Kleg)/vertical stiffness (Kvert) in recreationally trained runners. Thirty-one male runners were recruited and underwent three separate tests. In the first session, the body composition, Kleg, and Kvert at running speeds of 12 and 14 km⋅h–1 were measured. In the second session, isokinetic knee and ankle joint PT at 60°⋅s–1 were tested. The force-time characteristics of the IMTP were evaluated in the final session. Pearson’s product-moment correlations, with the Benjamini–Hochberg correction procedure, showed that the knee flexor concentric and eccentric and extensor concentric PT  were moderate to largely correlated with Kleg and Kvert at 12 and 14 km⋅h–1. The knee extensor eccentric PT  was moderately correlated with the 14 km⋅h–1Kvert. The ankle plantar flexor concentric and dorsiflexor eccentric PT  were largely correlated with Kleg at 12 km⋅h–1. The ankle plantar flexor concentric and eccentric and dorsiflexor eccentric PT  were moderate to largely correlated with Kvert at 12 and 14 km⋅h–1. For IMTP testing, high correlation was only found between the IMPT peak force (PF) and Kvert at 14 km⋅h–1 . Thus, superior leg-spring stiffness in recreational runners may be related to increased knee and ankle joint strength, eccentric muscular capacity, and maximal force production.Neuromuscular characteristics, such as lower-limb joint strength and the ability to rapidly generate force, may play an important role in leg-spring stiffness regulation. This study aimed to investigate the relationship between isokinetic knee and ankle joint peak torque (PT), the force-time characteristics of isometric mid-thigh pull (IMTP), and leg stiffness ( Kleg) and vertical stiffness (Kvert), are typically used to characterize the mechanical properties of a “leg-spring,” representing the lower limbs during running  and the maximum compression in the lower-limb length   . Greaterss (COM) . Previouss (COM)  and thatss (COM) . Thus, eKleg and Kvert after training  are mostly due to neuromuscular adaptation (Neuromuscular strength is an important factor in determining leg-spring stiffness. Improvements in aptation . It has aptation . For exaaptation . The plaaptation  and are aptation . Meanwhiaptation . The kneaptation  and helpaptation . Therefor = 0.50) in older adults, whereas Kleg/Kvert during practical running.However, the relationship between knee and ankle strength and leg-spring stiffness is a topic of debate in scientific and sports communities. As current literature commonly used the ultrasonography technique to measure tendon stiffness or tendinous tissues stiffness, which cannot directly reflect leg-spring stiffness during running. For example, Kleg at 12 km⋅h–1. Notably, the time required to make contact with the ground and propel the body forward is >200 ms at sub-maximal speed (12 km⋅h–1) (The runners’ ability to generate force and power within a transitory period during landing could effectively regulate leg-spring stiffness . The iso km⋅h–1) . It is, Kleg and Kvert) in recreational male runners.Thus, in the present study, we aimed to evaluate the relationship between isokinetic lower-limb joint peak torque (PT) (such as knee and ankle joint concentric and eccentric PT) and IMTP force-time characteristics , and leg-spring stiffness  from a collegiate running club volunteered to participate in this study. The basic information and physical characteristics of the subjects are presented in Kleg, and Kvert at running speeds of 12 and 14 km⋅h–1 were measured. In the second session, PT of concentric and eccentric action at 60°⋅s–1 velocity of isokinetic knee flexor and extensor muscles, and that of ankle plantar flexor and dorsiflexor muscles, was conducted. Force-time characteristics  of the IMTP were evaluated in the final session.This was a cross-sectional study involving recreational runners to investigate the association between leg-spring stiffness and isokinetic lower-limb joint strength, and isometric time force characteristics. Each subject performed three separate test sessions in the laboratory with a minimum rest interval of 48 h. Before each session, participants were asked to refrain from strenuous exercise for 24 h and acquire more than 8 h of adequate sleep; it was also ensured that they did not experience any muscle soreness or fatigue. The researcher examined the subjects’ commitment to the instructions with the help of the questionnaire when the subjects came to the laboratory for testing. In the first session, the body composition, Body composition was measured prior to the leg-spring stiffness test. Subjects were required to achieve a rested and hydrated state on the day before the body composition test. On the measurement day, they were required to avoid drink and food intake 2 h before testing. Standing height was measured with a wall-mounted measuring device , and the measurement accuracy was within 0.1 cm. Bodyweight, fat mass, and fat-free were measured using a bioimpedance analyzer . Body mass index (BMI) was calculated by dividing weight in kilograms by height in square meters.–1, the subjects performed running at speeds of 12 and 14 km⋅h–1 for 4 min, during which time data were recorded for analysis. An 8-camera, 3-dimensional (3D) motion capture system  was used to record the position of 36 reflective markers (100 Hz) to accurately obtain the vertical displacement of the COM [calculated by the body segmental analysis technique , using inverse dynamics. The vGRF was filtered with a cut-off frequency of 200 Hz, and the marker trajectories were filtered with a cut-off frequency of 7 Hz using a fourth-order Butterworth low-pass filter . Based oKleg) and vertical stiffness (Kvert) were calculated according to Morin’s method  between 0.86 and 0.99. Kleg (kN⋅m–1) is defined as the ratio between the maximal vGRF and the maximum compression in the lower-limb length , Δy is the maximal displacement of the COM during ground contact, v is the running velocity (m⋅s–1), and tc is the ground contact time (s) (a threshold of 50 N vGRF was set when the foot contacted the force plate).In this study, Kvert (kN⋅m–1) is the ratio of the maximum vGRF to the vertical displacement when the COM reaches its lowest point , eccentric (Kflex–ecc), knee extensor concentric (Kex–con), and eccentric (Kex–ecc) PT at 60°⋅s–1.In the knee joint isokinetic strength test, the subjects sat on the testing table with the knee at 90° flexion. The shaft of the dynamometer was visually aligned with the lateral condyle of the femur and remained constant during the concentric and eccentric contractions . The shoAdors–con), eccentric (Adors–ecc), plantar flexor concentric (Aplan–con), and eccentric (Aplan–ecc) PT were measured at 60°⋅s–1.In the ankle joint isokinetic strength test, subjects laid supine on the dynamometer seat with their hips and knees fully extended, and the right foot was placed on the foot plate. The shaft of the dynamometer was visually aligned with the lateral malleolus of the foot and kept constant during the concentric and eccentric contractions . The rigThe IMTP test was performed using a portable test rack and two force platforms at a sampling frequency of 1,000 Hz . This test has consistently proven to be highly reliable for ICCs ranging from 0.80 to 0.97 . Before F50,F100,F150,F200,F250,F300, and F350) from the initiation of the pull were collected for every trial. The calculation formula for the RFD is:Force-time curves were collected at rest for 2 s and at full pull for 5 s during the IMTP test, with the highest force generated reported as the absolute PF. In addition, the time-specific forces at 50–350 ms  (The RFD was applied to pre-determined time bands: 0–50 to 0–350 ms (RFD–350ms) . These t–350ms) .Kleg and Kvert at 12 and 14 km⋅h–1) were analyzed using Pearson’s product-moment correlations. Magnitudes of correlation (r) were defined as “small” when r = 0.1–0.3, “moderate” when r = 0.3–0.5, “large” when r = 0.5–0.7, “very large” when r = 0.7–0.9, and “extremely large” when r = 0.9–1.0  and the statistical programming language R.1The Shapiro–Wilk test was used to test data normality. Values are expressed as mean ± SD. The correlations between isokinetic strength (knee and ankle joint concentric and eccentric PT), IMTP force-time characteristics , and leg-spring stiffness ( 0.9–1.0 . All reprocedure . StatistKleg and Kvert at 12 and 14 km⋅h–1) are shown in All isokinetic strength, IMTP time force characteristics, and leg-spring stiffness values are listed in Kflex–con , Kflex–ecc , and Kex–con  was moderately to largely correlated with Kleg at 12 and 14 km⋅h–1. The PT of Aplan–con  and Adors–ecc  was largely correlated with 12 km⋅h–1Kleg after Benjamini–Hochberg adjustment , Kflex–ecc , Aplan–con , Aplan–ecc , and Adors–ecc  was moderately to largely correlated with Kvert at 12 and 14 km⋅h–1. The PT of Kex–con  and Kex–ecc  was moderately to largely correlated with the 14 km⋅h–1Kvert  and Kvert at 14 km⋅h–1, after Benjamini–Hochberg correction  is an important parameter regulating running mechanics because it reflects the amount of energy storage and recoil for a given compression. However, scientific literature on the relationship of neuromuscular strength with leg-spring stiffness remains unclear. This study aimed to investigate the relationship between isokinetic lower-limb joint strength, isometric force-time characteristics, and leg-spring stiffness in recreationally trained male runners. Our primary finding was that the maximum torque of knee and ankle production was strongly correlated with Kleg and Kvert. Furthermore, only the IMTP PF was correlated with Kvert at 14 km⋅h–1. No correlations were found between leg-spring stiffness and specific time force at 50–350 ms or RFD at 0–50 to 0–350 ms. The potential mechanisms of how these neuromuscular characteristics regulate leg-spring stiffness are shown in Leg-spring stiffness  and Kvert values (26.89–29.26 kN⋅m–1) reported are similar to those of previous investigations at similar running speeds , and those of previous studies  between IMTP PF and eccentric strength . At a higher speed (14 km⋅h–1), the muscle activity during the stance phase may reinforce the driving force in the forward direction  was relatively low (–1) and did not require explosive contraction, which may have resulted in a limited correlation with leg-spring stiffness. By contrast, the PF of IMPT was largely correlated with vertical stiffness, as Kvert was calculated using Fmax. Although no statistical correlation was found, the ability to quickly generate a higher force is essential for runners. Theoretically, it enables runners to rapidly push off the ground, thereby decreasing ground contact and resulting in a quicker transition from the braking to the propulsive phase of the running gait . In addition, a large correlation does not imply a cause-effect relationship. Therefore, studies with a longitudinal study design should be performed in the future to confirm the effects of isokinetic joint strength, isometric force-time characteristics, and their manipulation through intervention on leg-spring stiffness.–1 were significantly correlated with Kleg and Kvert. Furthermore, the IMTP PF was largely correlated with Kvert at 14 km⋅h–1. Therefore, leg-spring stiffness is associated with specific neuromuscular characteristics in recreational runners. Runners should focus on improving their knee and ankle joint strength, eccentric muscular capacity, and maximal force production. Although we did not find a correlation between specific time interval force or RFD and leg-spring stiffness, a previous study indicated that quicker force production facilitates leg-spring stiffness regulation . The participants provided written informed consent to participate in this study. Written informed consent was obtained from the participants for the publication of any potentially identifiable images or data included in this manuscript.SC and QZ conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents, materials, and analysis tools, prepared figures and/or tables, authored or reviewed drafts of the manuscript, and approved the final draft. DW conceived and designed the experiments, and authored or reviewed drafts of the manuscript. YS performed the experiments, and prepared figures and/or tables. HD guided the writing of the manuscript, critically revised the manuscript, and approved the final draft. FL conceived and designed the experiments, and approved the final draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


