Κ) values of the emulsion samples were found to be 4.01–26.05 Pasn and were significantly affected by optimization parameters (p < 0.05). The optimum formulation was determined as 0.29% XG, 2.5% CSOB, 2.5% fat. The low-fat (LF-IC) and full-fat control samples (FF-IC) were compared to samples produced with an optimum formulation (CBLF-IC) based on the steady shear, frequency sweep, and 3-ITT  rheological properties, thermal properties, emulsion stability, light microscope images, and sensory quality. CBLF-IC showed similar rheological behavior to FF-IC. The mix of CBLF-IC showed higher emulsion stability and lower poly-dispersity index (PDI) value and fat globule diameters than those of FF-IC and LF-IC. The thermal properties of the samples were significantly affected by the addition of CSOB in an ice cream mix. CBLF-IC exhibited a lower temperature range (ΔT), enthalpy of fusion (ΔHf), and freezing point temperature (Tf) than those of FF-IC and LF-IC. While CBLF-IC exhibited a higher overrun value than other samples, it showed similar sensory properties to the FF-IC sample. The results of this study suggested that CSOB could be used successfully in low-fat ice cream production. This study also has the potential to gain new perspectives for the evaluation of CSOB as a fat substitute in a low-fat ice cream.This study aimed to investigate the utilization of cold-pressed chia-seed oil by-products (CSOB) in a low-fat ice cream formulation as a fat replacer and stabilizer. In the study, ice cream emulsion mixtures were formulated by using 0.2–0.4% xanthan gum (XG), 2.5–12.5% fat, and 1–3% CSOB. Optimization was performed using the response surface methodology (RSM) and full factorial central composite design (CCD) based on the flow behavior rheological properties of the emulsions obtained from 17 different experimental points. All of the emulsion samples showed non-Newtonian shear-thinning flow behavior. The consistency coefficient ( Ice cream is a frozen milk-based dessert as a frozen aerated emulsion (O/W) containing partially combined fat globules, air bubbles, and ice crystals. Ice cream includes milk, milk cream, emulsifiers, sweeteners, stabilizers, and flavorings . Ice creStabilizers are preferred in ice cream production because they provide specific and important functions such as increasing the viscosity and smoothness of the ice cream mix, improving aeration, reducing ice recrystallization, and the rate of structural collapse during melting ,6. EmulsSalvia hispanica L.) seed, belonging to the Lamiaceae family, has a high amount of oil (25–38%), carbohydrates (26–41%), high dietary fiber (18–30%), and protein (19–23%) [Chia ((19–23%) . Chia se(19–23%) ,13. Rese(19–23%) ,16,17,18Fat substitutes can be used to reduce the amount of fat in foods and replace it with oil-like substances and reduce the caloric value with new additives. Fat substitutes can provide many of the properties that fat gives to foods and be classified as carbohydrate-derived fat substitutes, protein-derived fat substitutes, synthetic fat substitutes, and fat compound substitutes. When these substances are used instead of fat in foods, the fat in the food can be partially or completely reduced, and the energy from fat is minimized but provides palatable products . RecentlChia-seed by-products derived from the cold-press oil industry have the potential to be utilized as a building strengthener and fat replacer due to their ability to develop relatively viscous solutions. Furthermore, Capitani et al.  reportedThe main purpose of this study is to produce ice cream with an optimum feature of low-fat content by using cold-pressed chia seed oil by-products without causing any change in the rheological and melting properties of ice cream. In this study, the optimization process was carried out based on the rheological analysis results of the ice creams produced with different formulations. The rheological and melting properties of the ice creams produced after the optimization process were investigated. Thus, production parameters with optimum rheological and melting properties were determined.In this study, CSOB, xanthan gum (XG), egg yolk powder (EYP), milk cream (35% milkfat), pasteurized milk (1.5% milk fat), sugar, and water were used for producing ice cream samples. CSOB, 8.07% fat, 56.10% total carbohydrate including fiber, 23.50% protein, 29.26% fiber, 5.21% ash, 6.85% moisture, was supplied from ONEVA Food Co. . XG and egg yolk powder (EYP) were obtained from Sigma-Aldrich , and the sugar, milk, and milk cream were purchased from the local market.The study was carried out in two stages. In the first stage, the responses were obtained at 17 different points for ice cream samples through the Design Expert Software , and the formulation was optimized according to flow behavior characteristics. In the second stage, the dynamic rheological and melting profile properties of the ice cream samples produced according to the optimum formulation were examined and characterized.X1), CSOB concentration (1–3 g/100 g) (X2), and gum concentration (0.2–04 g/100 g) (X3), using an unblocked full factorial central composite design (CCD). Seventeen different experimental points were obtained by using Design Expert Software  to determine the optimum amount of fat, CSOB, and gum content. For the estimation of the error, the design consists of three of the factorial points. A quadratic model was fitted to the experimental data for each response. Model applicability was evaluated based on the R2, R2-adj, lack of fit, F, and p-values obtained from ANOVA. Response surface plots showing the effect of model parameters on the K value were built by Design Expert Software. The optimization was performed based on the highest desirability value. The formulation with the lowest fat content with a desirability value of 1 was determined as the optimum formulation. Three central points were used. Analysis of all points was performed in triplicate, and the results were reported as mean values and standard deviations. The formulation of the samples has been determined in accordance with the ice cream standard. Accordingly, 2.5% fat ratio representing low-fat ice cream (0.5 ≤ milk fat < 3), 7.5% fat ratio representing semi-fat ice cream (3 ≤ milk fat < 8), and 12.5% fat ratio representing fat ice cream (8 ≤ milk fat ≤ 13) were used. Based on these fat ratios, the amount of milk and milk cream to be used has been calculated. The percentage of fat, XG, and CSOB vary in formulations, and the amounts of sugar (12%) and EYP (3%) remain constant. Seventeen formulations obtained in this way are shown in The formulation of ice cream samples was determined using the Design Expert Software . Response surface methodology (RSM) was carried out for the study of three factors, namely fat concentration (2.5–12.5 g/100 g) (Ice cream formulations consisted of 2.5–12.5% fat (derived from 1.5% milk fat and 98.5% milk cream), 12% sugar, 3% egg yolk powder (EYP) and 0.2–0.4% xanthan gum (XG), 1–3% CSOB. XG and milk were weighed according to the formulations determined in the first stage of ice cream production. XG was slowly added in order to dissolve it thoroughly and kept in a magnetic stirrer at 1000 rpm. Then, CSOB, which was weighted according to the formulation, was added and mixed. Then milk cream was added sequentially. The EYP and sugar were added and mixed. Then, the samples were homogenized with ultra turrax (Daihan. HG-15D) at 1000 rpm for 3 min. After this process, the ice cream mixture samples were kept at 0–4 °C for 2 days to mature. Afterward, ice cream was produced from the mixes using an ice cream machine . Ice cream samples were stored at −18 °C for analysis.Flow behavior, dynamic, and 3-ITT rheological properties of ice cream mixes were determined using a stress and temperature-controlled rheometer . A parallel plate probe  was used for rheological measurement. All measurements were performed at 25 °C and duplicated for the accuracy of the results.−1). The measurement was carried out at a constant temperature of 25 °C, and 3 parallel studies were carried out for each sample. The data obtained from the rheological analysis were fitted to the power law model, and nonlinear regression was used to calculate model parameters;Flow behavior rheological properties of ice cream samples prepared using CSOB were determined using a parallel plate probe  with a shear rate in the range 0.1–100 , K the consistency coefficient (Pasn), γ the shear rate (s−1), and n the flow behavior index.In Equation (1), the −1 (ω). Elastic modulus , n) and In Equations (2) and (3), the −1 as constant shear rate value and 150 s−1 as variable shear rate value. The linear viscoelastic region has been taken into consideration in the selection of the values, and the linear viscoelastic region of the samples ends at 50 s−1. The ice cream samples were subjected to a very low shear rate (0.5 s−1) for 100 s during the first time interval. In the second time interval, 150 s−1 was exposed to the determined cutting force for 40 s. In the third time interval, the dynamic rheological behavior of the ice cream in the second time interval was tested by subjecting the samples to the low shear rate level in the first time interval. For this purpose, the change in the viscoelastic solid structure . The samples were diluted 500-fold with ultrapure water before homogenization by stirring in an ultrasonic water bath for 1 min. The particle size of the samples was determined according to the dynamic light scattering technique .W1, and W2 represent the weight of a unit volume of ice cream mix and the weight of a unit volume of ice cream after freezing, respectively.The volumes of ice cream mixtures (just before freezing) and ice cream samples (immediately after freezing) were weighed, and values were recorded . Overrunzero), and the thermal data were recorded from −20 to +50 °C in a nitrogen atmosphere with a heating rate of 1 °C/min. An empty pan was used as the reference. The flow rates of nitrogen gas for cooling were 50 mL/min. The onset temperatures (T0), Tf, and ΔHf of the transitions of ice formation and ice melting were determined. The onset temperatures are considered as the intersection of the tangent and baseline to the left side of the melting peak. Freezing points were determined by using the temperature of the steepest slope. The enthalpy of fusion was calculated by extrapolating the baseline under the peak by connecting the flat baseline before and after the melting peak and integrating the peak above the baseline. The amount of ice formed per gram of sample (freezable water) was determined by the method described by Soukoulis et al. [The thermal properties of ice cream samples were analyzed by a differential scanning calorimeter (DSC) by A DTA-DSC  operating at atmospheric pressure  according to the method reported by Hwang et al. . Ice cres et al.  by dividA trained panel of 10 members  assessed the sensory characteristics of ice cream samples. Panel members were instructed on the ice cream samples prior to the commencement of the tests. The training consisted of a two-hour thorough presentation to the panelists on the purpose of the study and the characteristics of the samples. The panelists were asked to identify the optimal persimmon content for the required enhanced ice cream in terms of look and color, odor, taste and flavor, texture, melting resistance, and overall acceptability. Ice cream samples were evaluated using a scaling method of descriptive attributes for all parameters  .L*, a* and b* parameters were evaluated. L*, a* and b* parameters represent whiteness/darkness, redness/greenness, and yellowness/blueness, respectively.The color parameters of ice cream samples were measured using a colorimeter . The CIELAB coordinate system was used, and the p < 0.05). As a result of the applied rheological analysis, power law model parameters were calculated with the help of nonlinear regression analysis. The Statistica software program  was used for nonlinear regression analysis.Ice cream samples were produced in three replications, and three parallel measurements were performed from each replication. The mean and standard