt the helical connections that are required for coupling between PhoQ’s domains. Gly7 insertions were introduced just before the HAMP domain (Gly7 –219/220) as the TM helix exits the membrane and just after the HAMP signal transduction domain within a short helical connection to the autokinase domain (Gly7 –260/261).We examined the intrinsic activities of the sensor and autokinase domains when decoupled from the HAMP domain, by inserting a stretch of seven helix-disrupting glycines , the sensor behaved normally, being efficiently crosslinked in a [Mg2+]-dependent manner , but does not consider similar hybrid activation states in the autokinase.In the following sections, we consider thermodynamic two-state allosteric signaling models of increasing complexity to understand the coupling of the sensor to the autokinase of WT PhoQ and our set of point-mutants. In these models, we assume that Mgtivation , we are tivation . We alsoractions . Therefo2+] ([Mg2+]>> Kd). At low [Mg2+], the activity of PhoQ is determined by the partition of PhoQ into the low and high affinity Mg2+ bound states according to the ratio of the two dissociation constants, KdOFF versus KdON. The midpoint of transition is dictated by the relative magnitudes of K, which reflects PhoQ’s preference for the ‘kinase-on’ vs. ‘kinase-off’ state, as well as the two Kds, as shown in 2+], demonstrating that even at the low-[Mg2+] conditions in which the sensor is fully in the crosslinked ‘kinase-on’ state, there remains a significant fraction of the WT autokinase that remains in the ‘kinase-off’ state. Therefore, this fully concerted signaling model is insufficient to describe the full range of activities of PhoQ variants.The simplest model for signaling in HKs is one in which the entire HK exists as one concerted domain in a two-state equilibrium of ‘kinase-on’ and ‘kinase-off’ states (equilibrium constant = K) which is then modulated by ligand binding . In suchThe next model we considered is one in which two domains, sensor and autokinase exhibit two-state equilibria and communicate allosterically. A ligand-dependent sensor can be allosterically coupled to an autokinase domain with a tunable coupling strength to allow for the desired degree of communication between the sensor and the autokinase. In such a scheme, the sensor would be a ligand-binding domain with all the properties previously described for a fully concerted HK. The autokinase on its own would have a constant activity level based on its own intrinsic ‘kinase-off’ to ‘kinase-on’ equilibrium. The sensor is then connected to the autokinase in a manner that biases the intrinsic autokinase equilibrium differently depending on which signaling state the sensor is in. A ligand-dependent allosterically modulated HK results from such a coupling, so long as sensor ‘kinase-on’ and ‘kinase-off’ states of the sensor alter the autokinase equilibrium differently .Sen is the ‘intrinsic’ equilibrium of the sensor domain when connected to an autokinase in the ‘kinase-off’ state, and KAK is the ‘intrinsic’ equilibrium of the autokinase domain when connected to the sensor in the ‘kinase-off’ state. When coupled to the ‘kinase-on’ state of either domain, KSen and KAK are scaled by a new factor, ⍺. 2+ dose-response curves. When ⍺ = 1, the two domains are fully uncoupled, and the binding of Mg2+ to the sensor is unable to affect the autokinase domain  with equilibrium constants as shown in e domain . A valuee domain . If 0<⍺ e domain  and the 2+], the low-crosslinking ‘kinase-off’ state of the sensor becomes dominant, and the corresponding activity of the autokinase will be dictated by the autokinase equilibrium when coupled to this ‘kinase-off’ state, KAK. At low [Mg2+], the high-crosslinking ‘kinase-on’ state of the sensor will be dominant, and the corresponding activity of the autokinase will be dictated by ⍺*KAK. The midpoint of transition will depend on the relative magnitudes of all the parameters. However, the range of behaviors possible by this model of coupling depends heavily on the intrinsic equilibria of the sensor and autokinase themselves . We observed that the two-domain model in Coupling provides a robust mechanism for setting both the upper and lower activity asymptotes of the WT sensor kinase. At high enough  dependent state transition of the sensor through coupling via ⍺1 without altering the basal autokinase activity, as shown in 1 = 1, the sensor is decoupled from the HAMP+ autokinase, and the HAMP domain can modulate the basal (and ligand-insensitive) activity of the autokinase through coupling via ⍺2, as shown in 1, ⍺2 ≠ 1), we can potentiate the ‘kinase-on’ or ‘kinase-off’ states of the sensor and autokinase in a manner that depends on both KHAMP and ⍺n’s, as shown in 1, ⍺2 <1, which enables the simultaneous potentiation of the ‘kinase-off’ state, while maintaining the correlated sensor-autokinase behavior of PhoQ. This matches our observation that the ‘kinase-off’ states of both the sensor and autokinase were potentiated by the HAMP domain in our Gly7 insertion experiments , as well as between the HAMP and autokinase in two locations  to disrupt interdomain coupling. We chose to insert Gly7 both before and after the S-Helix motif (res 261–270) since this entire region is considered a coupling motif between the HAMP and Autokinase. We also included Gly4 insertion at 260/261 which suffices as an alternate insertion for decoupling HAMP from autokinase. Finally, we combined Gly7 insertions with some point mutations that show behavior markedly different from WT  to further differentiate between changes in domain two-state equilibria and changes in interdomain allosteric coupling.In order to fit our semi-empirical models to experimental observations, we generated a set of 35 single-point mutants and GlySen, KHAMP, KAK, ⍺1, ⍺2), and the dissociation constants for Mg2+ to the two sensor states  by the global average activity in our dataset. This normalizes the range of autokinase activity to one that is similar to crosslinking fractions (range 0–1) and gives both types of experimental measurements similar weights in our global fits. We give additional weight to data with experimental replicates (and hence greater certainty) by simply treating each replicate as an independent data set, with all the variables held constant between replicates during fit. Each parameter is allowed to sample a 10-log range of possible values, and the best fit is determined by minimizing the sum of residuals across the entire dataset. In order to avoid getting trapped in any local minima of the parameter space, we repeat the fit 125,000 times using randomly generated starting values for each parameter and determine confidence intervals for our parameters using bootstrapping to generate over 3000 synthetic dataset fits . Where mutations or insertions have been introduced, we allow the parameters expected to be affected by the mutation to vary locally for the corresponding data set. Moreover, six mutants can be fit with fewer local parameters than were utilized in the fit, as the values for some of these locally fit parameters remain close to the globally fit value (within 10%), as highlighted in dOFF, KdON, KSen, KHAMP and ⍺1 show excellent convergence to the ‘best fit’ value, with well-defined minima in the sum of residuals as we explore parameter value. Three parameters, KAK, S and ⍺2 show strong signs of covariability, and wider confidence intervals. In the fully activated state, the observed signal is defined by the product of the scaling factor, S, and the fraction of the protein in the ‘kinase-on’ state (approximately S*KAK). This product is well-defined and converges to a value of ≈ 1.02. However, as KAK is lowered below this value, S increases in parallel to maintain a constant value for the product of S*KAK. In AK are also restrained, and vice versa. Nevertheless, we can place a functionally meaningful upper limit on KAK, of approximately 0.1. Similarly, we can place an upper limit on the value of 0.1 for ⍺2, which represents the negatively cooperative coupling of KAK to the parameters defining the other domains. ⍺2 describes the difficulty of the autokinase to transition into the ‘on-state’ when coupled to the HAMP2 state vs the HAMP1 signaling state. Our data show that this transition is disfavored. 2 values centered around 10–2 and 10–7, we converge to similar parameter fits for the other global parameters since both values of ⍺2 establish a tightly coupled ‘kinase-off’ state within the sensitivity of our experiments. Therefore, these uncertainties do not affect any of our conclusions below, which depend on presence of strong versus weak and negative versus positive coupling. We also examined the ability to fit a simpler two-domain allosteric coupling model (7 insertions (We are able to obtain a remarkably good fit for our entire dataset with the aforementioned considerations. ng model  to our dsertions .AK was unfavorable towards forming the ‘kinase-on’ versus ‘kinase-off’ states (KAK <1), even at limiting low concentrations of Mg2+. This indicates that the observed activity for the WT protein is less than what is observed for some of the mutants, and what might be observed in a hypothetical state in which the autokinase is unfettered by connections to HAMP and the membrane. Although unexpected, this finding is consistent with a large body of data  = 25. The HAMP1 state is weakly coupled to the autokinase, which allows the autokinase to sample both kinase-off and kinase-on state, with an effective equilibrium of S.KAK = 1.02. This allows for the partial decoupling of the sensor and the