SEM micrographs showed the presence of rough surface topology after blending the two polymers, with the appearance of small pores. In addition, the presence of surface cracks indicates the biodegradability of the prepared membranes that would result after enzymatic treatment. These results are supported by surface roughness results that show the surface of the κ-carrageenan/starch membranes became rougher after enzymatic treatment. The hydrophilicity of the prepared membranes was evaluated from contact angle (CA) measurements and the swelling ratio. The swelling ratio of the prepared membranes increased gradually as the starch ratio increased, reaching 150%, while the water-uptake capacity increased from 48 ± 4% for plain κ-carrageenan to 150 ± 5% for 1:2 κ-carrageenan/starch blends. The amylase enzyme showed an effective ability to degrade both the plain κ-carrageenan and κ-carrageenan/starch membranes, and release glucose units for up to 236 and 563, respectively. According to these results, these blends could be effectively used in making safe and biodegradable molded toys with superior water-absorbing capabilities.κ-carrageenan is useful for its superior gelling, hydrogel, and thickening properties. The purpose of the study was to maximize the hydrogel properties and water-absorbing capacity of κ-carrageenan by blending it with starch from potato peels to be used as safe and biodegradable water-absorbent children’s toys. The prepared materials were analyzed using FTIR and Raman spectroscopy to analyze the functional groups. Results showed that there was a shift in the characteristic peaks of starch and κ-carrageenan, which indicated their proper reaction during blend formation. In addition, samples show a peak at 1220 cm Hydrogels are commonly used for bio-fabrication, as they are biocompatible materials ,2. Histoκ-carrageenan is a family of polysaccharides extracted from a specific red seaweed species in the Rhodophyceae family, formed by alternate units of D-galactose and 3,6-anhydro-galactose  joined by 1,3 and β-1,4-glycosidic linkage. κ-carrageenan has the ability to form gels and membranes with better mechanical properties . It has Starch is an inexpensive and readily available material that is produced from renewable resources and can be efficiently used ,11,12. IStarch comprises linear amylose and branched amylopectin structures. Amylose is composed of α-1,4 glucose units. This moiety supports the ability for film-forming. Starch-based films are often brittle and present weak mechanical properties. Therefore, starch can be mixed with other polymers to produce blends with excellent mechanical traits after solvent evaporation. Polymer blending is a well-used strategy whenever a change or improvement of properties is necessary. Starch-based films were created by blending starch with gelatin , lignoceFew studies have reported on starch/κ-carrageenan blends. κ-carrageenan has a linear sulfated structure obtained from red seaweed. It is composed of D-galactose and 3,6-anhydro-D-galactose units linked by α-1,3 and β-1,4-glycosidic linkages. κ-carrageenan has gelling and film-forming features. These traits allow the production of biodegradable films .The aims of the present work were to develop blends composed of mixtures of κ-carrageenan and starch to make safe and biodegradable molded toys as a novel application. κ-carrageenan was combined at a high temperature with various percentages of starch in the presence of glycerol to form a viscous liquid solution that can be poured into a mold, cooled at room temperature till complete solidification, and finally reshaped using shaped molds . An evaluation was carried out by analyzing the films’ mechanical and swelling properties, these being the chemical interactions between the two polysaccharides, assessed by Fourier-transform infrared (FTIR) spectra, Raman spectra, and a scanning electron microscope (SEM). In addition, the hydrophilicity of the prepared membranes was evaluated from contact angle (CA) and water uptake (WU) measurements, and their potential biodegradability was also studied.κ-carrageenan was obtained from Across Organics, and the α-amylase enzyme (700 U/mL) was purchased from Sigma-Aldrich. RPI Glycerol Liquid, USP grade, and molecular weight (92.1).The potato peels, which were collected from different restaurants in Borg El-arab city, were washed with tap water, then cut into 1 × 1 cm square pieces. The formed pieces were submerged in tap water and homogenized using a kitchen blender, then sieved using stainless-steel mesh (kitchen stainless steel sieve with filter sifter). The obtained large particles were discarded, while the starch-containing filtrate was left at room temperature till full evaporation of its water content. The dried starch molecules were collected, ground, and kept in a dry place till use.κ-carrageenan solution (5%) was prepared by dissolving in boiling water. Starch solution was prepared by dispersing varying proportions (2.5–10%) of starch in distilled water. Blended solutions were prepared by mixing their pre-prepared solutions while stirring at 80 °C to obtain κ-carrageenan/starch blends at different ratios of 2:1, 1:1, and 1:2 with dropwise addition of glycerol (50 µm), which make membranes more elastic and enhances their mechanical characteristics. The prepared solutions were cast in a dolphin-shaped mold and kept at 27 °C for 48 h under 35% humidity . It is wκ-carrageenan and κ-carrageenan/starch blend were characterized via several characterization methods, described as:The presence of the functional groups of both mixed and individual polymers was verified by Fourier-transform infrared (FTIR) technique   and RamaWater uptake of the prepared membranes was measured by the change in the weight between the wet  measurements . For eacThe tensile strength of the polymer electrolyte membranes was measured using a Universal Testing Machine  at room temperature. The specimens were 50 × 10 mm. Measurements were carried out at a constant speed of cross heads movement 5 mm/minute. Three measurements were taken for each sample, and the mean values were considered .This test has been performed according to with some modifications. The biodegradability of the prepared membranes was investigated through the incubation of 0.07 g of each membrane with 100 µL amylase enzyme in 1400 µL acetate buffer (pH 5.5). After 72 h incubation at 30 °C and 150 rpm, SEM, Raman spectroscopy, and FT-IR analysis were investigated using the solid residuals of each membrane. On the other hand, the concentration of the liberated glucose units was determined spectrophotometrically using glucose kit .−1 was attributed to O-H stretching vibrations, formed due to the vibrational stretching associated with free inter- and intra- molecular bonds in -OH groups [−1 was attributed to C-H stretching vibration. Samples also show a band in the region of 750–1300 cm−1 that corresponds to the carbohydrates region [−1 corresponds to the ester sulfate groups, and the peak at 1670 cm−1 is due to the carbonyl group contained in D-galactose [−1, and the band at 2930 cm−1 is attributed to the O–H stretching and the C–H stretching vibrations, respectively [−1. Shifting of bands in this region is due to interactions between the charged functional groups of the C-O stretching band and the C-O-H group band from 1162 cm−1 (κ-carrageenan) to 1151 cm−1 (κ-carrageenan/starch 2:1) that can be related to the interaction (possibly through hydrogen bridges) of the OH groups of starch with the κ-carrageenan structure [−1 (s), due to N-H stretching of secondary amides, and 2980 cm−1, due to C-H stretching of the amylase enzyme, in addition to the characteristic peaks of κ-carrageenan/starch membrane that appeared [Infrared spectroscopy is an effective method commonly used for polysaccharide characterization to study molecular interactions distinguished by the change of absorption bands. The fingerprint region of typical spectra of κ-carrageenan is presented in H groups , and thes region ,33. The ectively , as showtructure . On the appeared .−1, which are due to the stretching of C-O, C-C, and C-OH bonds belonging to starch [−1 was due to the C-H stretching. Absorption at 1238 cm−1 was due to the ester sulfate group, and absorption at 1048 cm−1 was due to glycosidic linkage [−1, which was due to the formation of the carbonyl group. This peak was initially thought to have resulted from the oxidation of carbohydrate radicals generated inside the κ-carrageenan polymer. However, as shown in −1 due to NH2 stretching, which confirms the effect of the amylase enzyme on the degradation of the membranes [Raman spectroscopy results confirmed the interactions of starch with κ-carrageenan. o starch . Absorpt linkage . Howeverembranes ,40.The investigation of the membranes’ biodegradability was dependent on the ability of the amylase enzyme to release glucose units from these membranes. As shown in SEM microphotographs of starch granules, as well as the surface of the κ-carrageenan/starch membranes are shown in Surface roughness values of the κ-carrageenan and κ-carrageenan/starch membranes with different ratios before and after enzymatic treatment are shown in Water uptake of the κ-carrageenan/starch membranes increased gradually as the starch ratio increased. As shown in However, regarding the viscoelastic properties of the hydrogel: the gelation of κ-carrageenan has been widely studied using various methods, such as rheological experiments, to understand the properties of the mixed carrageenan gels.The viscoelastic behavior of κ-carrageenan reveals a fluid-like character. At 9 °C, κ-carrageenan molecules are in the ordered state, but neither aggregation nor gelation takes place. Moreover, κ-carrageenan molecules being in the ordered state are obviously stiffer than in the disordered one.In cases where κ-carrageenan is blended with starches, a synergistic effect on the rheological properties of the pastes and gels exists. Starch pastes and gels are considered a biphasic system, with the continuous phase consisting of the solvent and dissolved starch during gelatinization, and a disperse phase consisting of swollen granules. This viewpoint emphasizes the presence of swollen particles in starch suspensions. The swollen granules are not only deformable, but also compressible and elastic.On the other hand, the hydrophilic/hydrophobic nature of the membranes’ surfaces is often indicated by the contact angle. Water contact angle values of the prepared membranes are listed in The tensile strength and elongation at break for the prepared membranes are given in Blends of κ-carrageenan and potato peel starch were prepared in three different ratios. Both polymers were characterized before and after blending, using SEM, FTIR, Raman, etc., in addition to testing their biodegradability through enzymatic treatment. The results provide useful information on the structural properties of κ-carrageenan and starch and the structural changes in the network induced when mixing different ratios of the two polysaccharides, before and after enzymatic treatment. The FTIR spectra of κ-carrageenan/starch blend-based membranes showed stretching and shifting of different bands that can be related to their interaction. Additionally, SEM showed the presence of rough surface topology after the blending of starch and κ-carrageenan, indicating the presence of starch granules and the interaction of starch and κ-carrageenan. After enzymatic treatment, multiple cracks were noticed on each membrane surface, indicating potent enzymatic degradation of the prepared membranes. The swelling ratio and water contact angle results of the κ-carrageenan/starch membranes illustrate hydrophilic properties that increased gradually as the starch ratio increased. However, after enzymatic treatment, the swelling degree increased to reach 180% for the κ-carrageenan/starch (1:2) membrane due to the increased porosity of the membranes’ surfaces, which allowed more water molecules to penetrate the matrix. According to these results, it is proposed that κ-carrageenan/starch blends can be effectively used as a super-absorbent hydrogel that can be used as biodegradable children’s toys.


