Abstract

DNA methylation-based age estimators (DNAm aging clocks) are currently one of the most promising biomarkers for predicting biological age. However, the relationships between objectively measured physical fitness, including cardiorespiratory fitness, and DNAm aging clocks are largely unknown. We investigated the relationships between physical fitness and the age-adjusted value from the residuals of the regression of DNAm aging clock to chronological age (DNAmAgeAcceleration: DNAmAgeAccel) and attempted to determine the relative contribution of physical fitness variables to DNAmAgeAccel in the presence of other lifestyle factors. DNA samples from 144 Japanese men aged 65–72 years were used to calculate first- (i.e., DNAmHorvath and DNAmHannum) and second- (i.e., DNAmPhenoAge, DNAmGrimAge and DNAmFitAge) generation DNAm aging clocks. Various surveys and measurements were conducted, including physical fitness, body composition, blood biochemical parameters, nutrients intake, smoking, alcohol consumption, disease status, sleep status, and chronotype. The peak oxygen uptake (VO_2peak) per kg body weight had a significant negative correlation with GrimAgeAccel (r= -0.222,p= 0.008). A comparison of the tertile groups showed that the GrimAgeAccel of the highest VO_2peakgroup was decelerated by 1.6 years compared to the lowest group (p= 0.035). Multiple regression analysis suggested that rather than physical fitness, serum triglycerides, carbohydrate intake, and smoking status, were significantly associated with DNAmAgeAccel. In conclusion, the contribution of cardiorespiratory fitness to DNAmAgeAccel was relatively low compared to lifestyle factors such as smoking. However, this study reveals a negative relationship between cardiorespiratory fitness and DNAmAgeAccel in older men.

This study aimed to examine the effects of hydrogen gas (H2) produced by intestinal microbiota on participant conditioning to prevent intense exercise-induced damage. In this double-blind, randomized, crossover study, participants ingested H2-producing milk that induced intestinal bacterial H2 production or a placebo on the trial day, 4 h before performing an intense exercise at 75% maximal oxygen uptake for 60 min. Blood marker levels and respiratory variables were measured before, during, and after exercise. Visual analog scale scores of general and lower limb muscle soreness evaluated were 3.8- and 2.3-fold higher, respectively, on the morning after treatment than that before treatment during the placebo trial, but not during the test beverage consumption. Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) concentrations and production rates significantly increased with placebo consumption; no changes were observed with test beverage consumption. After exercise, relative blood lactate levels with H2-producing milk consumption were lower than those with placebo consumption. A negative correlation was observed between the variation of 8-OHdG and the area under the curve (AUC) of breath H2 concentrations. Lipid oxidation AUC was 1.3-fold higher significantly with H2-producing milk than with placebo consumption. Conclusively, activating intestinal bacterial H2 production by consuming a specific beverage may be a new strategy for promoting recovery and conditioning in athletes frequently performing intense exercises.

Introduction The muscle mechanoreflex has been considered to make a small contribution to the cardiovascular response to exercise in healthy humans because no pressor response has been observed during stimulation of mechanosensitive receptors, such as static passive stretching, during many human studies. There is room for rethinking this consideration since the pressor response to upper limb exercise is greater than that to lower limb exercise. We examined whether static passive stretching of the forearm muscles causes a muscle mechanoreflex-induced pressor response in humans. Methods Eighteen healthy men were recruited for this study. After a 15-min rest period in the supine position with a neutral (0°) wrist joint angle, all participants completed static passive stretching of the forearm for 60s at four different intensities: minimal painful passive stretching (PPS); moderate-intensity passive stretching (MPS); low-intensity passive stretching (LPS); and no load (NL). During the procedure, beat-to-beat arterial blood pressure was measured using finger photoplethysmography. The force generated between the passively stretched hand and the experimenter's hands was recorded using a force transducer. Results Mean arterial pressure (MAP) during PPS and MPS significantly increased from baseline during the last 40s (P < 0.05). MAP was significantly greater at 50s and 60s, depending on the intensity. MPS induced a greater peak response in MAP than lower intensities (P < 0.05). None of the subjects reported pain during the MPS and LPS trials. Conclusion Static passive stimulation of the forearm is an effective method of isolating the muscle mechanoareflex-induced pressor response in humans.

