However, this oat product usually has poor sensory acceptance, which is directly related to the texture properties. This study aimed to use the milling method to improve the oat rice texture. The nutrient component, microstructure, pasting, and thermal properties of oat treated with different degrees of milling  were researched. The results showed that milling would damage the bran layer of oat rice, increasing starch, β-glucan, total leached solids content, and the gelatinization enthalpy (ΔH). Meanwhile, oil, protein content, the pasting viscosity, and the pasting temperature were decreased. Milling made oat rice sticky and soft, and the bound water and non-flowing water migrated like flowing water. The cross-section of oat rice showed that milling damaged the surface of oat rice, which was beneficial to water entry and starch dissolution, and increased the viscosity of oat rice. When the milling time was 40 s and 60 s, the appearance, aroma, taste, texture, and overall acceptability of oat porridge were better. Moreover, rapid digestion fraction (k Avena sativa) contain large amounts of starch (about 60%), protein (13%–20%), oils (2%–12%), and β-glucan (2.0%–7.5%) (Oats (0%–7.5%) . There i0%–7.5%) . Studies0%–7.5%) . It has 0%–7.5%) .In Europe, North America, and other countries, oats are usually processed by baking bread or biscuits, while oats are usually used to prepare oat rice or noodles in China . Naked oin vitro starch digestibility of oat rice. The changes in nutrient composition, water absorption index (WAI), SP, and total solids leaching during the cooking of oat rice with different DOM were also studied. The moisture distribution and molecular chain interaction of oat rice under DOM provided the basis for the WAI and SP of oat rice. The microstructure of the oat rice was observed by scanning electron microscope (SEM). Determination of functional group changes of oat rice with milling treatment by Fourier transform infrared (FTIR) spectroscopy. Differential scanning calorimeter (DSC) and Rapid Visco-Analyzer (RVA) techniques were used to study the pasting and thermal properties of oat during different DOM. Pearson correlation analysis was used to analyze the relationship between the correlation index and texture index in the cooking process. The appearance, aroma, taste, texture, and overall acceptability were evaluated through sensory evaluation experiments to determine the optimum milling time. Combined with the conclusion of sensory evaluation and in vitro digestion of starch, aiming to develop nutritionally fortified oat rice with both slower starch digestibility and desirable textural attributes. This study provided the theoretical basis for oat in the food industry.The purpose of this paper is to investigate the effects of milling treatment on the texture, gelatinization properties, and Whole-grain oat was purchased from Yangufang Ecological Agricultural Science and Technology Co., Ltd. . Total starch, AM/AP, and mixed-linkage beta-glucan kits were purchased from Megazyme International Ireland Ltd. . The BCA protein assay kit was purchased from Biyuntian . Other chemicals were analytical grades.The oat was milled by a grain polisher  for 0 s, 20 s, 40 s, 60 s, and 80 s, respectively. These samples are named DOM-0, DOM-20, DOM-40, DOM-60, and DOM-80. The oat rice was ground into powder through a 100 mesh sieve and stored at 4 ℃.Oat rice samples were rinsed three times with distilled water in and 45 ℃ oven for 24 h drying. Place the oat rice in a petri dish about 3 cm high. A tristimulus colorimeter  was used to obtain lightness (L), redness (a*), and yellowness (b*) . The calThe protein and fat contents were evaluated by the Kjeldahl method  and SoxhThe transverse section of oat rice with different DOM was evaluated using an SEM  . After s−1 to 4000 cm−1  was used to scan the spectrum from 400 cm000 cm−1 .Accurately weigh a 3.0 g (dry basis) sample in RVA  tank, add distilled water, and quantify to 28.0 g. The pasting characteristics of oat rice were determined according to the research method proposed by Qian et al  samples were accurately weighed, pressed with 6 µL of distilled water, and at room temperature stand for 12 h. The change of ΔH, onset temperature (To), peak temperature (Tp), and conclusion temperature (Tc) were measured according to the method of Yang et al (r) and dry supernatant (Ws) were weighed.The Li method has been slightly modified F. . The oat2 was determined by the GPMG sequence. Each sample was repeated twice. CPMG sequence parameters are set as spectrometer frequency (SF) 23 MHz, offset frequency (O1) 312.3755 kHz, 90° pulse time P90 14 µs, 180° pulse time P180 28 µs, number of sampling points (TD) 40020, repeated sampling waiting time (TR) 4500 ms, scanning number (NS) 64, echo time (TE) 200 µs, echo number Echo Count 1000, analog gain (RG) 20.0 dB, digital gain (DRG) 3  3 .The previous research methods have been slightly modified H. Y. . Add disPlaced 2.5 g of cleaned oat rice in a beaker, added distilled water at 1:6 (W/V), and cooked in an induction cooker (1600 W) for 60 min. Take the middle of the beaker and mix the oat rice for determination. The TPA was measured by a P/36R cylindrical probe. Pre-test speed 10.0 mm/s, Test speed 1.0 mm/s, Post-test speed 1.0 mm/s, Target mode Strain, Strain 75%, Time 5.0 s, Trigger force 5.0 g. The hardness, stickiness, chewiness, and elasticity were selected as the detection indexes of the texture of oat rice . Each meOat porridge was cooked in the same way as 2.9 TPA. 15 panelists  had background and knowledge in food science or had experience in sensory analysis and were selected to participate in this study. Rank products based on appearance, taste, texture, aroma, and overall acceptability on a scale of 1 (strongly dislike) to 9 (strongly like). Each reviewer provided mineral water for mouthwash.50 mg oat porridge powder and 2 mL distilled water were placed in a centrifuge tube and bathed at 37 ℃ for 5 min, and 8 mL of trypsin and starch glycoside enzyme buffer were added, then shaken in a 37 ℃ water bath shaker. At 0 min, 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, 90 min, 120 min, 180 min, 240 min, and 300 min, take 100 μL and immediately mixed with 900 μL anhydrous ethanol and centrifuge the supernatant. Glucose oxidase/peroxidase reagent (GOPOD) was used for determination .The logarithm formula of the logarithm of slope (LOS) plot was used to fit the starch digestion map of milled and defatted oat rice.t is the starch fraction digested at time t (min), while C0 is the starch fraction digested at t = 0. C∞ is the estimated maximum starch digestibility, and k is the digestion rate constant.CThe LOS curve can determine the number of starch components with different digestible rate constants. CPS model can distinguish various digestive patterns in the starch digestive system .Ct=C0+C∞1 and k2 respectively represent the digestion rate coefficients, and C∞1 and C∞2 respectively represent the maximum digestibility of the two starch components. t2start represents the point in time at which partial digestion of the second starch begins. The digestive patterns of rapid and slow starch components could be determined by t2start. When t2start = 0, t2start ≥ t1 (completion time of rapid digestion), and 0 < t2start < t1, represent parallel mode, sequential mode, and combination mode respectively . A descriptive analysis of ANOVA, correlation analysis, and heat map clustering analysis was performed using SPSS Statistics software version 19.0  and Origin 2021 .Table S1 shows that there has been an increase in the number of DOM, whiteness, β-glucan, and starch level of oat rice with the milling time increased. However, the content of protein and oil were reduced. The sub-aleurone layer and endosperm of oats contain a lot of starch and protein. Increasing DOM removed pigment, protein, fiber, and oil from oat bran, therefore, the cooking time was reduced. Meanwhile, oat rice or oat porridge became soft, thick, and waxy. Li and Xu et al. researched that whiteness and stickiness could increase by milling treatment, and confirmed that the increase of starch pasting degree and the reduction of leached AM would produce this condition .Milling destroyed the layer, and increased the diffusion of water and the pasting of starch during cooking , while t3 and –CH2 at absorption peaks near 2850 cm−1. The vibration of C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="pc-E00C" format="PBM" resolution="300" x-size="12" y-size="9">000000000000000000000000000000000000111111111111000000000000111111111111000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="20.666667pt" height="16.000000pt" viewBox="0 0 20.666667 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg>O is around 1750 cm−1, and the peak of CO of fatty acids is around 1700 cm−1 , T22 (1–10 ms), and T23 (10–1000 ms)  can represent the signal amplitudes of water in various states. The proportions of M21, M22, and M23 to the total area are S21, S22, and S23, respectively, which can indirectly reflect the water in different states and moisture content (1000 ms) . The tramulation . Studies to flow . T2 is p content .2 relaxation spectrum pushes to a longer relaxation time. This result indicates that the strength of organic material binding to water generally tends to weaken with the entry of water molecules , the statistical results are shown in 2 of all samples was greater than 0.99, indicating that the fitting results were credible. According to the CPS model fit, t2start was within 0 min of the end of digestion in the first digestive phase. This indicated that the digestion of samples was carried out by a CPS digestion. As 1) rate and SDF (k2) rate constant, especially for DOM-0 k1, which was significantly 3 times higher than that of DOM-40. The maximum starch digestibility