autokinase at low Mg2+ concentrations observed in wild type PhoQ , potentially owing to our choices of parameters to locally float for these mutants (-E) as discussed in Materials and methods. Significantly better fits were obtained by altering the parameters varied for these mutants from KHAMP and α2 to KAK and α2. Thus, these residues may be involved in the underlying equilibrium of the autokinase domain itself due to their proximity to the ‘S-helix’ that connects the HAMP domain to the autokinase. Finally, double mutants are not fit well, especially when the two sites of mutation are in close proximity . This is likely because the thermodynamic effects of double mutations are often non-additive in structurally and sequentially proximal positions that interact directly. Additionally, while we observe relatively invariant expression of almost all variants , some variants, particularly double mutants required slight induction of expression with 10 µM IPTG for observable levels of membrane-inserted PhoQ by western-blotting . In summary, the crosslinking and transcriptional activity data are very well fit for the entire set of mutants, except for a fraction of the nonfunctional mutants in which Mg2+ binding and transcription were significantly decoupled. Even for these mutants, however, there is a qualitative fit to the data, and possible reasons for the deviation.We obtained fits within experimental error for the /Kd)2 term, likely to a minor extent.In a similar vein, for the final models of Figure 6,7, why is Mg binding only considered to selected states ? And in Figure 6A what does AK "on/off" signify?Line 549 – Discussion of the setpoint of the autokinase domain depends on the "reference point" given that KAK and alpha2 are correlated parameters. For example, one could view the intrinsic activity of the autokinase as being the fully uncoupled state, with KAK defined closed to 1.0 and alpha2 having a smaller value that currently modeled in the case of the Y60C (WT) protein. Could one fix KSen and KAK at the values for the Gly-decoupled systems and allow the shifts in equilibrium owing to HAMP coupling to be compensated solely for by alpha1 and alpha2? This framing might be more straightforward for understanding the HAMP coupling. Although the reference position is largely arbitrary and in any given fitting scheme likely depends on the choice of constraining and fixing parameters, it does alter how one views the role of kinase-activating mutations. i.e. with the fully decoupled state as the reference, the HAMP is always deactivating, with different variants (including the WT) deactivated to varying extents. Some additional comments on this issue may help readers understand the range of kinase behavior and how it is influenced by HAMP.Related to the previous point, in Figure 7 the alpha2 parameter seems to have a large amount of uncertainty, and appears biphasic in the fits, this behavior deserves a comment as to its impact in the model. How much would the interpretations change if alpha2 is considered to hold its extreme values?p. 30 line 587 – It's unclear what is meant by the statement that the HAMP domain "serves to tune the ligand-sensitivity amplitude of the response" (p. 30 line 587). In this model, the HAMP domain does alter the sensitivity of the sensor domain by favoring the sensor OFF state (even though it does not directly modulate KdOFF), but what is meant by "sensitivity amplitude".Has the MD model referenced in Figure 2 been published previously? If not, some information on its production should be provided.Representative data for the cross-linking and western blotting should be shown in the supplemental.eLife. Your revised article has been evaluated by Volker Dötsch (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Allosteric mechanism of signal transduction in the two-component system histidine kinase PhoQ" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed – see the comments from Reviewer 2 and 3 below.Reviewer #1 (Recommendations for the authors):The authors answered satisfactorily my commentaries and concerns. The way in which the authors wrote the revised manuscript is much more ordered and comprehensive. This work represents an interesting attempt to explain the mechanism of signal transduction by sensor kinases in two-component systems.Reviewer #2 (Recommendations for the authors):The authors have in large part addressed the concerns I had expressed about the initial version of this submission. Most importantly, these include a major reorganization of the manuscript by presenting experimental data before discussing models that could explain the data. This reorganization has resulted in an improved manuscript. However, the reorganized manuscript includes text that is now unnecessary, given the new order of presentation. Eliminating