Preservation of hamstring tendon insertion at the time of anterior cruciate ligament (ACL) reconstruction is a well-known technique; however, its effect on graft integration is not well studied. The present study was conducted to study the graft integration inside the tibial and femoral tunnels, respectively, after ACL reconstruction using hamstring tendon graft with preserved insertion.Twenty-five professional athletes who underwent ACL reconstruction using hamstring tendon graft with preserved tibia insertion were enrolled in the study. Functional outcomes were checked at final follow-up using Lysholm score and Tegner activity scale. Magnetic resonance imaging (MRI) was done at 8 months and 14 months follow-up to study the graft tunnel integration of the ACL graft at both tibial and femoral tunnels.p = 0.02). The mean FI score  decreased from 3.04 (2–5) at 8 months to 2.57 (2–4) at 14 months (p = 0.02).The mean Fibrous interzone (FI) score  decreased from 2.61 (1–5) at 8 months to 2.04 (1–4) at 14 months follow-up (Graft integration occurs early in the tibial tunnel as compared with the femur tunnel with preserved insertion hamstring tendon autograft.Trial registration CTRI/2019/07/020320 [registered on 22/07/2019]; http://www.ctri.nic.in/Clinicaltrials/pdf_generate.php?trialid=33884&EncHid=&modid=&compid=%27,%2733884det%27 Anterior cruciate ligament (ACL) reconstruction is a commonly performed orthopedic procedure . The goaThe hamstring tendon graft is a soft tissue graft that needs to be integrated within the bone tunnels (graft tunnel integration) . Graft iGraft tunnel integration is an important factor affecting graft maturation, and hence it is crucial for the proper functioning of reconstructed ACL. Therefore, the present study was conducted to compare the graft integration inside the femoral and tibial tunnel after ACL reconstruction using STGPI graft.It was hypothesized that preservation of hamstring tendon insertion hastens the process of graft integration in the tibial tunnel.This was a prospective study conducted in a regional sports injury center after institutional ethical committee approval (GMC/12C/2018/277). The present study is registered with the Clinical Trials Registry-India (CTRI/2019/07/020320). Twenty-five elite male sportspersons of age 18–35 years who underwent ACL reconstruction using STGPI graft were enrolled in the study. Exclusion criteria were female patients, multi-ligament injury, history of chronic inflammatory disease, previously operated on the same knee, smokers, alcohol, and history of steroid intake.Data for this study were collected prospectively from 25 patients involved in different sporting activities who had undergone primary ACL reconstruction. ACL reconstruction was done using a quadruple hamstring tendon graft using the transportal technique. Both the semitendinosus and gracilis tendon insertions at the tibia were preserved, and their free ends were sutured back to the insertion site . EndobutThe semitendinosus and gracilis (STG) grafts were harvested through a 3–5 cm incision centered 2 cm medial to the tibial tuberosity. The proximal free ends of the tendons were sutured together using Ethibond no. 5 suture . The tendons were looped around an Ethibond No. 5 suture placed at their middle, thus creating a quadrupled graft. The graft was sized with graft sizers of 0.5 mm increments.The femoral tunnel was drilled using the transportal technique. A 4.5 mm canulated drill bit was used to create a tunnel in the femur. The length of the tunnel was measured with depth gauze. The reaming of the tunnel was done with a femoral reamer corresponding to the diameter of the graft. The no. 5 Ethibond was passed into the tunnel, and the loop was parked inside the joint. The tibial tunnel was drilled using a tibial tunnel guide (Smith and Nephew India Ltd.) with the angle of the guide kept at 55°. A tibial reamer with a diameter equivalent to the size of the graft was used to drill the tunnel. The loop of the Ethibond no. 5 parked inside the knee joint was retrieved from the tibial tunnel with the help of a suture grasper. The length of the tibial tunnel and the intraarticular part of the proposed graft was measured with a depth gauge, which was added to the already measured length of the femoral tunnel to determine the exact length of both the tunnels plus the intraarticular part of the graft. An endobutton was selected so that at least 15 mm of the graft remained inside the femoral tunnel. Quadruple graft length plus loop length was kept equal to total tunnel length plus intraarticular length Fig. .Fig. 1ThOn postoperative day 0, knee range of motion and static quadriceps exercises were started as per pain tolerance. From postoperative day 1, straight leg raising and full weight-bearing with brace were started. This regimen was continued for up to 6 weeks. At 6 weeks, the use of brace was discontinued and static cycling and half squatting were introduced in addition to the existing physiotherapy. At 3 months, light jogging and full squatting were allowed. During 3–6 months, patients underwent conditioning, endurance, proprioception enhancement, and sport-specific training exercises. After 6 months of the rehabilitation program, limb symmetrical index  was calculated using single hop test, thigh wasting, and knee laxity. If the limb symmetrical index (LSI) was more than 85%, athletes were allowed to return to sports in practice games and subsequently to professional matches; however, if LSI was less than 85% in any of the parameters, players were asked to continue physiotherapy and revisit monthly till LSI > 85% was achieved.Knee laxity was checked by anterior drawer test and Lachman test at 8 months and 14 months.MRI was done at 8 months and 14 months follow-up to study the graft tunnel integration of ACL graft in both the tibial and femoral tunnels. The MRI follow-up time was decided after considering the timing of return to sports. All the patients in the present study were professional athletes, and previous studies have shown that the mean time to return to sports was around 8–9 months , 17. TheIn the present study, MRI was performed on Achieva 1.5 T model  using a standard polarized knee coil supplied by the manufacturer. Coronal and sagittal short tau inversion recovery (STIR) imaging with 3 mm slice thickness with 0.3 mm gap with field of view (FOV) 252 × 154 mm and number of signal averages (NSA) 1.9 were obtained. The sagittal images were placed along the longitudinal axis of the ACL graft using an axial scout view. Three-millimeter slice thickness with 0.3 mm gap, one acquisition, and a 504 × 237 matrix was used. Total imaging time for this protocol was 12–20 min. The local ethics committee approved the MRI study protocol.The images were processed and evaluated on OsiriX software for the Apple Mac system. The MR images were interpreted by a consensus of two readings, and the following findings were recorded:Graft tunnel integration was analyzed by visually assessing the signal intensity of the fibrous interzone on sagittal STIR image Figs. , and 5, p < 0.05.Data were coded and recorded in the MS Excel spreadsheet program. SPSS v23 (IBM Corp.) software was used for data analysis. Descriptive statistics were elaborated in the form of means/standard deviations and medians/interquartile ranges (IQRs) for continuous variables, and frequencies and percentages for categorical variables. Correlation of FI score with timing and site  was assessed using the Wilcoxon test. Statistical significance was determined at 2. Eleven out of 25 patients had dominant limb involvement, and 14/25 patients had nondominant involvement.The mean age of the patients in the present study was 25.1 ± 5.1 years. The mean BMI was 23.8 kg/mV = 69.0, p = 0.017). The mean FI score  decreased from 3.04 (2–5) at 8 months to 2.57 (2–4) at 14 months .Nonparametric tests were used to make a statistical inference as data were not normally distributed. Paired Wilcoxon test was used to explore the difference in fibrous interzone score at the two time points. The mean FI score  decreased from 2.61 (1–5) at 8 months to 2.04 (1–4) at 14 months follow-up . At 14 months follow-up, mean FI score at tibial end was 2.04 ± 0.82 and at femoral end was 2.57 ± 0.66 .On comparing the graft tunnel integration in femoral and tibia tunnel Table , it was n = 11) and 2.4 in patients having graft diameter > 7.5 . The mean femur FI score at 8 months was 2.45 in patients having graft diameter ≤ 7.5 (n = 11) and 2.64 in patients having graft diameter > 7.5 (p = 0.5).The mean graft diameter was 7.8 ± 06 mm. The mean FI score (tibia) at 14 months was 1.55 in patients having graft diameter ≤ 7.5 (p = 0.03). Similarly, the mean difference in pre-injury and post-surgery (14 months) Tegner activity scale in patients with FI score (tibia) ≤ 2 and FI score ≥ 3 was 0.3 and 1.3, respectively (p = 0.02). The mean Lysholm score (Table p = 0.047).The mean Lysholm score Table  in patieThe main finding of the present study was that graft incorporation happened early in the tibial tunnel compared with the femoral tunnel. Many factors can affect the graft tunnel integration such as type of graft , size of the graft, fixation method, preservation of muscle fiber, BMPs, etc. , 20. HamRuffilli et al. in a meta-analysis study concluded that, despite promising results of preserving the hamstring tendon insertion during anterior cruciate ligament reconstruction (ACLR), a prospective MRI evaluation study was required . TherefoThe possible reason for early graft tunnel integration in the tibial tunnel compared with the femoral tunnel could be preserved hamstring tendon tibial insertion and the presence of remnant muscle fibers. In preserved insertion ACLR, blood supply at insertion end is preserved, which could facilitate early graft incorporation. Secondly, despite the best efforts of removing muscle fibers from the tendon, there are always some fibers that remain attached to the tendon, and the presence of these muscle fibers facilitates graft tunnel integration. Cuti et al.  examinedIn the present study, it was observed that patients with better graft integration had better clinical outcomes  to detect the correlation between the graft integration and functional outcomes, as the study was powered to detect the graft integration inside the tunnels; therefore, further studies are required to establish the correlation between graft integration and functional outcomes. Second, there was no control group in the present study; therefore, comparative study between STG free graft and preserved insertion graft will be needed in the future. Third, all the patients in the present study were males; therefore, the effect of gender on graft integration could not be studied. Fourth, the effect of graft fixation methods on the graft tunnel integration was not studied; in the present study, the femoral end of the graft was fixed using endobutton, and the tibial end of the graft was sutured back to the insertion. Although previous studies have observed that this method of fixation result in satisfactory functional outcomes , 30], thGraft tunnel integration occurs early in the tibial tunnel compared with the femoral tunnel after anterior cruciate ligament reconstruction with preserved insertion hamstring tendon graft. Clinical outcomes after ACL reconstruction are directly correlated to graft tunnel integration. Level of study—4.