of RDF (C∞1) increased and then decreased, while that of SDF (C∞2) was just the opposite. The content of resistant starch (RS) in oat porridge significantly increased with milling treatment. Therefore, the initial digestion time of SDF (t2start) increased first and then decreased. Due to the increased starch and reduced lipid content in the milling of oat rice, amylose–lipid complexes will be formed during the cooking process, which will affect the digestibility of starch  inside . SDF staf starch . Researcf starch . Interesf starch .Table 2F1 and k2 are the lowest, while t2start is the highest, indicating that the digestion rates of RDF and SDF are lower at this time, which is suitable for patients with hyperglycemia. Consequently, proper milling can improve the texture properties and starch digestibility of oat. Combined with the conclusion of sensory evaluation and in vitro digestion of starch. The present study designed oat rice with higher nutrient intensity, better texture, and slower starch digestibility.This study set out to impact DOM on nutrient ingredients, pasting, thermal, texture properties, and starch digestion. The milling process increased stickiness, β-glucan, and the whiteness of oat rice, but decreased hardness, chewiness, elasticity, protein, and lipids content. Milling also increased the total solid leached and leached starch content but decreased the leached protein content. It is shown that milling damaged the oat bran layer, reduced AM and starch-lipid interaction, and reduced pasting temperature and gelatinization viscosity. DOM increased the level of starch and β-glucan leaching while reducing the bran layer at the oat, resulting in increased viscosity during rice milling. Through sensory evaluation experiments, it was finally determined that 40 s and 60 s were the most suitable milling periods, with higher relative scores for appearance, taste, aroma, texture, and overall acceptability. Pearson analysis found that the WAI, SP, and total solid leached of oat rice were related to the texture properties. According to the analysis of starch digestion in oat porridge with milling treatment, when the milling time is 40 s–60 s, kThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The effects of treatment time of continuous annealing (ANN) and cycle numbers of cycled ANN on the structural, physicochemical, and digestive properties of water caltrop starch were studied under 70% moisture at 65 °C. It was found that continuous and cycled ANN have no significant effects on the morphology of starch granules. However, the relative crystallinity and content of resistant starch increased pronouncedly, possibly due to crystalline perfection, which also led to the rise in gelatinization temperature and the narrowed gelatinization temperature range of starch. The treatment time in continuous ANN generally showed a pronounced effect on the rheological properties of water caltrop starch. During pasting, the breakdown viscosity and setback viscosity of all treatment decreased, implying that ANN modified starch was less susceptible to the condition in heating and continuous shearing, and less likely to cause short-term retrogradation. In contrast, peak viscosity decreased with increasing treatment time of continuous ANN, indicating crystalline perfection restricted the swelling of starch granules and viscosity development during pasting process, which was consistent with the results of steady and dynamic rheological evaluation. All ANN-modified samples showed pseudoplastic behavior with weak gel viscoelastic characteristic. Under a total annealing time of 96 h, the pasting and rheological properties of water caltrop starch were essentially less affected by annealing cycle numbers. However, multistage ANN showed stronger resistance to enzyme hydrolysis. Trapa taiwanensis Nakai) is an annual herbaceous aquatic plant that is suitable for a growing environment with a high temperature and abundant moisture and light. Water caltrop is rich in nutritional value and has been shown to have some physiological benefits, for example, the shell has anti-inflammatory [Water caltrop , under a temperature above the glass transition but below the gelatinization temperature of starch for a period of time from minutes to days. It can be regarded as an eco-friendly and cost-effective process compared with other technologies, such as chemical and enzymatical modification, due to the fact that ANN does not involve the use of chemical reagents [Annealing (ANN), a type of physical modification, is generally performed in excess moisture content  was harvested in the Xiaying district of Tainan, Taiwan. The resistant starch assay kit was purchased from the Megazyme International Ireland, Co. . All chemicals and buffers used were of analytical grade.Water caltrop (w/v) sodium hydroxide solution. After discarding the brown supernatant, the wet starch sediment was mixed with ten times the amount of distilled water to remove impurities. This washing process was repeated several times until the pH value of the supernatant reached to 7. Afterwards, the starch precipitate was dried, milled, sieved, and stored in plastic containers at room temperature.Water caltrop starch was isolated essentially according to the methods of Liu et al. . BrieflyWater caltrop starch was annealed according to the methods of Chung et al. and Su et al. with some modification ,21. WateStarch samples were attached to a double-sided carbon tape with a platinum film coated on the surface via a metal ion coating instrument , then examined with a scanning electron microscope .The crystalline structure and relative crystallinity of starch samples were analyzed according to Liu et al. . Sampleso), peak temperature (Tp), conclusion temperature (Tc), and gelatinization enthalpy (ΔH), were measured using a differential scanning calorimeter  [The thermal properties of the starch samples, including the onset temperature (Tzerland) . Two milA total of 6% starch suspension was prepared with deionized water and placed in a rapid visco analyzer  for gelatinization. A typical heating/cooling profiles for starch analysis was adapted . The pas−1. The Herschel–Bulkley model was used to describe the steady shear rheological behavior of water caltrop starch paste:σ and σ0 are the shear stress and yield stress, respectively; K is the consistency index; n is the flow behavior index.The steady shear properties of starch samples were evaluated according to Tsai and Lai . SamplesThe dynamic rheological properties were measured using the same rheometer as described in F is the conversion from absorbance to micrograms; and W is the weight of the sample in gram.The in vitro digestibility of starch samples was analyzed by using the resistant starch assay kit (K-RSTAR) from Megazyme International Ireland, Co. , which essentially determined the hydrolysis of starch by a mixture of α-amylase and amyloglucosidase ,25. Starp < 0.05) by using IBM SPSS Statistics 20 software.The data were analyzed with one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test at a significance level of 95%  was used to analyze the thermal characteristics of starch gelatinization transition. As shown in o), peak (Tp), and conclusion (Tc) temperature, as well as the transition enthalpy (ΔH), for native and various annealed water caltrop starches during gelatinization transition are summarized in c–To) implied that annealing can promote the formation of homogeneous microcrystalline and crystalline stability [The onset , indicating a pudding-like texture. After undergoing various annealing modification, the viscosity decreased to 0.540–0.969 Pa∙s (=540–969 cP), resulting in a honey-like texture [50 (viscosity at 50 s−1) in relation to dysphagic diets may not cover the entire range of shear rate variations during the swallowing process. Our results showed that the value of η50 could only differentiate the textures between native water caltrop starch and annealed water caltrop starch. The difference in viscosity  represents elastic and solid-like properties, and loss modulus (G″) refers to viscous and liquid-like properties. Based on the in vitro digestibility determined through the hydrolysis of starch by a mixture of α-amylase and amyloglucosidase, starch can be classified into rapid digestible starch , slowly digestible starch , very-slowly digestible starch , and resistant starch  ,27,33,38This study showed that both the treatment time of continuous and the cycle number of cycled ANN had a pronounced impact on the relative crystallinity and starch compositions based in-vitro digestibility, particularly the sum of very-slow digestible starch and resistant starch (RS) content. These results suggest that the continuous and cycled ANN conditions applied in this study, particularly ANN 48 × 2, can produce modified water caltrop starch with lower digestibility, which may be beneficial for people who have concerns about controlling blood sugar level. Moreover, the continuous and cycled ANN conditions applied in this study pronouncedly improved the thermal stability, shear resistance, and resistant starch contents of water caltrop starch due to crystalline perfection, which enhanced the application range of water caltrop starch. The impact of the treatment time of continuous ANN on the properties of water caltrop starch was generally greater than that of the cycle numbers of cycled ANN. In contrast, the increase in RS content is more effective for cycled ANN. However, the in vitro digestibility, determined through the hydrolysis of starch by a mixture of α-amylase and amyloglucosidase, cannot completely represent the exact digestion in vivo, and may also be altered during processing. Therefore, more research on real systems is needed for a better understanding of the effect of processing and in vivo conditions.