such text would shorten the paper and make it easier for an interested reader to follow the essential line of reasoning. The authors should critically edit a clean copy of the revised text for brevity with the aim of eliminating unnecessary repetition. For instance, there is no need for the text in lines 127-143 since similar statements are made in the Introduction, lines 121-125 or in the section that begins on line 183. A second example is lines 570-584 where there is no need to describe again the mutations that have already been explained in previous sections .Reviewer #3 (Recommendations for the authors):The authors have done a good job of responding to my comments. The new discussion in lines 254-267 regarding the Mg binding (Lines 423-438 in the edited version) is very helpful. I do, however, still think that it should be explicitly stated somewhere that there are no hybrid subunit states (one subunit on and one subunit off) considered in the model. The data may indeed not distinguish fully cooperative from non-cooperative behavior in terms of subunit activation, but the readers should realize that the subunits are considered independent for Mg binding but not so for activation. Essential revisions:This paper, and the proposed model of transmembrane signaling mediated by allosteric coupling, was enthusiastically received by the reviewers and editor. Although generally positive, the reviewers raised a number of concerns (see below) that should be addressed in a revision. In particular, the authors should:1) Revise the writing substantially to make the paper more accessible to non-enzymologists. The last paragraph of Reviewer #2's public review provides a useful roadmap to a more accessible manuscript.Per Reviewer #2’s suggestion, we have made the following changes to bring forward and emphasize the experimentally generated results and consolidate modeling work in the latter half of the manuscript. We have also added several clarifications to make text more accessible to non-enzymologists.1. New Figure 2 now contains an expanded set of experimental data  to provide motivation for the subsequent mathematical analysis.2. New Figure 2—figure supplement 1 provides examples of western blots and crosslinking quantifications.7 insertions in PhoQ forward to Figure 3 along with the associated Results section.3. We have moved the former Figure 4 on Gly7 insertions in other HKs forward to Figure 4 along with the associated Results section.4. We have moved the former Figure 5 on Gly7 insertion choices.5. We have moved Figure 7A into figure 7—figure supplement 1 and expanded on the justification of mutations and Gly6. We have consolidated the concerted (1-domain) signaling model from Figure 2  and the 2-domain allosteric model from the former Figure 3 into a new figure, Figure 5. Certain elements of this figure have been migrated to Figure 5—figure supplement 1 to aid in visual clarity. The Results sections for these parts have been consolidated to lines 250-3552) Revise their model based on the feedback of Reviewer #3 about how Mg binding is handled.2+ binding is handled in the model (Lines 254-267). We discuss the factors that are built into the “observed” Kd that is reported in the manuscript, as a result of the formulation of our model.We have elaborated on how MgReviewer #1 (Recommendations for the authors):The model in which is suggested that PhoQ transmit signals via interdomain allostery is statistically justified? The other possible models do not fit statistically?We have added Figure 7—figure supplement 4 showing poor fitting results for the simpler 2-domain model. We have made additional comments in Results  and Methods (lines 831-832) on this subject, and supplied the full list of fit values for the 2-domain 2-state model as “Figure 7—figure supplement 4 Source data file 1”.It is not clear to me why in Figure 2A and 7A is necessary a MD model to indicate the different domains of the protein. It seems that will be sufficient a sketch of the different domains of the protein.7 insertions using this MD model. We have provided a paragraph in the Methods section detailing the generation of the model (lines 878-894).We have now place nine selected mutants in diagrammatic form in Figure 2B, as suggested by the reviewer, but it became too busy when all were included. For the full set of mutants we felt it useful to present mutation locations on an MD model particularly to show buried vs. solvent accessible positions. Furthermore, this representation better illustrates the tertiary context of mutations at the HAMP/autokinase junction. We have migrated Figure 7A to Figure 7—figure supplement 1 and highlighted the justification for our choice in mutations and GlyReviewer #2 (Recommendations for the authors):1. How were the positions of the mutational