Noniridescent and nonfading structural colors generated from metallic and dielectric nanoparticles with extraordinary optical properties hold great promise in applications such as image display, color printing, and information security. Yet, due to the strong wavelength dependence of optical constants and the radiation pattern, it is difficult and time-consuming to design nanoparticles with the desired hue, saturation, and brightness. Herein, we combined the Monte Carlo and Mie scattering simulations and a bidirectional neural network (BNN) to improve the design of gold nanoparticles’ structural colors. The optical simulations provided a dataset including color properties and geometric parameters of gold nanoparticle systems, while the BNN was proposed to accurately predict the structural colors of gold nanoparticle systems and inversely design the geometric parameters for the desired colors. Taking the human chromatic discrimination ability as a criterion, our proposed approach achieved a high accuracy of 99.83% on the predicted colors and 98.5% on the designed geometric parameters. This work provides a general method to accurately and efficiently design the structural colors of nanoparticle systems, which can be exploited in a variety of applications and contribute to the development of advanced optical materials. Color generation from the assembly of nanometer- or submicron-sized structures has been of great interest for various fields including chromatic displaying ,2,3, colThe structural colors of nanoparticle systems depend on the material, size, shape, and volume fraction of nanoparticles, and the dielectric properties of the surrounding environment. Therefore, by adjusting the parameters as mentioned above, the corresponding spectra, either scattering, transmission, or reflection modified by nanoparticles, can be manipulated to obtain a variety of colors. However, the design of specific properties of nanoparticle systems is usually based on a trial-and-error method , which rThe above studies have mainly focused on the periodic systems whose optical response is obtained using the time-consuming electromagnetic simulations; yet, disperse nanoparticle systems have received little attention. In this work, a multilayered BNN model was built to accurately predict the color generation of gold nanoparticle systems and to inversely design the geometric parameters for the desired colors. The single scattering properties of nanoparticles were calculated by using the Lorenz–Mie theory, the multiple scattering problems between nanoparticles were considered by solving the radiative transfer equation, and the spectrum-to-color conversion was obtained based on the International Commission on Illumination (CIE) color spaces. Our approach captures the complex relationships between the geometric parameters and structural colors with high reliability and accuracy, and can be used for the analysis and optimization design of structural color in nanoparticle systems.r, particle volume fraction fv, and layer thickness h, and three color property parameters:  in the CIE 1976-Lab color space, as key features to establish the dataset for the prediction and inverse design of structural colors. The color property parameters of nanoparticle systems were obtained from a series of simulations, which include Mie scattering calculation, Monte Carlo simulation, and spectrum-to-color conversion. Then, a multilayered BNN was developed and trained to predict the structural colors of nanoparticle systems and inversely design the geometric parameters for the desired colors.Our data generation process and machine learning strategy for structural colors of nanoparticle systems are schematically illustrated in r was set to 5–100 nm in 5 nm intervals, the range of fv was set to 1 × 10−6–3 × 10−5 in 1 × 10−6 intervals, and the range of h was set to 0.5–10 mm in 0.5 mm intervals. A total of 12,000 parameter combinations were generated and the corresponding transmission spectra from 360 to 830 nm  were obtained by combining the Mie scattering and Monte Carlo calculation. Then, the color property parameters  were obtained by conducting the spectrum-to-color conversion. This dataset was divided into three groups for training (9600), validation (1200), and testing purposes (1200). All colors generated in the training and validation dataset are plotted in the CIE 1931-XYZ chromaticity diagram in The range of μext, scattering coefficients μsca, and scattering phase functions Φ, are calculated as [μext,m= 4πκ/λ is the extinction coefficient of water, κ is the imaginary part of the refractive index of water, λ is the incident light wavelength, and μext,p, μsca,p, and Φp are the extinction coefficient, scattering coefficient, and scattering phase function of gold nanoparticles, respectively. In the framework of the independent scattering approximation, the optical properties of dilute monodisperse gold nanoparticles can be calculated as [Qext,p, Qsca,p, and Φp,s are the extinction efficiency factor, scattering efficiency factor, and scattering phase function of single gold spheres, respectively, and can be calculated using Lorenz–Mie theory [For dilute gold nanoparticles embedded in water, the total optical properties of nanoparticle systems, which include the extinction coefficients lated as :(1)μext=lated as :(4)μext,e theory . The diee theory  dataset,e theory  was usedIλ is the spectral radiative intensity in the direction of s, bIλ is the spectral blackbody intensity, and T(λ) of the layer can be calculated as:N0 is the total number of the photons that are incident on the layer, and tN(Ωt) is the number of photons that are collected with the use of detectors positioned in the hemispherical space outside the lower surface in the solid angle Ωt. In this study, 106 photons were used for each wavelength to obtain accurate results without using too much computation time.Given the optical properties of the nanoparticle systems, the radiative transfer problems  can be accounted for by solving the radiative transfer equation (RTE) ,41:(7)dIo method ,42,43. Ao method . After iX, Y, and Z are defined by [ID65(λ) is the spectral power distribution of the standard D65 illumination. The colors perceived by the human eye result from the interplay between the sensitivity of the eye’s three cone cells, the spectral intensity of the light source, and the spectral reflection or transmission from the object. The CIE-1931-XYZ and CIE 1976-Lab color spaces were used to assess the generated colors of nanoparticle systems ,45,46. Afined by ,45,47: and greenness (−), and b represents the yellowness (+) and blueness (−). The conversion relation between  and  is stated as [nX, nY, and nZ being the tristimulus values of a reference white object, and:The normalized values fication . The CIEtated as :(15)L=1165 and a 2° standard colorimetric observer, they amount to nX = 95.049, nY = 100, and nZ = 108.891. In contrast to X, Y, and Z, the tristimulus values L, a, and b allow the quantitative comparison of colors. The CIE color difference function ΔE can be defined as the Euclidean distance between two vectors  and  [E ≤ 1.0 usually represents identical colors [For the standard illuminant D a′, b′) ,48.(20)l colors ,48.A multilayered BNN model, first proposed by Liu , was utiL, a, b) based on the input geometric parameters . The number of layers and neurons in the hidden layer are determined by neural network training and construction, and the training is performed by minimizing the loss function, which is defined as the mean square error (MSE) between the predicted values :N is the number of the entries of color data. The training of the INN subsequently uses the fixed pre-trained FNN model as a color predictor to evaluate the inverse design output. Note that the loss function of INN compares the output color properties  rather than the designed geometric parameters , as shown in Prior to training, the geometric properties of nanoparticle systems and the corresponding color properties are normalized to improve the convergence rate and reduce the training error. In the training process, the FNN is first trained to achieve accurate prediction of the structural colors  and true values . The results obtained by the FNN show a high consistency with those obtained by the theoretical simulations, which demonstrates that the FNN has a good prediction ability for the sample of the test set. To better evaluate the performance of the FNN, the color differences ΔE between the predicted colors and true colors and their statistical distributions are illustrated in E < 1.0 [In order to test the accuracy and generalization ability of the FNN, 1200 groups of completely new test data were used and analyzed.  E < 1.0 ,48. As sL, a, and b are 0.036, −0.75, and −0.701, respectively, which indicates that the particle radius shows strong negative correlation with the chromaticity and has weak correlation with the lightness. On the contrary, the layer thickness and particle volume fraction show strong negative correlation with the lightness and weak positive correlation with the chromaticity. These results are consistent with the theoretical analysis in To further investigate the relationship between the geometric parameters and color properties, a heat map of the Pearson correlation coefficient matrix is illustrated in Overall, the application of FNN can perform the accurate prediction of structural colors of nanoparticle systems with high reliability and efficiency. Although the acquisition of 12,000 groups of training data requires a certain computational cost, after the initial one-time computation investment, the FNN method has a great advantage in the computational efficiency and can be easily and flexibly used in the related applications.L, a, b) are taken as the input, and the output is the designed geometric parameters  denoted as the intermediate layer. The output of the pre-trained FNN is the predicted colors  obtained based on the designed geometric parameters . The weights in the INN are optimized by minimizing the loss function defined as the mean square error between the output predicted colors  and the input desired colors . After optimization design, an INN model, which has 4 hidden layers with 100 neurons each layer, is constructed, as shown in Inverse design is much more complicated because different geometric parameters of nanoparticle systems may yield the same color, and “one-to-many” results may lead to untrained models. Introducing a bidirectional neural network with a tandem strategy is an effective way to solve the “one-to-many” problem ,49. As sth epoch. After a sudden drop, the downward trend of the loss function gradually slows, and the values of training and validation loss are 8.03 × 10−5 and 0.0399, respectively, at the 5000th epoch. Note that the value of validation loss 0.0399 corresponds to the color difference ΔE = 0.1997, showing that the INN performs well in the training process. We again use 1200 groups of new data to test the accuracy and generalization ability of the INN. The desired color properties  are input into the INN to predict the designed geometric parameters . Then, the data of  are fed to the FNN to obtain the designed color properties . L, a, b) and inversely designed color properties , and the corresponding results of color differences ΔE. Good consistency between the desired and inversely designed color properties is observed. Most of the color difference values (90%) are below 0.5, and only 1.5% of the values are larger than 1.0, indicating that the inverse design of structural colors of nanoparticle systems based on the INN is highly accurate (with a high accuracy of 98.5%).As shown in E between the desired and designed colors are very small, as shown in Furthermore, we randomly used three groups of data in the test set to evaluate the performance of the INN. To summarize, our inverse design strategy based on a BNN bypasses the complicated simulations in searching for geometric parameters of nanoparticle systems for the desired colors, and so greatly accelerates the design of structural colors with high accuracy and efficiency. Our work provides a general method to accurately and efficiently design the structural colors of nanoparticle systems. Meanwhile, our model can be further extended to more complex nanoparticle systems by using additional information such as particle type, shape, and size distribution.In summary, we combined the Monte Carlo, Mie scattering simulations, and bidirectional neural network model to successfully improve the design of structural colors in gold nanoparticle systems. A FNN model with 3 hidden layers including 200 neurons each layer, and an INN model of 4 hidden layers containing 100 neurons each layer were constructed. The proposed neural network could accurately capture the complex relationships between the geometric parameters and structural colors. Taking the human chromatic discrimination ability as a criterion, our proposed neural network achieved a high accuracy of 99.83% on the predicted colors and 98.5% on the designed geometric parameters. Our work demonstrates the applicability of the deep learning method for the prediction and inverse design of structural colors in nanoparticle systems, and will open a new way for the further exploration of the related applications about nanoparticle-based materials.