Oryza sativa), especially its reproductive stages, and can lead to significant crop losses. Among its physiological consequences, heat stress can increase reactive oxygen species (ROS) levels  levels . In matu) levels . ConceivOsFLO6 (FLOURY ENDOSPERM 6) is a non-enzymatic protein with a carbohydrate binding domain that is important for proper starch biosynthesis in rice leaves, endosperm, and pollen , an ortholog of FLO6 is needed for starch granule initiation in the leaves, possibly by its direction of glucan substrates to a glycosyltransferase enzyme , have α and β subunits, likely forming heterooligomeric complexes. However, different species have different numbers of Cpn60α and Cpn60β isoforms, and the exact oligomeric assembly of these subunits is not known. In rice, 3 Cpn60α and 3 Cpn60β genes have been identified, of which OsCpn60α1, OsCpn60α2/TCD9, and OsCpn60β1 have been characterized  that aid in protein folding and stability by acting as chaperonins. HSP60s are one of the 5 major families of HSPs, with one of the best studied HSP60s being GroEL from cterized , all of Plant Physiology, OsHSP60-3B, which encodes a plastidial HSP and is important for pollen viability under heat stress. Using a radiation screen, the authors identified oshsp60-3b as a heat-sensitive, male sterile mutant, and its allelic identity was confirmed using map-based cloning and sequencing. As revealed by quantitative reverse transcription (qRT)-PCR and GUS staining, OsHSP60-3B is widely expressed across plant tissues. OsHSP60-3B expression increased over anther development and was induced by heat stress. However, in contrast to previously characterized OsHSP60s, the loss of OsHSP60-3B neither impacts the development of vegetative tissues nor influences the plant phenotype under ambient temperatures (22 to 28 °C). Instead, OsHSP60-3B had specific roles in regulating pollen starch synthesis and ROS under heat stress.In this issue of oshsp60-3b mutant resembled the wild type under ambient temperatures but had pale anthers, inviable and shrunken pollen, and impaired pollen starch synthesis at high temperatures. This was seen under field conditions as well as in controlled environments. The early development of anther, tapetum, and microspore was not affected, suggesting heat stress specifically affected starch accumulation in the pollen at later stages. At pollen maturity, oshsp60-3b had smaller starch granules than the wild type at 28 °C and was missing starch entirely at 34 °C. Using a yeast two-hybrid screen for OsHSP60-3B interactors in the anther, the authors identified OsFLO6 as the only plastid-localized interaction candidate. They confirmed this interaction with yeast two-hybrid, split luciferase, and bimolecular fluorescence complementation. Also, an immunoblot showed that OsFLO6 protein abundance decreased at high temperatures in oshsp60-3b anthers compared with the wild type, supporting the role of OsHSP60-3B in maintaining starch synthesis by stabilizing OsFLO6. Also, OsHSP60-3B localized to the plastid stroma in a diffuse pattern, which resembled the known localization of OsFLO6 (OsHSP60-3B stabilizes OsFLO6 to maintain starch synthesis under high-temperature conditions (>34 °C).The f OsFLO6 . Based oOsHSP60-3B may prevent the accumulation of ROS and cell death at high temperatures. The authors performed a comparative RNA-sequencing analysis of oshsp60-3b and the wild type at 3 different anther developmental stages at 34 °C and found 298 differentially expressed genes between the mutant and wild type common to all 3 stages. Gene Ontology analysis revealed that oxidation reduction was the most enriched biological process and oxidoreductase activity the most enriched molecular function at all 3 developmental stages. Trypan blue and 3,3′-diaminobenzidine staining confirmed that oshsp60-3b had increased H2O2 accumulation and cell death at high temperatures compared with the wild type.Another interesting possibility raised by this work is that OsHSP60-3B is a unique HSP involved in the rice male gametophyte heat stress response. OsHSP60 improves rice pollen heat tolerance. Further exploration of the molecular mechanisms could reveal the specificity of OsHSP60-3B regulation, such as whether other starch synthesis proteins are also stabilized by OsHSP60-3B, how OsHSP60-3B prevents excessive ROS accumulation, and whether there are additional OsHSP60-3B heat response pathways that have yet to be identified. Further studies should explore how the different subunits of Cpn60s function together to regulate protein folding under various environmental conditions.The findings presented here suggest that


In this mini-review, we highlight key themes and concepts that have emerged related to how tissue stiffness, a fundamental material property, guides various morphogenetic processes in living organisms.From a physical perspective, morphogenesis of tissues results from interplay between their material properties and the mechanical forces exerted on them. The importance of mechanical forces in influencing cell behaviour is widely recognised, whereas the importance of tissue material properties  On Growth and Form posited that biological form by morphogenesis is a direct reflection of physical and mathematical notions, thereby setting forward the principles of mechanobiology . As. As31]. A similar story of tissues buckling as they grow within rigid borders is found during the morphogenesis of pavement cells within the outer epidermal layer of plants  and it hDrosophila gastrulation, the rectangular shape (i.e. an anisotropic constraint) of the ventral furrow means more cells generate tension along the long (anteroposterior) axis than the short  axis . Th. Th36]. As we discussed earlier, material properties and forces are intrinsically linked, and this has implications for morphogenetic robustness, such as in the well-studied model system of the ventral furrow. Mesodermal cells across the ventral portion of the tissue are connected by a supracellular cytoskeletal network. The connectivity of the network determines the tissue's rigidity. In a poorly connected network, the tissue is floppy and contractile forces are insufficient to enable folding, whereas a well-connected network can fold normally thanks to actomyosin contraction. Interestingly, stiffness (network connectivity) increases at the onset of invagination, beyond what would be minimally needed; allowing the system to be robust because if contractility was lower than normal, the morphogenetic process of tissue bending would be unimpeded . MoreoveFinally, another concept that may widely explain the ability of developing tissues to overcome noise was discovered in a study of plant morphogenesis. Sepal epidermal cells show significant variability in their stiffness, both in space and in time . SpatiotMany aspects of morphogenesis emerge from self-organisation. The oldest and most established explanation of self-organisation is Turing's reaction-diffusion system, in which patterns arise from diffusion of activator and inhibitor, namely, long-range inhibition and short-range activation . Such a Theoretical work suggests that if a cell layer is highly contractile, it can cross an instability threshold, whereby the forces are so large that it ‘pulls itself apart’ ,46. ThisTheoretical models of this idea were hypothesised decades ago. George Oster hypothesised that changes in cell motility and stiffness could give rise to different self-emergent patterns from a uniform field of cells . ExperimThese recent findings reveal how mechanical feedback in a Turing-style reaction-diffusion model enables self-organisation, implying that stiffness, and potentially other mechanical cues, help orchestrate morphogenesis in a robust, reproducible manner and enable efficient patterning.During animal development, cells are often not specified where they are later destined to be. Hence, a key aspect of morphogenesis involves the reorganisation of tissues and migration of cells from one location to another. The potential for cells to begin migrating is usually preceded by an epithelial-to-mesenchymal transition (EMT), the process by which epithelial cells lose their apicobasal polarity in favour of front-rear polarity, weaken their intercellular adhesions and become motile. Cellular EMT is impacted by cell's mechanosensing changes in the rigidity of their surroundings, with an increase in stiffness triggering EMT  49,50].,50.49,50Once motile, cells physically pull on their surroundings. In doing so, they detect the amount of resistance that is being provided to the pulling force, thereby allowing them to sense the stiffness of nearby fibres and cells. Because stiff materials deform less than soft materials, cells are typically naturally guided to follow stiffness gradients toward stiffer substrate, a process called durotaxis . An analTissue invasion is also mediated by the presence of absence of perforations in the basement membrane. For example, reductions in stiffness permit the formation of salivary gland epithelial end buds and subsequent branching morphogenesis . In tumoBasement membrane stiffness also controls the cellular architecture and growth of cancer progression. When the basement membrane is soft, and as benign carcinomas become overcrowded as they grow and proliferate, the basement membrane buckles, creating a bud-shaped, benign tumour , top 5959. On thDuring embryogenesis, tissue layers undergo dramatic changes that change their topology and shape. These processes involve a coherent flow of cells and cell rearrangements, a process generally termed morphogenetic flows. Morphogenetic flows contribute to rearranging and folding of tissues into specific shapes, and regional spatially varying control of tissue rigidity is a key physical mechanism that guides morphogenetic flows.In the case of vertebrate axis elongation, anterior tissues are stiff, mechanically supporting the extending body axis, whereas posterior tissues are fluid, exhibiting zero stiffness, which enables the tissue to locally remodel in the elongating tailbud region, with cells able to move freely and deform easily  60]. Th. Th60]. Tribolium beetles the morphogenetic flows are characterised by a large-scale posterior flow of dorsal tissue, with blastoderm sliding freely underneath the vitelline membrane, whereas anteroventral tissue (on the opposite side of the embryo) remains stationary , mu, mu68], We introduced this review by explaining how morphogenesis is a consequence of material properties and forces, and we then described several ideas related to how stiffens can direct morphogenesis. With reference to examples we mentioned earlier, we would now like to briefly address feedback in the opposite way: how the forces of morphogenesis affect material properties. Although stiffness is, in many cases, an intrinsic property of tissues, there is growing evidence of how forces can modulate stiffness. We explored some of these ideas earlier, in the context of external stress, during which tissues can respond by changing their material properties, thereby buffering the forces and preserving tissue integrity .Mechanical forces regulating stiffness is not only limited to external physical perturbations, but also happens during normal morphogenesis too. Earlier, we mentioned that neural crest cells follow a gradient of stiffness  in the developing embryo. This stiffness gradient emerges because the neural crest cells themselves soften the adjacent placodal tissue via forces from cell–cell adhesions , creatinForces can also affect stiffness of extracellular material, although, it should be noted that mechanical properties of ECM in general are complex, and the ECM is viscoelastic, meaning it deforms over time under an applied stress and can return to its original shape when the stress is removed. Thus, stiffness alone is only one metric amongst many that should be considered. For example, if degradation occurs slowly and in a controlled manner, as it does during processes like tissue repair, mechanical changes may be subtle or even negligible.We also highlight the model system of placodal patterning, which arises from mechanical self-organisation. Stiffness in the dermis  arises from reciprocal cell-ECM interactions: cells pull on fibres, which makes them more contractile, leading to higher rigidity and contractile instability . We alsoCollectively, these examples highlight the complexity of the mechanics of morphogenesis.Morphogenesis across species gives rise to an enormous array of tissues and organs. We hope that this mini-review has highlighted the critical importance that material properties, like stiffness, has on orchestrating morphogenesis and the formation of diverse tissues.We anticipate further research on the role of material properties will provide a cohesive physical explanation underpinning morphogenesis. Morphogenetic events do not happen in isolation, but require large-scale co-ordination so understanding the rigidity changes locally is insufficient to explain such processes. One notable example in which there has been considerable progress to gain a coherent view is in the ventral furrow. We have discussed many of the local behaviours at play, but there is also an appreciation that other parts of the embryonic epithelium must respond or contribute to the movement of ventral invagination in a concerted manner. Tissues on the lateral sides of the embryo need to move toward the furrow and the tissue on the (opposite) dorsal side needs to stretch and expand, made feasible by softening, which is molecularly explained by a reduction in actomyosin levels ,72. ThisGiven the impressive amount we have learnt from studying stiffness, there are plenty of other material properties that can garner attention in the next few years. These include viscosity (a strain rate dependent on time) stress-relaxation (the observed time-dependent reduction in stress in response to constant strain), creep (a time-dependent strain at constant stress), strain-rate sensitivity (change in strain over time) and hysteresis (a difference in the stress-strain relation when loading/unloading due to viscoelasticity). Assaying material properties in developing organisms is very difficult, though, and progress will be catalysed by the rate of development of new technologies.Further progress in this field will be accelerated by methods that enable rapid, non-invasive, non-destructive mechanical imaging. Considerable progress has been made with techniques like Brillouin microscopy, which offers the ability to study the viscoelastic properties of biological samples in 3D over space and time. Brillouin microscopy has recently been impressively applied to various model systems in the context of morphogenetic events like tissue folding . AlthougMaterial properties of tissues have been increasingly well recognised as an important component for understanding how form of tissues emerges. In many cases, this has resolved discrepancies that have existed purely based on gene expression patterns.Stiffness of tissues plays an active role in morphogenetic form. It can constrain or permit growth and migration, dictate morphogenetic flows, enable Turing-like patterning and contribute to robustness.The development of new technologies will spur research into other material properties in attempt to gain a holistic understanding of the physical basis of morphogenesis.