The mitochondrial genome (mitogenome) has been extensively used for phylogenetic and evolutionary analysis in Diptera, but the study of mitogenome is still scarce in the family Chironomidae.Here, the first complete mitochondrial genomes of four Chironomid species representing Diamesinae, Orthocladiinae, Prodiamesinae and Tanypodinae are presented. Coupled with published mitogenomes of two, a comparative mitochondrial genomic analysis between six subfamilies of Chironomidae was carried out.Mitogenomes of Chironomidae are conserved in structure, each contains 37 typical genes and a control region, and all genes arrange the same gene order as the ancestral insect mitogenome. Nucleotide composition is highly biased, the control region displayed the highest A + T content. All protein coding genes are under purifying selection, and the ATP8 evolves at the fastest rate. In addition, the phylogenetic analysis covering six subfamilies within Chironomidae was conducted. The monophyly of Chironomidae is strongly supported. However, the topology of six subfamilies based on mitogenomes in this study is inconsistent with previous morphological and molecular studies. This may be due to the high mutation rate of the mitochondrial genetic markers within Chironomidae. Our results indicate that mitogenomes showed poor signals in phylogenetic reconstructions at the subfamily level of Chironomidae. The typical mitochondrial genome (mitogenome) of insects is a double-strand circular molecule ranging from 14kb to 20kb in size, which encodes 37 genes  and a control region . Due to The dipteran family Chironomidae is a diverse aquatic insect group, and are important bioindicators for freshwater ecosystem monitoring. Within Chironomidae, several phylogenetic studies have been conducted based on morphological characters or combining genetic markers to reconstruct the evolutionary history of subfamilies , but no In the present study, we provide complete mitogenomes for four species representing the subfamilies Diamesinae, Orthocladiinae, Prodiamesinae, and Tanypodinae. Along with the published mitogenomes of subfamilies Chironominae and Podonominae, the first comparative analysis of the genome structure, base composition, substitution and evolutionary rates among six chironomid subfamilies is presented. In addition, a phylogenomic analysis covering six chironomid subfamilies was carried out.Potthastia sp. (Diamesinae), Rheocricotopus villiculus (Orthocladiinae), Prodiamesa olivacea (Prodiamesinae) and Clinotanypus yani (Tanypodinae) are documented for the first time. The mitogenomes of Chironomus tepperi (Chironominae) and Parochlus steinenii (Podonominae) were retrieved from GenBank (Propsilocerus akamusi (MN566452) (Culicoides arakawae and Forcipomyia makanensis)   was exclanensis)  were selanensis) . DetaileFor the newly sequenced species, total genomic DNA was extracted from the body,  using a General AllGen Kit . The entire mitogenome of each species were sequenced using the Illumina NovaSeq 6000 platform with an insert size of 350-bp and a paired-end 150-bp sequencing strategy by the Allwegene Company and Novogene Co., Ltd. at Beijing, China. About 2 Gb clean data were obtained from each library after trimming using Trimmomatic .de novo assembly was performed using IDBA-UD  (https://www.ncbi.nlm.nih.gov/orffinder/) and then compared with published mitogenomes of insects using the program BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Newly sequenced mitogenomes were submitted to GenBank (accession numbers: MW373523–MW373526).Genome annotation was conducted as previously described in ndex.py)  with invCGView Server V 1.0  was usedPhylogenetic analyses were conducted using the sequences of 13 PCGs and two rRNAs. The PCGs were aligned based on amino acid sequences using Muscle implemented in MEGA X . The rRNChironomus tepperi, Potthastia sp., Rheocricotopus villiculus, Parochlus steinenii, Prodiamesa olivacea, and Clinotanypus yani are 15,652, 15,913, 15,985, 16,803, 16,190, and 16,247 bp in size, respectively  in the remaining four species.Nucleotide composition  of the sChironomus tepperi, Potthastia sp., Rheocricotopus villiculus and Prodiamesa olivacea. The start codon of ND5 in Chironomus tepperi, Potthastia sp., Rheocricotopus villiculus, Prodiamesa olivacea and Clinotanypus yani was GTG. ND1 started with TTG in Potthastia sp., Rheocricotopus villiculus, Parochlus steinenii, Prodiamesa olivacea, and Clinotanypus yani. Most PCGs have complete termination codons (TAA or TAG), however, COII in Parochlus steinenii and Clinotanypus yani has an incomplete termination codon (T-).Among Chironomidae species, most PCGs initiate with the standard start codon ATN. The start codon of COI was TTG in Chironomus tepperi, Potthastia sp., Rheocricotopus villiculus, Parochlus steinenii, Prodiamesa olivacea, and Clinotanypus yani were 3,730, 3,743, 3,726, 3,729, 3,729, and 3,709, respectively. The most frequently codon families are Ile, Leu2, and Phe (>300), while the least used codon family is Cys (<50) in all six Chironomidae species  in  species . The rel species .ω) was used to test for signatures of natural selection , positive AT-skew, and negative GC-skew . All theChironomus tepperi: the AT-skew of 12S rRNA and 16S rRNA in Chironomus tepperi is −0.01 and 0.00, respectively , mainly due to the variation in control region size. All Chironomidae mitogenomes contain 37 typical genes and a control region, the order and arrangement of these genes are completely accordant with the ancestral insect gene arrangement . The whoParochlus steinenii and Clinotanypus yani has an incomplete termination codon (T-) that probably completed by post-transcriptional polyadenylation  lacks the dihydrouridine (DHU) arm. This could be commonly found in published insect mitogenomes . The A+TIn this study, we applied a variety of strategies to explore the phylogenetic relationships of six subfamilies within the Chironomidae, and confirmed the monophyly of Chironomidae . HoweverIn this study, we sequenced four complete mitogenomes representing four subfamilies of Chironomidae by whole genome sequencing technologies and did the first comparative analysis of mitogenome base composition and evolutionary history in Chironomidae. The study shows that mitogenomes of Chironomidae are conserved in structure, gene order and nucleotide composition. Our results revealed that mitogenomes have poor phylogenetic signals for subfamily level relationships in Chironomidae.10.7717/peerj.11294/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11294/supp-2Supplemental Information 2Parochlus steinenii | KT003702, Forcipomyia sp. | MK000395, Culicoides arakawae | NC_009809, Chironomus tepperi | JN861749, Prodiamesa olivacea, Clinotanypus yani, Potthastia sp., and Rheocricotopus villiculus.The names of the eight sequences, from top to bottom are Click here for additional data file.10.7717/peerj.11294/supp-3Supplemental Information 3Chironomus tepperi; Sheet 2, Potthastia sp.; Sheet 3, Rheocricotopus villiculus; Sheet 4, Parochlus steinenii; Sheet 5, Prodiamesa olivacea; Sheet 6, Clinotanypus yani.Sheet 1, Click here for additional data file.