deviation values were presented. Statistical applications were carried out with the Statistica software package . The Duncan multiple comparison test was used to compare the mean values of the parameters after optimization  and determination coefficient (R2) calculated for 17 different points created with the trial design are shown in R2 values of the power law model were higher than 0.99. This shows that the power law model is suitable for determining the flow behavior properties of ice cream mixes. According to the ice cream formulations, K and n values differed and were found as 4.01–26.05 Pasn and 0.23–0.38, respectively. n values lower than 1 indicated that all ice cream mixtures exhibited the non-Newtonian pseudoplastic flow behavior , while the sample-11  exhibited the highest K value (26.05 Pasn). The K value of the samples increased with increasing CSOB, gum, and fat content. These results showed that increasing the K value with the increase in CSOB can improve the shear-thinning properties of ice cream mixes. The desired consistency values can be obtained by using CSOB even at low-fat and gum content. Thus, CSOB can be used for improving rheological properties in low-fat ice cream.Power law model parameters  of ice-cream mixtures. As seen in K value of the ice cream mixes. R2 and Adjusted R2 values of the model used were determined as 0.9864 and 0.9618, respectively. The differences between Adjusted R2 and predicted R2 was lower than 0.2, and the lack of fit value was found as insignificant. These results indicated that the quadratic model could be successfully used to describe the effect of formulation on the K value of the samples. The p-value of the model was lower than 0.05, indicating that the model terms significantly affected the K value. In this model, A, B, C, AB, C² are significant model terms. The linear effect of all independent variables was significant. The interaction and model terms A and B, and the quadratic effect of C was also be found as significant.The K values of the samples. Chia seeds affect fat binding and gel-forming properties due to the functional properties of dietary fiber. Olivos-Lugo et al. [K) and can be used in the production of low-fat ice cream. With the increase in the K value of the mixtures, the n value decreases and shows pseudoplastic behavior, which is the typical flow behavior characteristic of ice cream. Due to the solid particles in CSOB, it significantly increases the K value by affecting the viscosity of the ice cream mixture. The fat, emulsifier, stabilizer, and CSOB used in ice cream mixtures significantly increase the K consistency coefficient of the mixtures. The main reason for this is the synergistic interaction between the components. The K value of the full-fat control sample was used to determine the optimum formulation. The sample with the highest desirability value and minimum fat content was selected as the optimum formulation. The optimum formulation was determined as 2.5% fat, 0.29% XG, 2.51% CSOB.Steady shear rheological analyzes showed that the increase in the amount of CSOB in the formulation resulted in a significant increase in the o et al.  reportedo et al.  suggesteo et al. . Based oK) and flow behavior index (n) values of the optimal and control ice cream mixtures. K and n values) as well as the determination coefficient (R2). K values varied from 3.46 to 5.66 Pasn, n values from 0.30 to 0.33, and R2 values higher than 0.99. While the K value of the full-fat control samples and the K value of the sample containing CSOB were found to be statistically insignificant, the K value of both samples was found to be significantly higher than the low-fat control. This result showed that an ice cream mix similar to the consistency properties of full-fat ice cream could be produced with the use of CSOB.The steady shear, frequency sweep, and thixotropic properties of the samples produced based on optimum formulation were compared with the values of the full-fat and low-fat control sample. The flow curves of optimum and control ice cream samples were given in G″, indicating that the solid character of all ice cream samples is more dominant than the liquid character. As seen in The dynamic rheological properties of ice creams produced from optimum and high-fat and low-fat control ice cream formulations were investigated. The frequency sweep test can simulate the liquid behavior of ice cream samples during chewing in the mouth , which h samples . As seenR2 values of the model were found in the range of 0.97–0.98. As can be seen in K′ and K″ values of the samples were in the range of 3.09–23.41 and 2.46–8.46, respectively; the values of n′ and n″ were found in the range of 0.268–0.535 and 0.212–0.288, respectively. The K′ values were higher than the K″ values for all samples. Accordingly, all of the ice cream samples showed a viscoelastic solid character. When CSOB was added to low-fat ice cream, the K′ and K″ values increased when compared to the low-fat ice cream sample.The dynamic rheological parameters  were obtained with the second-order structural kinetic model. G0′, Ge′, G0′/Ge′, k × 1000, and R2 values were 8.42–21.12, 14.81–38.73, 1.760–1.834, 11.10–27.00, and 0.995–0.996, respectively. FF-IC showed the highest G0′, Ge′, G0′/Ge′, and k × 1000 values, indicating that the full-fat control sample (FF-IC) showed the highest thixotropic behavior. The amount of fat in an O/W emulsion has a significant impact on its rheological characteristics. Therefore, the decrease in the fat content of the ice cream samples causes weak rheological properties, especially the recoverable character, as the fat content of the ice cream samples decreases. Although CBLF-IC has low-fat content (2.5%), FF-IC and CBLF-IC showed similar thixotropic behavior and viscoelastic solid character so the higher recoverable behavior obtained with CSOB addition can be explained by more intermolecular interactions by the formation of small aggregates of hydrocolloid. These rheological properties indicate that CSOB can be utilized to enhance the rheological properties and thixotropy of low-fat ice cream samples.Parameters  from cycle to cycle. G* value after applied thermal stress. As can be seen, after 10 applied thermal cycles, a slight increase in the G* value of all samples was observed. This insignificant change indicates that all samples are resistant to thermal stress and have high emulsion stability. The percent change (ΔG*) in the G* values of the samples was calculated as 14.37, 10.10, 8.37 for FF-IC, LF-IC, and CBLF-IC, respectively. This may indicate that the sample containing CSOB may show higher stability than other samples.The emulsion stability of the ice cream mixes was determined by the thermal loop test. The thermal loop test could be used as a fast method to measure emulsion stability by subjecting to different numbers of thermal cycles. The temperature fluctuations during processing, production, storage, and transportation stages were simulated in this test . The str32), PDI value, and zeta potential values of the samples were found as 0.494–1.305 μm, (−39.13)–(−28.90) mV and 0.493–0.741, respectively (The fat particle size (dectively . As can f) of FF-IC, LF-IC, and CBLF-IC were obtained as −3.66, −3.35, and −3.99 °C, respectively. The freezing points of the ice cream samples were significantly differed (p < 0.05). The freezing point temperature of ice cream is closely related to the serum phase concentration and the soluble biopolymer concentration. Generally, the Tf value decreases as the serum phase concentration increases or the molecular weight of the soluble biopolymers decreases [f values of ice creams when 2% wheat and oat fiber were added. Researchers explained this result by increasing the serum phase concentration due to the high water holding capacity of wheat and oat fiber. They reported an increase in the Tf value with the addition of apple fiber and inulin, which have higher soluble fiber content. The high water holding capacity of the insoluble fibers in the CSBO content may have caused a decrease in the Tf value. In addition, the use of less XG in the sample containing CSOB may have caused a decrease in the less soluble biopolymer substance in the serum phase, thus significantly reducing the Tf value. The higher Tf value obtained from LF-IC could be due to a decrease in solid concentration by reducing fat contents.The thermal properties of the ice cream samples are presented in ecreases ,44. Soukecreases  investigThe melting resistance of ice cream represents the ability of ice cream to resist melting when exposed to high temperatures. The heating system in the DSC provides the temperature that causes the formation of an endothermic peak. The melting enthalpy (ΔH) is calculated by integrating with the area of the endothermic peak and is the amount of energy leaving the system. It occurs as a change in the overall internal energy of food . In our ΔT values were found as 14.16, 15.95, and 17.05 for the samples CBLF-IC, LF-IC, and FF-IC, respectively. The sample containing CSOB had the lowest ΔT value than control samples. The temperature range (ΔT) could be used as an indicator of the uniformity of the size distribution of ice crystals. Therefore, a narrow melting temperature range indicates a more homogeneous distribution of ice crystals melting in a smaller temperature range . The enrThe overrun values are shown in L*, a* and b* color values of the samples are presented in L*, a* and b* values of the samples were found as 77.08–83.42, 4.23–4.93 and 13.63–15.41, respectively. As can be seen, a significant difference was detected between the color values of the samples. The highest L* value was measured from the FF-IC sample, while the lowest L* value was obtained from the CBLF-IC sample. The high L* value of the FF-IC sample can be explained by the higher fat content than the other two samples. In addition, with the introduction of CSOB into the ice cream formulation, a decrease in L*, a* and b* values was observed. This result shows that CBLF-IC will cause a significant change in the color values of the ice cream samples.Sensory scores of ice cream samples are shown in K value of the ice cream mix samples with the increase in CSOB in the formulation. The low-fat sample (CBLF-IC) containing CSOB, produced as a result of the optimization, showed similar rheological properties to the full-fat sample (FF-IC). Emulsion stability, fat globule size and distribution, thermal properties, and overrun analyzes indicated that the quality of ice cream mix and ice cream could be improved with the addition of CSOB. According to the results of the sensory analysis, the addition of CSOB did not cause a significant decrease in the sensory qualities of ice cream. This study has shown that CSOB can be used successfully in the production of low-fat ice cream and can provide a new perspective for the evaluation of this by-product with low economic value.The consumption of chia seed oil has been increasing in recent years due to its polyunsaturated fatty acids, especially linolenic acid. As a result of the production of cold- pressed chia seed oil, waste rich in carbohydrates, fiber and protein emerges. Evaluation of this waste is an important issue for cold press manufacturers. In this study, the potential for the use of CSOB in the production of low-fat ice cream was investigated. A significant increase was observed in the