Further studies showed that SCU1 and RCU1 increased yield, amylose content, amylopectin and total starch, and the number of starch particles in lotus, and also significantly reduced peak viscosity, final viscosity and setback viscosity of lotus rhizome starch. To account for these changes, we measured the activity of key enzymes in starch synthesis and the relative expression levels of related genes. Through analysis, we found that these parameters increased significantly under SCU and RCU treatment, especially under SCU1 and RCU1 treatment. The results of this study showed that the one-time application at the erect leaf stage (SCU1 and RCU1) could improve the physicochemical properties of starch by regulating the key enzymes and related genes of starch synthesis, thus improving the nutritional quality of lotus rhizome. These results provide a technical choice for the one-time application of slow-release fertilizer in lotus rhizome production and cultivation.Slow-release fertilizer is an environmentally friendly fertilizer that is widely used in crop cultivation instead of traditional nitrogen fertilizer. However, the optimal application time of slow-release fertilizer and its effect on starch accumulation and rhizome quality of lotus remains unclear. In this study, two slow-release fertilizer applications  were fertilized under three fertilization periods  to study the effects of different application periods. Compared with CK (0 kg∙ha Agriculture is an important industry in the national economy, and lotus has been dominant in the production and consumption of aquatic vegetables in China . By 2021In the past decade, slow-release fertilizer (SRF), as an environmentally friendly fertilizer, has been widely used in crop production; because it can improve the nutrient absorption efficiency of crops, the one-time application of slow-release fertilizer can also reduce the burden of fertilizer on the environment and reduce production and labor costs ,8. The mThere have been many studies on the effects of nitrogen fertilizer on crop yield and starch quality. In rice, nitrogen application can improve rice yield by increasing SPAD and net photosynthetic rate of rice leaves, increasing panicle number, grain number and grain weight ,14, and TaSBE and TaSSS in wheat were significantly increased, the proportion of B-type small starch granules in endosperm was increased, the relative expression levels of TaGBSSI and TaGBSSII were decreased, the biosynthesis of starch and amylose was reduced and the physicochemical properties of wheat starch were significantly affected [Starch is the main component of lotus rhizome, accounting for about 70% of dry weight . There aaffected . The resPrevious studies have focused only on the effects of slow-release fertilizer on crop agronomic traits, yield and yield components. However, the effects on the quality of lotus rhizome starch remain unclear. In addition, it is still unknown whether the application time of slow-release fertilizer will affect the yield and starch quality of lotus rhizome. This study investigated the effect of two type slow-release fertilizer application times on the yield and starch quality of lotus rhizome under the same fertilization level. It explored the choice for the one-time application of slow-release fertilizer in lotus production and planting.Significant differences were observed in the N release characteristics of SCU and RCU in the pool . The N r−2 s−1) at 40 days, which were significantly higher than CK, SCU2 and SCU3 (−2 s−1) that was significantly higher than CK, RCU2 and RCU3 at 70 days of RCU1 treatment. The results showed that SCU and RCU application in the erect leaf stage (SCU1 and RCU1) could significantly improve the photosynthetic capacity of lotus plants.Dynamic changes in SPAD and Pn of lotus leaves after fertilization were determined. Over time, SPAD value and Pn of lotus leaves first increased and then decreased under SCU and RCU treatments . After Sand SCU3 . Under RTo explain the effect of increased photosynthetic capacity on starch accumulation and starch content in lotus, we measured starch accumulation and starch content at three expansion stages. Starch content gradually decreased with the postponement of fertilization stage . In thisLotus rhizome yields from SCU treatments showed a trend of SCU1 > SCU3 = SCU2 > CK, while SCU1 (44.35 kg) was 16.94%, 24.49% and 58.85% higher than SCU3, SCU2 and CK, respectively. After treatment with RCU1, lotus rhizome yields reached a peak value (47.06 kg), which was 25.39%, 46.97% and 68.55% higher than RCU2, RCU3 and CK, respectively. The results showed that both SCU1 and RCU1 treatments could significantly increase lotus rhizome yield. Compared to SCU1 treatment, lotus rhizomes yield under RCU1 treatment was significantly higher than under SCU1 treatment .In order to explore the change in starch content, we selected SCU and RCU treatments of starch granules in the late swelling stage for morphological observation and we examined the number of starch granules by using statistics . SCU andThe proportion of amylose and amylopectin in the total starch content is closely related to the pasting properties of starch. Therefore, we determined the pasting properties of starch in rhizome of lotus at the later swelling stage under SCU and RCU treatments. Under SCU and RCU treatments, the starch paste performance of the lotus rhizome at the late swelling stage was significantly different in seven fertilization stages . With th−1, respectively), 142.07%, 92.54% and 183.33% higher, respectively, than CK treatment. SCU and RCU treatments had no significant effect on GBSS, SBE and SSS activity at the late swelling stage of the lotus rhizome. These results indicate that the application of SCU and RCU in the erect leaf stage can significantly increase the activities of GBSS, SBE and SSS.In order to explore the effects of the SCU and RCU application stage on the mechanism of starch synthesis and accumulation, we determined the activities of key enzymes in starch synthesis. The application of SCU and RCU at different stages had a significant impact on GBSS, SBE and SSS activities. GBSS, SBE and SSS activity showed a single peak curve and increased in the middle swelling stage and then decreased in the late swelling stage . After SNnGBSS, NnSBEI, NnSBEII and NnSSSIV) in starch synthesis  under SCU and RCU treatments all changed according to unimodal curves, which was consistent with rice results [NnGBSS were higher under SCU1 than under RCU1. After RCU1 treatment, SBE activity and relative expression of relevant isoforms (genes) NnSBEII were significantly higher than after SCU1 treatment. However, application at SCU and RCU  significantly increased enzyme activity and gene expression of key enzymes in starch synthesis, but the effect was not as good as that of a one-time application of SCU and RCU in the erect leaf stage (SCU1 and RCU1), while delayed application of fertilizer would only increase labor costs. The results of this paper show that SCU1 and RCU1 significantly increased the activity of the key enzyme and gene expression of the key enzyme in starch synthesis, which promoted starch accumulation and increased starch content. SCU1 treatment promoted amylose accumulation and significantly increased amylose content in lotus rhizome.Previous studies have shown that GBSS, SBE and SSS are key enzymes in starch synthesis  and that results ,44. In p results ,46. HoweCrop yield comes from biomass accumulation, and starch accumulation is one of the main methods of biomass accumulation, which is closely related to photosynthetic capacity ,48. NumeStarch pasting properties reflect the degree of expansion of starch granules . At high2O5/K2O = 20%/10%/18%) and RCU: resin-coated urea (N ≥ 43%). SCU and RCU release periods were 60 and 90 days.The field experiment was carried out at the aquatic vegetable test field of Yangzhou University in Yangzhou City, Jiangsu Province, in 2022. The lotus cultivars used in the experiments were ‘MRH’. Two SRFs were selected for the experiment, including SCU: sulfur-coated compound fertilizer (the main slow-release component is sulfur-coated urea) (N/P2 (5.8 m × 1.9 m). The nutrient content of the soil prior to sowing was as follows: 12.8 g·kg−1 organic matter, 1.04 g·kg−1 available nitrogen, 9.4 mg·kg−1 available phosphorus, 54.4 mg·kg−1 available potassium. The available nitrogen, available phosphorus, available potassium and organic matter in soils were measured with alkaline hydrolysis diffusion method, molybdenum-antimony colorimetry method, flame photometric of ammonium acetate method and potassium dichromate oxidation method, respectively [−1) was selected by referring to our previous method [−1  sulfur-coated compound fertilizer was applied once at May 25 (erect leaf stage), June 20 (erect leaf completely covering the water stage) and July 10 (swelling stage of lotus rhizomes), respectively. (5) RCU1, (6) RCU2 and (7) RCU3: 482 kg·ha−1  resin-coated urea was applied once at May 25 (erect leaf stage), June 20 (erect leaf completely covering the water stage) and July 10 (swelling stage of lotus rhizomes), respectively. The phosphorus and potassium levels of the seven treatments were the same: total P = 103.5 kg·ha−1 and total K = 186.3 kg·ha−1, respectively. The difference part was supplemented by superphosphate (P2O5 ≥ 16%) and potassium sulfate (K2O ≥ 52%).The experiment was conducted in a pool with an area of 11.02 mectively . Lotus ws method . Seven fN release from SCU and RCU was studied in a pool under the same conditions as the lotus planting. Nitrogen release after fertilization was studied by soil embedding . The ferFive identical erect leaves were selected for each treatment, and three points were randomly selected for each erect leaf, and their growth status was measured at 0, 10, 20, 30, 40, 50 and 60 days after SCU application and 0, 10, 20, 30, 40, 50, 60, 70, 80 and 90 days after RCU application to measure the leaf net photosynthetic rate using the LI-6400 xt photosynthetic apparatus . Leaf SPAD values were measured using a chlorophyll meter .Lotus rhizomes were collected in three stages: early , middle  and late  swelling stage, and sample harvesting times were 10 August, 30 August and 25 September, respectively in 2022 . A portiExtraction of total starch and amylose content refers to the anthrone-sulfuric acid colorimetry method with slight modification . The lotThe starch was isolated with reference to Zhou et al., 2020 with minor modifications . Lotus rThe starch was placed in the alcohol and dispersed evenly with an ultrasonic oscillator. Next, a little alcohol was glued to a toothpick to leave a uniform layer of powder on the surface of the sample table and the powder was dried with hot air electricity. The morphology of starch particles was observed by environmental scanning electron microscope  and we examined the number of starch particles by using statistics.w/w, dry basis and mixed with 25 g H2O. The programming cycle for pasting was set at 14 min. Starch samples were started at 50 °C for 1 min and then heated from 50 °C to 95 °C, held at 95 °C for 2.5 min, cooled to 50 °C at 12 min and finally held for 2 min [Starch pasting properties were determined using a rapid viscosity analyzer . Starch samples were 2.5 g total weight; 7%, g centrifuged at 4 °C for 10 min, supernatant was discarded, 1 mL of extract was added to precipitation and thoroughly mixed and then placed on ice for use in the test [The activities of GBSS, SBE and SSS were measured by starch synthase kits . About 0.1 g lotus rhizome sample was weighed into the mortar, then 1 mL extract added, homogenize fully in the ice bath; 10,000× ® Universal Blue qPCR SYBR Green Master Mix , 0.4 μL forward primer, 0.4 μL reverse primer, 1 μL diluted cDNA and 8.2 μL RNase-Free ddH2O). The RT-qPCR data were analyzed using the 2−ΔΔCT method [Lotus samples were ground into fine powder in liquid nitrogen, loaded into a centrifugal tube and stored in a −80 °C refrigerator. Three biological replicates were used for RNA extraction per treatment. RNA was obtained using the RNA extraction kit . Then, HiScriptII RT SuperMixfor qPCR  was used for reverse transcription into cDNA. The RT-qPCR was conducted on a CFX96 real-time PCR system  on a total reaction volume of 20 μL  and p values of <0.01 were considered highly statistically significant (**). All data were expressed as mean ±SD.Microsoft Excel 2016 software was used to organize the data. Graphpad Prism 8.0 was used to construct the graphs. Data were analyzed statistically using IBM SPSS Statistics 26.0 software, A one-time application at the erect leaf stage of both SCU and RCU is suitable for high quality and high yield of lotus rhizome production. These results provide a technical choice for the one-time application of slow-release fertilizer in lotus rhizome production and cultivation.