High hydrostatic pressure (HHP) treatment is a non-thermal processing technology, which is widely used in the food processing field at present. In this study, the effects of HHP treatment (100~500 MPa for 5 min) on the physicochemical properties, texture parameters, and volatile flavor compounds of oysters were investigated. The results showed that HHP treatment increased the water content while reducing the crude protein and ash content of the oyster. Texture parameters showed that HHP treatment improved the hardness, springiness, chewiness, and cohesiveness of oysters, compared with the control group. In addition, the saturated fatty acid (SFA) content was slightly increased after HHP treatment, while the difference in monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) content was not significant. Furthermore, HHP increased hexenoic aldehyde, 2,4-heptadienal, 1-octene-3-ol, and 2-octen-1-ol and decreased the contents of 3. 6-nadien-1-ol, 3-octanone, and 2-undecanone, suggesting that HHP might inhibit the fishiness of oyster and showed a positive effect on its flavor. Based on the above results, HHP improved the edible qualities such as texture properties and volatile flavor of oysters. This meets the requirements of consumers on the edible quality of seafood and provides new ideas for the development of seafood. Crassostrea gigas) is a kind of nutritious food, which constitutes a necessary ingredient for a healthy diet [Aquatic food is rich in nutritional value, widely consumed in the world, and has great consumption potential ,2. Oystethy diet ,4. The fthy diet . In addithy diet ,7. Howevthy diet . Every ythy diet ,10. Oystthy diet ,11.Vibrio vulnificus and other pathogenic microorganisms in oysters were eliminated [High hydrostatic pressure (HHP) treatment is a non-thermal processing technology that has attracted widespread attention in the field of food processing and preservation ,13,14,15iminated ,22. Moreiminated . HoweverBesides nutrition and freshness, texture characteristics and volatile flavor are also important factors for consumers’ acceptability of seafood products . The forThe proximate composition changes after different pressure treatments are shown in The pH value of aquatic products is an important indicator to evaluate the quality of food . As showp < 0.05). HHP treatment has been reported to cause degeneration and deformation of myofibrillar protein. With the increase of pressure, the morphology of myofibrillar protein usually changes from tight aggregation to dense uniformity [Hardness is an internal binding force for food to maintain its own shape . As showiformity . During iformity . Moreoveiformity .p < 0.05). Springiness has been reported to be closely related to sarcoplasmic proteins. Sarcoplasmic proteins form insoluble precipitates at a pressure above 140 MPa. In addition, when the mass concentration of sarcoplasmic protein was higher than 50 mg/mL, the protein formed a gel, and the strength of the gel increased with the increase of pressure, which directly led to the increase of springiness [Springiness reflects the degree of recovery of the oyster after being deformed by an external force, and the springiness of oyster closed muscles is positively related to the binding force between muscles . Table 2inginess . Furtheringiness .p > 0.05). However, as the pressure increased to 400 MPa, the chewiness of oysters increased significantly (p < 0.05). It was reported that the chewiness of chicken breast was improved after high-pressure treatment (300–600 MPa), which is similar to the present results [Chewiness represents the tenderness of food. The effect of HHP treatment on the chewiness of oysters is shown in  results . In this results .p < 0.05) when the pressure was greater than 300 MPa . Therefore, the ratio of n-3 PUFA to n-6 PUFA in the HHP treatment group was significantly higher than that in the control group (p < 0.05). The levels of long-chain n-3 and n-6 fatty acids  and their ratios (n-3/n-6) are considered important to human health [Marine lipids contain large amounts of long-chain triglycerides with high levels of unsaturated fatty acids, as well as high levels of unsaturated phospholipids, which are usually closely related to total lipid levels . In addin health .Principal component analysis (PCA) is a method used to determine the similarity between objects in a two-way data set (samples of sensors). The DI value indicates the degree of separation between samples. As shown in The GC–MS elution peak was searched through the database NIST0 8.L spectral library, and the composition and concentration of volatile components during different HHP treatments were further obtained through quantitative analysis. Twenty-nine volatile compounds were detected by HS–SPME/GC–MS, including seven aldehydes, five alcohols, four esters, two ketones, seven hydrocarbons, and four other classes of compounds . A totalThe amount and relative contents of volatile components of HHP-treated oysters and the control group are shown in Aldehydes play an important role in the flavor of oysters . In the Alcohol compounds have a higher odor threshold, so they contribute less to flavor unless the concentration is high or unsaturated alcohol . A totalCompared to alcohols, ketones have a lower threshold. Microbial, lipid oxidation, or amino acid degradation may produce volatile ketones . In thisThe highest content of volatile substances in the detected oyster samples are hydrocarbons. However, due to the higher odor threshold of hydrocarbons, they have little effect on the overall flavor of oysters .Crassostrea gigas) were purchased from the aquatic product market near Ocean University of China in December 2019. All oyster samples were processed experimentally within 24 h after harvest. Then, 48 oysters  with full gonads were selected for subsequent experiments. They were all washed with purified water, put into polyethylene plastic bags, and then vacuum-heat-sealed.Adult oysters , with water as the pressurized medium. Single-cycle treatments with different pressure, at 100, 200, 300, 400, and 500 MPa, were applied to oyster samples, the holding time was 5 min, and 0.1 MPa was used as the control group. The speed of pressure regulation was 100 MPa/min, and the time for maintaining the pressure was 5 min. Subsequently, one part was used for the determination of texture, and the other part was stored at −80 °C for the determination of biochemical and volatile components. Each HHP treatment condition was completed in three separate runs.Moisture was determined by drying (at 60 °C) to constant weight. The crude protein content was determined by using a fully automatic Kjeldahl analyzer . Crude fat was extracted through a Soxhlet extractor, according to previous reports . GlycogeThe oyster sample was homogenized with deionized water. The pH value of oysters after HHP treatment was measured with a pH meter .Fatty acids were determined according to previously reported methods, which were slightly modified . BrieflyThe adductor muscle part of the oyster was selected for measurement to ensure the uniformity of the sample. The texture meter , equipped with an aluminum cylindrical probe (diameter P/36–50 mm), was used to tested texture changes of the oyster. The oyster samples were compressed at a compression speed of 5 mm/s to 80% of the initial sample, and the test time was 5 s. Each oyster sample was evaluated by four texture parameters, including hardness, springiness, chewiness, and cohesiveness [Oyster flavor substances were determined according to previous methods . PEN 3 eThe headspace solid-phase microextraction (HS-SPME) was used to extract the volatile compound components of oysters based on the previous method with slight modification . Brieflyp < 0.05) of all data were obtained through the Turkey test. The iMPact of HHP treatment was evaluated by using ANOVA and SPSS14.0 software .The effect of different HHP treatments  on oyster samples was analyzed by one way of variance. To analyze the effect of HHP treatment on the physical and volatile substances of oyster samples, the mean differences (Overall, HHP treatment increased the water content of the oysters, which led to a decrease in the crude protein and ash. HHP also slightly changed the composition of fatty acids. Texture parameters showed that HHP significantly changed the texture of oysters by increasing hardness, springiness, chewiness, and cohesiveness. Moreover, HHP treatment increased the quantity and concentration of volatile components and improved the flavor of raw oysters. The present research results provide a reference for the influence of HHP on oyster flavor and also provide a new idea for the development of aquatic products.


Furthermore, there was no significant difference in the normalized peak torques, maximum work done and muscle thickness of the PL and TA between groups. The CAI side demonstrated a delayed muscle contractile response of the PL when compared with the healthy group although there was no difference in muscle strength and muscle size. Clinicians should consider the muscle contractile response of the PL for rehabilitation of the ankle evertor with CAI.This study aimed to investigate the muscle contractile response of the peroneus longus (PL) and tibialis anterior (TA) in groups with and without chronic ankle instability (CAI) using tensiomyography. Twenty-three adults, 12 with CAI and 11 healthy participants, participated in this study. All subjects underwent a tensiomyographic assessment of the PL and TA to measure delay time, contraction time and maximal displacement. The ankle evertor and invertor normalized peak torques, maximum work done and muscle thickness of the PL and TA were calculated. The delay time and contraction time of the PL in the CAI side were significantly higher than those in the healthy group ( Lateral ankle sprains are one of the most frequent injuries in trauma ,2. Many Unintentional ankle perturbations such as a trapdoor with the use of electromyography (EMG) is a popular way to test the function of muscle responses in individuals with CAI ,14. SuchTensiomyography (TMG) has recently been applied to evaluate the mechanical characteristics of skeletal muscles in a convenient and non-invasive way . With thHowever, to the best of our knowledge, there are no studies that have examined the muscle contractile response of the lower extremities of CAI subjects with TMG. The purpose of the present study was to investigate the muscle contractile response in the PL and TA muscles of subjects with CAI. Our hypothesis was that the PL of the CAI group would be lacking muscle response and contraction compared with the healthy group.We conducted a cross-sectional study to investigate neuromuscular responses on the CAI and non-CAI side of subjects with CAI. This study was done in the laboratory of Hiroshima University. The participants in this study included physically active university students. All dependent variables including the TMG scores for the PL and TA, the evertor and invertor mean normalized peak torques and the mean normalized maximum work done at 60°/s and 180°/s and the muscle thickness for the PL and TA were compared between the CAI and non-CAI sides of the CAI group and the healthy group (the independent variables).Twelve young subjects (11 men and 1 woman) with CAI and 11 healthy subjects (9 men and 2 women) with no history of a lateral ankle sprain were included in this study . In the This study was authorized by the Institutional Review Board at Hiroshima University (ID number: E-1905) and all of the subjects provided signed informed consent to participate in this study.