AbstractThe present study aimed to test the hypothesis that changes in the dermal tissue dielectric constant (TDC) and biomechanical properties of the skin would be correlated with the indicators related to dehydration. Ten healthy adult men were enrolled in three trials: no fluid intake (DEH), ad libitum fluid intake (AL-HYD), and programmed fluid intake (P-HYD) after exercise in a randomized crossover design. The participants performed a pedaling exercise at 60% heart rate reserve until 2% body mass loss. At 120 min after exercise, an incremental exercise test was carried out. Aerobic capacity, body composition, TDC, biomechanical properties of the skin (pliability, viscoelasticity, and total recovery), and indicators related to dehydration in the serum and urine were measured before and 120 min after exercise. Higher values of the pliability and viscoelasticity, and lower value of the total recovery on the hand were demonstrated in the P-HYD trial compared to the DEH trial (all P < 0.05). Changes in the TDC were significantly correlated with changes in body mass (P < 0.05), total body water (P < 0.05), serum osmolarity (P < 0.05), and hematocrit (P < 0.01). Changes in the biomechanical properties of the hand were significantly correlated with changes in body mass, hematocrit, and urine specific gravity (all P < 0.05). The present study showed that the changes in skin characteristics correlated with the body water and dehydration-associated indicators in the serum and urine, thus suggesting that skin characteristics may be useful in the assessment of dehydration.

Acute exercise can improve vascular stiffness in the conduit artery, but its effect on the retinal arterioles is unknown. The present study investigated the effects of acute dynamic exercise on retinal vascular stiffness. In experiment 1, we measured the cardio-ankle vascular index (CAVI), carotid artery intima-media thickness (carotid IMT), and retinal blood velocity by laser speckle flowgraphy in 28 healthy old and 28 young men (69 ± 3 and 23 ± 3 yr, respectively). Pulse waveform variables, which were used as an index of retinal vascular stiffness, were assessed by retinal blood flow velocity profile analysis. In experiment 2, 18 healthy old and 18 young men (69 ± 3 and 23 ± 3 yr, respectively) underwent assessment of pulse waveform variables after a 30-min bout of moderate cycling exercise at an intensity of 60% heart rate reserve. There was a significant difference in the baseline pulse waveform variables between the old and young groups. Pulse waveform variables in the retinal arteriole did not significantly change after acute dynamic exercise, whereas CAVI significantly decreased. These findings suggest that retinal vascular stiffness does not change by acute exercise. The effect of exercise on vascular stiffness in the retinal arterioles might be different from that in the conduit artery.NEW & NOTEWORTHY Acute dynamic exercise is well known to improve vascular stiffness in the conduit artery while its effect on the retinal arterioles has been unknown. This study showed that an acute dynamic exercise does not change vascular stiffness in the retinal arteriole in healthy humans. Different responses to acute dynamic exercise in vascular stiffness in retinal arterioles and conduit arteries are suggested.

PURPOSE: Although the muscle mechanoreflex is an important mediator to cardiovascular regulation during exercise, its modulation factors remain relatively unknown. Therefore, the purpose of this study was to investigate the effect of muscle stiffness on the muscle mechanoreflex. METHODS: Participants were divided based on their median muscle stiffness (2.00 Nm/mm) into a low group (n = 15) and a high group (n = 15), and the muscle mechanoreflex was compared between the groups. After a 15-min rest in the supine position, heart rate (HR), blood pressure (BP), stroke volume (SV), and cardiac output (CO) were measured at rest for 3 min and during static passive dorsiflexion (SPD) at 20° for 1 min. Following a 15-min re-rest, muscle stiffness and passive resistive torque were evaluated in the distal end of the muscle belly of the medial gastrocnemius. RESULTS: Peak relative changes in HR (low group: 6 ± 4% and high group: 12 ± 4%) and CO (low group: 8 ± 10% and high group: 13 ± 9%) were greater in the high group than in the low group (both, P < 0.05). A significant positive correlation was found between resistive torque during SPD and muscle stiffness and peak relative changes in HR (r = 0.51 and 0.61, both P < 0.05). However, there was no correlation between muscle elongation during SPD and peak relative changes in HR (r = - 0.23, P = 0.20). CONCLUSION: These findings suggest that muscle stiffness may be modulatory factor of muscle mechanoreflex.

BACKGROUND: Cerebral blood flow (CBF) pulsatility is dampened by large and small arterial buffer functions. Resistance training (RT) improved small arterial buffer function regardless of impaired large arterial compliance. This response to small arterial buffer function with RT may be compensatory adaptation to impaired large arterial compliance for protecting end-organs such as the brain from excessive pulsatile stresses. This study aimed to investigate the effects of changes in large arterial compliance and small arterial buffer functions with RT on CBF pulsatility.METHODS: Twenty healthy men were assigned to either the RT group (N.=10) or the control (CON) group (N.=10). Subjects in the RT group exercised three times/week for 8 weeks. Arterial compliance of common carotid artery was measured as an index of large arterial compliance. Dampening factor (DF) in the cerebral artery and pulsatility index (PI) in the middle cerebral artery (MCA) were used to evaluate small arterial buffer function and CBF pulsatility, respectively.RESULTS: In the RT group, arterial compliance significantly decreased (P<0.05) and DF in the cerebral artery signifi-cantly increased (P<0.05) after intervention compared to baseline. However, there was no change in PI in MCA in both groups. A negative correlation was found between Delta arterial compliance and Delta DF in the cerebral artery (r=-0.671, P<0.05).CONCLUSIONS: These results may suggest that the response of small arteries to RT is a compensatory adaptation to impaired large arterial compliance for protecting end organs from excessive pulsatile stresses.