Starch is a principal dietary source of digestible carbohydrate and energy. Glycaemic and insulinaemic responses to foods containing starch vary considerably and glucose responses to starchy foods are often described by the glycaemic index (GI) and/or glycaemic load (GL). Low GI/GL foods are beneficial in the management of cardiometabolic disorders . Differences in rates and extents of digestion of starch-containing foods will affect postprandial glycaemia.In vitro studies of starch digestion by α-amylase model early stages in digestion and can suggest likely rates of digestion in vivo and expected glycaemic responses. Reports that metabolic responses to dietary starch are influenced by α-amylase gene copy number, heightens interest in amylolysis.Amylolysis kinetics are influenced by structural properties of the food matrix and of starch itself. Native (raw) semi-crystalline starch is digested slowly but hydrothermal processing (cooking) gelatinises the starch and greatly increases its digestibility. In plants, starch granules are contained within cells and intact cell walls can limit accessibility of water and digestive enzymes hindering gelatinisation and digestibility. Km and kcat/Km) for evaluating catalytic efficiency and ease of digestibility of starch by α-amylase. Suitable kinetic methods maximise the information that can be obtained from in vitro work for predictions of starch digestion and glycaemic responses in vivo.This review shows how enzyme kinetic strategies can provide explanations for differences in digestion rate of different starchy foods. Michaelis-Menten and Log of Slope analyses provide kinetic parameters (e.g.,   •Michaelis-Menten kinetics determines catalytic efficiency of amylase action on starch.•Km and kcat/Km are useful for estimating ease of digestibility.Kinetic parameters •Log of slope analysis can quantify starch fractions digested at different rates.•Categorisation of Resistant Starch can be based on types of interaction with amylase.•Enzyme kinetic studies of amylolysis are useful for predicting postprandial glycaemia. It is cTypically, native (raw) starch represents only a small percentage of the total starch intake. Some raw starch granules from bananas, peanuts and uncooked vegetables may be eaten, but most dietary starch originates from bread, pasta, rice, potato and yams that have been hydrothermally processed (by cooking in water). This treatment significantly increases starch gelatinisation and its digestibility . Starch An account of starch digestion in the upper gut appears in a recent review . The firin vivo . Such findings are valuable for predicting potential behaviour lsewhere . Values 2Interest in salivary α-amylase has increased greatly. A nineteenth century demonstration that human saliva contains an agent that catalyses starch breakdown was important for studies of enzyme catalysis . Kinetic2.12.2.).The contribution of salivary amylase to the digestion of starch is of considerable conjecture . For insAMY1 and AMY2 that code for salivary and pancreatic α-amylases, respectively. The number of copies of the genes in individuals is variable  is known to affect appetite and the mass of body fat. Studies in mice have shown that high GI diets result in increased Fto expression that can lead to raised levels of body fat , insulin resistance and type-2 diabetes . Obesitybody fat . Diets cbody fat . Moreovebody fat .Undigested starch, termed ‘resistant starch’ (RS), reaches the colon together with other non-digestible carbohydrates, notably non-starch polysaccharides of plant cell walls (fibre) . This main vitro strategies for assessing starch digestibility are valuable, not just for evaluating how starch-containing foods affect postprandial metabolism, but also for providing insight into molecular mechanisms of amylolysis , complexation with protein and lipid or through chemical modification of starch molecules. The current classification of resistant starch as RS 1,2,3,4 etc, developed by Englyst takes account of these different types of RS and is a useful way of identifying many different forms of RS in the context of complex food matrices, but offers no mechanistic description for resistance (viz: (a) causing interference of starch-enzyme binding or (b) inhibition of the catalytic event itself  is a physiological description of the fraction of dietary starch that enters the large intestine, i.e., material that escapes digestion during transit from the mouth to the terminal ileum . Resistasistance . The kinsistance . Thus, tt itself . Interesc health .4The enzyme kinetics of α-amylase action on starch are complex . In addiGiven the complexity of starch structure and the knowledge that α-amylase can attack a starch α-1-4 linked chain at numerous sites within the chain, there is no certainty that each site is equally reactive given any constraints introduced by an ordered starch structure. Therefore, the initial reaction velocities on which the Michaelis-Menten model is based cannot necessarily be extrapolated to the hydrolysis kinetics for the whole of the starch substrate. Hence the interest in extended hydrolysis investigations discussed in the section below dealing with digestibility curves.Nevertheless, it has been known for many years that the relation between starch concentration [S] and the initial rates of digestion by amylase [v] can be fitted by the familiar Michaelis-Menten equation :(1)v=Vma, Vmax, is reached when the enzyme is saturated with its substrate . Vmax is directly related to the total enzyme concentration [E] and can be written as k[E] where the proportionality constant k is frequently referred to as the catalytic rate constant (kcat) with dimensions of Time−1.The use of software for fitting to a rectangular hyperbola by non-linear regression allows determination of Because of substrate depletion during enzyme assays and competitive inhibition of amylase by its product maltose, an integrated form of the Michaelis-Menten equation incorporating a function for competitive inhibition has been recommended for data analysis . MaltoseA popular method of data analysis involves a fit to the double reciprocal (Lineweaver-Burk) form of the Michaelis-Menten equation:v will be magnified when the reciprocal is taken. In Hanes-Woolf plots of S/v against S . This second-order rate constant relates the reaction rate to the concentration of free enzyme. CE allows comparison of the relative rates of reaction of different substrates that are acted upon by the same enzyme. For amylase, starches of different botanical and/or food sources exemplify varieties of substrate for the enzyme. If the concentration of enzyme is unknown, kcat cannot be determined directly, but Vmax/Km values can be used as indicative of the respective CE of amylase for each substrate, provided that differences in the amount of enzyme (units of activity etc.) used for each substrate are accounted for. Because of variations in starch structure arising from botanical source and/or hydrothermal processing, CE values obtained in vitro of different starch forms can provide explanations for the known variations of in vivo digestion rates of different starchy foods. Michaelis-Menten studies of CE have yielded useful data on the relative ease of digestion of different botanical starches  for starch-amylase complexes  can identify and estimate the potency of dietary materials that could slow intestinal digestion of starch and attenuate postprandial peaks in glycaemia. Some authors prefer to express inhibition data in terms of IC50 values  to indicate the inhibitory efficacy of an agent at concentrations likely to be experienced in vivo. Use of IC50 values may be well intentioned but unless it has been established that the inhibitor does not act competitively (either fully or in part), IC50 values determined in vitro will be dependent on the substrate concentration used in the enzyme assays (in vitro-derived IC50s become somewhat arbitrary due to difficulties in estimating concentrations of starch and inhibitor(s) in the intestinal lumen.Extensions of the Michaelis-Menten equation to account for the effects of reversible inhibitors  have beee assays . Additio5in vivo following dietary starch loads. From estimations of RDS and SDS defined in this way, predictions of likely in vivo glycaemic indices (GI) of various carbohydrate-containing foods have been made  is the slope of the digestibility curve and so a plot of the estimated slopes against t at various time points is linear with a slope of –k and an intercept on the Y axis equal to Ln(C∞k) (The term (Ln(C∞k) .Fig. 3LoC∞ without the need of extended incubations with their inherent risks of loss of catalytic activity. Prolonged exposure of most enzymes at raised temperatures is likely to lead to a loss of catalytic activity. If experimenters are using pancreatin preparations that contain proteases, there is an even greater chance of inactivation because of proteolysis of amylase. C∞ may also provide an approximate indication of the quantity of RS by subtraction from the total amount of starch added to the reaction mixture at the start of the incubation. In any experiment, the digestibility constant, k, will be directly proportional to the concentration (or units of activity) of α-amylase used in the reaction mixture. Therefore, when comparing the digestibility constants obtained for a variety of different starches and starch-rich foods, allowance must be made for any differences in enzyme concentration used in the series of experiments.If data points are determined at relatively small intervals of time (see above), the plot between adjacent points can be regarded as linear for easy estimation of the slope at various time points throughout the digestibility curves . This loLOS plots are very sensitive to changes in the digestibility behaviour of the substrate so if a starch/food sample contains α-glucan fractions digested at different rates, discontinuities appear in linear plots and the relative amounts of the fractions and their susceptibility to amylolysis can be estimated . DigestiC∞ and k values derived from the LOS plot AUC=Cin vitro digestibility curves obtained with finely milled endosperm flour to create a smooth porridge, were 33% higher than the equivalent values for coarse endosperm flour. The 33% difference correlated closely with differences in AUC of the glycaemic responses observed in the in vivo study of ileostomates after consumption of the smooth or coarse wheat endosperm  and C∞ were the most strongly correlated with GI rankings for matched starchy food products. The authors concluded that the in vitro method for starch digestibility showed potential for rapid prediction of GI values.More recently, it was reported that data obtained from digestibility curves and LOS analysis, correlated reasonably well with literature values of  vivo GI . However7A comparison of various kinetic methods, including the LOS model, for identifying fractions of starch of varying digestibility rate, has been published and the LOS system was reported to be generally reliable . In spitin vivo and whether its consumption is likely to produce sharp peaks in glycaemia and insulinaemia. Recent developments in our understanding of amylolysis, however, emphasise the importance of basing estimations of RDS and SDS on sound data obtained from full digestibility curves rather than two time points of 20 and 120 min. A recent publication by The relative proportion of RDS in starchy foods is often taken as indicative of how rapidly the starch will be digested In vitro digestibility measurements continue to be of considerable use in predicting in vivo glycaemia to allow identification and recommendation of suitable foods and diets for management of obesity and diabetes and for the development of novel ingredients and food products with enhanced nutritional qualities. The importance and potential significance of amylase measurements has been emphasised in a recent publication that relates the value of in vitro screening of digestibility profiles of food products for prediction of likely glycaemic responses in vivo ( in vivo . It is b in vivo , 2022.It is very important therefore, that methods used in the analysis of digestibility are rigorous. We hope that use of the kinetic methods described here means that digestibility data can be interpreted with greater precision and mechanistic insight, forming the basis of reliable nutritional opinion and future national dietary guidelines.Peter J. Butterworth: Conceptualisation and development of review, and writing original draft and subsequent revisions; Balazs H. Bajka: Contributed to review development, revisions of article and preparation of Figures; Cathrina H. Edwards: Contributed to review development, revisions of article and preparation of Figures, including art work for The 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.