Children and adolescents with adiposity excess are at increased risk of future cardiovascular (CV) disease. Fat accumulation promotes the development of elevated blood pressure (BP) and arterial stiffness, two main determinants of CV risk which are strongly inter-related. We aimed at investigating whether the association between overweight and arterial stiffness, taken at different arterial segments, is mediated by increased BP or is BP-independent.Three hundred and twenty-two Italian healthy adolescents  attending the “G. Donatelli” High School in Terni, Italy, underwent measurement of arterial stiffness by arterial tonometry (aortic stiffness) and semiautomatical detection of pressure–volume ratio of the common carotid (carotid stiffness). The mediator effect of BP was tested for each anthropometric or biochemical measure of fat excess related to arterial stiffness.z-to-R 2.07, P = 0.04), and independent from BP.Both carotid and aortic stiffness showed positive correlations with body mass index, waist, hip, and neck circumferences (NC). Only carotid stiffness, but not aortic stiffness, was associated with serum markers of fat accumulation and metabolic impairment such as insulin, homeostatic model of insulin resistance (HOMA-IR), serum gamma-glutamyl transferase (sGGT) and uric acid. The association with NC was stronger for carotid than for aortic stiffness (Fisher In healthy adolescents, fat accumulation is associated with arterial stiffness. The degree of this association differs by arterial segments, since carotid stiffness is more strongly associated to adipose tissue excess than aortic stiffness and shows a BP-independent association with NC whereas aortic stiffness does not. The rate of children and adolescents with overweight or obesity is increasing worldwide . An exceAn accelerated vascular ageing, expressed by an increase in arterial stiffness, is one of the pathophysiological pathways linking adiposity to future CV risk in children and adolescents . At thisTo better address this issue, it should be considered that the physical properties of the arteries are affected not only by the structural characteristics of the vessel wall, such as elastin and collagen content , but alsIt should also be considered that measures of arterial stiffness from different arterial segments (e.g. aortic stiffness and carotid stiffness) could variably respond to the effects of metabolic and CV risk factors , and thaTo take due account all these aspects, with the aim of gaining better knowledge on the association between fat accumulation and various measures of arterial stiffness, and to properly assess the mediating role of BP in such association, we analysed data from a cohort of Italian healthy adolescents undergoing an extensive evaluation of cardiovascular and metabolic status enrolled in the MACISTE study . In the Details of the MACISTE study were reported elsewhere . BrieflyParticipants and their parents  were notified about study procedures and were asked to give informed consent. All measurements and procedures were carried out at the high school, in order to provide the best approach for motivating individuals to participate to the project. The present study was approved by the Local Ethic Committee and conducted in accordance with the Declaration of Helsinki.All measurements were performed on each participant within the same day. All participants were evaluated during the early morning and after at least 13 h of overnight fasting. Smoking or caffeine consumption was not allowed during the day before.et al.[Blood samples were drawn after 13-h overnight fasting conditions and delivered to the centralized laboratory of the Terni University Hospital for the analysis. Low-density lipoprotein (LDL) cholesterol was calculated by the Friedewald formula. Fasting serum insulin, the HOMA-IR calculated from the model described by Matthews et al., serum u2) and expressed as percentile values based on age and sex, according to the Centers for Disease Control and Prevention (CDC) Growth Charts. Overweight was defined as BMI ≥85° percentile or as BMI >25 kg/m2[Body weight was measured to the nearest 0.5 kg, with participants wearing light clothing and no shoes. Height was measured to the nearest 0.5 cm. BMI was calculated as follows: weight (in kg)/height  , as prevA) and lumen area (A), which is the diastolic lumen area evaluated from the diameter values assuming that the cross-section of the artery is circular, were also calculated. The instantaneous diameters flanked by carotid SBP/DBP were used to calculate carotid stiffness (CS). Specifically, the cross-sectional distensibility coefficient was calculated as DC = ΔA/(A × PP), where PP is carotid PP. Bramwell–Hill equation, derived by the Moens–Korteweg equation, converted DC into a carotid stiffness parameter according to the relationship CS = (DC × ρ)(−1/2), where ρ represents the blood density. CS was expressed with the same measurement units of pulse wave velocity (m/s) thus enabling a direct comparison between the two techniques [Carotid stiffness was assessed by means of the Medical Device Software Carotid Studio  which allows the estimation of vascular parameters by ultrasound imaging, based on a validated contour tracking algorithm ,37. The chniques .Z test. Continuous variables were expressed as mean ± standard deviation (SD) if normally distributed, or as median and interquartile range (IQR) if nonnormally distributed. Categorical variables were expressed by their relative (%) frequencies. If nonnormally distributed, variables were log-transformed before entering into linear models. Pearson's R and Spearman's rho correlation coefficients were used to describe the correlation between parametric and nonparametric variables. The Fisher r-to-z transformation was applied to determine the significance of the difference between two correlation coefficients. Given that a sex-specific susceptibility of weight induced arterial stiffening and BP rise was described in previous literature [The assumption of satisfactory normal distribution was tested for all the examined variables by the Kolmogorov–Smirnov terature , we testMultiple adjustments were made through stepwise linear multivariate models where carotid stiffness and cf-PWV were introduced as dependent variables in separate models. In each model, a measure of body fatness was introduced (one at a time) along with age, sex, heart rate, total cholesterol, Tanner state and levels of physical activity. Further adjustment was also made by introducing also brachial MAP or (aortic or carotid) PP to the fully adjusted models. Before performing multivariate analyses, all variables were tested for collinearity through appropriate statistics.N = 5000). If the 5000 bootstrap 95% bias-corrected confidence intervals (CIs) did not cross 0, the indirect effect was taken as significant. The covariates of age, sex, height, heart rate, physical activity and Tanner status were introduced as independent variables in the mediation models. P < 0.05 (two-tailed) was considered statistically significant. Assuming from previous literature [α = 0.05.To address the mediating effect of BP in the relationship between fat accumulation and arterial stiffness, we first run the Sobel's test , which wterature  that theFrom an initial population of 539 participants, 44 individuals were excluded due to low quality of arterial waveform signal by applanation tonometry, 64 individuals were also excluded due to low quality carotid ultrasound images, and 109 individuals refused to undergo blood test.P = 0.89). As expected, all anthropometric and biochemical measures related to fat excess were higher in participants with overweight than normal weight  with the exception of HDL-cholesterol, which was lower in participants with overweight than in normal weight (P = 0.04), and glucose and triglycerides serum levels, which were not significant by groups.Three hundred and twenty-two participants  were evaluated, 40 (12%) were found with overweight. The clinical, anthropometric and laboratory characteristics of the study population according to overweight status are summarized in Table P < 0.01), with the exception of peripheral and central DBP, which were significantly lower in overweight vs. normal weight, and brachial PP, which was comparable between the two groups. The aortic-to-brachial PP amplification was reduced in participants with overweight than in lean participants . When the association between the examined variables and overweight status was analysed according to sex , the main results did not substantially change. Specifically, sex was not an effect modifier of the relationship between all the examined variables and overweight status .Measures of central (aortic) and peripheral BP were higher in participants with overweight as compared to normal weight .Both carotid and aortic stiffness showed a positive correlation with BMI and with measures of local adiposity such as WC and HC, WHR and NC Table . The corThe correlation between measures of adiposity, metabolic status, BP and arterial stiffness were tested in separate multivariate models. In each mode, a measure of arterial stiffness  was introduced as the dependent variable, and each measure of adiposity or metabolic status (one per each model), together with other fixed independent variables following three steps. In the first step  and aortic stiffness . When MAP was replaced with carotid PP (Model 3), NC was the only measure to remain associated with carotid stiffness , whereas its association with aortic stiffness lost significance .Many of the observed univariate associations of carotid and aortic stiffness with measures of adiposity lost its significance when MAP was introduced to the model (Model 2). Only NC remained associated with carotid . NC remained significantly associated with carotid stiffness even after accounting for the mediating effect of carotid PP , although the strength of this association was very poor. The bootstrapping analysis overall confirmed these results .In a mediation analysis model, built on the general conceptual model that adiposity could negatively be associated with arterial stiffness through direct effects and through increased BP Fig. , MAP wasIn our study we showed that, in a cohort of healthy adolescents, indexes of body fat accumulation such as BMI, WC, HC, waist/hip ratio and NC were positively associated with measures of aortic and carotid stiffness. Carotid stiffness showed overall more robust associations with body fat accumulation than aortic stiffness, as it was demonstrated for NC, and was also significantly associated with laboratory biomarkers of impaired metabolic status, such as serum insulin, HOMA-IR, SUA and sGGT, whereas aortic stiffness did not.Our study investigated the mediating role of BP in the association between fat accumulation and arterial stiffness in adolescents. In line with previous literature, showing that fat excess was closely linked to increased BP in adolescence , we demoIn comparison to previous findings , the novelty of our approach was that each measure of arterial stiffness was evaluated not only according to MAP values, which are known to be constant along the arterial tree, but also according to measures of local pulsatility (e.g. aortic and carotid PP) to eliminate the possible residual confounding effect related to overweight status. It has been described, indeed, that overweight in adolescence is an amplifier of the PP increase that follows body growth . InteresOur mediation analysis was based on the general conceptual model that adiposity in adolescence is a key antecedent of both elevated BP and increased arterial stiffness. Although limited by the cross-sectional nature, this model was supported by results from previous animal experiments , human oet al.