® . Based on a study by Delagi et al., the anatomical position of the sensor was normalized to all subjects [The method of measuring TMG was the same for both groups. The investigator, T.T., with three years or more of TMG experience, took all of the measurements. A portable device was percutaneously applied to generate electrical stimulation, which induced a muscle contraction that was detected by a digital transducer placed on the muscle belly. The delay time (Td), contraction time (Tc) and muscle displacement (Dm) of the PL and TA were collected by TMG  . The TMGsubjects . The selsubjects . Our resWe investigated the isokinetic evertor and invertor mean normalized peak torque (Nm) using the Biodex System 4 . Each subject sat on the adjustable seat of the dynamometer and elevated the test leg with the support arm below the knee. The foot was positioned on a footboard and fixed by two Velcro straps. All of the subjects underwent the test barefoot. The subtalar joint was placed in a neutral position by aligning the axis of the dynamometer to cross the sagittal axis of the joint. A rubber heel cup provided stability on the footboard and adjustable thermoplastic stays were put on the medial and lateral boundaries of the foot. A pair of standard Velcro straps placed diagonally held the trunk steady and a single strap fixed the hip. The arms were placed crossed on the chest and the opposite leg was put on the support arm attached to the chair. The ankle joint range of motion was measured with the subject on the dynamometer. The setting of the end range was normalized for all test subjects from a 40° eversion to a 45° inversion. To eliminate the influence of gravitational effects on the data, the weight of the leg of each subject was measured and the data were corrected within the Biodex software. Isokinetic testing of the evertor and invertor mean normalized peak torque was performed at a velocity of 60°/s and 180°/s with a concentric contraction of the ankles. A 2 min interval was provided between the warm-up and the test. The subjects were instructed to test their best effort. All subjects were tested five times and the evertor and invertor mean normalized peak torques and mean normalized maximum work done were recorded by the Biodex software. Each value calibrated with body weight (Nm/kg) was employed during the data analysis to correct for body weight.An ultrasound unit  with an 8 to 12 MHz range linear transducer, 8 MHz frequency, 4 cm image depth and 1.5 cm depth of focus was used to longitudinally visualize and obtain images of the muscle thickness in the PL and TA. Each subject was evaluated in the prone position for the PL muscle and in the supine position for the TA muscle. The PL muscle thickness images were captured at 50% of the distance from the fibular head to the lateral malleolus ,25. The 2) statistics and the post-hoc observed power was generated by G*Power 3.1 . For the follow-up, a Tukey post-hoc analysis was carried out to statistically analyze the difference between the CAI side, non-CAI side and the healthy group. Cohen’s d statistics were calculated as the effect sizes for post-hoc comparisons. The significance level was defined at 5% (p < 0.05). SPSS version 23.0 for Windows  was used for analyzing the data. A pre-study sample size was estimated from the Td of the PL of three legs in the CAI side (mean 18.66), three legs in the non-CAI side (mean 16.30) and three legs in the healthy group (mean 16.12) with an alpha level p < 0.05 and power of 0.8 using G*Power 3.1 because there was no previous study that used the TMG value of the PL as an outcome. A minimum of 33 subjects was calculated to be needed.All outcomes were analyzed using a normal analysis with a Shapiro–Wilk test. An unpaired t-test was used to compare the demographic information of the subjects between the CAI and healthy groups. The comparisons of the TMG value, mean normalized peak torque, mean normalized maximal work done, muscle thickness of the PL and TA between the CAI side and non-CAI side and healthy subjects were conducted using a one-factor ANOVA test. The effect sizes were calculated using eta-squared (ηThe study ultimately included 12 subjects with CAI and 11 healthy subjects. Of the demographic characteristics, there were significant differences only in the CAIT and number of lateral ankle sprains in both groups. The comparison of the TMG values of the PL and TA between the CAI and healthy groups are shown in The comparisons of evertor and invertor mean normalized peak torques and the mean normalized maximum work done at 60°/s and 180°/s between the CAI and healthy groups are presented in The most important result of this study was that the Td and Tc of the CAI side were significantly higher than those of the healthy group in the PL, even though there were no differences in the muscle strength or muscle size. In contrast, no significant difference in any of the TMG values of the TA could be found among the two groups. According to our knowledge, this is the first study to compare the TMG value of the PL and TA between CAI and healthy populations. This novel investigation of the muscle contractile response in CAI subjects can provide insights for therapists and researchers who treat patients with lateral ankle sprains and CAI.In the present study, the TMG values of the PL in the CAI side were significantly higher in Td and Tc compared with those of healthy subjects. Previous investigations have shown that Td indicates the muscle fiber conduction velocity. Tc is the time from the onset of the muscle contraction to the peak contraction and it reflects the generation of the speed of force ,27. ConsThere were no significant differences between the mean normalized peak torques and the mean normalized maximum work done of the ankle evertor and invertor at both 60 and 180 degrees between the CAI and healthy groups. As a lateral ankle sprain is due to an over-inversion of the ankle, most investigators involved in the study of CAI have given attention to the ankle evertor strength . Cho et No differences between the CAI and healthy groups were observed for the muscle thickness of the PL and TA. Our results were similar to those of studies that evaluated muscle volume and strength of the lower legs through magnetic resonance imaging in subjects with/without CAI . AdditioThis study has a few limitations. The first limitation was the small sample size. Experiments with a sufficient number of subjects are necessary for future research. Second, all measurements were performed in stationary conditions and therefore differed from dynamic characteristics on the field. Third, we focused on the PL as an evertor muscle and the TA as an invertor muscle but other muscles also perform these movements. Fourth, it is important to note that this was a cross-sectional study; thus, it is unclear whether the larger Td and Tc in the PL were the result of CAI or whether athletes with a larger Td and Tc in the PL are predisposed to CAI. Prospective studies may provide an answer to this question. Given that current research has identified muscle contractile response deficits in CAI individuals primarily through methods that confirm the muscle contractile response to the direct electrical stimulation of the skeletal muscle, advanced experiments investigating particular neuromuscular functions in subjects with CAI could be insightful.In conclusion, the present findings demonstrated the impaired muscle response and muscle contraction of the PL, which indicated the requirement for ankle evertor training for CAI. Furthermore, the clinical implications of this study suggested the significance of rehabilitation of the muscle response and contraction speed. Additional studies are required to determine the appropriate rehabilitation program for improving the muscle contractile response in CAI.


Slitrk5), a transmembrane protein with few identified functions, as a negative regulator of hedgehog signaling in osteoblasts. Slitrk5 is selectively expressed in osteoblasts and loss of Slitrk5 enhanced osteoblast differentiation in vitro and in vivo. Loss of SLITRK5 in vitro leads to increased hedgehog signaling and overexpression of SLITRK5 in osteoblasts inhibits the induction of targets downstream of hedgehog signaling. Mechanistically, SLITRK5 binds to hedgehog ligands via its extracellular domain and interacts with PTCH1 via its intracellular domain. SLITRK5 is present in the primary cilium, and loss of SLITRK5 enhances SMO ciliary enrichment upon SHH stimulation. Thus, SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts that may be attractive as a therapeutic target to enhance bone formation.Hedgehog signaling is essential for bone formation, including functioning as a means for the growth plate to drive skeletal mineralization. However, the mechanisms regulating hedgehog signaling specifically in bone-forming osteoblasts are largely unknown. Here, we identified SLIT and NTRK-like protein-5( Hedgehog signaling is essential for bone formation. Here, the authors show that the transmembrane protein SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts, suggesting it may be a potential therapeutic target to enhance bone formation. As many of these effects appear to be inherent to the molecular targets of these agents, ultimately addressing this issue will require identification of new therapeutic targets to increase bone formation. In this respect, it is notable that while the Hedgehog (Hh) pathway plays a fundamental role in bone development and homeostasis, it has yet to be therapeutically harnessed for skeletal disorders. The Hh pathway is initiated through the binding of Hh ligands to the transmembrane receptor Patched1 (PTCH1), relieving its repressive effects on Smoothened (SMO). Activated SMO moves to the primary cilium where it acts through several intermediates to ultimately activate GLI family member transcription factors2. GLI transcription factors, primarily GLI1 and GLI2, subsequently enter the nucleus to activate downstream target gene expression, which includes transcriptional feedback regulation of Hh pathway components themselves 3. Disruption of Hh signaling leads to multiple bone defects. Ihh deficient mice display dwarfism with disruptions in growth plate structure4. Ptch1 haploinsufficiency leads to increased bone mass in mice, which is driven by enhanced osteoblast responsiveness to Runx25. Conversely, Gli1 haploinsufficiency causes decreased bone mass and impaired fracture healing in mice6. In addition, mice with an upregulation of Hh signaling in mature osteoblasts display both increased bone formation and excess RANKL-driven osteoclastogenesis, which results in enhanced bone resorption and reduced bone mass7. Given the central role of the Hh pathway in osteoblasts, proper regulation of this pathway is essential. However, little is known about how the responses of osteoblasts to Hh ligands is “fine-tuned”.Current treatments for osteoporosis all have major limitations, which includes rare but severe toxicities, limits on maximum duration of therapy, efficacy in certain anatomic sites, or inducing a low bone turnover state that is undesirable in some contexts, such as during repair of skeletal injury8. While there have been relatively few studies of SLITRK family members, studies to date show that Slitrks are highly expressed in the central nervous system and play roles in neuronal survival, neurite growth and synapse formation11. In mice, genetic deletion of Slitrk5 leads to defects in corticostriatal neurotransmission and obsessive–compulsive disorder-like behaviors12. Here we find that loss of Slitrk5 enhances osteoblast differentiation and function in vitro and in vivo by directly regulating Hh signaling in osteoblasts. Slitrk5 represses the expression of downstream Hh target genes by its direct interactions with Shh and Ptch1. Taken together, this identifies Slitrk5 as a novel Hh co-receptor that represses Hh signaling specifically in osteoblasts to regulate bone formation.We performed an initial screen for