This n = 20 per group) including low translucent (LT) , high translucent (HT) , and multilayered system (ML) . Each monolithic zirconia group was equally subdivided according to be either air-abraded with 110 µm aluminium oxide particles or left untreated (control). After thermocycling, the flexural strength was measured by using a universal testing machine. Two-way ANOVA followed by Tukey's post hoc and independent samples t-test were used for the statistical analyses (P < 0.05).  Sixty bar-shaped specimens were fabricated from three different types of presintered monolithic zirconia (P = 0.010). Having controlled the effect of surface treatment, the flexural strength of HT and LT zirconia was found to be significantly higher than the ML zirconia system (P ≤ 0.001). Airborne particle abrasion could significantly decrease the flexural strength of monolithic zirconia only in ML zirconia (P = 0.002).  Surface treatment and types of zirconia were found to have a significant interaction ( Multilayered zirconia system had the lowest flexural strength among all groups. Moreover, the flexural strength of this system was attenuated by surface treatment with airborne particles abrasion. Its Bilayered core-ceramic systems were designed to cover the opaque face of zirconia under a tooth-like appearance while having the strength of yttria-stabilized tetragonal zirconia polycrystalline (Y-TZP). A common complication of bilayered zirconia is porcelain veneer chipping due to the dense and unreactive surface of zirconia and its lower adhesion to ceramic veneers , 4, 5, aThe inherent semitranslucency of monolithic zirconia impedes a natural tooth-like appearance ; howeverAttaining information regarding the flexural strength is of importance, particularly the mechanical strength of monolithic zirconia after surface treatments , 13. Lon2O3) on the flexural strength of aged monolithic zirconia. The null hypothesis was that different translucencies and surface treatment would not affect the flexural strength of thermocycled monolithic zirconia.Not enough studies have addressed the effect of different translucencies and types of monolithic zirconia especially multilayered system and the combined effect of translucency and surface treatment on the flexural strength of monolithic zirconia. This in vitro study was designed to evaluate the effect of three different translucencies and air abrasion with aluminum oxide (Aln = 20 per group) including low-translucent (LT) monolithic zirconia , high-translucent (HT) zirconia , and multilayered (ML) system of monolithic zirconia . The specimens were manufactured according to ISO 6872, with ± 0.02 mm accuracy [In this experimental in vitro study, a bar-shaped specimen (25 × 5 × 2 mm) was designed , based on which 60 specimens were milled  out of three different types of presintered monolithic zirconia (accuracy  and sint2O3 particles (n = 10 per subgroup). Both sides of the specimens were air-abraded with 110 µm laboratory Al2O3 particles  for 10 seconds at 400 kPa air pressure from a 10 mm distance. The particle size was adopted with respect to the previous studies claiming that larger particles enhance the surface abrasion, wettability, and cement bond strength [Each group was subdivided to be either left untreated as the control or air-abraded with Alstrength , 23 ; images were taken at ×500 and ×1500 magnifications to check the surface properties.M = 3Wl/2bd2, where W is the applied load (N), l is the test span (mm), b is the specimen width (mm), and d is the specimen thickness (mm) [The specimens were, then, subjected to 1000 thermal cycles  between 5 °C and 55 °C, ultrasonically rinsed with distilled water for 10 minutes, and air-dried for 20 seconds. To measure 3-point flexural strength, a force  was applied on a universal testing machine in the middle of the specimen on a 20 mm fixture at a speed of 1 mm/min . The foress (mm) .t-test for pairwise comparisons (α = 0.05).Statistical analyses were done by IBM SPSS for Windows . Kolmogorov–Smirnov and Shapiro–Wilk tests were used to confirm normal distribution and the homogeneity of variances. Two-way ANOVA was used for the descriptive data (mean and standard deviation (SD)), and one-way ANOVA was used to evaluate the effect of each factor, followed by Tukey's post hoc test and independent samples P ≤ 0.001), while the effect of surface treatment with Al2O3 was insignificant (P = 0.249). Moreover, a significant interaction existed between the surface treatment and zirconia type (P = 0.01). Having controlled the effect of surface treatment, one-way ANOVA showed that zirconia type significantly affected the flexural strength (P ≤ 0.001). Tukey's post hoc test showed that, in both the control and APA subgroups, the flexural strength of both HT and LT zirconia specimens was significantly higher than that in the ML system (P ≤ 0.001) (≤ 0.001) .P = 0.978) and HT (P = 0.182) groups in neither the control nor the APA subgroups. Independent t-test showed that the surface treatment had no significant effect on the flexural strength in the HT (P = 0.176) or LT zirconia group (P = 0.110). But, in the ML zirconia system, air-abrasion significantly reduced the flexural strength compared with the control group (P = 0.002)  .2O3 created porous and irregular surfaces in all types of zirconia. In the control group without surface treatment, ML system and HT zirconia had the most and least irregular surfaces, respectively. However, after airborne abrasion with Al2O3, LT and HT zirconia showed rougher surfaces , which included three grades as ultratranslucent multilayered zirconia, supertranslucent multilayered zirconia, and multilayered zirconia . Flinn eμm are not usable in smaller dimensions due to the impossibility of phase transition [Pereira et al.  and Parkansition . Spyropoansition  evaluateThe current findings showed that the flexural strength was not significantly different between the LT and HT zirconia. Likewise, Matsuzaki et al.  found thIn line with the present findings about ML zirconia compared with HT and LT zirconia, Mao et al.  found thμm Al2O3 particles on the ﬂexural strength of monolithic zirconia [Another finding of the present study was the significant interaction between the surface treatment and zirconia type. APA creates the clean and rough surface required for higher adhesion to dental cement or veneering porcelains , 33, 34.zirconia , 23, 34.2O3 particles (0.28–0.40 MPa) [Contrary to what the present study found about LT and HT zirconia, some studies showed concerns about the superficial damage after airborne particle abrasion with 50–120 µm Al.40 MPa) –38. Howe.40 MPa) , 39. Dis.40 MPa) , 39, 40.Mao et al.  examinedA systematic review detected that the translucency and flexural strength were significantly influenced by the composition, microstructure, and surface treatment . ReviewiKurtulmus et al.  investigAmong the limitations of the present study, was the nonclinical in vitro nature, which restricts free interpretation of the results for the clinical conditions. Further studies are recommended to consider other types of zirconia with different surface treatments.2O3.Within the limitations of this study and considering the in vitro conditions, it can be concluded that, between the three different translucencies of monolithic zirconia, the multilayer zirconia system had the lowest flexural strength than the two other types of monolithic zirconia. Besides, the flexural strength of this system decreased by surface treatment with airborne particle abrasion with Al


BAM gene family diversified, giving rise to isoforms with different compartmentation and biological activities. Here, we characterized BETA-AMYLASE 9 (BAM9) of Arabidopsis . Among the BAMs, BAM9 is most closely related to BAM4 but is more widely conserved in plants. BAM9 and BAM4 share features including their plastidial localization and lack of measurable α-1,4-glucan hydrolyzing capacity. BAM4 is a regulator of starch degradation, and bam4 mutants display a starch-excess phenotype. Although bam9 single mutants resemble the wild-type (WT), genetic experiments reveal that the loss of BAM9 markedly enhances the starch-excess phenotypes of mutants already impaired in starch degradation. Thus, BAM9 also regulates starch breakdown, but in a different way. Interestingly, BAM9 gene expression is responsive to several environmental changes, while that of BAM4 is not. Furthermore, overexpression of BAM9 in the WT reduced leaf starch content, but overexpression in bam4 failed to complement fully that mutant’s starch-excess phenotype, suggesting that BAM9 and BAM4 are not redundant. We propose that BAM9 activates starch degradation, helping to manage carbohydrate availability in response to fluctuations in environmental conditions. As such, BAM9 represents an interesting gene target to explore in crop species.β-Amylases (BAMs) are key enzymes of transitory starch degradation in chloroplasts, a process that buffers the availability of photosynthetically fixed carbon over the diel cycle to maintain energy levels and plant growth at night. However, during vascular plant evolution, the  A conserved nonenzymatic beta-amylase protein promotes starch breakdown in leaves; mutation of the gene can cause starch accumulation while overexpression can increase nighttime starch use. Arabidopsis thaliana; Starch is the major storage carbohydrate in plants and is composed of α-1,4- and α-1,6-linked glucose polymers. In leaves, starch is synthetized in chloroplasts during the day and degraded at night, when energy from photosynthesis is unavailable. This pattern of diel turnover is important for optimizing plant growth  and is cBAM genes  are required to phosphorylate the surface of this structure, thereby disrupting it to facilitate access and activity of glucan hydrolyzing enzymes . HoweverAM genes . Their cAM genes .bam1, bam2, and bam6 single mutants have starch contents similar to wild-type (WT) plants, but the loss of BAM3 leads to starch-excess phenotype, which is further exacerbated by the loss of BAM1 (Five of the nine proteins (BAM1-3 and BAM5-6) are enzymatically active, four of which are targeted to the plastid . The maj of BAM1 . InteresBAM1 is highly expressed in guard cells, where the starch turnover is different from the rest of the leaf, with appreciable starch remaining at the end of the night, when mesophyll cells have nearly exhausted their supplies. At the beginning of the day, BAM1 contributes to the rapid degradation of guard cell starch releasing osmolytes and respiratory substrates needed to bring about stomata opening . BAM1 dibam4 mutants have a starch-excess phenotype and reduced nighttime maltose levels without change in total BAM activity (Four of the nine proteins (BAM4 and BAM7-9) appear to have lost enzymatic activity. Two—BAM7 and BAM8—are nonplastidial and are remarkable examples of neo-functionalization. In addition to their BAM2-related BAM domain, these proteins possess a BRASSINAZOLE-RESISTANT1-type DNA binding domain. They are located in the nucleus where they act as transcription factors regulating shoot growth, but do not influence starch metabolism . The BAMactivity . This leBAM9 promoter, which comprised 2.3kb of sequence upstream of the start codon  coding sequence (CDS) was placed after and in frame with the 612 first nucleotides of BAM9 CDS. The resulting construct was driven by the native ear dawn , confirmear dawn .BAM9 gene encoding the first 71 amino acids of the protein and inserted it upstream and in-frame with the CDS of YFP. As for the full-length fusion protein, the YFP signal from expression of this construct was detected in chloroplasts . This construct, driven by the Arabidopsis Ubiquitin10 promoter, was used both for transient expression studies in leaf mesophyll protoplasts from WT plants and for stable transformation of WT plants. In both