<title>Abstract</title><sec>
<title>Background</title>
Compared with age-matched untrained men, resistance-trained men who have undergone long duration training (&gt; 2 years) at a high frequency (&gt; 5 days/week) may be lower cardiovagal baroreflex sensitivity (BRS) because of central arterial stiffening. Therefore, the purpose of this study was to examine the effect of greater central arterial stiffness in resistance-trained men on cardiovagal BRS in a cross-sectional study to compare resistance-trained men with age-matched untrained men.


</sec><sec>
<title>Methods</title>
This cross-sectional study included resistance-trained men (<italic>n</italic> = 20; age: 22 ± 3; body mass index: 26.7 ± 2.2) and age-matched untrained men (control group: <italic>n</italic> = 20; age: 25 ± 2; body mass index: 23.7 ± 2.4). The β-stiffness index and arterial compliance were assessed at the right carotid artery using a combination of a brightness mode ultrasonography system for the carotid artery diameter and applanation tonometry for the carotid blood pressure. And, the cardiovagal BRS was estimated by the slope of the R–R interval and systolic blood pressure during Phase II and IV of Valsalva maneuver (VM). The participants maintained an expiratory mouth pressure of 40 mmHg for 15 s in the supine position.


</sec><sec>
<title>Results</title>
The β-Stiffness index was significantly higher in the resistance-trained group than in the control group (5.9 ± 1.4 vs. 4.4 ± 1.0 a.u., <italic>P</italic> &lt; 0.01). In contrast, the resistance-trained group had significantly lower arterial compliance (0.15 ± 0.05 vs. 0.20 ± 0.04 mm²/mmHg, <italic>P</italic> &lt; 0.01) and cardiovagal BRS during Phase IV of VM (9.0 ± 2.5 vs. 12.9 ± 5.4 ms/mmHg, <italic>P</italic> &lt; 0.01) than the control group and. Moreover, cardiovagal BRS during Phase IV of VM was inversely and positively correlated with the β-stiffness index (<italic>r</italic> = − 0.59, <italic>P</italic> &lt; 0.01) and arterial compliance (<italic>r</italic> = 0.64, <italic>P</italic> &lt; 0.01), respectively.


</sec><sec>
<title>Conclusion</title>
Resistance-trained group had greater central arterial stiffness and lower cardiovagal BRS Phase IV compared with control group. Moreover, the central arterial stiffening was related to cardiovagal BRS Phase IV. These results suggest that greater central arterial stiffness in resistance-trained men may be associated with lower cardiovagal BRS.


<italic>Trial Registration</italic> University hospital Medical Information Network (UMIN) in Japan, UMIN000038116. Registered on September 27, 2019.


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Strenuous exercise induces organ damage, inflammation and oxidative stress. To prevent exercise-induced organ damage, inflammation and oxidative stress, rehydrating may be an effective strategy. In the present study, we aimed to examine whether beverage intake after exhaustive exercise to recover from dehydration prevents such disorders. Thirteen male volunteers performed incremental cycling exercise until exhaustion. Immediately after exercise, the subjects drank an electrolyte containing water (rehydrate trial: REH) or did not drink any beverage (control trial: CON). Blood samples were collected before (Pre), immediately (Post), 1 h and 2 h after exercise. Urine samples were also collected before (Pre) and 2 h after exercise. We measured biomarkers of organ damage, inflammation and oxidative stress in blood and urine. Biomarkers of muscle, renal and intestinal damage and inflammation increased in the blood and urine after exercise. However, changes in biomarkers of organ damage and inflammation did not differ between trials (p > 0.05). The biomarker of oxidative stress, thiobarbituric acid reactive substances (TBARS), in plasma, showed different changes between trials (p = 0.027). One hour after exercise, plasma TBARS concentration in REH had a higher trend than that in CON (p = 0.052), but there were no significant differences between Pre and the other time points in each trial. These results suggest that beverage intake after exercise does not attenuate exercise-induced organ damage, inflammation or oxidative stress in healthy males. However, rehydration restores exercise-induced oxidative stress more quickly.