Lameness in sheep continues to be a global health, welfare and economic concern. Damaged, misshapen or overgrown feet have the potential to cause lameness either directly, or indirectly. There is a lack of understanding of the predisposing factors for different hoof conformation traits in sheep. Our exploratory study aimed to investigate the prevalence of, and risk factors for, three distinct hoof conformation traits relating to the sole and heel, hoof wall, and hoof wall overgrowth. Feet of 400 ewes from four UK commercial sheep farms were inspected at four time points across 12 months. For each conformation trait, a four-point ordinal system was used to score each individual claw, and foot-level scores were calculated. We present 92.4% of foot-level observations to be affected by ≥1 conformation traits. Whilst hoof conformation traits were correlated to some degree, a unique set of sheep-, foot- and farm-level factors were associated with each distinct conformation trait. We provide, for the first time, key insight into the multifaceted and multifactorial aetiology of hoof conformation in sheep, building upon previous landmark studies. Our results inform hypotheses for future prospective studies investigating the risk factors for adverse hoof conformation in sheep. Dichelobacter nodosus. Footrot has two clinical presentations representing a continuum of infection: interdigital dermatitis (ID), which presents as inflammation of the interdigital skin, and severe footrot (SFR), which presents as the aggressive separation of hoof horn from the underlying sensitive tissue. Contagious ovine digital dermatitis (CODD) is also an infectious disease of significant concern, affecting approximately 50% of UK flocks [Lameness is one of the most significant health and welfare challenges facing sheep farmers worldwide. Sheep can become lame through infectious and non-infectious origins, but the majority of lameness in English sheep flocks is caused by footrot , an infeK flocks ,3, and wK flocks . Non-infThe ruminant hoof is a specialised epidermal structure, acting as an interface between the animal and its environment. The hoof is comprised of four key regions: periople, heel, sole horn, and wall horn . The perD. nodosus present, with these sheep acting as subclinical carriers of infection [D. nodosus found on infected feet [The significance of hoof conformation traits, or the shape, size and condition of hooves, has received little attention in sheep. It is known that sheep can display signs of impaired locomotion and stance due to poor hoof conformation, such as overgrown hooves ,15, and nfection . Moreoveted feet , which cted feet . D. nodosus infection pressures at pasture to increase risk of poor conformation development. Conversely, they postulated lower ground and air temperatures, or dry underfoot conditions, to improve conformation. However, this landmark study did not differentiate between conformation of the sole and heel, and hoof wall, nor explicitly investigate a range of sheep-, and farm-level putative variables associated with distinct conformation traits. In cattle, studies investigating the environmental factors have made significant contributions to our understanding of the mechanical moderators of hoof conformation [Given the association between poor hoof conformation traits, bacterial load and lameness, understanding the risk factors for misshapen, damaged or overgrown hooves is integral to reducing lameness risk. Knowledge of risk factors could help predict changes to hoof integrity and higher risk of lameness, whilst also informing measures to promote good-structured and optimally-functioning hooves. To the authors’ knowledge, there have been no on-farm epidemiological studies explicitly investigating the risk factors for the prevalence nor severity of specific hoof conformation traits in commercial flocks. One 18-month study investigated the development of poor hoof conformation (or misshapen or damaged hooves) in sheep . They spormation ,20,21,22The aims of our study were to provide robust observational evidence of the prevalence of and potential risk factors for distinct hoof conformation traits in sheep from four commercial flocks over a 12-month period. To our knowledge, this is the first study of its kind.The study was a longitudinal, repeated cross-sectional field survey of four commercial sheep farms (identified as A–D) in England and Wales. Farm characteristics, study design and data collection are described in detail in . BrieflyFarm-level data were obtained from retrospective management practice questionnaires completed by each farmer. Data included farm soil type and flock footrot vaccination  status, in addition to estimates of pasture moisture, quality, type and sward height for each calendar month between August 2019 and September 2020. Meteorological data  were extracted from publicly available local UK MET Office station archives, as described in .All data were manually entered into Microsoft Excel 2016 . The final dataset comprised of 5672 foot-level observations and 1418 sheep-level observations obtained from 400 ewes across four farms. Total score at foot-level for each conformation trait was calculated by taking the sum of scores for the paired claws  of that foot. The maximum score at foot-level per conformation trait was 6. Total score at sheep-level for each conformation trait was calculated by taking the sum of scores for all four feet. The maximum score at sheep-level per conformation trait was 24. Hoof conformation scores were treated as continuous data. The locomotion score was excluded from analyses due to the low prevalence of lameness in the ewes sampled. Due to low prevalence of SFR, presence of ID and SFR were combined into one category  for analysis. s). Probability values of <0.05 were considered significant. All analyses were performed in Genstat , and R statistical software package v 3.5.3 . Associations between continuous and categorical variables were investigated using Kruskal–Wallis tests, and associations between categorical variables were investigated using Chi-squared tests. The relationship between hoof conformation scores were assessed by Spearman’s rank correlation coefficients  remained in the final model. Collinearity among fixed-effects was assessed using variance inflation factors (VIF) from the “car” package [Univariable and multivariable linear mixed effects models were constructed to investigate the associations between sheep-, foot- and farm-level variables and each of the three hoof conformation traits at foot-level. The foot-level continuous outcome variables were: (1) sole and heel conformation, (2) hoof wall conformation and (3) hoof wall overgrowth, all coded 0–6. Models were constructed using the “lmer” function from the “lme4” package in R . Models  package . Collinen = 569/1418) of all sheep-level observations scored ≥ 1 for all three conformation traits concurrently. Almost 50% (n = 674/1418) of all sheep-level observations had ≥1 feet with misshapen and/or damaged sole and heel area. Over 95% (n = 1359/1418) had ≥1 feet with misshapen and/or damaged hoof wall, and over 89% (n = 1264/1418) had ≥1 feet with hoof wall overgrowth present. A summary of the scores for three hoof conformation traits at sheep-level are found in Ewes were persistently affected by ≥1 conformation trait; no ewes scored 0 for all three conformation traits throughout the duration of the study. Approximately 40% (n = 432/5672) of all foot-level observations scored 0 for all three conformation traits. Of those affected, approximately 13.4% (n = 700/5240) of foot-level observations scored ≥ 1 for all three conformation traits concurrently. Over 20% (n = 1182/5672) of foot-level observations had misshapen and/or damaged sole and heel areas. Almost three-quarters had misshapen and/or damaged hoof wall  or hoof wall overgrowth present . A summary of the scores for three hoof conformation traits at foot-level are found in Individual feet were persistently affected by ≥1 conformation trait (scores ≥ 1). Only 7.6% (p < 0.001) and had higher mean wall overgrowth scores (p < 0.001). Back feet were more often affected by hoof wall conformation (p < 0.001) and had higher mean hoof wall scores (p < 0.001). No difference in sole and heel conformation prevalence was observed between front and back feet (p > 0.05), but back feet had higher mean sole and heel scores (p = 0.054), albeit a trend association.Front and back feet were disproportionately affected by hoof conformation traits (scores ≥ 1) . Front fp < 0.001); higher sole and heel scores were observed on Farm A, higher hoof wall scores were observed on Farm C, and higher hoof wall overgrowth scores were observed on Farm B. Foot-level scores also changed over time (p < 0.001); highest scores for sole and heel conformation were observed at Visit 2, highest scores for hoof wall conformation were observed at Visit 4, and highest scores for hoof wall overgrowth were observed at Visit 3 . Sole and heel conformation and hoof wall overgrowth scores showed a weak, positive association , similar to hoof wall conformation and hoof wall overgrowth scores .Sole and heel conformation and hoof wall conformation scores at foot-level showed a fair, positive correlation  were more likely to have feet with lower wall overgrowth scores, compared to fit ewes (BCS = 3.0). This is biologically plausible considering greater body weight, estimated to be around 12% of liveweight for every BCS score , exertedFor all hoof conformation traits, a foot was more likely to have higher scores when ≥1 other feet of the ewe had scores ≥ 1. This suggests that feet are not independent, and sheep- or farm-level factors are likely to play more important roles. Nonetheless, foot position was a significant risk factor for all three conformation traits, but varied between traits. Back feet were more likely to have higher scores for sole and heel, and hoof wall conformation. Back feet are typically exposed to higher levels of moisture and deeper faecal matter than front feet , and thiD. nodosus presence and load [Interestingly, feet with clinical disease were more likely to have lower scores for wall overgrowth. It is widely recognised that wall overgrowth proceeds a non-weight bearing event, such as footrot infection . Consideand load . It is iWe report variation in the risk of hoof conformation traits over time. Feet of ewes at Visit 2 were more likely to have higher sole and heel scores than those at Visit 1. Three-quarters of the flocks studied were housed during Visit 2, which reflects the increase in risk during this time. Whilst warm, damp bedding is likely to cause the softening and maceration of sole and heel horn, other factors around parturition, such as hormone or nutrition, could play a role in reducing sole horn integrity . InteresThis study describes in detail the prevalence of, and risk factors for, three distinct hoof conformation traits at foot-level in commercial ewes from four UK sheep flocks, and as such, represents the largest such study of its kind. As with any longitudinal field survey, we cannot guarantee that the results are generalisable to all flocks. Due to the nature of the study, we cannot confirm the direction of causality of the associations between risk factors and hoof conformation trait score, but our findings provide reasonable insight into the likely candidates at play. Whilst the collection of pasture-based variables provided insightful data into specific conditions experienced by ewes, farmers’ estimations of variables may be impacted by recall bias, interpretation, and resultant observer bias. Future studies warrant the measurement of pasture conditions by a single observer at multiple time points across the month, in addition to objective measurements of soil moisture and temperature. Intensive observations of sheep at uniform sampling points could also prove valuable in further understanding the temporal dynamics of hoof conformation traits. Further measurements of hoof integrity, such as hoof hardness, could help elucidate the dynamics between hoof conformation, pasture moisture and the mechanical properties of hoof horn.The aetiology of hoof conformation in sheep is complex. We have provided a detailed description of the hoof conformation traits affecting commercial ewes and have updated our understanding of the risk factors involved. We highlight a number of different sheep-, foot- and farm-level factors as key influences affecting the properties of the hoof. We propose wet underfoot conditions to be the most important environmental factor affecting hoof conformation in sheep, influencing the dynamics between moisture, abrasion, growth and wear. Further work is required to fully elucidate the causality of the risk factors identified, in order to inform early-intervention managements aimed at improving hoof conformation and increasing resilience to opportunistic infection and lameness.