transmembrane proteins displaying selective expression in osteoblasts, as this gene set will be enriched for novel receptors or co-receptors that may be druggable, and identified SLITRK5 as a co-receptor that regulates Hh signaling. The SLITRK family, composed of SLITRK1 through SLITRK6, are type I single pass transmembrane proteins. Their nomenclature is based on containing N-terminal extracellular leucine-rich repeat (LRR) domains, similar to those of Slit proteins, and an intracellular carboxyl terminus that has sequence similarity to neurotrophin receptors (Trks)14. Slitrk5 was identified as specifically expressed in osteoblasts, apart from its robust expression in the nervous system, and it has only very modest expression in osteoclasts and bone marrow cells   Fig. . To inveing Fig. . Likewissts Fig. . In addists Fig. . Thus, SSlitrk5 is critical for brain-derived neurotrophic factor (BDNF)-dependent signaling in neural cells15. To assess whether Slitrk5 regulates osteoblast differentiation through BDNF signaling, WT and Slitrk5−/− calvarial osteoblasts were treated with BDNF over the course of osteoblast differentiation. BDNF did not impact osteoblast differentiation in either WT or Slitrk5−/− cells . Loss of Gnas increases Hh signaling, leading to enhanced osteoblast differentiation21. Another regulator named speckle-type POZ protein (Spop) is an E3-ubiquitin ligase adapter, regulating the ubiquitination and degradation of GLI322. Spop-deficient osteoblasts showed increased GLI3 repressor levels and decreased Hh signaling, resulting in osteoblast differentiation defects23. Here, we identify Slitrk5 as a novel regulator of Hh signaling in osteoblasts. Slitrk5 acts at the most proximal steps in Hh signaling, acting upstream of SMO at the level of SHH and the PTCH1 receptor complex, enhancing SHH-induced osteoblast differentiation.Hh signaling is progressively decreased as osteoblasts mature. This prevents excessive bone resorption as activation of Hh signaling in mature osteoblasts upregulates RANKL expression which drives osteoclastogenesis and bone resorption26. Hedgehog interacting protein (HHIP) attenuates Hh signaling by competing with PTCH1 for binding to HH ligands29. HHIP is induced by Hh signaling, forming a negative regulatory feedback loop27. Similarly, Glypican-3 (GPC3) competes with PTCH1 for Hh ligand binding, inhibiting Hh signaling30. The binding affinity of SLITRK5 for SHH (Kd ≈ 40 nM) is similar to that of GPC3 for SHH (Kd ≈ 32 nM), arguing that SLITRK5 binds to Hh ligands at a physiologically relevant concentration. Both of these negative regulatory co-receptors have slightly lower affinity than that of positive regulatory co-receptors such as HHIP-SHH (Kd ≈ 6 nM)30, which would suggest that with increasing ligand titration, first positive and then negative-regulatory pathways are engaged. This would allow productive signaling to be engaged first and then negative regulatory pathways are increasingly recruited with higher ligand concentrations. In addition, Slitrk5 is also transcriptionally upregulated by Hh signaling, suggesting that SLITRK5 forms a negative regulatory feedback loop in response to Hh stimulation. This also fits the overall pattern that many Hh pathway components, such as Gli1 and Ptch1, are themselves upregulated in response to Hh pathway activity.Here we identify SLITRK5 as a Hh co-receptor that acts at the level of the primary Hh receptor PTCH1 to regulate Hh signaling. SLITRK5, therefore, joins other Hh co-receptors including BOC, CDO, and GAS1Cdo and Boc displayed different expression patterns during mouse embryonic development and mice with mutation of Cdo exhibited holoprosencephaly while Boc mutants showed defective commissural axon guidance33, suggesting the regulation of Hh signaling is cell context-dependent and Hh co-receptors may serve to provide tissue and context-specific “tuning” of Hh responsiveness. In line with this, we show here that, outside of the central nervous system (CNS), Slitrk5 expression is largely restricted to osteoblasts. Accordingly, Slitrk5−/− mice displayed increased bone formation but no other obvious Hh signaling related phenotypes outside of bone.Slitrk5 is also expressed in neural tissues, it is possible that Slitrk5 also modulates Hh signaling in neural cells and is therefore relevant to the oncogenesis of Hh pathway driven CNS tumors such as medulloblastoma. Supporting this, Slitrk5 expression is upregulated in tumors marked by active Hh signaling, such as Hh dependent medulloblastoma16. Similarly, missense mutations in SHH and Slitrk5 have been implicated in the attention deficit hyperactivity disorder35, raising the possibility that SLITRK5-regulation of Hh signaling contributes to disease processes outside of bone.Given that 37 and that this study nominates SLITRK5 as a candidate therapeutic target relevant to this context. Hh signaling is activated during fracture healing and expression of a constitutively active SMO mutant in Col1-cre targeted osteoblasts led to enhanced bone formation in the fracture callus, suggesting that augmented Hh signaling can promote fracture healing36. A decrease in total Hh signaling activity has also been implicated as contributing to the age-related declines in fracture healing capacity and local osteoblast generation37. In addition, the hedgehog agonist purmorphamine enhanced bone regeneration in a calvarial defect mouse model36. Meanwhile, we here report that deletion of the negative Hh regulator Slitrk5 improved bone fracture healing. Thus, Hh signaling may have therapeutic importance for fracture healing. Given the narrow and osteoblast selective expression pattern of Slitrk5 alongside the absence of Hh associated phenotypes in nonskeletal tissues, targeting SLITRK5 is promising for offering an osteoblast-specific means to modulate Hh responses to enhance osteoblast differentiation and skeletal repair.There are currently very limited therapeutic drug options for bone repair, thus it is notable both that prior studies identify Hh as a key pathway regulating fracture repair−/− mice were previously described12. All experiments were performed according to the guidelines approved by the Animal Care and Use Committee of the Weill Cornell Medical College. All relevant ethical regulations have been complied with for animal testing and research. All mice were maintained in 12/12 light/dark cycle at room temperature of 20.5–22.5 °C and humidity of 30–70% and had ad libitum access to dry laboratory food and water.Slitrk5hSLTRIK5-ECD (2 µg/ml) or BSA (2 µg/ml) was added to the 96-well microtiter plates. The plates were incubated at room temperature for 2 h before being washed four times. SHH-His  or control His peptide was added and the plates were incubated for another 2 h at room temperature. After 4 times wash, the plates were incubated at room temperature for 30 min. After 4 times wash, substrate solution was added, and Relative Light Units was measured at 450 nM using a luminometer.Surface plasmon resonance experiments were performed using a Sierra Sensors MASS-1 . Human SLITRK5  was immobilized on a research-grade high-capacity amine sensor chip  using the amine coupling method. SHH was diluted in PBS-T  and flowed over the surface at a rate of 10 µl/min.For analysis of gene expression, total RNA from cultured cells or tissues was extracted using TRIzol(Qiagen) according to the manufacturer’s instructions. cDNA was then obtained using a High-Capacity cDNA Reverse Transcription Kit (Invitrogen). An SYBR Green polymerase chain reaction (PCR) Master Mix Kit (Applied Biosystems) was used for real-time PCR. QuantStudio 6 Flex RT-PCR Software v1.3 was used for mRNA analysis. Sequences of the PCR primers used are shown in Supplementary Table C3H10T1/2 cells were transiently transfected with GLI1 responsive reporter plasmid (GLI1-BS-Luc) and Renilla luciferase plasmid together with control or Slitrk5 plasmid. Twenty-four hour after transfection, cells were treated with vehicle or SHH in serum-free medium for 36 h. Cells were then lysed and luciferase activity was measured using the Dual-luciferase reporter assay system (Promega).38. Static and dynamic histomorphometric analyses were performed using the Osteomeasure Analysis System (Osteometrics).Mice were injected with 20 mg/kg calcein (Sigma) at 10 days and 2 days before scarification. Plastic embedding, TRAP staining, and toluidine blue staining of the undecalcified lumbar region were performed as previously describedGli1 and Col1a1 RNA in formalin-fixed, paraffin-embedded tissues, ISH was performed using the BOND RNAscope Detection Reagents kit  according to the manufacturer’s instructions. Briefly, 20 ZZ probe pairs targeting the Gli1 and Col1a1 mRNA were designed and synthesized by Advanced Cell Diagnostics . Tissue samples were incubated with Leica Epitope Retrieval 2 for 20 min at 95 °C, then pre-treated using Leica Protease at 40 °C for 20 min, then incubated with Gli1 or Col1a1 RNA probe at 40 °C for 240 mins. ACD AMP 1–6 was applied at 40 °C for 60–120 min for signal amplification before application of 3,3′-Diaminobenzidine (DAB).To detect 39. For each cell, the ciliary fluorescence intensity was normalized to cell body intensity.Hind limbs from mice were dissected and fixed in ice-cold 4% paraformaldehyde solution for overnight and then decalcified in 0.5 M EDTA solution at 4 °C for 2 weeks. Samples were either embedded in optimal cutting temperature compound (Leica) and sectioned at 20 µm thickness or embedded in paraffin and cut into 5-µm-thick sections. Immunostaining for β-galactosidase and OPN was performed using an anti-β-galactosidase antibody  and an anti-OPN antibody . For cilium staining, bone marrow stromal cells were harvested from 3-week-old mice and osteoblast differentiation was induced with ascorbic acid and β-glycerophosphate. Cells were then infected with lentiviral constructs encoding Slitrk5-flag, Ptch1-flag, or Smo-GFP. Cells were cultured under serum starvation conditions ) for 48 h before 4 h SHH (300 ng/ml) treatment. After SHH treatment, cells were fixed with 4% paraformaldehyde (PFA) for 10 min, permeabilized with 0.1% (v/v) Triton X-100 for 10 min, blocked with 5% for 1 hr. Antibodies were used as follow: rabbit anti-Flag  and mouse anti-acetylated tubulin . Imaging was performed with a Zeiss LSM 880 with an Airyscan high-resolution-detector confocal microscope. Carl Zeiss Zen 2.3 SP1 FP3  was used for immunofluorescence imaging analysis. To quantify the relative fluorescence of protein in the primary cilia, the CiliaQ ImageJ plugins were used for image segmentation and ciliary fluorescence quantificationl-glutamine, 1% penicillin/streptomycin, 1% HEPES, and 1% nonessential amino acids, and differentiated with ascorbic acid and β-glycerophosphate. For ALP activity, osteoblast number was assessed quantitatively using Alamar blue assay. Cells were then washed, fixed in 10% neutral-buffered formalin, and incubated with a solution containing 6.5 mM Na2CO3, 18.5 mM NaHCO3, 2 mM MgCl2, and phosphatase substrate (Sigma-Aldrich). ALP activity was measured by a luminometer. For extracellular matrix mineralization staining, cells were fixed in 10% neutral-buffered formalin and stained with alizarin red.Primary osteoblasts were isolated from 5–7 days old mice by triple collagenase/Dispase II digestion. Cells were cultured in α-MEM medium (Gibco) containing 10% FBS, 2 mM 3VO4, 0.5 mM PMSF, 1 μg/ml leupeptin, 5 μg/ml aprotinin, and 1% Triton X100]. Flag or HA agaroses were used in the immunoprecipitation and immunoprecipitated proteins were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes . The membranes were then blocked with 5% nonfat milk and incubated with specific antibodies.HEK293T cells or C3H10T1/2 cells were transfected with the indicated DNA plasmids. Cells were lysed in RIPA buffer or TNT lysis buffer [10 mM Tris, 50 mM NaCl, 5 mM EDTA, 2 mM NaF, 30 mM sodium pyrophosphate, 100 mM Na™ Red Anti-HA Affinity Gel , EZview™ Red ANTI-FLAG® M2 Affinity Gel , Flag peptide  and HA peptide  were used for immunoprecipitation experiments. The following primary antibodies were used: anti-acetylated tubulin , anti-tubulin , anti-HA , anti-DYKDDDDK , anti-GFP , anti-β-galactosidase antibody , anti-SHH , anti-SHH , anti-Osteopontin  and anti-Flag . Cell lines were purchased from ATCC: C3H10T1/2 cells clone 8 (CCL-226), 293T (ATCC® CRL-3216™), Saos-2 (ATCC® HTB-85™).BDNF  was used at a concentration of 40 ng/ml. Purmorphamine (540220-5MG) was purchased from EMD Millipore K252a (11338) was purchased from Cayman. SHH (8908SH) was purchased from R&D systems. EZviewSurgery was performed under isoflurane (2.5%) anesthesia via nosecone. The surgical sites were sterilized, and an incision was made on the right femur. The fracture was made using a Dremel saw with a diamond thin cutting wheel  and a 25-gauge syringe needle was inserted from femoral condyles to stabilize the femur. Muscles were reapproximated, and the skin was closed using wound clips.The mouse femurs were skinned and fixed in 70% ethanol. Femurs were scanned using a Scanco Medical uCT 35 system with a spatial resolution of 7 μm. The X-ray tube potential of 55 kVp, an xray intensity of 0.145 mA, and an integration time of 600 ms. For analysis of femoral bone mass, a region of trabecular bone 2.1 mm wide was contoured, starting 280 microns from the proximal end of the distal femoral growth plate. For analysis of fracture callus bone volume, the whole callus was contoured.t test. One-way Anova followed by Tukey’s post hoc test was used when the data involves multiple group comparisons. GraphPad PRISM v.8.4.3 was used for statistical analysis.All results are presented as the mean ± SD. Comparisons between two groups were analyzed using a two-tailed, unpaired Student’s Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review File


Endometrial cancer (EC) is a deleterious condition which strongly affects a woman’s quality of life. Although aggressive interventions should be considered to treat high-grade EC, a conservative approach should be taken into consideration for women wishing to conceive. In this scenario, we present an overview about the EC fertility-sparing approach state of art. Type I EC at low stage is the only histological type which can be addressed with a fertility-sparing approach. Moreover, no myometrium and/or adnexal invasion should be seen, and lymph-vascular space should not be involved. Regarding the pharmaceutical target, progestins, in particular medroxyprogesterone acetate (MPA) or megestrol acetate (MA), are the most employed agent in conservative treatment of early-stage EC. The metformin usage and hysteroscopic assessment is still under debate, despite promising results. Particularly strict and imperious attention should be given to the follow-up and psychological wellbeing of women, especially because of the double detrimental impairment: both EC and EC-related infertility consequences. Endometrial cancer (EC) is the fifth most common cancer in women worldwide and the most common malignancy of the female genital tract in developed countries ,2. Most Most patients with EC typically show high BMI or are obese, and they also have often some components of metabolic syndrome, such as hypertension and/or diabetes. EC can be distinguished in two main clinic and pathological/molecular types: I and II. Type I is the endometrioid type (EEC) and is the most common. It tends to develop in a younger cohort of women and includes the perimenopausal group . This tyType II endometrial cancer is typically found in older patients. This cancer is not estrogen dependent, usually is aggressive, and of high stage and grade. It presents a non-endometrioid histology, deeply invades the myometrium and has the worst prognosis .Most endometrial cancer cases are sporadic and only 10% of them are considered familiar. Particularly, patients with EC at a younger age may have a genetic etiology. Usually, EC is associated to Lynch syndrome . DiagnosWhen considering a conservative management approach, the characteristics of the disease should be evaluated to select the therapeutic approach that perfectly fits for the patients. Fertility sparing could be considered for patients who have interest to preserve their fertility and plan to conceive as soon as possible after remission, with no medical contraindication to the medical treatment and favorable histopathological characteristics of the cancer. However, patients should be informed that the fertility-sparing option is not a standard management and treatment for EC, and they must be encouraged to undergo a TH-BSO after pregnancy . TraditiThus, the ideal candidates for fertility-sparing treatment have been suggested to be young women with G1, no myometrium and/or adnexal invasion, and without lymph-vascular space involvement ,16. ThesChae et al.  reportedThe Japan Society of Gynecologic Oncology, the European Society of Gynecological Oncology and the Korean Society of Gynecologic Oncology have established that fertility-sparing treatment should be considered only for patients with grade 1 EEC confined to the endometrium . The BriMRI is known to be the best method and offers the highest efficacy to determine the myometrial invasion . AccordiAlthough literature exists regarding medical treatment for fertility sparing, it is necessary to specify that there is no consensus on which agent, dose, or duration of treatment is most effective. Generally, in accord with the present published literature, progestin, in particular medroxyprogesterone acetate (MPA) or megestrol acetate (MA) at 400–600 mg/daily and 160–320 mg/daily, respectively, tends to be the most employed agent in conservative treatment of early-stage EC . A compl2 usually have a higher risk for EC developing [There is evidence that suggests that diseases associated with insulin resistance are also a risk factor for EC. It has been reported that patients with BMI >25 kg/mveloping  and a baveloping ,41. Metfveloping  demonstrveloping . Obese pveloping ,45. Furtveloping . Mu et aveloping  conducteveloping  reportedveloping  conducteveloping  retrospeAlthough recent studies  showed tPatients who have chosen fertility sparing have to understand that a strict follow-up to assess the response is necessary. Complete remission for patients undergoing fertility-sparing treatment was described as 72% after 6 months and 78% after 12 months, suggesting that to have marginal benefits, hysteroscopy and imaging must be performed after 6 months and not before ,56. CurrGynecological chronic conditions could be heavily stressful for women . The sexThe majority of breast cancers and type-1 endometrial cancers show a similar overexpression of the estrogen receptors. To continue with, the risk of these types of tumors is higher in women who present elevated systemic estrogen levels . MoreoveBoth MI and DCI are second messengers, after conversion, inside the cell as inositolphosphoglycans (IPGs) in their form as MI-IPG and DCI-IPG. These products both mediate the insulin signaling pathway but with some marked differences . MI and an indirect steroidogenic effect mediated by the insulin pathway;an independent steroidogenic direct effect through downregulation of the aromatase gene expression and cytochrome P450 side-chain cleavage (P450scc) genes. These actions lead to an increase of testosterone synthesis.In particular, this ratio has been found around 40:1 in the peripheral blood , about 2DCI could synthetically act in the following ways. First, DCI causes a decrease in androgens in the short term followed by a decrease in estrogen . SecondlIt is now elucidated that low-grade EECs and SECs have distinguishing molecular features. Type I EC, also known as endometrioid endometrial cancer, presents PTEN mutation, the most frequent somatic mutation. Moreover, this mutation commonly co-occurs with PIK3CA and PIK3R1 mutations in human EECs. In addition, a co-operativity has been proved between Pten loss and Ctnnb1 (which encodes β-CATENIN) mutation or Mlh1 inactivation. On the contrary, serous endometrial cancers (SEC) present as first event of the mutation of p53. Moreover, the frequent occurrence of FBXW7, PIK3CA and PPP2R1A somatic mutations, as well as CCNE1 amplification have been proved to be associated with SEC carcinogenesis .Nevertheless, epigenetic cell modifications are gaining importance in cancer etiopathogenesis and characterization. In particular, a group of molecules could be used as marker of clinical prognosis: the non-coding RNAs (ncRNAs). Moreover, ncRNAs can have also a diagnostic role. New insights propose a therapeutic marker role, helping in building an “epigenetic profile” . ActuallYoung women with stage IA and low-grade, positive progesterone receptors EEC and non-metastatic involvement or risk factors may require conservative management for fertility sparing if the motherhood desire is not yet satisfied. Uterine preservation is a feasible option that also seems to be safe until conception. The management may be various. The use of oral hormone therapy, the LNG-IUD and the GnRHa, alone or combinate between each other, offer good results for fertility preservation. In the last years, the data on metformin combined with hormone therapy are extremely encouraging, especially for obese patients, but more data are needed. Hysteroscopic surgery plus progestin or LNG-IUD may reduce the recurrence rate and should be preferred to other treatments, thanks to this high remission rate, although deserving further studies. Therefore, the optimal treatment is still debated and no consensus has been reached yet. Guidelines for managing conservatively the fertility of these patients should be drawn up without ever forgetting that an individual approach for this problem is mandatory since each patient has different characteristics and expectations regarding motherhood.


The salivary gland (SG) microvasculature constitutes a dynamic cellular organization instrumental to preserving tissue stability and homeostasis. The interplay between pericytes (PCs) and endothelial cells (ECs) culminates as a key ingredient that coordinates the development, maturation, and integrity of vessel building blocks. PCs, as a variety of mesenchymal stem cells, enthrall in the field of regenerative medicine, supporting the notion of regeneration and repair. PC-EC interconnections are pivotal in the kinetic and intricate process of angiogenesis during both embryological and post-natal development. The disruption of this complex interlinkage corresponds to SG pathogenesis, including inflammation, autoimmune disorders (Sjögren’s syndrome), and tumorigenesis. Here, we provided a global portrayal of major signaling pathways between PCs and ECs that cooperate to enhance vascular steadiness through the synergistic interchange. Additionally, we delineated how the crosstalk among molecular networks affiliate to contribute to a malignant context. Additionally, within SG microarchitecture, telocytes and myoepithelial cells assemble a labyrinthine companionship, which together with PCs appear to synchronize the regenerative potential of parenchymal constituents. By underscoring the intricacy of signaling cascades within cellular latticework, this review sketched a perceptive basis for target-selective drugs to safeguard SG function. The salivary gland (SG) microvasculature represents a kinetic cellular system, crucial for maintaining tissue vitality and homeostasis. One of the key components of this complex are pericytes (PCs), mural cells that ensheath the abluminal interface of the endothelial lining, while sharing the same basement membrane (BM) ,2,3. TheWith the intention of discerning the traditional