substitutions chosen and how are they related to the protein structure? Except for the introduction of Trp at membrane boundaries and noting for other substitutions their general positions in the three-dimensional structure in Figure 7A and that they were "expected to be on the interior of the protein", no information is provided. More information would help an interested reader understand the logic of mutational placement.7 insertions. The choice of single point mutations is further discussed in lines 136-146, 189-194, 395-407. We also provide additional references (Refs 44-54).We have moved Figure 7A to Figure 7—figure supplement 1 and expanded on it to include rationale for choice of mutations and Gly2. Why were so few substitutions placed in the sensor domain since there were many possibilities away from the Mg2+-binding site? It seems as if they would have been informative.2+) binding remains the most robust way of altering the sensor domain equilibrium to assess its coupling to the rest of PhoQ. We incorporate some sensor mutants near the dimeric interface to show how the sensor’s intrinsic equilibrium can be altered by mutation; however, more sensor mutations would not add information content to examining the allosteric coupling to HAMP and autokinase beyond what is achieved by ligand binding. This rationale is discussed in lines 136-139.In this work, we primarily try to understand how changes in the sensor domain are transduced across the HAMP domain to alter the activity of the autokinase domain. Ligand  as discussed in Results (lines 593-599) and Methods (lines 874-875).4. What about the role of the transmembrane domain? In the manuscript, this region is considered part of the sensor domain. What is the basis for this consideration? Could it be a distinct domain with its own equilibrium between on and off? This issue should be discussed in the text.We note that our allosteric coupling model can be extended to include more modular domains . However, such granularity rapidly inflates the number of parameters needed to describe the corresponding model (number of states = # of domains ^ # of states). Given the already established structural coupling of ligand-binding to TM geometry in phoQ ), we opted to treat it as part of the same ligand-sensing module (i.e. ‘sensor’) as discussed in Results (lines 560-563)5. The mutant Gly7 270/271 is shown in Figure 7A, listed in Table 2 alone and in combination with single-site substitutions, and cited on line 522 but is not otherwise discussed in the text. It should be.7 270/271 mutant is now discussed in Results (lines 399-403).The GlyReviewer #3 (Recommendations for the authors):This manuscript describes a comprehensive study of kinase activation and allosteric coupling in the sensor histidine kinase (SHKs) PhoQ. Quantitative assays for sensor domain activation and kinase response are used to evaluate a large number of variant proteins that display a range of properties with respect to ligand binding, interdomain coupling and kinase activity. The data is used to construct and fit a conceptually elegant model that provides a thermodynamic explanation for domain interactions, allostery and sensing responses in SHKs. The experiments also demonstrate that sensor kinase domains intrinsically favor their "on" states and that HAMP domains act to deactivate both the sensor and the kinase units. In all it is a very impressive study that sets the bar for enzymatic approaches aimed at understanding signaling by multidomain transmembrane kinases. Generality of key principles are explored by examining several SHKs related to PhoQ. The paper is well written and the complex data and their interpretation are for the most part clearly discussed. That said, there are some issues the authors should address:The model applied for Mg binding should be described to a greater extent. The equations of Figures 1, 2,3,6,7 represent a situation more complex than the accompanying schematics portray. Even the simplest equation of 1B implies sequential binding of 2 Mg ions to one PhoQ dimer (presumably 1 site per subunit). Furthermore, the binding sites are assumed to be independent and, importantly, there are no intermediate states in the model in which one subunit is "on" (in either its sensor or kinase domain) and the other is "off". Is it known experimentally that the two subunits act independently and what is the consequence of not allowing for hybrid activation states within the dimer?Addressed in response to public review #3In addition, there may be a factor of 2 missing in treating the relative dissociation constants for Mg binding to an empty PhoQ or to a singly Mg-occupied PhoQ. Because the multiplicity changes by a factor of 2 in going from both the empty to the half-occupied state and again by 2 in going from the