[et al.[et al.[et al.[Our results, if viewed within the context of previous literature, generally confirm that the aortic and carotid arterial pathways differently respond to the detrimental effect of excess adiposity. Paini et al. first del.[et al. evaluatel.[et al. found thl.[et al. showed tOther results deserve to be commented in our study. We found that participants with overweight showed a reduced PP amplification, thus suggesting that adiposity is associated with increased PP more strongly at the central level than in periphery. This feature also confirms that adiposity has a negative impact on arterial function through its effects on central large arteries . In our Overall, these results could inform targeted therapeutic interventions to young individuals, in the setting of primary prevention with the aim of reducing risk of future CV disease. Our study also confirms previous evidences in favour of the evaluation of NC as a proxy measure of metabolic impairment and unhealthy CV health in adolescence, supporting its use in clinical practice due to its simplicity of measurement , and alsStrengths of our study were the use of validated and up-to-date technologies for the assessment of aortic and carotid stiffness and the availability anthropometric and laboratory parameters of body fat accumulation and metabolic status, combined with accurate noninvasive estimates of BP at the level of arterial stiffness measurement. It should be acknowledged, however, that in our study the use of different technologies for the assessment of aortic and carotid stiffness might have impacted on results. Carotid stiffness was measured locally at a single arterial point through a nonpropagative model based on the volume/pressure relationship of a cross-sectional arterial segment. Aortic stiffness was measured by the propagation velocity of the pulse wave along two arterial points, classically along the carotid-femoral segment. Previous studies reported that different methodologies for arterial stiffness assessment could provide different results because of different BP and HR dependencies . As prevWe acknowledge that metrics adopted for defining overweight derived from a population percentile distribution approach rather than the association with disease outcomes. Despite we attempted to select variables reflecting body fat distribution to those with documented relationship with insulin resistance and CV risk, we could not exclude that other biological or behavioural components might display stronger association with arterial stiffness than those documented in our study. Anatomical and structural neck differences could have also impacted on results; in order to minimize this effect and to reduce variability, a validated medical device software (Quipu srl) based on a robust contour-tracking algorithm was adopted flanked by properly set and standardized ultrasound measurement protocols. Finally, the small sample size, also determined by the lower than the expected number of participants, requires that results of our study should be confirmed in larger datasets.In conclusion, in line with previous literature, our results support the view that adiposity is a determinant of early vascular ageing in adolescence, and that increased BP is a significant mediator of such association. Our results could therefore raise the hypothesis that therapeutic interventions aimed at reducing the burden of increased adiposity in adolescents could positively impact on CV health through preservation of normal arterial function and reduction of BP values.The authors express their deepest gratitude to Professor Giuseppe Schillaci for his inestimable contribution to the conception, design and development of the MACISTE Study.Sources of funding: The MACISTE study was funded by “Regione Umbria − research project for programming health, prevention of chronicity and model organization in the context of dysmetabolical diseases”, year 2013E.B. and V.G. are co-founders of QUIPU s.r.l., Pisa, Italy, a spin-off company of the Italian National Research Council and the University of Pisa developing software medical devices.


The pattern of substance use in Iran is characterized by a high prevalence of opioid use and opioid use disorder (OUD). Although opioid maintenance therapy (OMT) has been introduced in Iran, approximately 50% of people with opioid use disorder remain unreached. Moreover, psychosocial treatment of OUD and common mental health symptoms during OMT is limited. Digital interventions have been shown to improve psychological distress, depression, anxiety, and post-traumatic stress disorder symptoms. In addition, providing psychoeducation and risk reduction counseling to prevent communicable diseases like HIV and infectious hepatitis is common via the Internet. However, despite these promising advances, no smartphone intervention in OMT has been investigated for the treatment of OUD and common comorbid mental health symptoms.We examine the effectiveness of adding a blended smartphone intervention based on community reinforcement approach, motivational interviewing- and cognitive behavioral therapy compared to OMT as usual that aims to improve OMT outcomes and addresses common mental health symptoms in OMT patients in Iran.Adults with opioid dependence entering 8 treatment centers in Tehran, Iran will be randomly assigned to receive either OMT plus a smartphone intervention or OMT as usual. The primary outcomes will be the percentage of negative urine tests for illicit, non-prescribed use of opioids  and treatment retention. Secondary outcomes will include the longest period of abstinence from the illicit, non-prescribed use of opioids  confirmed by urine samples, changes in communicable disease risk-taking behaviors, changes in stress and common mental health symptoms, and client satisfaction. Data analysis will follow the intention-to-treat principle and employ  linear mixed models.This study will provide substantial knowledge for designing effective blended interventions for OUD. Moreover, it will investigate if treatment retention and OMT-related outcomes and common mental health symptoms can be improved by adding a smartphone intervention to OMT.https://en.irct.ir/trial/53578.The online version contains supplementary material available at 10.1186/s12888-023-05007-0. Iran has a high prevalence rate of opioid use. The 12-month prevalence of opioid use in the population aged between 15 and 64 is reported to be over 3.0%, and the 12-month prevalence of DSM-5 diagnosis of opioid use disorder (OUD) is 2.23% , ; the numFurthermore, any negative effects due to the smartphone intervention will be measured via the Negative Effects Questionnaire  at all fData will be analyzed according to the intention-to-treat principle (ITT). We will examine for differences in all primary and secondary outcome variables between the two study arms at baseline and follow-up assessments using  linear mixed models. We will specifically model repeated measures nested within participants and participants nested within treatment centers by defining respective random intercepts and slopes. Study conditions and time will be modeled as fixed effects. Appropriate distributions for non-normal continuous and/or count outcomes will be specified . Subject to the sample’s actual gender distribution, we will investigate the intervention’s gender-specific effects by including a gender*treatment*time interaction term. If this is not possible due to inadequate sample size , separate subgroup analyses will be run, which will allow the determination of differential treatment effects.This study aims to investigate the effectiveness of blended OMT treatment in which OMT as usual, is delivered in combination with a smartphone intervention for patients with opioid dependence in Iran.The prevalence and burden of OUD in Iran, both on an individual and societal level, warrant a variety of effective treatment strategies. So far, research on the effectiveness of digital interventions for the treatment of OUD is primarily limited to add-on interventions that have been investigated in the United States . HoweverThrough the integrated approach of blended interventions, we expect to provide our participants with both the benefits of digital interventions and the advantages of face-to-face therapy. However, there are many unknowns regarding blended interventions, such as how to combine these treatment modalities and the characteristics of therapist support. Our study will attempt to provide relevant answers to these questions. Furthermore, we will better understand to what extent the blended intervention suits patients in OMT settings by exploring treatment adherence, dropout, and satisfaction with the blended intervention. In addition, by monitoring the activities of our patients in the smartphone intervention , we will be able to get insights into what happens during the treatment period. This will further increase our knowledge of the suitability of blended treatments for patients in OMT.We will not interfere with the current practices of OMT as usual. By permitting the practice of usual care for OMT patients, the generalizability of our results is enhanced. Given the heterogeneity of OMT as usual, this could also be regarded as a limitation. However, since our study is designed to be a pragmatic trial, we aim to compare the effectiveness of the blended intervention with the current OMT as usual practice. Therefore, OMT as usual will be monitored carefully to control for potential confounding effects.This study will provide substantial knowledge to aid in developing effective blended interventions delivered in OMT for Persian-speaking populations. In addition, to aid the implementation and ensure treatment fidelity, therapists will receive training on how to deliver a blended intervention and technical support from the research team. Usage will be facilitated via the implementation of open-source software that can be used and maintained with few resources. Thus, long-term sustainability will be achieved.Below is the link to the electronic supplementary material.Supplementary Material 1: SPIRIT 2013 Checklist