Adverse food reactions (AFRs) are abnormal clinical responses related to food or the ingestion of a food component, including drinks, food additives, and dietary supplements ,2. AFRs On the other hand, a food allergy (FA) is a reproducible adverse health effect arising from a specific immune system response. Based on the immunological mechanism involved, an FA may be classified as: (a) IgE-mediated, which is one that is mediated by antibodies belonging to immunoglobulin E (IgE); (b) non-IgE, a heterogeneous group of food allergies in which there is an immune reaction against food components, but where the primary pathogenesis is not a product of IgE and thought to act mainly through cell-mediated mechanisms; and (c) mixed, in which both IgE-mediated and cell-mediated immunological mechanisms are involved in the reaction ,2,4.Non-IgE-mediated and mixed FAs are still a tough challenge for clinicians. The diagnosis of non-IgE and mixed FAs remains one of exclusion and is mainly clinical, except for food-protein-induced enteropathy (FPE) and eosinophilic gastrointestinal disorder (EGID), in which histological confirmation is required. Unfortunately, non-IgE and mixed FAs are frequently implicated in causing gastrointestinal symptoms in children and adults but remain likely underdiagnosed, and evidence-based protocols with which to diagnose and treat these diseases are lacking .The clinical presentation of non-IgE and mixed FAs is characterized by significant gastrointestinal and extraintestinal symptoms that disappear after starting an elimination diet  and reappear when the culprit allergen is reintroduced . In thisNutrients [Looking at pediatric practices, an interesting review published by Calvani et al. in utrients  thoroughThe clinical scenario of non-IgE and mixed allergies in both the pediatric and particularly the adult population is not limited to the six thoroughly described diseases presented in the paper by Calvani , which aIn recent years, recent advances in FGIDs have demonstrated micro-organic alterations at the biopsy level with different characteristics from organic intestinal diseases , which pathologists often do not recognize in a routine examination, thus labeling them as “normal mucosa” or “mucosa with non-specific chronic inflammatory infiltrate”. An increase in eosinophil and mast cell count was demonstrated in a subgroup of patients with FGIDs along with local and systemic cytokine changes ,11,12,13These insights fuel a contagious enthusiasm in the scientific community. Eliminating some key food allergens, demonstrated to be involved in acute microorganic changes in intestinal barrier function via CLE analysis, could be a way to manage patients more effectively in the future. Researchers should approach this intriguing topic by leaving behind the confines of classic allergology and gastroenterology “dogmas” and preconceptions, and a joint effort involving allergists, gastroenterologists, and immunologists on these topics is needed.


The patient took analgesics once a day before the surgery and three times a day after the surgery until POD11. Pyrexia (37.5–39.5 degree Celsius) in synchronization with nonnephrogenic VARP remitted on POD18. Intravenous antibiotics had little effect on changes of pyrexia. Serum procalcitonin values (reference range <0.5 ng/mL) are 0.07 ng/mL on POD12 and 0.06 ng/mL on POD18. His polyuria came to react with sublingual desmopressin after alleviation of pyrexia. He left the hospital under hormone replacement therapy without newly added neurological sequelae other than hypopituitarism. After GTR of an infundibular epidermoid cyst, based on values of serum procalcitonin, post-eTSS MM can be distinguished from infection and can be treated with symptomatic treatments. The postoperative transient nonnephrogenic VARP that differs from usual central diabetes insipidus can react with sublingual desmopressin after alleviation of PPP in the clinical course of post-eTSS MM. An infundibular epidermoid cyst should be sufficiently resected in one sitting to minimize comorbidities, its recurrence, or postoperative MM to the utmost.Prolonged postoperative pyrexia (PPP) due to Mollaret's meningitis following endoscopic transsphenoidal surgery (eTSS) for an intracranial epidermoid cyst can be confused with postoperative meningeal infection after transsphenoidal resection, especially in the middle of the COVID-19 pandemic. Anosmia, as well as dysgeusia, cannot be evaluated in patients of eTSS for a while after surgery. We report a case of an infundibular epidermoid cyst with post-eTSS Mollaret's meningitis (MM). The post-eTSS MM caused vasopressin-analogue-resistant polyuria (VARP) in synchronization with PPP. A 59-year-old man experiencing recurrent headaches and irregular bitemporal hemianopsia over three months was diagnosed with a suprasellar tumor. The suprasellar tumor was an infundibular cyst from the infundibular recess to the posterior lobe of the pituitary, which was gross-totally resected including the neurohypophysis via an extended eTSS. Since awakening from general anesthesia after the gross total resection (GTR) of the tumor, the patient continuously had suffered from headache until the 13 Mollaret's meningitis, a benign recurrent aseptic disease, is known to be associated with intracranial and intraspinal epidermoid cysts –7. In prA 59-year-old man presented with a three-month history of gradually progressive blurred vision with headaches (mainly retrobulbar pain). The headaches were frequently associated with coughs or sneezes. Magnetic resonance (MR) imaging demonstrated a sellar-suprasellar tumor compressing the optic chiasm superiorly Figures . Postconth postoperative day (POD13). The patient took analgesics once a day before the surgery and three times a day after the surgery until POD11. The patient's diabetes insipidus (DI) became stronger after the eTSS. This DI was controlled with sublingual desmopressin tablets until POD11. Pyrexia did not emerge until the patient's peroral analgesic dose decreased on POD11. This pyrexia (37.5–39.5°C) continued until POD18. This prolonged postoperative pyrexia (PPP) could be controlled with peroral nonsteroidal antiinflammatory drugs (NSAIDs). In addition, hard-to-control polyuria , in other words, vasopressin-analogue-resistant polyuria (VARP), emerged in synchronization with PPP and continued with some alterations until POD35. This PPP was unresponsive to intravenous pitressin or sublingual desmopressin tablets. The plasma natrium fluctuated from 123 mEq/L to 135 mEq/L, which was below the normal range (138 mEq/L–145 mEq/L), during the VARP. Water supply and sodium correction were performed on the basis of laboratory data. Although his nuchal stiffness was not observed, his neck was not supple retrospectively. Although anosmia and dysgeusia could not be estimated because of postoperative nasal packing, leukocytopenia was not found in the patient .–15.10–15From this case, we have found two things that the previous reports did not refer to –15; the The PPP should be distinguished from a symptom of postoperative bacterial meningitis. Because healthcare-associated meningitis or ventriculitis is a serious complication in different neurosurgical procedures and is associated with significant morbidity and mortality , in the Fever is a relatively common occurrence among patients in the intensive care setting. Although the most obvious and concerning etiology is sepsis, drug reactions, venous thromboembolism, and postsurgical fevers are all on the differential diagnosis . In the Serum procalcitonin levels seem to be the best marker in differentiating between bacterial and viral meningitis in adults . In addiThe postoperative hard-to-control polyuria: pitressin/desmopressin-resistant polyuria, which should instead be called vasopressin-analogue-resistant polyuria (VARP), differs from usual central diabetes insipidus (DI). VARP came to react with sublingual desmopressin after alleviation of pyrexia in the clinical course of post-eTSS Mollaret's meningitis in our case. The postoperative VARP is something different from usual DI: hypernatremia associating polyuria was not observed, indicating excessive excreted urinary natrium quantity during VARP. The VARP interacts with central hyperthermia (PPP), which suggests an involvement of the abovementioned hypothalamic dysfunction during the transient VARP. This VARP may belong to the category of cerebral salt wasting syndrome coexisting with DI , or is VPPP in this case was linked with the patient intake of NSAIDs. The VARP settled as the PPP remitted. Based on these findings and the clinical course, transient hypothalamic dysfunction (HD) is probably inflammation-induced. If so, NSAIDs and corticosteroids might be effective for HD after resection of infundibular epidermoid cysts.This case is the first report of transient nonnephrogenic VARP synchronizing with PPP during Mollaret's meningitis after resection of an infundibular epidermoid cyst. As a similar mechanism, Rathke's cleft cysts (RCCs) sometimes present with acute onset, and the presentation is consistent with the features of pituitary apoplexy caused by pituitary adenoma . As wellIn our case, even under GTR of an infundibular epidermoid cyst, a post-eTSS Mollaret's meningitis continued until POD18 and had settled after POD19. If the eTSS resulted in insufficient resection, the Mollaret's meningitis would not have settled and been long lasting. Thus, surgical gross total resection remains the mainstay for treatment of intracranial epidermoid cysts , 33. HerIn the middle of the COVID-19 pandemic, prolonged pyrexia should be appropriately diagnosed and distinguished from coronavirus infection or other diseases. Physicians need to correctly approach this new disease in daily clinical practice, often representing a challenge in terms of differential diagnosis .After GTR of the infundibular epidermoid cyst, based on values of serum procalcitonin, post-eTSS Mollaret's meningitis can be distinguished from infection and can be treated with symptomatic treatments despite PPP. The postoperative VARP differs from usual central DI and can react with sublingual desmopressin after alleviation of PPP in the clinical course of post-eTSS Mollaret's meningitis. An infundibular epidermoid cyst should be sufficiently resected in one sitting on the basis of its recurrence rate.


Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test. Rho family GTPases are implicated in a wide range of basic cellular processes including cell morphology, polarity, motility, division, and defence . These eAlthough Rho-type GTPases are evolutionarily conserved in eukaryotes , they caDespite the evolutionarily conservation of all Rho-type GTPases, plant ROPs and their partners have many unique characteristics ,11,12,13Rho GTPases, like almost all Ras-related proteins, contain a C-terminal cysteine-containing motif  GTPase was mutated to alanine (S74A) or glutamic acid (S74E) [Interestingly, the ROP-specific R76 residue that is required for the PRONE-mediated nucleotide exchange is close to an evolutionarily conserved serine residue  . The latd (S74E) . These ed (S74E) . Recentld (S74E) .Here, we report that although our approaches to identify Akt-related plant AGC kinases potentially phosphorylating the S74 residue of plant ROPs failed, we could demonstrate that plant calcium-dependent protein kinases (CPKs) accept the Arabidopsis ROP1 GTPase as in vitro substrate in a conformation-dependent way. The phosphorylation takes place at several Ser/Thr residues, excluding S74, and the phospho-mimetic mutation of one of these residues affects the protein–protein interaction capability and the function of the GTPase. These data open new avenues to investigate the interlink between kinase and ROP GTPase signalling pathways in plants.Medicago sativa ROP6 protein interfered with the regulation and consequently with the in vitro as well as in planta function of the GTPase [We have previously demonstrated that the S74E phosphorylation-mimic mutation of the e GTPase . The S74e GTPase  is knowne GTPase ,26. To ie GTPase ,32. To ee GTPase ,34,35,36In order to test whether animal AGC kinases can phosphorylate the evolutionarily conserved phosphorylation site, the commercially available murine cAMP-dependent protein kinase (PKA) catalytic subunit was used in a similar kinase assay. It was found that although this kinase domain was able to phosphorylate the AtROP1 protein, this phosphorylation was neither dependent on the conformation nor the presence of the S74 residue of the GTPase . TherefoThe amino acid residues surrounding S74 of AtROP1 can also serve as a recognition site for the plant-specific calcium-dependent protein kinases (CPKs)  Figure . MoreoveIn the Arabidopsis pollen tube, there is a feedback regulation between the AtROP1 GTPase and a tip-localised calcium gradient . TherefoTo determine the site of phosphorylation, kinase assays were made using the CPK34 kinase in the presence of “cold” ATP and the WT and DN forms of the AtROP1 protein as substrate. The phosphorylated proteins were cut out of CBB-stained gels and subjected to trypsin digestion followed by partial acidic cleavage, phosphopeptide enrichment, and mass spectrometry analysis to detect phosphopeptides. Several serine and one threonine residues were found to be phosphorylated in both ROP1 versions . The S97in planta ROP function.Nevertheless, we mutated it into alanine (S97A) or glutamic acid (S97E) to test whether its phosphorylation might have an effect on ROP1 function. Beside introducing into the wild type, the S97 phosphorylation-related mutations were combined with the constitutive active (CA) and dominant negative (DN) mutations. The interactions of the mutant versions with an upstream regulator GEF2 and a downstream effector RIC2 were tested in the yeast two hybrid system. The S97A and S97E mutations weakened the interaction of WT or CA ROP1 with GEF2 but strengthened it with RIC2. Since both the S97A and the phospho-mimic S97E mutation affected the interactions in a similar way B, the reThe other sites phosphorylated by CPKs on ROP1 resided in the N-(S4) or C-terminal regions  of the protein. It has to be mentioned that the region between T20 and D60 was not detected by MS, probably because this region contains neither tryptic cleavage site nor basic amino acids promoting ionisation . To deciRho-type GTPases of animals are well-known to be integrated into both upstream and downstream kinase-related signal transduction pathways . They arThe AGC-type kinase phosphorylation consensus sequence around S74 of AtROP1 (RXRXXS) due to a plant-specific conserved arginine residue overlaps with one of the predicted phosphorylation site of calcium-dependent protein kinases (CPKs)  exhibited a stronger phosphorylation signal than the C-terminal part including most of the sites identified by MS .Cloning of AtROP1 and its DN (T20N) and CA (G15V) mutant versions into the bacterial protein expression vector pET26b has been described earlier . The S97A and S97E mutations were introduced by the overlap extension polymerase chain reaction approach  using the 5′ AtR1 NdeI forward and the AtROP1S97A BamHI Rev or AtROP1S97E BamHI Rev primers , respectFor the polymerase chain reaction (PCR), Phusion high-fidelity polymerase was used , according to the manufacturer’s instructions. The constructs were verified by Sanger sequencing of the inserts.v/v) glycerol at −20 °C for later use.Plasmid constructs for protein expression were transformed into BL21(DE3)Rosetta  or ArcticExpress(DE3)RIL  competent cells, respectively. Proteins were induced to be expressed using 1mM IPTG  during an incubation for 45 min to 12 h according to the competent cell’s description. The expressed proteins were purified with HIS-Select Nickel Affinity Gel  or Ni-IDA Agarose gel , following the manufacturer’s instructions. Purified proteins were concentrated using Amicon Ultra 0.5 mL filters  and then stored in 10%  with app. 5 pmol kinase protein . The buffer to test AGC1.7 kinase activity included 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2+ ions to Fe3+ ions. Protein digests were loaded onto the Fe-NTA beads in 0.1% trifluoroacetic acid and 80% acetonitrile, the beads washed with loading buffer, then phosphopeptides eluted by 1% ammonium hydroxide and 50 % acetonitrile, and dried. Digests with or without phosphopeptide enrichment were analysed using LC-MS/MS on a Waters Acquity nanoHPLC  on-line coupled to a LTQ Orbitrap Elite mass spectrometer  in data-dependent acquisition mode using HCD and ETD fragmentation. Database search was performed by Protein Prospector (v 5.12.3), first against the complete Swissprot 2014.6.10 database (545388 entries) considering only tryptic peptides, and then against the identified proteins also considering cleavage after aspartic acid and glutamic acid. Precursor mass tolerance was 5 ppm and fragment mass tolerance was 20 ppm for HCD fragments and 0.8 Da for ETD fragments. Carbamidomethylation of cysteine residues was set as fixed modification, and oxidation of methionine residues, pyroglutamic acid formation from peptide N-terminal glutamine residues, methionine loss and acetylation of protein N-termini and phosphorylation of serine, threonine, and tyrosine residues as variable modification. Protein and peptide hits were accepted with a minimum score of 22 and 15 and a maximum E value of 0.01 and 0.05 . Phosphopeptide matches were also inspected. The mass spectrometric analysis of phosphorylation sites was described elsewhere in even more detail [Non-radioactive kinase assays were performed parallelly to determine phosphorylation sites by mass spectrometry. The Coomassie Brilliant Blue stained gel bands were cut and in-gel digested by trypsin. Disulphide bridges of the proteins were reduced by dithiothreitol and free sulfhydryls alkylated by iodoacetamide. Digestion with side-chain-protected porcine trypsin  was performed for 4 h at 37 °C. The extracted peptide mixtures were split, and half of the digests was submitted to acidic cleavage by formic acid. Phosphopeptide enrichment was performed by immobilized metal affinity chromatography on magnetic Fe-NTA agarose beads that were prepared from Ni-NTA beads  by replacement of Nie detail .https://www.takarabio.com/documents/User%20Manual/PT3024/PT3024-1.pdf—last accessed 20 July 2021). Transformants were grown on appropriate drop-out media in order to follow up the activation of the ade2 and/or the his3 reporter genes  where it was required).Yeast two-hybrid screening was carried out using the Saccharomyces cerevisiae yeast strain, with AH109 as the host. The yeast strain was co-transformed with pDest-GADT7 (AtRop1 and its mutants) and pDest-GBKT7 or pAD-GAL4 (Rop effectors and regulators) constructs based on the Yeast Protocols Handbook  pollen grains were shaken in liquid pollen germinating medium [http://rsbweb.nih.gov/ij/ (last accessed on 15 September 2021.)) in 20-20 randomly chosen pollen tubes expressing the given transgene. The significance of difference between sets of data was determined by one-way analysis of variance (ANOVA) following Duncan’s multiple range tests; a P value of less than 0.05 was considered significant.Freshly collected or frozen tobacco ; CPK17 (AT5G12180); CPK34 (AT5G19360); CPK30 (AT1G74740), GEF2 (AT1G01700); RIC2 (AT1G27380); AGC1.7 (AT1G79250);


Integrins serve as conduits for the transmission of information between cells and their extracellular environment. Signaling across integrins is bidirectional, transducing both inside-out and outside-signaling. Integrin activation, a transition from a low affinity/avidity state to a high affinity/avidity state for cognate ligands, is an outcome of inside-signaling. Such activation is particularly important for the recognition of soluble ligands by blood cells but also influences cell-cell and cell-matrix interactions. Integrin activation depends on a complex series of interactions, which both accelerate and inhibit their interconversion from the low to the high affinity/avidity state. There are three components regarded as being most proximately involved in integrin activation: the integrin cytoplasmic tails, talins and kindlins. The participation of each of these molecules in integrin activation is highly regulated by post-translation modifications. The importance of targeted phosphorylation of integrin cytoplasmic tails and talins in integrin activation is well-established, but much less is known about the role of post-translational modification of kindlins. The kindlins, a three-member family of 4.1-ezrin-radixin-moesin (FERM)-domain proteins in mammals, bind directly to the cytoplasmic tails of integrin beta subunits. This commentary provides a synopsis of the emerging evidence for the role of kindlin phosphorylation in integrin regulation. Integrins, the subject of this collection, serve as primary portals of communication between cells and their extracellular environment. Information transfer across integrins