immunophenotype (CD146+/endoglin+/Platelet-derived growth factor receptor beta (PDGFRB+)) of PCs from other mesenchymal cells, a myriad of biomarkers have been identified ,24. It iThe SG vasculature is an indispensable complex of an accurately organized hierarchical tree of diversified cellularity, decisive in establishing and preserving tissue health by virtue of blood perfusion and through the potent reciprocity of the cells and the extracellular matrix (ECM). The build-up added to regeneration and/or repair of effective blood vessels turn out by intussusceptive angiogenesis, the chief event involving proliferation and migration of ECs and equilibrium of the microvascular lattice via coverage with PCs, archetypal multipotent stem cells (SCs), as a stringent fundamental ingredient . The proWithin the framework of this complex organization, PCs are closely associated with endothelial coating, act as co-regulators of ECs, and are phenotypically divergent, being contingent upon the anatomical region . On accoAccording to our understanding, PCs exhibit two types of physical contacts with ECs: “peg-and-socket” and adhesion plaques . Within modus operandi of the radiation process with respect to puzzling, distinct radiosensitivity of salivary cells, Radfar et al. [WNT and PDGF genes, thereby supporting the idea that both blood vessel-framing ECs (CD34+CD31+ cells) and PCs may have a common mesenchymal precursor [Although several pathological conditions including autoimmune diseases (Sjögren’s syndrome), irradiation, malignant tumors, and acute or chronic inflammation bring about disorders of the SGs, owing to the acinar cells hypofunction, and even apoptosis, all these pathologies exhibit corresponding microvascular dysfunction ,38,39. Sr et al.  noted thr et al. . Hakim er et al.  found our et al. ,34. Condrecursor ,46,47. Irecursor .Salivary gland tumors (SGTs) represent an assemble of histologically miscellaneous neoplasms that possess low prevalence among HNCs . In the 3 and metastasize in the company of new vasculature that develops from five steps: (1) the increase of hypoxia-inducible factor 1-alpha HIF1Awhich attaches to hypoxia-response elements in the VEGF promoter that induces EC proliferation, followed by (2) the discharge of matrix metalloproteinases (MMPs) by both PCs and ECs, and degradation of the ECM/BM, (3) activation and migration of ECs, (4) formation of the capillary lumen, and ultimately (5) steadiness of tumor neovessels [Crosstalks between PCs and ECs are acknowledged as culminate interlinkages in the labyrinthine marvelous process of angiogenesis. Straightforwardly, tumors surpass 2–3 mmovessels ,55,56,57ovessels ,59.Here, to make the landscape more complex, we reviewed the framework of elaborate protein-protein interrelatedness among decisive signaling pathways in a paracrine or autocrine manner. We underscored how the crosstalk intermingles to promote vascular development and the misregulations that can balance the homeostasis state into a tumorigenic program, as well as how the signaling components interface with each other to exert aversion to pharmacological approaches.VEGFR1 knockout leads to ECs hyperplasia and ectopic vascular morphogenesis [RAF, MEK1, and RAS-GAP causes defective angiogenesis during embryogenesis [VEGF is an assertive signaling factor that belongs to the PDGF supergene family and governs the angiogenic events during embryogenesis and adulthood in both pathological and physiological conditions ,60. The ogenesis . The sigogenesis ,66,67,68ogenesis ,70,71. Togenesis ,73,74. Nogenesis ,76. Instogenesis . Meanwhiogenesis ,79,80. Wogenesis ,79. AKT ogenesis ,82,83. Pogenesis ,85. Corrogenesis ,87. Basiogenesis ,89. Introgenesis  and the ogenesis ,91,92,93ogenesis  targetinogenesis .We brought together the interlinking latticework among VEGF, NOTCH, PDGF, TGFB, and downstream signaling pathways in a diagram . BrieflyHES1/5, HEY [mTORC1/2 [PI3K [TGFB [c-MYC [The NOTCH pathway represents an evolutionary thoroughly conserved pathway that regulates fundamental cellular processes, inclusive of cell-to-cell communication, tissue differentiation, SCs maintenance, proliferation, and development, as well as cell fate ascertainment of vascular ECs and the regulation of angiogenesis ,95,96. T1/5, HEY , mTORC1/mTORC1/2 , PI3K [9B [c-MYC  to contrB [c-MYC .NOTCH3 gain-of-function and loss-of-function denote the fact that NOTCH3 is indispensable for PCs proliferation and limitation of blood vessel permeability [NOTCH/c-MYC activates the AKT pathway via PTEN phosphorylation [ECs mainly express DLL4, JAG1, and JAG2, in addition to NOTCH receptors 1–4, whilst PCs possess NOTCH receptors 1–3 on their surface and JAG1 and DLL1 ,108,109.eability ,110, theeability . Thus, ieability ,119,120.eability ,122,123.rylation ,123. In PGDFb or PDFGRB genes is lethal, assignable to vascular dysfunction, and caused by PCs deficiency [The PDGF family of chemokines and mitogens is known to possess four members which assemble into homo- or heterodimer forms: PDGF-a, -b, -c, -d that bind to two tyrosine kinase receptors, PDGFRA and PDGFRB ,125. PDGficiency ,129. Addficiency . The comficiency ,132. Losficiency . Activatficiency ,133; eveficiency ,135. At ficiency . Interesficiency . Additioficiency . Intriguficiency . Likewisficiency ; thus, fstricto sensu, Bone morphogenetic proteins (BMPs), activin, and nodals families [c-MYC, pointed to proliferation, HES1, and JAG1 [The TGFB superfamily members comprise more than thirty constitutional related signaling molecules, counting TGFBs families ,139 thatfamilies . TGFB fafamilies . The funfamilies . The TGFfamilies . Typicalfamilies ,140. Thefamilies ,140,143,families ,121. Oncfamilies . Finallyand JAG1 ,146,147.and JAG1 . To dissand JAG1 ,150,151.and JAG1 ,153. In and JAG1 ,150. Intand JAG1 . The KO and JAG1 . Recent and JAG1 ,155. Anoand JAG1 . The exiand JAG1 . Additioand JAG1 . Of inteBCL2), cell cycle (CyclinD1(CCND1)), and N-MYC [The HH signaling pathway plays an imperative role in a multiplicity of developmental and postnatal processes, including cell proliferation and differentiation, orchestrating the regulation of angiogenesis, blood vessel maturation, repair of normal tissues, and survival of normal/malignant SCs ,162,163.nd N-MYC ,173,174.VEGFR2 levels. Likewise, SPOP constitutes a transcriptional target of HIFs and hypoxia determines the accumulation of SPOP into the cytosol, which is satisfactory for the instauration of the tumorigenic program. The tumorigenesis materializes via ubiquitination and degradation of tumor suppressors like PTEN, ERK phosphatases, DAXX, and GLI2 [Adding to this puzzle, Shh represents the most significantly expressed Hh within the vasculature, along with Ihh, expressed by ECs, and also by cancer cells in Oral squamous cell carcinomas (OSCCs) ,176,177.and GLI2 . Finally2+ pathways and coordinates actin cytoskeletal rearrangements. In WNT/PCP signaling, upon RAC GTPase actuating, JNK settles c-JUN transcription. In turn, RHO GTPase activates ROCK. Besides, the WNT/Ca2+ pathway turns on PLC, which activates IP3 to release Ca2+. DKKs, as WNT antagonists, associate with LRP5/6 and Kremen receptors and dictate the withdrawal of LRPs from the plasma membrane. In the canonical WNT pathway, if a WNT ligand is absent, beta catenin is phosphorylated by a destruction complex , which is discerned by UBE3 ligase B-TRCP and targeted for proteasomal degradation, so the target genes are repressed by TCF/LEF. Once the pathway is activated by a ligand, the stabilization of beta-catenin is promoted, and it moves to the nucleus where it activates TCF/LEF and transcribes the target genes. WNT/beta-catenin collaborates with HH signaling in a positive feedback loop. The two pathways are mediated by GSK3, CK1, SUFU, PTEN, and SMO. SUFU negatively regulates GLI signaling and beta-catenin. Additionally, the loss of PTEN could switch on both beta catenin and GLI. Prominently, DAXX associates with AXIN to stimulate the tumor suppressor P53 to prompt apoptosis.We summarize the crosstalk between HH, WNT, VEGF, and NOTCH signaling pathways in 2+ pathways [2+ pathway turns on the Phospholipase C (PLC), which in turn activates Inositol 1, 4, 5-trisphosphate (IP3) to increase intracellular Ca2+ [The WNT signaling pathway dictates a plethora of cellular events, including formation and remodeling of vessels, cell fate specification, proliferation, survival, and apoptosis ,191. Thepathways . The nonpathways . The trapathways ,194,195.lar Ca2+ ,197,198.lar Ca2+ . DKKs colar Ca2+ .c-MYC [JAG1 [CCND [CCN1 gene-a negative regulator of VEGF, in ECs, enhancing proliferation and EC hyperplasia. Prominently, WNT5a signals through FZD-ROR-RAC receptors and regulates the angiogenesis, the vascular morphogenesis via PCP, and ECs proliferation, being overexpressed in HPC/SFT [PTEN could activate both beta catenin and GLI [SMO KO downregulates beta catenin levels, which is autonomous of the GLI effect [DLL-4 expression [betacatenin or VEGFR2 from ECs leads to hypovascularization and rescinds angiogenesis following DLL4 downregulation in tip cells [The canonical WNT/- beta catenin signaling pathway governs multiple developmental events, including renewal and regeneration processes, and also the regulation of ECs growth and angiogenesis ,201. In c-MYC ,205, JAGYC [JAG1 , and CCNG1 [CCND ,207. IntG1 [CCND ,209. FurG1 [CCND . The WNTG1 [CCND . AdditioG1 [CCND . WNT5a iG1 [CCND . Of noteG1 [CCND  demonstrG1 [CCND  by ECs iG1 [CCND . The WNT HPC/SFT ,216. To  HPC/SFT . SUFU re HPC/SFT . Accordi and GLI . Several and GLI ,220,221.I effect . On the I effect . Moreovepression . The depip cells . The croip cells . Notwithip cells  , also other non-coding RNAs (ncRNAs), and signaling molecules for interchanging information with recipient cells ,228,229.+ CK7+ alpha-SMA stromal cells in the Schneiderian membrane may be involved in EMT-related regenerative processes [There is substantial therapeutic potential of EXOs from EC-PC communication. It was shown that activation of the HIF pathway induces an angiogenic response from PCs through the exosomal bidirectional interconnection between both cell types . Of noterocesses . Considerocesses . In detarocesses  and remarocesses ,247,248.Congruous with the desideratum for novel therapeutic perspectives to reimpose SG function and along with the knowledge that radiation therapy brings about deleterious side effects , the goaAlthough several approaches aim at fundamentally regenerating the duct and acinar cell lineages, it is attractive to speculate that, in fact, restoration of microvasculature after SG damage  is the primary target ,22, inasSalivary gland (SG) microvasculature constitutes an indispensable cellular organization that possesses specialized features to maintain tissue stability and homeostasis. Pericyte-endotelial cell (PC-EC) interconnections are instrumental for vascular development, maturation, and remodeling in both physiological and pathological conditions. As mesenchymal stem cells (MSCs), PCs are widely explored in the field of regenerative medicine, as they represent an impregnable candidate ingredient not only for enhancing vascular integrity and angiogenesis but also to reinstitute SG function after damage, thereby accomplishing tissue regeneration and repair. The molecular events appertaining to PC-EC sophisticated interconnections were meticulously characterized to unravel the phenomena that bring about SG disorders. Biological operations, including cell proliferation, SC renewal, and differentiation, are orchestrated by a plethora of signaling pathways that cooperate with each other to harmonize the developmental and postnatal equilibrium status. Consequently, an exquisite modulation of these molecular pathways can provide a plan of action to develop novel target-selective drugs to overcome SG dysfunction.