This study aimed to evaluate the color stability and translucency of various types of monolithic zirconia after immersion in chlorhexidine and Listerine mouthwashes.n = 12 per group). Each group was equally divided and immersed in either 2% chlorhexidine (CHX) or Listerine mouthwash for 2 min daily over 7 days. Changes in color (ΔE) and the translucency parameter (ΔTP) were evaluated and compared. The data were analyzed with one-way ANOVA followed by Tukey’s post-hoc tests (α = 0.05).This experimental study was performed on 36 disk-shaped specimens fabricated from low-translucent, high-translucent, and multilayered monolithic zirconia  and the high-translucency zirconia group . Listerine caused a significantly higher ΔE in the high-translucency zirconia group compared to the multilayered zirconia group (P = 0.0165). It also resulted in a significantly higher mean ΔTP in the multilayered zirconia group compared to the low-translucency (P = 0.0003) and high-translucency zirconia groups (P = 0.019).Chlorhexidine caused a significantly lower ΔE and a significantly higher ΔTP in multilayered zirconia compared to the low-translucency (ΔE: In both mouthwashes, multilayered monolithic zirconia exhibited the highest color stability among the tested materials; albeit with the most pronounced changes in translucency. Meanwhile, high-translucency monolithic zirconia was more prone to discoloration when exposed to both mouthwashes. A ΔE≥3.7 is the value for a color difference visible to the naked eye, rendering ΔE = 3.7 the established clinically-acceptable threshold for color change .The data were analyzed by using SPSS software . The results were presented as mean ± standard error (SE) for ΔE and ΔTP. The Shapiro-Wilk test was used to evaluate normal distribution, and Levene’s test was used to assess the equality of variance. One-way ANOVA with Tukey’s post hoc was used to compare the groups (α = 0.05).The Shapiro-Wilk test approved the normal distribution and Levene’s tests confirmed the equality of variances .P<0.001) and ΔTP . The results of Tukey’s post hoc test showed that ΔE of the ML zirconia was significantly lower than that of the LT zirconia (P = 0.0027) and HT zirconia groups (P<0.001). The mean ΔTP of ML zirconia was significantly different from that of the LT zirconia (P<0.001) and HT zirconia (P = 0.022). Chlorhexidine mouthwash increased the translucency parameter in the LT zirconia and decreased it in the HT zirconia and ML zirconia groups  and ΔTP . The results of Tukey’s post hoc test showed that ΔE of the HT zirconia was significantly higher than that of the ML zirconia (P = 0.0165). Moreover, the mean ΔTP of ML zirconia was significantly different from that of the LT (P = 0.0003) and HT zirconia groups (P = 0.019). Listerine mouthwash increased the translucency parameter in the LT group and decreased it in the HT and ML zirconia groups  . In termAmid concerns regarding color and translucency in monolithic zirconia, several studies have assessed the influence of mouthwashes on these aspects , 37, 41.Sasany et al.  detectedThe variations in ΔE and ΔTP among different monolithic zirconia materials in the current study may stem from factors such as variations in chemical structures, grain size and shape, crystalline phase distribution, porosity, and thickness, all of which can affect zirconia’s optical properties –46. The 2O3 dopants, whereas multilayered specimens were 5Y-TZP. Adding La2O3 to conventional zirconia increases translucency by promoting smaller grain sizes and narrowing grain boundaries. However, in such materials, the reduction of alumina content is essential to preserve mechanical properties. This trade-off can decrease aging stability, as evidenced by the high-translucency zirconia group in the present study [Multilayered zirconia mimics the shade gradient of natural teeth, with the incisal area of a crown displaying the highest translucency and gradually increasing in opacity and chroma towards the gingival area . The laynt study , 50, 51.Consistent with the lowest color change observed in multilayered zirconia specimens in the current study, similar research showed that higher 5Y-TZP cubic zirconia yielded lower ΔE compared to 3Y-TZP tetragonal specimens. Existing literature suggests that elevated yttria content can minimize surface-level low-temperature degradation, potentially reducing surface roughness and solution infiltration , 11, 52.In the present study, the multilayered zirconia specimens were pre-colored by the manufacturer, while the low-translucency and high-translucency zirconia specimens were shaded with coloring liquids in the laboratory. In line with the current findings indicating the lowest ΔE in multilayered zirconia specimens, Subaşı et al.’s findings  demonstr2) compared to 3Y-TZP zirconia . This discrepancy might arise from the utilization of distinct protocols and solutions.Unlike the present study, Lee et al.  reportedChlorhexidine mouthwash is usually recommended for a usage period of 7 to 14 days , 57. HowThe pH of the solution in which the acrylic resin is immersed has been reported as a significant factor influencing discoloration . AdditioThe present study assessed the color-difference by using a formula based on CIELAB, incorporating lightness, chroma, and hue weighting functions, along with an interactive term addressing chroma and hue differences to enhance the accuracy of assessment for blue color as well as a scaling factor for CIELAB a* scale to enhance the performance for gray colors. Nowadays the CIEDE2000 color difference formula has emerged and is recommended for its enhanced applicability and reliability in dentistry. Moreover, it offers improved adjustments for determining color differences by addressing non-uniformities in the CIELAB formula .in-vitro nature, which allowed staining on both sides of the specimens, unlike clinical conditions. This factor also precluded the consideration of oral hygiene practices, like tooth brushing, which can affect restoration color stability in vivo [Among the limitations of the current study was its  in vivo . FurtherWithin the limitations of the present study and with respect to the evaluated materials, it can be concluded that high-translucency monolithic zirconia is more susceptible to discoloration when exposed to both chlorhexidine and Listerine mouthwashes than the other two monolithic zirconia systems (ML and LT). Meanwhile, multilayered monolithic zirconia is the most color stable material. Nevertheless, color changes in all groups were below the clinically acceptable threshold (ΔE< 3.7) Moreover, multilayered zirconia exhibits the greatest changes in translucency when exposed to chlorhexidine and Listerine mouthwashes compared to the other two monolithic zirconia systems (HT and LT).S1 Table(XLSX)Click here for additional data file.


To investigate the changes in macular cystic schisis (MCS) and sensitivity during the day in X-linked retinoschisis (XLRS) patients.Treatment-naïve patients with genetically verified XLRS underwent best-correlated visual acuity (BCVA) testing with ETDRS charts, spectral domain optical coherence tomography, and microperimetry (MP) twice a day, at 9 a.m. and 4 p.m., to measure changes in central retinal thickness (CRT), macular volume (MV), average threshold (AT), and fixation stability parameters (P1 and P2).p = .021), the AV improved in 1.84 dB , the CRT decreased in 24.43 µm , and the MV dropped in 0.27 µm3 . P1 and P2 did not variate. The collapse of the MCS led to the reduction of macula thickness. CRT at baseline correlated with the decrease of CRT (Spearman’s ρ: − 0.83 [p = .001]). Age and change of BCVA, CRT, and AV did not correlate among one another. Eyes with disrupted ellipsoid zone showed a more prominent change in CRT (p = .050). Photoreceptor outer segment length and integrity of the external limiting membrane and cone outer segment tips were not associated with BCVA, AT, or CRT variation.At baseline, the BCVA of the 14 eyes of 8 patients amounted 0.73 (± 0.23) LogMAR. Between timepoints, the BCVA increased in 3.21 letters : 2020–10,328.Institutional Review Board of the Hamburg Medical Chamber (The online version contains supplementary material available at 10.1007/s00417-023-06106-0. X-linked retinoschisis  is a monogenetic hereditary retinal dystrophy with an estimated prevalence between 1 in 15,000 and 1 in 30,000 caused by mutations of the retinoschisis-1 gene (RS1)) at baseline and with the PROS length (Spearman’s ρ: 0.69 [p = 0.006]). Furthermore, the PROS length also significantly correlated to the average threshold at baseline (Spearman’s ρ: 0.69 [p = 0.009]). The average threshold at the morning control correlated to fixation stability P2 at 9 a.m. (Spearman’s ρ: 0.48 [p = 0.018]), which, in turn, correlated to fixation stability P1 (Spearman’s ρ: 0.95 [p = 0.0001]).The mean BCVA was 0.73 (± 0.23) LogMAR at 9 a.m. and 0.74 (± 0.33) LogMAR at 4 p.m. . An improvement of the average threshold was observed from 19.70 (± 2.57) dB at 9 a.m. to 21.62 (± 2.81) dB at 4 p.m.  and its mean relative change was 9.73 (± 9.72) % (p = 0.003). Fixation parameters P1 and P2 did not significantly vary between timepoints: P1 was 56.92 (± 25.7) % at 9 a.m. and 59.67 (± 27.05) % at 4 p.m. , while a P2 of 84.92 (± 15.01) % at 9 a.m. and 87.08 (± 18.37) % at 4 p.m. was measured (mean change: 2.85 [± 13.23] % p = 0.45).The diurnal changes in macular anatomical and functional parameters are illustrated in Fig. p = 0.007). The mean relative change of CRT was − 4.05 (± 5.53) % (p = 0.013). The collapse of MCS led to the reduction of macular thickness, as demonstrated in Fig. 3 to 8.76  µm3 between the intraday controls was − 0.27  µm3 and also statistically significant (p = 0.016). The median relative change of the macular volume amounted to − 2.68  % (p = 0.009). Age was associated with the variations of CRT, BCVA, and average threshold  µm at baseline to 410.50  µm at the afternoon control was shown . Eyes with disrupted EZ showed a higher diurnal variation of CRT compared to eyes with intact EZ . The diurnal CRT variation did not significantly correlate to PROS length or ELM and COST’s integrity , no microperimetry and BCVA measurements were performed as training before the functional test of this study. Furthermore, the function and macular thickness variation was sampled only at two timepoints and not throughout the day. In conclusion, treatment-naïve XLRS patients experience diurnal changes in macular thickness and function with improved macular sensitivity (BCVA and average threshold assessed by MP) and reduced macular thickness (CRT and macular volume). Therefore, we advocate a time-adapted protocol in patients with MCS in XLRS, as visual function and retinal morphology measurements are essential endpoints in clinical trials. The diurnal decrease of CRT correlated only with the CRT at baseline and the disruption of the EZ. Notably, the diurnal changes of functional parameters and MCS were not associated with the age or among each other. The PROS length and the integrity of COST and ELM did also not influence the circadian changes of macular function and thickness. Further investigations with a higher number of participants are needed to confirm these results.Supplementary file1 (DOCX 31.1 KB)Below is the link to the electronic supplementary material.