is bidirectional; signaling is both inside-out and outside-in. Productive inside-out signaling results in integrin activation, a transition from a low affinity/avidity state to a high affinity/avidity state for cognate ligands. Such activation is particularly important for the recognition of soluble ligands by blood cell integrins and for certain cell–cell and cell–matrix interactions e.g., ,2,3,4,5),3,4,52,3Bidirectional signaling across integrins relies heavily upon post-translational modifications (PTMs) with protein phosphorylation being particularly prominent. Activation of serine, threonine and tyrosine phosphokinases are associated with ligation of integrins as is the activation of phosphatase dependent pathways. Within the integrin activation triumvirate of integrin β CT, talin and kindlin, each component is subject phosphorylation. Both the α and β CT are susceptible to phosphorylation e.g., ,19). The. The19])612, K2QW615 and K3QW598 to alanines [The three kindlins (kindlin-1 (K1), (FERMT1), kindlin-2  and kindlin-3 , found in mammals are products of separate genes ,26. Theyalanines ,30. The alanines ,32 help alanines ,34, intealanines ,36,37. Palanines .Kindlins are adapter proteins with many different binding partners giving rise to many different cellular responses ,40. The Crystal structures of kindlins, including their complex with integrin β CT have been reported ,53,54,55Several different mechanisms have been proposed as to how the binding of kindlins to beta subunits activates integrins, but consensus has yet to be reached. Ye et al.  used nan481 of K3 to integrin β3 CT [Mass spectrometry and antibodies specific for phosphorylated amino acids have been used to identify a number of phosphorylation sites in each of the three kindlins. These studies were either targeted, focused on a particular kindlin or derived from global phosphoproteomic studies in specific cell types. Multiple phosphorylation sites have been identified in each of the three kindlins; and serine, threonine and tyrosine modifications have all have been identified . To datein β3 CT .8 and T30 within the F0 subdomain in the regulation of mitotic spindles [8 and T30 is pivotal for normal spindle formation, these PTM are not required for integrin activation as demonstrated using a phosphorylation resistant K1T8T30/AA mutant. K2 has also been shown to control mitotic spindle assembly through a mechanism distinct from that of K1 and depends on α-tubulin acetylation, AKT serine/threonine kinase or paxillin [The eight phosphorylation sites in kindlins depicted in spindles . Orientapaxillin .193 by Src was shown to be an important event in regulating integrin outside-in signaling which controls cell migration and proliferation. Focal adhesion kinase-induced recruitment of Src to focal adhesions leads to Src activation and K2 phosphorylation and in turn regulates tyrosine phosphorylation of paxillin and cell behavior [193 also promotes recruitment of migfilin to focal adhesion, and this event plays an important role in regulation of Src activity in focal adhesions [159, S181 and S666. Mutation of these serines to alanines inhibited the formation of invadopodia in MDA-231 cells on dense fibrillary collagen; conversely, phospho-mimetic mutants stimulated the formation of invadopodia even on low-density collagen [159 by serine/threonine kinase IkB kinase subunit epsilon (IKKe), and K2 phosphorylation-promoted invadopodia formation and metastasis of colorectal cancer [Several functionally important phosphorylation sites have been identified in K2 . Cell adbehavior . K2 phosdhesions . K2 phosdhesions . Phosphocollagen . In colol cancer .482 or S484, which reside in a hyper-variable region of K3, was first identified in hematopoietic cells including human platelets, human erythroleukemia HEL cells and mouse macrophage-like RAW 267.4 cells [484 reacted with agonist-stimulated platelets and HEL cells, whereas an antibody to pT482 did not [482 was identified in the T lymphocyte Jurkat cell line [484 was dependent on integrin-linked kinase binding to K3, and K3 phosphorylation was crucial for αLβ2-induced neutrophil adhesion and recruitment [482 and/or S484 is also critical for its function as a tumor promoter in breast cancer cells, where K3 and its phosphorylation contributed to its tumor promoter activity [Agonist-induced K3 phosphorylation on T.4 cells . Two ind.4 cells  although did not . Howeverell line . In neutruitment . K3 phosactivity .® ). The proteolysis of kindlins represents another line of PTM. Calpain-induced cleavage of K3 in leukocytes was reported [The importance of phosphorylation in controlling the functions of kindlins and their role in integrin activation is just beginning to be recognized. It remains to be seen if phosphorylation of individual residues differs in different cell types and exerts cell-specific effects. This is likely to mark the next step in this emerging area. This commentary focused on phosphorylation per se but other PTM of kindlins can also be anticipated. One such PTM, ubiquitination of K2, which has functional consequences, has already been reported . Talin areported , and, in


These data revealed that Ser324 (KM ∼6 μM) and Thr649 (KM ∼25 μM) were preferential sites for phosphorylation by AKT, whereas Ser348, Ser577, Ser595 (KM ∼10 μM), Ser711 (KM ∼79 μM), and Ser764 were found to be preferred targets for AMPK. Phosphorylation of TBC1D4 by AKT or AMPK did not alter the intrinsic RabGAP activity, but did disrupt interaction with insulin-regulated aminopeptidase (IRAP), a resident protein of GSVs implicated in GLUT4 trafficking. These findings provide evidence that insulin and contraction may regulate TBC1D4 function primarily by disrupting the recruitment of the RabGAP to GLUT4 vesicles.TBC1D4 is a 160 kDa multidomain Rab GTPase-activating protein (RabGAP) and a downstream target of the insulin- and contraction-activated kinases AKT and AMPK. Phosphorylation of TBC1D4 has been linked to translocation of GLUT4 from storage vesicles (GSVs) to the cell surface. However, its impact on enzymatic activity is not well understood, as previous studies mostly investigated the truncated GAP domain lacking the known phosphorylation sites. In the present study, we expressed and purified recombinant full-length TBC1D4 using a baculovirus system. Size-exclusion chromatography and coimmunoprecipitation experiments revealed that full-length TBC1D4 forms oligomers of ∼600 kDa. Compared with the truncated GAP domain, full-length TBC1D4 displayed similar substrate specificity, but had a markedly higher specific GAP activity toward Rab10. Using high-resolution mass spectrometry, we mapped 19 Ser/Thr phosphorylation sites in TBC1D4. We determined Michaelis–Menten kinetics using  Phosal state , 4. The al state , 6. TBC1al state , 7. Previn vitro  and in r. Baculovirus-infected Sf9 cells were cultured, harvested, and the recombinant protein was purified as described under “9Sf9 cells with a purity of ∼80% as determined by densitometry of Coomassie-stained gels. In SDS-PAGE the purified protein appeared as a single band with an apparent Mr of ∼170 kDa (B). In contrast, using size-exclusion chromatography (SEC), the apparent molecular mass of the purified protein corresponded to 608 kDa , or both tagged proteins. As shown in Figure 1D (lower panel), immunoprecipitation of HA-TBC1D4 with HA-antibodies resulted in coprecipitation of both HA-TBC1D4 and FLAG-TBC1D4. To map the interacting domains of TBC1D4, we coexpressed FLAG-tagged full-length TBC1D4 and different HA-tagged domain constructs of TBC1D4  and analyzed protein–protein interactions with immunoprecipitation using HA- and FLAG antibodies. As illustrated in Figure 1E, FLAG-tagged TBC1D4 coprecipitated with the HA-tagged N-terminal PTB domains , the residual CB/GAP domains with the C-terminal tail of TBC1D4  but not with the protein lacking both PTB domains and the 127 amino-acid-spanning C-terminal tail . We further investigated structural features of TBC1D4 complexes by limited proteolysis using low concentration of trypsin and analysis of partial digestion products by mass spectrometry. Interestingly, rapid fragmentation occurred predominantly at tryptic sites close to two phosphorylation sites in TBC1D4, Ser348 and Ser577  of the murine eviously . The conP domain A. Baculo∼170 kDa B. In conFig. 1A; .Figure 1in vitro. Purified GST-Rab10 was loaded with [γ-32P]GTP, incubated with purified full-length TBC1D4 protein, and the rate of [32P]phosphate release resulting from hydrolysis of [γ-32P]GTP was measured as described previously , consistent with previous studies that identified main Rab substrates using recombinant GAP domain expressed and purified from E. coli .We investigated the catalytic activity of TBC1D4 and performed RabGAP assays as described in “eviously . Figure  E. coli . We nextubstrate C.Figure in vivo at 16 Ser/Thr phosphorylation sites in response to insulin stimulation, muscle contraction, and simulation with the AMPK activator AICAR  where corresponding KM values for AMPK were about 20 times higher . Conversely, the AMPK KM value for phosphorylation of Ser595 was sevenfold lower than that for AKT , whereas KM values obtained for phosphorylation of Ser711 by AKT (79.4 μM) and AMPK (117.2 μM) were similar . Relative Vmax values for the phosphorylation sites were mostly in the same order of magnitude for AKT and AMPK  in TBC1D4, and overexpression of the mutant protein (“4P”) have been shown to impair GLUT4 translocation in 3T3-L1 adipocytes, suggesting an inhibitory effect of the TBC1D4 phosphorylation state on its RabGAP activity . We aimed to determine the stoichiometry of TBC1D4 phosphorylation by quantitative phosphopeptide mapping of selected phosphosites by mass spectrometry. Briefly, AKT-phosphorylated TBC1D4 was separated by SDS-PAGE, digested with trypsin, and the resulting peptides were determined as described in “Figure 4B, phosphorylation of TBC1D4 with AKT resulted in a substantial reduction in the abundance of the nonphosphorylated peptide RAHT649FSHPPSSSR and a concomitant increase in phospho-peptide form RAHpT649FSHPPSSSR, indicating that ∼80% of Thr649  in TBC1D4 has been phosphorylated in vitro. Phosphorylated TBC1D4 was then incubated with purified [32P]GTP-loaded Rab10, and the amount of released [32P]phosphate was determined as described under “Fig. 4C). Similar results were obtained with TBC1D4 phosphorylated by AMPK  and phospho-TBC1D4 IMAC-purified from okadaic acid treated Sf9 cells or coexpression of TBC1D4 with constitutively active AKT baculovirus . However, the amount of 14-3-3/TBC1D4 complexes recovered was similar after phosphorylation by AKT or AMPK. We then sought to address whether phosphorylation-induced 14-3-3 binding alters TBC1D4 GAP activity. TBC1D4 was coexpressed with constitutively active AKT2 kinase and purified. As shown in Figure 5C, the GAP activity of phosphorylated TBC1D4 toward Rab10 was not affected by the presence of 12 M excess of 14-3-3.Phosphorylation of TBC1D4 in response to insulin stimulation or muscle contraction has been shown to increase the binding of 14-3-3 proteins to the RabGAP , 21, 22. or AMPK B. HoweveD). As shown in Figure 5E, nonphosphorylated TBC1D4 displayed specific binding to cIRAP, which was completely abolished after phosphorylation with either of the kinases.The cytoplasmic tail of the GLUT4-vesicle resident protein, IRAP (insulin-regulated aminopeptidase), has been shown to interact with TBC1D4 in an insulin-dependent manner . To explFig. 