cases, YFP fluorescence was exclusively located in chloroplasts , consistroplasts , from whNext, we compared the predicted tertiary structures of BAM9, BAM4, and the catalytically active BAM3 proteins. As a template, we used the recently solved crystal structure of sweet potato BAM bound to maltotetraose  accessi. Three rSeparate multiple sequence alignments were made for each of the modeled BAMs using orthologous sequences encoded in the genomes of other vascular plants , respectively).We investigated the impact of losing BAM9 by isolating and analyzing  mutants . Two ind mutants . To conf alleles . The mutbam9 did not have a starch-excess phenotype, in contrast to previously described bam4, bam3, and the bam1bam3 mutant lines  of 4–8 experimental replicates, each of which had between 4 and 8 biological replicates. Even in the bam9 single mutant and in the bam1bam9 mutant, the end-of-night starch content was consistently higher than in the WT (144 ± 28%) and bam1 (106 ± 21%), although these differences were typically less than 1 mg g−1 FW, and no differences in starch content were apparent at the end of the day.We observed variation in the absolute starch contents between experiments, which could result from several factors . However, when we compiled the results of multiple experimental replicates where we measured starch content at the end of the day and at the end of the night, the result of the loss of BAM9 was consistent. The starch-excess phenotype of bam9 mutant, maltose levels were generally similar to or slightly lower than in the WT, whereas nighttime maltose levels were lower in the bam3, bam4, and bam1bam3, as previously reported .We quantified maltose in a subset of the mutants, given that it is a key intermediate of the starch degradation pathway . In thisreported . In bam4bam9 mutant with the lsf1 mutant. It has been shown that LSF1 interacts with BAM1 and BAM3  was not substantially different from the WT. Interestingly, in most BAM9-expressing lines, there was a decrease in starch content at the end of the night, while starch content at the end of day remained largely un-affected (bam9 mutant but the line overexpressing BAM9–YFP to the highest extent (OE B9 #2) had less starch in its root-tip columella cells, similar to the observation in leaves.To further investigate the importance of BAM9 in regulating leaf starch metabolism, we measured the starch content in transgenic plants constitutively expressing either the BAM9–YFP fusion protein  or BAM9 affected . Thus, waffected . No diffbam4 mutant to see whether it was able to rescue the starch-excess phenotype of that line. In parallel, we transformed bam4 mutant with a similar construct encoding BAM4–YFP. We used RT-qPCR to determine the level of expression of each construct and compared transformed lines with similar expression of either BAM4–YFP or BAM9–YFP  did not (bam4-OE9#2) with considerably stronger expression, but this line still had a starch-excess phenotype similar to bam4. This failure of BAM9 to rescue the bam4 phenotype suggests that BAM4 and BAM9 functions are nonredundant.We also overexpressed BAM9–YFP in the BAM9–YFP . While B did not . We analOur results establish BAM9 as a regulator of transitory starch metabolism in Arabidopsis leaf chloroplasts. Although BAM9 acts alongside the homologous protein, BAM4, the functions of the two proteins appear to differ.BAM9 expression in the leaves increased at night, peaking toward dawn, as was reported previously , by the circadian clock, and by light signaling , with a much lower specific activity than against soluble starch . Their expression increases during fruit ripening, when starch is being broken down into sugars, but is severely repressed by cold storage, when starch degradation is inhibited  and to evaluate homologous genes as potential targets to improve starch traits in crop species. plants (Col-0 ecotype) were grown in a climate-controlled chamber with a constant temperature of 20°C, 60% relative humidity, with a 12-h photoperiod with a uniform light intensity of 150 µmol photons m−2 s−1. Two mutant alleles of At5g18670  were characterized in this work: bam9-1 (SALK_021635), and bam9-2 (GABI_722D02). The following mutants characterized by bam1-1, SALK_039895; bam3-1, CS92461; bam4-1, SALK_037355 and lsf1-2, SALK_053285.Arabidopsis Total RNA was isolated from individual rosettes using GENEzol extraction buffer according to the manufacturer guidelines . Genomic DNA contamination was removed using DNase I . First-strand cDNAs were synthesized from 1 µg RNA using oligo(dT)18 primers and the RevertAid First Strand cDNA Synthesis Kit . Resultant cDNA was diluted 25-fold and 2.5 µL was used in a 10 µL reaction of KAPA SYBR FAST qPCR Master Mix (2×) Universal . RT-PCR reactions were performed with BAM9 gene-specific primers and control primers for the housekeeping gene ACTIN2. Real-time quantitative PCR reactions were performed using the LightCycler 480 II system (Roche). Efficiencies of each primer pair were similar. Primer sequences are listed in BAM9 gene was cloned in frame with the GUS reporter gene in the pGWB533 plasmid . These plasmids were then recombined into pUBC-YFP-DEST using an LR clonase (Invitrogen), resulting in an in-frame fusion of the CDS with a C-terminal YFP tag, under control of ubiquitin promoter. For stable expression of TAP-tagged BAM9 protein in WT plants, the BAM9 CDS was cloned downstream of the 35S promoter and in frame with a C-terminal myc tag by recombination of the aforementioned BAM9-pDONR221 into pYL436 . An argon laser was used for excitation (at 514 nm). Emission was captured between 462 and 500 nm for YFP and between 662 and 721 nm for chlorophyll autofluorescence.Structure predictions for AtBAM3, AtABM4, and AtBAM9 were performed using SWISS-MODEL. The crystallized BAM from sweet potato  was chosBAM9, lacking the first 71 amino acids, was amplified either with EcoRI and XbaI restriction sites from Arabidopsis cDNA and cloned into the pProEX Htb vector, or with EcoRI and SalI restriction sites and cloned into the pET28a+ expression vector, resulting in a protein with a 6× His-tag at the N-terminal and C-terminal ends of the protein, respectively. Similarly, we cloned BAM3 CDS lacking its predicted 49 amino acid chloroplast transit peptide in the same vector. All cloning primer sequences are given in The CDS of E. coli Arctic Express (DE3) RIL, transformed cells were grown at 30°C for 3 h prior induction by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside. Cells were then incubated for 18.5 h at 13°C, and subsequently harvested by centrifugation . Cells were resuspended in 50 mM Tris–HCl, pH 7.5, 300 mM NaCl, 40 mM imidazole, 1 mg/mL Lysozyme, 1× Protease Inhibitor Cocktail (Roche), 2 mM dithiothreitol (DTT), and lysed using a microfluidizer. Lysates were cleared by centrifugation  and filtering through a 0.45 μm mesh. Cleared lysates were loaded on HisTrap HP 1 mL columns , washed with 50 mM Tris–HCl pH 7.5, 300 mM NaCl, 40 mM imidazole, 2 mM DTT, followed by the same medium with 63 mM imidazole. Bound proteins were eluted using the same medium with a linear gradient of imidazole concentration increasing up to 500 mM. Eluted proteins were buffer-exchanged into 20 mM Tris–HCl pH 7.5, 50 mM NaCl, 2 mM DTT using NAP-25 columns, concentrated using Vivaspin 2—PES concentrators with a molecular weight cut-off of 50 kDa, and finally buffer-exchanged into 10 mM MOPS-NaOH pH 7.5, 50 mM NaCl, 2 mM DTT, aliquoted and snap frozen in liquid N2 prior to analysis.For expression in Far-UV CD spectra of recombinant BAM3 and BAM9 proteins were acquired at 25°C, a protein concentration of 0.2 μg/μL in 10 mM MOPS-NaOH pH 7.5, 50 mM NaCl, 2 mM DTT and using a cuvette with 1 mm path length. Data acquisition was done using a Jasco Spectropolarimeter  between 260 and 200 nm, using a data pitch of 0.5 nm and a band width of 2 nm. Recorded protein spectra were buffer-corrected and normalized to mean residue ellipticity according to 2, 0.1% [w/v] bovine serum albumin (BSA), 1 mM DTT, 0.01% [v/v] Triton X-100, 4 mM NaCl, with 30 mg of hydrated maize starch , or Sephadex G10 resin (Sigma-Aldrich) as negative control. After incubation on spinning wheel, the supernatant was collected, the pellet washed 3 times with the same buffer, and the pellet-bound proteins eluted with incubation medium, to which 2% (w/v) sodium dodecyl sulfate (SDS) was added.Starch binding capacity of BAM9 was assayed in vitro as described by BAM activities of recombinant BAM9 and BAM3 proteins were assayed using the Betamyl-3 assay kit  or by performing in-vitro digestion of soluble starch . For activity on Betamyl-3, recombinant proteins were assayed in final concentrations of 25 mM MOPS-NaOH pH 7.5, 25 mM NaCl, 1 mM DTT, 0.05% [w/v] BSA together with Betamyl-3 substrate at 25°C for either 10 or 20 min, as indicated. Complete deprotonation of the released 4-nitrophenol was achieved by raising the pH of the reactions to 8.5 by addition of 1 M Tris–HCl pH 8.5. Absorbance of the released 4-nitrophenol was subsequently measured at 400 nm using a Tecan M1000 plate reader. For activity measurements using soluble starch, the substrate was solubilized by boiling for 15 min, cooled to 25°C, and subsequently incubated with recombinant protein for 15, 30, or 60 min, as indicated, at 25°C. Reactions contained 25 mM MOPS-NaOH pH 7, 10% [v/v] ethylene glycol, 5 mM ethylenediaminetetraacetic acid .g, 4°C). BAM activity in the supernatant was assayed against soluble starch, as described above.To assay BAM activity in crude protein extracts, leaves were homogenized in extraction medium containing 50 mM MOPS, pH 7, 20% (v/v) ethylene glycol, 10 mM DTT, 10 mM EDTA, and the homogenate clarified by centrifugation  were frozen and pulverized using a Mixer Mill . Starch and soluble sugars were extracted from the frozen powder and quantified as described in For the microscopic analysis of starch in root tips, Arabidopsis seeds were surface-sterilized with 70% (v/v) ethanol and sown on agar plates containing half-strength Murashige and Skoog medium. Seedlings were harvested at midday (ZT7) after 13 d of growth in a 12-h light/12-h dark cycle. Root tips were immersed in Lugol’s iodine solution (Sigma) for 30 s, then immediately placed on a slide containing Hertwig’s Solution . Samples were examined with a Leica DFC 7000T microscope with a bright field filter .Total proteins were extracted from rosettes homogenized in 50 mM Tris–HCl, pH 8, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM DTT, 1× Complete Protease Inhibitor EDTA-free Mixture (Roche). Equal amounts of soluble proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and subject to immunoblotting. For stable lines expressing YFP-tagged proteins, anti-GFP/YFP  and anti-plant actin  were used. For immunoblotting of BAM9, we used antibodies raised in rabbits  against the recombinant purified protein. For immunoblotting of BAM3, we used antibodies described previously (Please refer to The following materials are available in the online version of this article. BAM9 expression over the diel cycle. Activity of recombinant BAM3 and BAM9 proteins when incubated with soluble starch. Binding of recombinant BAM3 and BAM9 proteins to starch granules. Analysis of the starch contents of multiple mutants produced using the bam9-2 mutant allele. Analysis of the starch contents of the bam9 lsf1 double mutant. Starch in the root tips of WT, bam9 mutant, and BAM9 over-expressing plants. Response of BAM9 and BAM4 gene transcription upon environmental perturbations. List of the oligonucleotide primers used for the different experiments of this study. List of the accession numbers of the BAM sequences used for structural modeling. List of the accession numbers of the BAM sequences used for the residue conservation analysis.kiab468_Supplementary_DataClick here for additional data file.