half-occupied to the fully occupied states, the effective Kd for binding to the singly occupied state is 4x larger than for binding to the empty state. It appears that all of the models accommodate only a factor of 2. This issue affects the (1 + [Mg]/Kd)2 term, likely to a minor extent.Addressed in response to public review #3In a similar vein, for the final models of Figure 6,7, why is Mg binding only considered to selected states ? And in Figure 6A what does AK"on/off" signify?Addressed in response to public review #3Line 549 – Discussion of the setpoint of the autokinase domain depends on the "reference point" given that KAK and alpha2 are correlated parameters. For example, one could view the intrinsic activity of the autokinase as being the fully uncoupled state, with KAK defined closed to 1.0 and alpha2 having a smaller value that currently modeled in the case of the Y60C (WT) protein. Could one fix KSen and KAK at the values for the Gly-decoupled systems and allow the shifts in equilibrium owing to HAMP coupling to be compensated solely for by alpha1 and alpha2? This framing might be more straightforward for understanding the HAMP coupling. Although the reference position is largely arbitrary and in any given fitting scheme likely depends on the choice of constraining and fixing parameters, it does alter how one views the role of kinase-activating mutations. i.e. with the fully decoupled state as the reference, the HAMP is always deactivating, with different variants (including the WT) deactivated to varying extents. Some additional comments on this issue may help readers understand the range of kinase behavior and how it is influenced by HAMP.Addressed in response to public review #3Related to the previous point, in Figure 7 the alpha2 parameter seems to have a large amount of uncertainty, and appears biphasic in the fits, this behavior deserves a comment as to its impact in the model. How much would the interpretations change if alpha2 is considered to hold its extreme values?Addressed in response to public review #3p. 30 line 587 – It's unclear what is meant by the statement that the HAMP domain "serves to tune the ligand-sensitivity amplitude of the response" (p. 30 line 587). In this model, the HAMP domain does alter the sensitivity of the sensor domain by favoring the sensor OFF state (even though it does not directly modulate KdOFF), but what is meant by "sensitivity amplitude".Addressed in response to public review #3Has the MD model referenced in Figure 2 been published previously? If not, some information on its production should be provided.A paragraph has been added to Methods section detailing generation of MD model (lines 878-894).Representative data for the cross-linking and western blotting should be shown in the supplemental.Figure 2—figure supplement 1 has been added with representative western blot and crosslinking quantifications.Reviewer #2 (Recommendations for the authors):The authors have in large part addressed the concerns I had expressed about the initial version of this submission. Most importantly, these include a major reorganization of the manuscript by presenting experimental data before discussing models that could explain the data. This reorganization has resulted in an improved manuscript. However, the reorganized manuscript includes text that is now unnecessary, given the new order of presentation. Eliminating such text would shorten the paper and make it easier for an interested reader to follow the essential line of reasoning. The authors should critically edit a clean copy of the revised text for brevity with the aim of eliminating unnecessary repetition. For instance, there is no need for the text in lines 127-143 since similar statements are made in the Introduction, lines 121-125 or in the section that begins on line 183.We have truncated the repeated statements in lines 127-143 of Results.A second example is lines 570-584 where there is no need to describe again the mutations that have already been explained in previous sections .7 260/261 is first introduced on line 184.We have removed redundant descriptions of mutations. GlyReviewer #3 (Recommendations for the authors):The authors have done a good job of responding to my comments. The new discussion in lines 254-267 regarding the Mg binding (Lines 423-438 in the edited version) is very helpful. I do, however, still think that it should be explicitly stated somewhere that there are no hybrid subunit states (one subunit on and one subunit off) considered in the model. The data may indeed not distinguish fully cooperative from non-cooperative behavior in terms of subunit activation, but the readers should realize that the subunits are considered independent for Mg binding but not so for activation.We have altered lines 255-258 to state this explicitly.