5via increasing the recruitment of GLUT4 transporters to the plasma membrane (in vitro by adding AMPK first for 5 min followed by adding of AKT for another 5 min and quantification of phosphosites by immunoblotting. Ser595 is a preferred site for AMPK (KM 10.5 μM) and comparably a minor target for AKT . Conversely, Ser324 is a preferential target for AKT (KM 6 μM) but a much poorer substrate for AMPK . Prior AMPK phosphorylation enhanced Ser595 phosphorylation by AKT in an additive manner (A). However, prephosphorylation of full-length TBC1D4 with AMPK markedly inhibited subsequent phosphorylation of Ser324 by AKT (B). In TBC1D4, the AKT target site Ser324 is flanked by other potential Ser/Thr residues  that, if phosphorylated, could alter its phosphorylation efficiency. However, using mass spectrometry, we failed to identify corresponding tryptic phosphopeptides that would indicate phosphorylation of any of these residues in addition to Ser324 (data not shown). We therefore investigated a possible interaction of phosphosites by using GST fusion proteins of the second PTB domain of TBC1D4 (PTB2) as substrate. Interestingly, inhibition of Ser324 phosphorylation by AKT after prephosphorylation with AMPK was not observed when purified GST-PTB2 of TBC1D4 was used as a substrate (C).Previous studies demonstrated that prior exercise enhances skeletal muscle insulin sensitivity , 24, 25 membrane , possiblmembrane . To stude manner A. Howeve4 by AKT B. In TBCubstrate C.Figure in vitro. Our results demonstrate that TBC1D4’s GAP activity is independent of its phosphorylation state but may be regulated through AKT/AMPK-dependent targeting to GLUT4 vesicles instead.The 160 kDa RabGAP TBC1D4 plays a critical role in insulin- and contraction-mediated GLUT4 translocation , 28. In 348 and Ser577, where the former is presumably involved in 14-3-3 binding  has been detected in at least two independent studies  and AMPK . Michaelis–Menten kinetics identified Ser324 (KM ∼6 μM) and Thr649. (KM ∼25 μM) as preferential sites for AKT, whereas Ser595 (KM 10 μM) and Ser711 (KM ∼79 μM) were found to be preferred targets for AMPK. The KM values of TBC1D4 sites for AKT and AMPK(α1β1γ1) are consistent with those reported for synthetic peptide substrates such as crosstide (∼16 μM) and SAMS peptide (∼27 μM), derived from AKT/AMPK phosphosites in glycogen synthase kinase-3 (GSK-3) and acetyl-CoA carboxylase (ACC), respectively , a reported target for IGF-1 and insulin  was shown to be phosphorylated in response to insulin  in 3-T3-L1 adipocytes . Importa binding , 41, 42.The insulin-regulated aminopeptidase IRAP is expressed in skeletal muscle and adipose cells and highly colocalized with GLUT4 as it constitutes a resident membrane protein of GLUT4 storage vesicles . Our dat711 induced a subsequent increase in TBC1D4 Thr649 phosphorylation  was cloned into the baculovirus expression vector pAcGS2-6xHis (BD Bioscience) and triple FLAG-pcDNA3.1. GST-cIRAP (Met1 - Thr110) and a baculovirus for a GST-tagged Rab10 were generated as described (Tbc1d4 (XP_006518825.1) was cloned into the pcDNA3.1-3xFLAG and pcDNA3.1-HA. HA-tagged proteins harboring different domains of TBC1D4  were generated by cloning respective PCR fragments into pcDNA3.1-HA. The second PTB domain of Tbc1d4 GST-PTB2; Ser189 - Leu478 was cloned into pGEX-4T.1. All constructs were confirmed by DNA sequencing.The long mouse isoform of murine escribed . Cloningescribed , 17. Thead libitum access to food  and water. Twelve-week-old mice were injected intraperitoneally with either 1 IU/kg Insulin, 250 mg/kg AICAR, or 0.9% sterile saline as control and sacrificed by cervical dislocation after 60 min of stimulation. Tissues were removed and immediately frozen in liquid nitrogen.Mice were kept in accordance with the NIH guidelines for the care and use of laboratory animals, and all experiments were approved by the Ethics Committee of the State Ministry of Agriculture, Nutrition, and Forestry . Three to six C57BL/6J mice per cage were housed at 22 °C and a 12-h light–dark cycle with 318, Ser588, and Thr642  antibodies were obtained from Cell Signaling Technology. Antibodies against FLAG and HA were from Sigma-Aldrich.TBC1D4 and phospho-TBC1D4 SerSf9 Grace supplemented and serum-free (Sf-900TM II SFM) media were obtained from Life Technologies. Sf9 cells (2.5 × 106 cells/ml) were infected with recombinant baculovirus at a multiplicity of infection (MOI) of 10, harvested 72 h postinfection, and lysed by mild sonication at 4 °C in lysis buffer . Coexpression of the His-tagged TBC1D4 and recombinant human constitutively active AKT2 (GST-AKT2ΔPH) was performed with AKT2 recombinant baculovirus at MOI of 7 and TBC1D4 recombinant baculovirus at MOI of about 3 to 5. The phosphatase inhibitor okadaic acid (OA) was purchased from Abcam and used as a concentration of 50 nM for 30 min before harvesting the Sf9 cells expressing TBC1D4. Recombinant His-tagged proteins were purified by IMAC using Ni-NTA resin (Qiagen), eluted with 100 mM imidazole, 150 mM NaCl, EDTA-free protease inhibitor, pH 8, and then dialyzed against 50 mM Tris-HCl, pH 7.4. Only freshly prepared TBC1D4 protein was used in our study. GST-γ14-3-3, GST-PTB2, and GST-TBC1D4 GAP domain were expressed in E. coli BL21-CodonPlus (DE3)-RIL and purified by GSH affinity chromatography (Cytiva) as described  in 50 mM Tris-HCl pH 7.5 at a flow rate of 1 ml/min. Standards used were blue dextrane, 2,000,000 Da for void volume (V0), bovine thyroglobulin, 670,000 Da; bovine γ-globulin, 158,000 Da; chicken ovalbumin, 44,000 Da; horse myoglobin, 17,000 Da; and vitamin B12, 1350 Da. Elution volumes (Ve) were recorded to determine partition coefficients Kav = (Ve − V0)/(Vt − V0), and the apparent Mr of affinity-purified TBC1D4 was calculated by regression analysis of Kav against log Mr of the standards.Proteins were separated by fast protein LC (FPLC) with an ENrich SEC650 column  in a buffer containing 50 mM Tris-HCl, pH 8, 2.5 mM DTT and, 5 mM MgCl2. The matrix was washed with the same buffer to remove unbound GTP, and [γ-32P]GTP bound Rab 10 was eluted in 20 mM Tris-HCl, pH 7.5, 1 mM DTT, 10 mM reduced GSH, 2.5 mM MgCl2 and then incubated with GST-full-length or TBC1D4 GAP domain, respectively, at room temperature (RT) for 30 min. The reaction was stopped with 0.5 M EDTA, radioactive [32P]phosphate was separated by filtration through activated charcoal and measured by scintillation counting as described , and 0.4 mM dithiothreitol (DTT) in a volume of 200 μl as described .Recombinant full-length TBC1D4 was purified by IMAC using Ni-NTA resins and used as a substrate in kinase assays. Constitutively active AKT2 was expressed as a GST fusion protein in eviously , and purescribed , and subvia 1 and 2 h gradients on 50 cm C18-LC columns EASY-Spray  or μPAC . Mass spectra were acquired in data-dependent acquisition mode and HCD as well as ETD fragmentation was used , a general contaminants list (245 sequences), and the His-tagged TBC1D4 sequence, with 10 ppm precursor mass and 0.02 Da fragment mass tolerance. Enzyme was set to trypsin with maximum two missed cleavage sites allowed. Carbamidomethylation of cysteine was set as fixed modification. N-terminal acetylation, methionine oxidation as well as phosphorylation (+79.966 Da) or “heavy” phosphorylation  of serine, threonine, or tyrosine were allowed as variable modifications. The fixed-value PSM Validator  and the ptmRS-node included in Proteome Discoverer were applied. FDR was set to 0.01 (strict). Only high confident peptides and if modified with a site probability of >75% were annotated to proteins within the result file.Mass spectrometry raw files were analyzed with Proteome Discoverer 2.3/2.4 software (Thermo Fisher Scientific). HTsequest search was done against protein FASTA files from 649, (core sequence AHT649FSHPPSSSR) before and after phosphorylation of 2 to 3 pmol TBC1D4 using purified AKT2 at an equimolar ratio for 5 to 10 min. Abundance of nonphosphorylated and phosphorylated peptides was determined by integrating the fractional abundances of all corresponding peptide species related to AHT649FSHPPSSSR and AHpT649FSHPPSSSR. For kinetic analyses, phosphorylation of Ser324, Ser595, Thr649, and Ser711 was determined by immunoblotting using phosphosite-specific antibodies and quantification by ECL (PerkinElmer) and data were used for nonlinear curve fitting to Michaelis–Menten kinetics to estimate apparent values for KM and Vmax.To estimate the phosphorylation stoichiometry of TBC1D4, we determined tryptic peptides and phosphopeptides comprising the major AKT2 phosphosite ThrTbc1d4 constructs and 3xFLAG-Tbc1d4 were lysed using lysis buffer containing 150 mM NaCl, 1% Triton X-100, and 50 mM Tris-HCl, pH 8. Cleared cell lysates containing HA-tagged TBC1D4 were labeled with anti-HA magnetic beads and incubated for 30 min at 4 °C. Subsequently beads were washed with 50 mM Tris, pH 8, and 150 mM NaCl, and the protein complexes were eluted using SDS sample buffer.Magnetic beads preconjugated with anti-HA monoclonal antibodies  were used as previously described . BrieflyE. coli BL21-CodonPlus (DE3)-RIL cells expressing GST-cIRAP and GST-γ14-3-3 were lysed and the cleared lysate was incubated with 100 μl of GSH-Sepharose beads (Cytiva) for 2 h at 4 °C with mixing as described previously  or paired, two-tailed Student’s t-test, as indicated in the figure legends. p values <0.05 were considered statistically significant.Data are expressed as mean ± S.E.M unless indicated otherwise. Nonlinear fitting to Michaelis–Menten was performed by using GraphPad Prism 9 . via the PRIDE (The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium he PRIDE  partner This article contains The authors declare no conflicts of interest in regard to this article.