Low back pain (LBP) is a common musculoskeletal symptom, which brings a lot of pain and economic loss to patients. One of the most common causes of LBP is intervertebral disc degeneration (IVDD). However, pathogenesis is still debated, and therapeutic options are limited. Insulin-like growth factor (IGF) signaling pathways play an important role in regulating different cell processes, including proliferation, differentiation, migration, or cell death, which are critical to the homeostasis of tissues and organs. The IGF signaling is crucial in the occurrence and progression of IVDD. The activation of IGF signaling retards IVDD by increasing cell proliferation, promoting extracellular matrix (ECM) synthesis, inhibiting ECM decomposition, and preventing apoptosis and senescence of disc cells. However, abnormal activation of IGF signaling may promote the process of IVDD. IGF signaling is currently considered to have a promising treatment prospect for IVDD. An in-depth understanding of the role of IGF signaling in IVDD may help find a novel approach for IVDD treatment. Intervertebral disc degeneration (IVDD) is a paramount contributor to low back pain and is a leading cause of disability that reduces the quality of life and causes economic loss . The strThe insulin-like growth factor (IGF) signaling pathways play an important role in regulating various cell activities, including proliferation, differentiation, migration, or cell death, which are critical to the homeostasis of tissues and organs . Severalin situ hybridization histochemistry detection. The staining of IGF1R was also positive in the disc cells by the avidin-biotin peroxidase complex method , receptors , IGF binding proteins (IGFBPs) one to seven, and IGFBP protease . IGF ligx method . Theoretx method . Therefox method . IGF1R ix method . When thx method . The autx method . IGF1 cax method .IGF1 and its binding protein and receptor system play an important role in the development and degeneration of the intervertebral discs. IGF1 has been shown to affect early dorsal (spine) development, which can promote chondrocyte differentiation and embryonic bone development . In respSeveral studies focused on the expression of IGF signaling in the intervertebral discs, and the results showed that it was closely related to IVDD. Osada et al. shown that the expression levels of IGF1 and IGF1R mRNA in the nucleus pulposus of fetal bovine intervertebral discs were higher than those of the adult discs . HoweverIVD is a complicated joint structure made up of three major components. The IVD’s center, NP, is gelatinous and resilient . The AF in vitro studies had found that IGF1 increased the synthesis of proteoglycans and inhibited the production of MMP2 (via the ERK/MAPK signaling pathway (Several studies have shown that IGF1 can promote the synthesis of ECM . It also of MMP2 . IGF1 ca of MMP2 . IGF1 in pathway . IGF1 al pathway . Okuda e pathway . Thus, t pathway .The underlying mechanism of IVDD is disc cell death and loss. Studies have shown that IGF1 can relieve the inhibition of intervertebral disc cells proliferation by the adverse microenvironment . Pratsin2O2 (As to programmed cell death, growing evidence indicates that IGF could retard cell apoptosis to delay IVDD . A study2O2 . In summ2O2 . HoweverIGF signaling is involved in various diseases and is considered to have pleiotropic effects . It has in vitro and in vivo studies have exhibited that IGF signaling may be a potential therapeutic target for the treatment of IVDD (in vitro and animals experements do not necessarily apply to humanity. Further research is needed to explore the exact role of IGF signaling in the treatment of IVDD using human disc cells.Both  of IVDD . The app of IVDD . The res of IVDD . The adm of IVDD . The add of IVDD . IGF1 tr of IVDD . In rece of IVDD . For exa of IVDD . HoweverIn recent years, the application of stem cells combined with IGF has provided new ideas for IVDD treatment strategies. Studies have confirmed that IGF1 helps MSC to differentiate into NP-like phenotypes . These Nin vivo and restricts its applicability (Although many studies have confirmed the promising results of the application of IGF in the treatment of IVDD, there are still challenges that limit its clinical application. The effects of IGF on cell proliferation, death, and matrix synthesis in intervertebral discs are complicated. The specific role of IGF signaling on intervertebral disc degeneration may be related to the dose and the time of application. Inappropriate use of IGF signaling may aggravate IVDD and contribute to the exacerbations of LBP. In the case of pathological nutritional insufficiency, exogenous injection of IGF1 is only beneficial to well-nourished areas in IVD, while in undernourished areas will increase cell mortality . The ovecability . There acability . Naturalcability . Small mcability .IVDD is the leading contributor to LBP which causes great economic loss and disability globally. Emerging evidence has highlighted the important roles of IGF signaling in the pathogenesis of IVDD. However, the precise mechanisms remain unclear. The activation of IGF signaling can retard IVDD by inhibiting cell apoptosis, cell senescence, matrix degradation, inflammatory and promoting cell proliferation, matrix synthesis. Unfortunately, the abnormal activation of IGF signaling can accelerate IVDD by stimulating excessive cell proliferation, increasing vascular and nerve growth, aggravating nutritional deficiency . Thus th


The liaison between Nitric oxide (NO) and phytohormones regulates a myriad of physiological processes at the cellular level. The interaction between NO and phytohormones is mainly influenced by NO-mediated post-translational modifications (PTMs) under basal as well as induced conditions. Protein S-nitrosylation is the most prominent and widely studied PTM among others. It is the selective but reversible redox-based covalent addition of a NO moiety to the sulfhydryl group of cysteine (Cys) molecule(s) on a target protein to form S-nitrosothiols. This process may involve either direct S-nitrosylation or indirect S-nitrosylation followed by transfer of NO group from one thiol to another (transnitrosylation). During S-nitrosylation, NO can directly target Cys residue (s) of key genes involved in hormone signaling thereby regulating their function. The phytohormones regulated by NO in this manner includes abscisic acid, auxin, gibberellic acid, cytokinin, ethylene, salicylic acid, jasmonic acid, brassinosteroid, and strigolactone during various metabolic and physiological conditions and environmental stress responses. S-nitrosylation of key proteins involved in the phytohormonal network occurs during their synthesis, degradation, or signaling roles depending upon the response required to maintain cellular homeostasis. This review presents the interaction between NO and phytohormones and the role of the canonical NO-mediated post-translational modification particularly, S-nitrosylation of key proteins involved in the phytohormonal networks under biotic and abiotic stresses. The transformation of NO into other redox forms under physiological conditions, is due to the susceptibility of free NO radical to oxidation and reduction. One of the key mechanisms by which NO mediates various pathways is through post-translational modification of various proteins. Among these, protein S-nitrosylation is perhaps the most prominent. It is the selective but reversible redox-based covalent addition of a NO moiety to the sulfhydryl group of cysteine (Cys) molecule(s) on a target protein to form S-nitrosothiols. The role of NO in signal transduction was first established in the animal system wherein the smooth vascular muscle cells it was found to bind to the heme group of guanylate cyclase (Nitric oxide (NO) is a multi-tasked, gaseous signaling molecule. This is due to its smallest diatomic molecular stucture which makes it highly diffusible across biomembranes. It is a key signaling molecule involved in numerous biotic and abiotic stress responses in plants . In plan cyclase  and cons cyclase . In plan cyclase  during vviz, S-nitrosylation (the formation of a nitrosothiol group on cysteine residues of target proteins), metal nitrosylation , and tyrosine nitration , metal-NO intermediates, SNOs, or ONOO–  transcription factor with AUXIN RESPONSE FACTOR (ARF) transcriptional activators that are present on the auxin-responsive promoter elements (ARE). However, in the presence of auxin, this transcription factor is subjected to ubiquitin (Ub)-mediated proteasomal degradation with the help of SCF-E3 ligase complex (steps 1–3), and auxin response initiates . The SCFassembly , and hen140 residue which is critical for TIR1 function and its interaction with AUX/IAA repressor.Independent studies on the cross talk between NO and auxin in controlling plant development (probably through S-nitrosylation) has been reported by TIR1/AFB E3 ubiquitin ligase complex assembly via S-nitrosylation of the ARABIDOPSIS SKP1-LIKE1 (ASK1) at Cys37 and Cys118. S-nitrosylation of ASK1 influences its binding with TIR1/AFB2 and Cullin1 (CUL1) which in turn promotes SCFTIR1/ and SCFAFB2 assembly, resulting in an impaired auxin signaling activation  genes  at Cys280 which not only suppresses its binding to SA but also inhibits its carbonic anhydrase activity , and Jasmonic Acid (JA) are key phytohormones that regulate plant responses to infection by a variety of pathogens including fungi, bacteria, viruses, and others . NO exersylation . The AraR) genes . Howeverrization . Furtheractivity . On the activity  and thusactivity . Notablyactivity  as shownatgsnor1-3 line compromised JA signaling. Her results from the biotin switch assay indicated that JAZ1 is S-nitrosylated at CyS229in vitro. Further investigations involving Flag-tagged JAZ1 over-expression in the atgsnor1-3 line also indicated the S-nitrosylation of JAZ1 in vivo. This implies that JA signaling during insect attack or infection by necrotrophs such as Botrytis cinerea is under redox control. She proposed that JAZ1 S-nitrosylation may block its interaction with the JA receptor component COI1, while enabling JAZ1 to recruit its co-repressors NINJA (Novel INteractor of JAZ) and TPL (TOPLESS) to inhibit turnover by the proteasome, consequently enabling prolonged JAZ1-mediated suppression of JA signaling, as shown in Jasmonic acid (JA) is an important phytohormone involved in plant development and response to injury, attack by insects, and necrotrophic pathogens. Jasmonic acid (JA) signaling depends on the interaction between JASMONATE ZIM DOMAIN (JAZ1) repressor proteins and CORONATINE-INSENSITIVE 1 (COI1), which is a subunit of the SCF ubiquitin E3 ligase complex for its proteasomal degradation . Ayyar , H2O2, and others  regulates a variety of physiological processes in plants including stomatal movement, protein storage, and adaptation to abiotic stresses such as cold, drought, and salt stresses. At the base of these events are complex signaling networks involving multiple components such as Kd others . NO is ad others . Ca2+ ret stress . On the t stress . S-nitro153 facilitates ABI5 degradation by enhancing its interaction with the E3 ligase complex promoting seed germination and seedling growth (137 in the guard cells. OST1 is a sucrose non-fermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6). Loss of GSNOR1 function in the Arabidopsis atgsnor1-3 plants results in NO overaccumulation in the guard cells, leading to S-nitrosylation of SnRK2.6 abolishing ABA-dependent stomatal closure.The basic leucine zipper transcription factor ABI5 (ABA-INDUCED 5) regulates ABA-mediated seed germination and early seedling growth and is considered as a NO sensor, as g growth , as show53 and Cys49/53, respectively, which abolishes their DNA binding ability  and is characterized by a vast production of ROS and NO. Both MYB2 and MYB30 have been shown to undergo S-nitrosylation at Cys ability . Taken tvia S-nitrosylation of AHP1 at Cys115 rendering it unable to phosphorylate, as shown in via S-nitrosylation coordinated plant growth and development.In plants, cytokinin is an essential phytohormone regulating plant growth and development. As 114. In addition, enzymes such as S-adenosylhomocysteinase and cobalamin-independent methionine synthase are also a part of the ethylene pathway and have been found to be the targets of S-nitrosylation by NO  . The acton by NO . This in162 site as indicted by an in vitro assay  in Arabidopsis involved in biosynthesis while MAX2 (MORE AXILLARY GROWTH2 in Arabidopsis), and D53 (Dwarf 53) and D5 (Dwarf 5) in O. sativa involved in strigolactone signaling pathway (in vitro and in vivo.Strigolactones belong to a more recently studied group of phytohormones involved in growth and development processes mainly in symbiotic mycorrhizal plant-fungi association . These a pathway . However2O2. NO is an essential signaling molecule in signaling