Few studies have explored the feasibility of shear-wave ultrasound elastography (SWUE) for evaluating the upper airways of patients with obstructive sleep apnea (OSA). This study aimed to establish a reliable SWUE protocol for evaluating tongue muscle elasticity and its feasibility and utility in differentiating patients with OSA. Inter-rater and intra-rater reliability of SWUE measurements were tested using the intraclass correlation coefficients. Submental ultrasound was used to measure tongue thickness and stiffness. Association between the ultrasound measurements and presence of OSA was analyzed using multivariate logistic regression. One-way analysis of variance was used to examine if the values of the ultrasound parameters varied among patients with different severities of OSA. Overall, 37 healthy subjects and 32 patients with OSA were recruited. The intraclass correlation coefficients of intra‐ and inter-rater reliability for SWUE for tongue stiffness ranged from 0.84 to 0.90. After adjusting for age, sex, neck circumference, and body mass index, the risk for OSA was positively associated with tongue thickness [odds ratio 1.16 ] and negatively associated with coronal imaging of tongue muscle stiffness [odds ratio 0.72 ]. There were no significant differences in tongue stiffness among OSA patients with varying disease severity. SWUE provided a reliable evaluation of tongue muscle stiffness, which appeared to be softer in patients with OSA. Future longitudinal studies are necessary to investigate the relationship between tongue softening and OSA, as well as response to treatment. Obstructive sleep apnea (OSA) is a breathing disorder characterized by frequent episodes of complete or partial upper airway collapse during sleep . Its preThe diagnosis of OSA is based on polysomnography (PSG), also called as a sleep study . Prompt Diagnostic ultrasound (US) has been widely used to assess musculoskeletal disorders in recent decades. It has several benefits, including portability, cost-effectiveness, and zero radiation exposure, and enables dynamic evaluation . Soft tiBetween October 2018 and May 2019, 32 OSA patients and 37 healthy subjects were recruited from the department of dentistry. All the subjects were evaluated by the STOP questionnaire, a validated tool to screen the presence of OSA . The heaEach subject underwent full-night PSG  in the sleep laboratory in accordance with the OSA diagnosis and management guidelines . MultiplSubmental US images of the tongue muscles were obtained using an US system  using a multi-frequency convex transducer . The scanning depth was set at 8 cm, and the focus at 4 cm. When using the SWUE mode, a color map representing the distribution of tissue elasticity was superimposed on a B-mode image. The assessed rectangular area was required to be homogenously filled with colors ranging from blue (soft) to red (stiff). The propagation mode was displayed simultaneously to demonstrate the contour lines of the radial pulse for ensuring data reliability . The musSubjects were positioned supine, with slight flexion in the neck and extending the head with a soft pad under the neck to expose the submental region. They were asked to close their mouth with the lips slightly in contact and to hold the breath and swallowing for 10 s during the examination.All measurements were performed by a single experienced researcher (except for reliability testing), who was not aware of the participants’ diagnosis. The B mode and SWUE images were obtained in the sagittal and coronal views. The transducer was held stationary to lightly contact the skin coupled by a thin layer of gel. To scan the tongue in the sagittal plane, the transducer was placed between the hyoid bone and symphysis of the mandible. The transducer was adjusted to permit the tongue base to appear in the middle of the screen and the acoustic shadow of the symphysis and hyoid bone at both sides of the scanning window. The dorsal surface of the tongue and soft palate were clearly identified ,B. ThereTongue thickness was measured from the deep fascia of the geniohyoid muscle to the thickest part of the dorsal tongue in both the sagittal and coronal planes. Measurements were performed five times using B-mode images and Image J software . Tongue stiffness was measured five times in a circular ROI positioned in the center of the tongue, where the contour lines of the shear waves in the propagation mode were the most parallel with the best reliability . Their aTongue muscle thickness and elasticity were measured twice in eight healthy volunteers by the primary investigator, with an interval of 24 h between the two examinations. The data were used for calculation of intra-rater reliability. The eight healthy volunteers were also evaluated by the second investigator, and the data were used for computing inter-rater reliability. The intraclass correlation coefficient (ICC) and corresponding 95% confidence interval (CI) were used to quantify the strength of reliability.The sample size was calculated based on a study in which magnetic resonance elastography was used to measure tongue elasticity . The meap were two-sided and differences with p < 0.05 were considered to be statistically significant.The Shapiro-Wilk test was used to test the distribution of the obtained data. Normally distributed continuous variables (expressed as mean ± SD) were compared using the independent t-test or the Mann Whitney U test for data that were non-normally distributed. Categorical variables (expressed as number and percentage) were compared using the chi-squared test or Fisher’s exact test in cases of sparse data. Associations between US measurements and the presence of OSA were analyzed using logistic regression and quantified as odds ratio (OR) and corresponding 95% CI. The confounders adjusted in multivariate analysis were selected according to previous studies  and inclThe intra-rater reliability values, reflected by ICC, were 0.98  for thickness in the sagittal plane, 0.97 (95% CI 0.84–0.99) for thickness in the coronal plane, 0.90 (95% CI 0.52–0.98) for stiffness in the sagittal plane, and 0.85 (95% CI 0.27–0.97) for stiffness in the coronal plane. The ICC values of inter-rater reliability were 0.95 (95% CI 0.74–0.99) for thickness in the sagittal plane, 0.92 (95% CI 0.62–0.98) for thickness in the coronal plane, 0.84 (95% CI 0.20–0.97) for stiffness in the sagittal plane, and 0.84 (95% CI 0.20–0.97) for stiffness in the coronal plane. Overall, the reliability measurements were classified as “excellent” for tongue muscle thickness and “good” for tongue stiffness .p = 0.022) and exhibited higher BMI values (p = 0.019). The data of PSG in the OSA group are detailed in Comparative data are summarized in Regarding US measurements, OSA patients exhibited significantly thicker tongues in both the sagittal and coronal views, with lower shear modulus (kPa) in the coronal plane. The distributions of all the individual US measurements were demonstrated on the dot plots . AssociaThe distinctive capabilities of US thickness and stiffness measurements for OSA are presented in Subgroup analysis was performed to examine the association between OSA severity and tongue muscle thickness/elasticity  in the OThis study aimed to assess tongue muscle stiffness in OSA patients using SWUE. First, we demonstrated that the stiffness of tongue muscles could be reliably measured using SWUE. Second, the US measurements demonstrated that OSA patients tended to have thicker and softer tongue muscles. Third, the thickness and stiffness of tongue muscles did not vary according to OSA severity.Our findings revealed that the reliability of US measurement ranged from 0.92 to 0.98 for tongue thickness and from 0.84 to 0.90 for tongue stiffness. Although several studies have investigated tongue thickness using US , only onTo the best of our knowledge, this study is the first to validate the reliability of US measurements for tongue muscle stiffness. Unlike strain elastography , which qAs SWUE was used in the present study, there was no need for the investigator to perform manual compression during the elasticity measurement. Therefore, this advantage allows the investigator to place the transducer and position the patient exactly like the protocol of most sub-mental US measurements . The onlOur study yielded increased tongue muscle thicknesses in patients with OSA compared with healthy subjects. Enlargement of tongue volume narrows the oral cavity and pharyngeal space and has been shown to be associated with OSA . Schwab According to our results, the tongue tissue of OSA patients appeared to be softer in the coronal view. This finding is consistent with a previous study, in which magnetic resonance elastography revealed that the shear modulus (kPa) of tongue tissue was approximately 10% lower in nine OSA patients compared with healthy controls . FurtherIn our study, the significant association between tongue stiffness and OSA was only found when SWUE was performed in the coronal – but not sagittal – view. Results of our study also revealed that tongue muscle stiffness did not vary according to the severity of OSA, which limited the usefulness of SWUE in aiding differential diagnosis. A recently published similar study  reportedIn the study by Our study has several clinical implications. First, considering the conflicting results of tongue muscle stiffness from two available papers , ours arThere were several limitations to this study that should be acknowledged. First, all subjects were examined while they were awake. As such, whether tongue stiffness changed during sleep could have not been determined in our protocol. Second, the healthy controls did not receive PSG examinations. However, they were required to answer “No” in all the questions in the STOP questionnaire. As answering “Yes” to 2 or fewer than 2 questions is considered to be low risk for OSA, the prevalence of asymptomatic OSA in our control group should be trivial. Furthermore, a separate additional analysis – to compare the US measurements between control subjects with and those without PSG findings – revealed no significant between-group difference . Third, Our findings suggested that the tongues of OSA patients were thicker and softer and the stiffness of tongue muscles did not vary according to the severity of OSA. Therefore, future longitudinal studies are necessary to explore causation between tongue thickening/softening and OSA; nevertheless, SWUE appears to be a reliable tool for evaluating the stiffness of tongue muscles.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Institutional Review Board of National Taiwan University Hospital (IRB number: 20180904RIND). The patients/participants provided their written informed consent to participate in this study.K-VC, C-AC, and Y-JC conceived and designed the study, recruited the study subjects, and planned and performed the statistical analysis. W-TW, C-AC, Y-JC, K-VC, and LÖ contributed to study supervision and critical revision of the manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.