Crustacean allergy, especially to shrimp, is the most predominant cause of seafood allergy. However, due to the high flexibility of immunoglobulin E (IgE), its three-dimensional structure remains unsolved, and the molecular mechanism of shrimp allergen recognition is unknown. Here a chimeric IgE was built in silico, and its variable region in the light chain was replaced with sequences derived from shrimp tropomyosin (TM)-allergic patients. A variety of allergenic peptides from the Chinese shrimp TM were built, treated with heating, and subjected to IgE binding in silico. Amino acid analysis shows that the amino acid residue conservation in shrimp TM contributes to eliciting an IgE-mediated immune response. In the shrimp-allergic IgE, Glu98 in the light chain and other critical residues that recognize allergens from shrimp are implicated in the molecular basis of IgE-mediated shrimp allergy. Heat treatment could alter the conformations of TM allergenic peptides, impact their intramolecular hydrogen bonding, and subsequently decrease the binding between these peptides and IgE. We found Glu98 as the characteristic amino acid residue in the light chain of IgE to recognize general shrimp-allergic sequences, and heat-induced conformational change generally desensitizes shrimp allergens. Th. Th24]. Here we aligned these TM epitope peptides, and found that there is a conservation of Glu (E), Arg (R), Ile(I), Gln (Q), Leu (L), and Asp (D) residues in these peptides . This in2 after heat treatment. The distance between the α-carbon of the N- and C-terminals was measured and compared as a rough indicator of how protein conformation deviated from its native form. Most of the TM epitope peptides curled up except for T3 and T9, whose terminal distances increased by ~1.5 Å. Most of the TM epitope peptides exhibited increased intramolecular hydrogen bonding after heat treatment except for T1, T5, T8 and T9, whose intramolecular hydrogen bonding did not change.All TM allergenic peptides exhibited considerate conformational changes compared to their native states (25 °C) with root mean square deviation (RMSD) ranging from 2.190 Å to 3.448 Å . Heat trHere a chimeric IgE that contains the variable region recognizing shrimp allergenic TM epitopes in the light chain of the Fab region was constructed . There i2 to −229.70 Å2 upon interaction with the complementary-determining regions.All the native TM allergenic peptides were recognized by chimeric IgE with various degrees of binding affinities , as suggHeat treatment indeed altered the interaction between allergenic peptides and IgE . The majGenerally, Glu98 in the light chain and Arg101 in the heavy chain formed hydrogen bonds with all TM allergenic peptides. Lys57 in the heavy chain formed hydrogen bonds with all TM allergenic peptides except T1, and His31 in the light chain formed hydrogen bonds with all the peptides except T6, T9, T10 and T11. These four amino acid residues play critical roles in IgE-recognizing allergens via hydrogen bonding. Glu98 and His31 in the light chain and Lys57 and Arg101 in the heavy chain form hydrophobic interactions with all the TM allergenic peptides. Leu97 in the light chain formed hydrophobic interactions with all peptides except T10. Tyr37 in the light chain formed hydrophobic interactions with all peptides except T8. Thr99 and Asn33 in the light chain formed hydrophobic interactions with all peptides except T5. His59 in the heavy chain formed hydrophobic interactions with all peptides except T9 and T11. Thr105 in the heavy chain formed hydrophobic interactions with all peptides except T6 and T9. These ten amino acid residues play critical roles in IgE recognition of shrimp allergens via hydrophobic interactions.As identified in previous studies, there are some critical TM amino acid residues involved in the recognition of these TM epitope peptides by IgE . General2) upon interaction with IgE while D142 and Q147 led to SASA exposure compared to the wild-type (as to 101.02 Å2).Here we also performed in silico mutations on some of these allergenic peptides and examined their interaction with IgE . For T7,It is reported that patients with shrimp allergy may also suffer from ingestions of other crustaceans such as lobster, crab, prawn, and crayfish, suggesting a significant similarity of the IgE-binding epitopes in these allergens . Among sAs for residue in the antibody, critical amino acid residues Glu98 and His31 in the light chain and Arg101 and Lys57 in the heavy chain recognize the allergenic peptides via hydrogen bonding. Glu98, Leu97, His31, Tyr37, Thr99, and Asn33 in the light chain, and Lys57 and Arg101 in the heavy chain recognize the peptides via hydrophobic interactions. Moreover, these amino acid residues are the molecular basis of shrimp allergen recognition by IgE. Currently, the majority of studies are focused on identifying the epitope sequences of TM and other allergens while there is no study on the IgE amino acid residues involved in shrimp TM recognition. Thus, to the best of our knowledge, this study offers the first insights into the structural basis of immune responses to shrimp allergenic proteins.in vivo experiments, explained the critical amino acid residues involved in IgE interaction with TM allergenic peptides, and thus offered a molecular view of thermal treatment on the shrimp allergens [Although invertebrate TMs are more stable to heat than non-allergenic vertebrate TMs, heat treatment in general did alter the conformational changes of shrimp TM allergenic peptides in all aspects in this study . This isllergens .In conclusion, there is a conservation of Glu, Arg, Ile, Gln, Leu, and Asp in the major shrimp allergenic TM. Among them, Arg, Glu, Asp, and Lys interact with IgE via hydrogen bonding while Arg, Glu, Leu, Lys, and Asp interact with IgE via hydrophobic interactions. In the shrimp allergic IgE, Glu98, and His31 in the light chain and Arg101 and Lys57 in the heavy chain recognize the allergens via hydrogen bonding while Glu98, Leu97, His31, Tyr37, Thr99, and Asn33 in the light chain and Lys57 and Arg101 in the heavy chain recognize the allergens via hydrophobic interactions. Heat treatment could alter the conformations of TM allergenic peptides, impact their intramolecular hydrogen bonding, and decrease the binding between these peptides and IgE. Our results not only revealed the molecular basis of IgE-mediated shrimp TM allergy but also the mechanism of heat-treated desensitization of TM epitope peptides.


Melospiza melodia) varied between an urban and a rural site. We determined song repertoire size of 24 individuals; 12 were at an urban (‘human‐dominated’) site and 12 were at a rural  site. Then, we compared song repertoire size, note rate, and peak frequency between these sites. Song repertoire size and note rate did not vary between our human‐dominated and agricultural sites. Peak frequency was greater at the agricultural site. Our finding that peak frequency was higher at the agricultural site compared to the human‐dominated site, contrary to many previous findings pertaining to frequency shifts in songbirds, warrants further investigation. Results of our pilot study suggest that song complexity may be less affected by anthropogenic factors in Song Sparrows than are frequency characteristics. Additional study, however, will be required to identify particular causal factors related to the trends that we report and to replicate, ideally via multiple urban–rural pairings, so that broader generalization is possible.Characteristics of birdsong, especially minimum frequency, have been shown to vary for some species between urban and rural populations and along urban–rural gradients. However, few urban–rural comparisons of song complexity—and none that we know of based on the number of distinct song types in repertoires—have occurred. Given the potential ability of song repertoire size to indicate bird condition, we primarily sought to determine if number of distinct song types displayed by Song Sparrows . Our results suggest that song type repertoire size was not affected by urban factors, though urban birds tended to sing songs with a lower peak frequency. Our primary variables of interest for this pilot study were song repertoire size and note rate, which were used to describe song complexity. We predicted that mean song repertoire size at the human‐dominated site would be smaller than at the agricultural site due to increased anthropogenic disturbance at the human‐dominated site. For the same reason, we predicted that individual songs from the human‐dominated site would be less temporally complex (fewer notes per second) compared to songs from the agricultural site. We also compared peak frequency between the sites in order to determine if birds at our human‐dominated site were experiencing selection similar to other urban populations, where peak frequency has shifted upward, probably in response to low‐frequency noise  that bordered Salt Creek and Brummett's Creek. Both creeks flooded during the spring (both years), covering the surrounding fields and likely affecting use by Song Sparrows. Aside from the activity of planting and harvesting crops, little human presence occurred at this site. We recorded Song Sparrows at our agricultural site within a 2‐km‐diameter area. The human‐dominated site was located on the campus of Indiana University, in Bloomington. Though the campus was covered by a relatively large area of green space, it was typical of many university campuses in that there were many sources of noise and artificial light at night, as well as a large proportion of area covered by impervious surface and regularly mowed areas. Humans generally occurred many times each day in Song Sparrow territories at this site. Most Song Sparrow territories on campus were centered along narrow (<10 m) riparian strips bordering Clear Creek or its tributaries, though some territories occurred adjacent to buildings where ornamental shrubs, primarily, provided cover. All of the Song Sparrows that we recorded on campus occurred within a 1.25‐km‐diameter area. We compared noise levels and impervious surface coverage between the human‐dominated and agricultural sites to confirm that the sites did vary regarding factors associated with urban impact.Our field sites were located in Indiana, at 39.17°N, 86.53°W. Field work occurred between April and July in 2018 and 2019. The agricultural site was located 6 km east of the city limits of Bloomington, which is well beyond the distance that young Song Sparrows likely disperse  and between 12:30 and 1:30 p.m. (afternoon session) in the center of five randomly selected Song Sparrow territories at both the agricultural and human‐dominated site. The same points were sampled during both the morning and afternoon at both sites on two different days, at least 4 days apart, when wind speeds were less than 25 km/h. Measurements were made using a ‘Radio Shack, 33–3042’ Super‐Cardioid Dynamic Microphone that was attached to a tripod, so that the top of the microphone was 1 m above the ground. The microphone was connected to a Tascam DR 100MKIII Linear PCM Recorder. For each recording, the gain of the recorder was set to the maximum (‘56.5’), the sample rate was 48 kHz, and the bit rate was 1152 kbps. We calculated ‘average power’ (dB) during a 2‐min period for each recording session by using Raven Pro 1.5  from our study showed that on average all new song types had occurred before 140 song instances were recorded  were visually analyzed to establish the number of song types in each bird's song repertoire. This general approach is commonly used for Song Sparrows . We measured the following variables: peak frequency (frequency with greatest energy), number of notes (note was considered a continuous trace on the spectrogram), and duration (to determine note rate). Because visual analysis of spectrograms is not appropriate for measuring minimum or maximum frequency in urban environments, due to the possibility of error . Statistical tests were considered to indicate a ‘significant difference’ if p was ≤.05.Because our data violated parametric statistical assumptions, we used two‐tailed Wilcoxon Rank Sum tests .33.1p = .01), but not during the afternoon session  and a mean impervious coverage area that was 600% more than at the agricultural site . Note that this p value was automatically ‘continuity corrected’ via the ‘wilcox.test’ function in R, to account for ties.The median song repertoire size at the agricultural site was 8 .The median note rate within songs at the agricultural site was 11.0 . The Hodges–Lehmann estimator indicated a peak frequency difference of 661.3 Hz .The median peak frequency for songs at the agricultural site was 5316.6 Hz  in 11 of 14 Oscine species investigated in one study  between our human‐dominated site and our agricultural site ; Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Project administration (lead); Writing – original draft (lead); Writing – review & editing . Adam M. Fudickar: Conceptualization (supporting); Funding acquisition (lead); Investigation (supporting); Methodology (supporting); Project administration (supporting); Writing – review & editing .