cascade interaction with almost all phytohormones. During the past few years, there is an extensive quest for search to explore the multiple and widely diverse mechanisms regarding plant hormones and NO interaction. Undoubtedly. NO has a dual role in the up and downregulation of plant hormones. The NO-phytohormonal interaction is mostly through NO-mediated post-translational modifications regulating the synthesis, distribution, degradation, and conjugation of the elements involved in the plant hormonal transport and signaling. However, there are still studies lagging in explaining how NO interacts with hormones and hormones-related proteins at translational and posttranslational levels. Moreover, the interplay among NO and phytohormones needs to be explored in future work. Overall, it requires extensive research and investigation to explore the coordination between NO and various phytohormonal signaling cascades operating in plants under the pressure of multiple stresses.Plants being sessile, are vulnerable to various environmental changes, challenging them to adjust and grow under such adversities. Plants can survive and grow under these challenging situations due to the coordinated signaling mechanisms involving phytohormones and other stress-responsive molecules like NO and HAP contributed to the writing—original draft preparation and images. BM contributed to the review, editing and gathering resources. WR contributed to the preparation of the table and its contents. MK and TA contributed to the section on S-nitrosylation of target proteins involved in phytohormonal network. DL and GL contributed to the illustration and visualization of the images. AH revised and improved the manuscript and images. CK critically discussed and revised the manuscript. BY revised the final draft and supervised the entire manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Preparations containing calcipotriol combined with betamethasone dipropionate  are available for the topical treatment of psoriasis. This review summarizes the differences in the efficacy and safety of these formulations, as well as the preferences of patients with various forms of psoriasis . It has been documented that foams provide higher bioavailability, resulting in increased efficacy in plaque psoriasis compared to ointments and gels. Gels or foams are preferred by patients for their different practical qualities . The available data indicate that ointments may be the most effective formulation in nail psoriasis, and gels are preferred by patients with scalp psoriasis because of their cosmetic features. Treatment with a foam formulation is associated with a lower number of medical appointments compared to treatment with an ointment and with a lower probability of developing indications for systemic treatment. The safety profiles of foams, ointments, and gels are comparable, with the most common adverse effect being pruritus at the application site (in 5.8% of the patients). A long-term proactive maintenance therapy markedly reduces the number of relapses and is likely to close the gap between topical and systemic treatment in psoriasis. Preparations containing calcipotriol combined with betamethasone (in the form of betamethasone dipropionate) are available for the topical treatment of mild psoriasis ,2. The tThe aim of this article is to review the similarities and differences between these three formulations. In this article, we use the terms “foam” and “foam containing calcipotriol with betamethasone” interchangeably as equivalent to “foam containing calcipotriol with betamethasone dipropionate”, the terms “ointment” and “ointment containing calcipotriol with betamethasone” as equivalent to “ointment containing calcipotriol with betamethasone dipropionate”, and the terms “gel” or “gel containing calcipotriol with betamethasone” as equivalent to “gel containing calcipotriol with betamethasone dipropionate”. All data refer to the approved calcitriol/betamethasone dipropionate concentration of 0.005%/0.064%. As of 2021, the concentration of active ingredients is identical in all formulations across all countries. The literature search for this article was performed until 31 January 2021 in PubMed and Scopus, using the search terms “calcipotriol” and “betamethasone” with “ointment” or “gel” or “foam”. All search results were analyzed in detail.Calcipotriol, a synthetic vitamin D3 analogue, has a similar mode of action to calcitriol—changing the expression of genes responsive to vitamin D. It binds to the retinoid X receptor and influences cell differentiation and growth regulation, immune functions, and the balance of calcium and phosphorus in the body . It alsoBetamethasone dipropionate belongs to the group of synthetic fluorinated glucocorticoids that exhibit anti-inflammatory and immunosuppressive effects by binding to glucocorticoid cytosolic receptors and then translocating to the nucleus where they regulate the transcription of numerous genes responsible for the immune response. It limits inflammatory infiltration, erythema, and edema, inhibits cell hyperproliferation, and improves the differentiation of keratinocytes in psoriasis ,9. Pharmacodynamic studies showed the anti-inflammatory and immunoregulatory synergy of the combination of calcipotriol and betamethasone dipropionate with respect to the effects of these active substances administered individually . The eff+ regulatory T cells and improved the balance between CD8+ or CD4+ regulatory T cells and pro-inflammatory CCR6+ γδ T17 lymphocytes in the lymph nodes. The data indicated that the synergistic antipsoriatic effect of Cal/BS was based on a reduction of the imbalance between regulatory CD8+ or CD4+ T cells and pro-inflammatory CCR6+ γδ T17 cells.The mechanism of calcipotriol/betamethasoneantipsoriatic activity has remained only partially known for a long time. However, in the last decade, a number of publications have started to discuss the immune background of psoriasis and the influence of T cells, B cells, dendric cells, as well as cytokines in its pathogenesis ,13,14,15Calcipotriol is stable in alkaline solutions with pH above 8, whereas betamethasone dipropionate requires an acidic environment with pH between 4 and 6. Therefore, the presence of both substances in an aqueous environment leads to interactions and to their decomposition . The devDermal and transdermal drug delivery is a continuous challenge for pharmaceutical technology. A number of various strategies of drug delivery across the skin barrier have been tested through the decades. A rich set of methods has also been proposed and tested for psoriasis treatment, e.g., laser-assisted drug delivery, foam formulations, nanoparticles, ethosomes, niomes . Among tSupersaturation, involving the increased concentration of a substance above its vehicle solubility threshold, was introduced for foams containing calcipotriol and betamethasone ,20. A suBoth in vitro and in vivo research showed the superiority in active substances’ speed of penetration and concentration reached when using foams compared to gels or ointments .The outcomes obtained with the use of foams support the view of an increasing number of dermatologists that foams leading to supersaturation of calcipotriol and betamethasone will change dermatology and clinical practice as regards the treatment of psoriasis ,22. p = 0.025). Moreover, assessment with the mPASI method  demonstrated a statistically significant advantage of foam over ointment at both assessed time points (1 week and 4 weeks). The authors concluded that the effectiveness of the foam formulation was markedly higher than that of the ointment, with a comparable safety profile.A double-blind multicenter phase II study  comparedp = 0.30) [The standard vasoconstriction test for the evaluation of glucocorticoid effects was used to compare the activities of a foam containing calcipotriol and betamethasone and an ointment containing betamethasone . The degree of vasoconstriction obtained with the foam was (median) 2.00 points, while that achieved with the ointment containing betamethasone but without calcipotriol was 1.75 points, with the difference being statistically insignificant ( = 0.30) .An analysis of the cost effectiveness of foams and ointments containing calcipotriol and betamethasone was conducted in Sweden . A relatA 12-week PSO-ABLE phase III study  was condThe secondary endpoints of effectiveness were the percentage of patients achieving at least 75% of modified PASI (mPASI75) reduction and the time to treatment success. A significant advantage of the foam was demonstrated also for those parameters. The authors emphasized that the median time for achieving the standard index of improvement of mPASI75 was 4 weeks for the foam and 12 weeks for the gel. p = 0.013) and at week 12 . The foam was also more effective as regards other parameters concerning the quality of life, including EQ-5D  and PQoL-12 , and in terms of the influence on pruritus, pruritus-related sleep deprivation, and work impairment. A phase III clinical trial also assessed the influence of using foam and gel formulations on patients’ quality of life . The folAnother phase III clinical trial  evaluateThe efficacy of gel, ointment, and foam formulations was not extensively studied in special locations which are difficult to treat. Some studies were conducted on the efficacy of calcipotriol/betamethasone in nail psoriasis. Rigopoulos et al.  treated In a study performed by Gregoriou et al. , a calciThere were no head-to-head studies, but indirect data may indicate that the ointment formulation is more effective in nail psoriasis compared to foam.The effect of a calcipotriol/betamethasone gel and foam in scalp psoriasis was extensively studied ,36,37,38A long-term study of a calcipotriol/betamethasone gel in adult patients performed by Saraceno et al.  indicateScalp psoriasis in adolescents was not extensively studied. An analysis of data from phase 2 studies was performed to evaluate the efficacy of a foam formulation in scalp psoriasis in adults and adolescents from the age of 12 years. An improvement was observed in PGA classification at week 4 in 73.6% of the adolescents and it was higher compared to that reached in the adults . There aThe safety profile of foams was compared to that of ointments by Koo et al.  and to tA study published in December 2020 evaluating foam containing calcipotriol with betamethasone indicated the latter as a drug that might influence the costs of the biologic treatment of patients with the moderate-to-severe disease . The autA recent meta-analysis was performed to compare literature data referring to the effectiveness of a treatment with a foam containing calcipotriol and betamethasone compared to that of classic methods for the systemic treatment of psoriasis . The timA similar presumption was the basis of a publication which analyzed the treatment costs of a foam containing calcipotriol and betamethasone and of systemic treatment . The autBagel et al.  investigThe chronic course of psoriasis and the tendency towards rapid relapses after topical treatment constitute a significant problem in the treatment of psoriasis. Topical corticoid treatments are approved for no more than 4–8 weeks, leaving the patient and physician with limited possibilities for treatment continuation. According to the registered posology regimen, the possibility of using a foam with calcipotriol and betamethasone as a proactive treatment for up to 52 weeks opens a new avenue for the long-term therapy of psoriasis. A phase III clinical trial (NCT02899962) described by Lebwohl et al.  and SteiIn this context, it is worth emphasizing that the clinical regression of lesions is not equivalent to the resolution of the inflammatory process in the skin. A study conducted with the use of noninvasive skin imaging techniques indicated that an inflammation persisted in the skin despite achieving the apparent regression of skin lesions . It was p < 0.001). The number of relapses during 52 weeks was 3.1 (study group), which was 35% lower than in the control group (4.8). Moreover, the tendency towards rebounds was lower compared to that at baseline (PASI ≥ 125%). The safety profile was comparable for both therapeutic methods. The phase III clinical trial which was mentioned above  includedIn 2020, Kircik et al.  summarizCalcipotriol/betamethasone foams show significantly higher efficacy compared to ointment and gel formulations in the treatment of plaque psoriasis. The higher clinical efficacy may be attributed to the supersaturation technique which was used for the production of the foam formulations. Gels and ointments have shown some benefits in the topical treatment of scalp and nail psoriasis, respectively. The available data indicate that the foam formulation may close the gap between topical and systemic therapy in plaque psoriasis, particularly when applied as a long-term proactive maintenance treatment.