Correction to: International Urogynecology Journal (2022) 34:1-4210.1007/s00192-022-05397-xThe authors regret that one of the article references has been erroneously replaced by a non relevant one.Reference 14 should be replaced by:Haylen, B. T. et al. An International Urogynecological Association (IUGA) / International Continence Society (ICS) Joint Report on the Terminology for Female Pelvic Organ Prolapse (POP). Neurourol. Urodyn. (2016) 10.1002/nau.22922.The authors would like to apologise for any inconvenience caused.


Knee stiffness is believed to occur to compensate for ankle joint instability in the supporting leg. Therefore, adjusting knee stiffness to accommodate ankle joint instability is crucial for maintaining kicking performance. Based on results of this study, it may be important to consider training and exercises focused on joint coordination to improve knee stiffness in soccer players with CAI.Soccer players with chronic ankle instability (CAI) may stabilize their supporting leg by the proximal joint to compensate for the ankle instability during kicking motion. This study aimed to investigate the characteristics of leg and joint stiffness of the supporting leg during side-foot kicking in soccer players with CAI. Twenty-four male collegiate-level soccer players with and without CAI participated in this study. The kinematic and kinetic data were obtained using a three-dimensional motion analysis system. Leg stiffness and joint  stiffness in the sagittal and frontal planes were calculated and analyzed. The results clarified that soccer players with CAI (0.106 ± 0.053 Nm/°) had greater knee stiffness in knee adduction during the kicking cycle compared to those without CAI (0.066 ± 0.030 Nm/°;  In soccer, musculoskeletal injuries occur frequently during games and practice . Among tA previous prospective cohort study found that 40% of patients with first-time ankle sprains developed CAI . TherefoKicking motions such as shooting and passing are integral to soccer games and have a significant effect on playing skills and performance . Among tPrevious studies have suggested that CAI in soccer players is associated with poor dynamic balance of the supporting leg  and footThis study included 24 male collegiate-level soccer players  with a soccer playing experience of 10.9 ± 2.0 years. For all participants, the preferred leg for kicking (kicking leg) was the right leg, with the left leg acting as the supporting leg. Regarding their main position, 9 participants were strikers, 7 were midfielders, and 8 were defenders. The inclusion criteria were (a) age > 18 years and (b) >6 years of soccer experience. We excluded participants with (a) histories of orthopedic surgeries, (b) current injuries in the lower extremities, and (c) pain in the lower extremities during daily activities and soccer practice.This study was conducted between August 2019 and March 2020 at the Kinematics Laboratory of the International University of Health and Welfare, Narita Campus, Chiba, Japan. Prior to testing, all participants volunteered to participate in this study and provided written informed consent. The study protocol was approved by the International University of Health and Welfare Ethics Committee  and was conducted in accordance with the tenets of the Declaration of Helsinki .The participants completed a questionnaire regarding their physical characteristics, medical history of ankle sprains, and current medical information before inclusion in this study. The Cumberland Ankle Instability Tool (CAIT) was used to identify CAI . The CAIThe participants were divided into CAI and no-CAI (N-CAI) (CAIT score ≥ 28) groups. Those who had a CAIT score of ≤27 ,26, a hiA three-dimensional motion analysis system  and AMTI force plates with an amplifier  were used to measure the kinetic data and GRF during testing, according to a previous study . The samAll the participants wore closely fitted dark shorts, and the measurements were taken barefoot to accurately measure the data. Thirty-nine reflective markers (14 mm) were placed to create the coordinate systems of the head, torso, arm, pelvis, and leg segments, according to the Vicon Plug-in Gait full-body model  . ReflectPrior to testing, all the participants performed the desired warm-up, including slow jogging and/or stretching. Subsequently, the participants performed side-foot kicking trials on the AMTI force plates. A ball  was placed at the desired position on the AMTI force plates. All participants were instructed to perform side-foot kicking with as much force as possible toward a net (width and height 0.6 m each), positioned 2 m in front of the ball. The approach velocity was arbitrary for all participants. The approach angle was set at 45° with respect to the direction of the net. Side-foot kicking was conducted to obtain five successful trials. If the ball was off-target, the trial was excluded. The intervals between trials were set at 3 min to check the acquired data, reorganize the measurement settings, and consider the participant’s fatigue.The Vicon Nexus software  was used to calculate all variable outcome measures based on the biomechanical model of the Vicon Plug-in Gait. The middle 3 trials of 5 successful kicking were used for data analysis. The vertical GRF, joint angles, and moments  of the supporting leg were obtained during a single side-foot kicking cycle. A single kicking cycle was defined as the duration from heel strike  to the frame in which the kicking leg reached its highest vertical position in the follow-through phase after ball contact. The kicking cycle was normalized as a percentage, that is, 0% and 100% represented the heel strike and frame of the highest vertical, respectively. Joint angles were derived using the Cardan y-x-z rotation sequence of the distal segment relative to that of the proximal segment. Positive joint angles represented flexion/dorsiflexion in the sagittal plane and abduction/inversion in the frontal plane. Joint moments were calculated as external moments; positive joint moments represented flexion/dorsiflexion in the sagittal plane and abduction/inversion in the frontal plane. The whole-body center of mass (CoM) was calculated from the weighted sum of all centers of mass of all segments defined by the markers. legK) was calculated as the ratio of the peak vertical GRF (peakvGRF) to the change in vertical displacement of CoM (∆CoML) [peakvGRF was obtained from the maximum value during the kicking cycle. ∆L was calculated as the lowest and highest position of CoM during the kicking cycle. Leg stiffness (legK) was normalized to the body mass (kg) and height (mm) of the participantsLeg stiffness ( (∆LCoM) , and forjointK) of the lower extremities was calculated by the ratio of the change in joint moments (∆peakM) to the change in joint angles (∆jointθ) [joint of the lower extremity joints were calculated as hip (hipK), knee (kneeK), and ankle stiffness (ankleK) in the sagittal and frontal planes. ∆jointM represented the change in joint moments between the moment at the heel strike and the maximum value during the kicking cycle. ∆jointθ represented the angular displacement between the angle at the heel strike and the maximum value during the kicking cycle. Joint stiffness (jointK) of the lower extremities was normalized to the body mass (kg) and height (mm) of the participantsJoint stiffness (∆θjoint) , and for® SPSS® Statistics for Windows version 27.0.  by an independent statistician. Prior to statistical analysis, all outcome data were averaged for the middle three of five successful trials, and the means and standard deviations were calculated. Normality of data was assessed by the Shapiro–Wilk test, and homogeneity of variance was verified with Levene’s test. The independent sample t-tests were used to identify differences in variables between the CAI and N-CAI groups, depending on whether these variables were normally distributed. Statistical significance was set at p < 0.05. Effect sizes (ESs) were calculated using Cohen’s d from raw data by all outcomes. ESs are considered small when d = 0.20, medium when d = 0.50, and large when d = 0.80 [t-tests were performed to compare each individual joint moment curve between groups using the open-source SPM1d code  in MATLAB® R2020b . Statistical analyses were conducted using the IBMd = 0.80 . Multiplp > 0.05). Patient characteristics of both groups are shown in p = 0.049). There were no significant differences in the other variables during the kicking cycle between the CAI and N-CAI groups (p > 0.05). The joint moment curves of the CAI and non-CAI groups during the kicking cycle are shown in p > 0.05).The peak joint angles and moments of the supporting legs in the lower extremities are shown in p = 0.36, 2 = 0.429, adjusted R2 = 0.344, p = 0.009).Leg stiffness did not differ significantly between the CAI (19.67 ± 2.85 kN/m) and N-CAI groups , whereas there was no significant difference in the sagittal plane (p > 0.05). Regarding hip and ankle stiffness, these variables showed no significant differences in either the sagittal or frontal planes (p > 0.05). The joint stiffness of the lower extremity joints is shown in This study aimed to investigate the stiffness characteristics of the supporting leg during side-foot kicking in soccer players with CAI. The results of this study showed that soccer players with CAI exhibit greater knee stiffness in the frontal plane during the kicking cycle compared to those without CAI; however, there were no characteristic differences in knee stiffness in the sagittal plane and hip and ankle stiffness. These results indicate that soccer players with CAI exhibit greater knee stiffness in knee adduction during kicking, potentially compensating for CAI-associated ankle instability.Previous studies have revealed that leg stiffness is adjusted to accommodate different surfaces ,32, incrAlthough the kicking motion was in the sagittal plane, the results of this study represented affected knee stiffness in the frontal plane. In a previous biomechanical study, the medial and lateral GRF components were generated in the supporting leg during the kicking cycle . In addiThere was no difference in leg stiffness, which represents stiffness of the entire lower extremity, between the CAI and N-CAI groups. This may be the result of ankle joint instability being compensated for by increased knee stiffness. Through the investigation of the angular displacements affecting leg stiffness among all players, it became clear that those at the hip and knee joints in the sagittal plane were the major contributors to leg stiffness. This suggests that knee and hip flexion are the most important factors in increasing leg stiffness during the kicking motion, similar to previous studies that investigated the main contributors of energy absorption during jump landing or side-stepping ,40,41. TChanges in the knee stiffness and kinematic/kinematic properties of the knee joint during landing motion are closely related to severe knee joint disorders such as anterior cruciate ligament injuries and knee osteoarthritis ,43,44,45This study had some limitations. Firstly, a previous study found that the CAIT measures the severity of CAI due to MAI, FAI, or a combination of these two phenomena . As a reThe results of this study clarified that soccer players with CAI exhibited greater knee stiffness in the frontal plane of the supporting leg during the kicking cycle, while no characteristic differences were observed in the sagittal plane, compared to those without CAI. This finding indicated a potential link between ankle instability in CAI with knee stiffness of the supporting leg during side-foot kicking. Therefore, adjusting knee stiffness to accommodate ankle joint instability may be a crucial mechanism for maintaining kicking performance. These findings have relevance for the development of training programs and exercises centered around joint coordination to enhance knee stiffness in soccer players with CAI.