Previous studies have not investigated the determinants of resting oxidative stress, including physical fitness, as it relates to redox regulation. The present study therefore was aimed at identifying lifestyle and biological factors that determine resting oxidative stress, including objectively measured physical fitness. In 873 middle-aged and elderly men and women, age and anthropometric parameters, lifestyle-related parameters, medication and supplementation status, physical fitness, biochemical parameters, and nutritional intake status, as well as three plasma oxidative stress markers: protein carbonyl (PC), F2-isoprostane (F2-IsoP), and thiobarbituric acid reactive substances (TBARS), were surveyed and measured. The determinants of PC, F2-IsoP, and TBARS in all participants were investigated using stepwise multiple regression analysis. In PC, age (β = -0.11, P = 0.002), leg extension power (β = -0.12, P = 0.008), BMI (β = 0.12, P = 0.004), and HDL-C (β = 0.08, P = 0.040) were included in the regression model (adjusted R 2 = 0.018). In the F2-IsoP, smoking status (β = 0.07, P = 0.060), BMI (β = 0.07, P = 0.054), and HbA1c (β = -0.06, P = 0.089) were included in the regression model (adjusted R 2 = 0.006). In TBARS, glucose (β = 0.18, P < 0.001), CRF (β = 0.16, P < 0.001), age (β = 0.15, P < 0.001), TG (β = 0.11, P = 0.001), antioxidant supplementation (β = 0.10, P = 0.002), and HbA1c (β = -0.13, P = 0.004) were included in the regression model (adjusted R 2 = 0.071). In conclusion, the present study showed that age, anthropometric index, lifestyle-related parameters, medication and supplementation status, objectively measured physical fitness, biochemical parameters, and nutritional intake status explain less than 10% of oxidative stress at rest.

Antimicrobial peptides (AMPs) play an important role in innate immunity in human skin. It is known that AMPs mainly function in the stratum corneum. Therefore, AMP concentrations in the stratum corneum need to be precisely measured to clarify functional and physiological importance of AMPs in cutaneous defence. Tape stripping (TS) is a well-established method by which components in the stratum corneum can be collected. However, the usefulness of the TS method for measuring AMP concentration in human skin remains unclear. Therefore, we compared it with another popular method, skin rinsing, which had been established as a method for measuring AMP concentration in human skin. When investigated on healthy medial forearm using RNase 7, which is one of the typical AMPs, as an index, there was a significant positive correlation between RNase 7 concentrations measured by the TS method at adjacent forearm sites, demonstrating the reproducibility of the TS method. Next, a significant positive correlation was detected in RNase 7 concentrations measured using the TS and the skin rinsing method, indicating that the TS method is comparable to the skin rinsing method. Thus, we speculate that the TS method is useful for measuring AMP concentration in human skin.

BACKGROUND: Preceding exercise enhances fat metabolism during subsequent endurance exercise. However, it is unknown which type of exercise most enhances fat metabolism during subsequent exercise. The aim of this study was to examine the influence of preceding resistance or endurance exercise on fat metabolism during subsequent endurance exercise and to compare the effect with that without a preceding exercise.METHODS: Nine male subjects performed the following trials: 1) endurance exercise (E trial), 2) endurance exercise preceded by resistance exercise (R+E trial), and 3) endurance exercise preceded by endurance exercise (E+E trial). There was a 20-min break after the preceding exercise. Subsequent endurance exercise was performed on a cycle ergometer at 80% of the individual ventilatory threshold. Blood and exhaled gas were analyzed respectively.RESULTS: The R+E and E+E trials showed a significantly lower respiratory exchange ratio (RER) and a higher amount of fat oxidation during endurance exercise than those in the E trial (P<0.05). Furthermore, the E+E trial showed a significantly lower RER (0.86 +/- 0.03 vs. 0.84 +/- 0.02) and a significantly higher amount of fat oxidation (0.38 +/- 0.05 g/min vs. 0.44 +/- 0.08 g/min) than those in the R+E trial.CONCLUSIONS: This study suggested that fat oxidation during subsequent endurance exercise was enhanced when a preceding exercise was performed and that endurance exercise as a preceding exercise enhanced fat oxidation more than resistance exercise did.

BACKGROUND: Increased arterial stiffness decreases cerebral blood flow (CBF), which plays a vital role in the maintenance of life and cognitive function. Previous studies have shown that high-intensity resistance exercise (HRE) increases arterial stiffness. HRE may decrease cerebral blood flow. However, the effect of increased arterial stiffness associated with HRE on CBF is not fully elucidated. Therefore, this study aimed to investigate the effect of increased arterial stiffness with HRE on CBF.METHODS: Study participants included 8 healthy young men. All subjects performed three exercise trials in a crossover design: HRE, low-intensity resistance exercise (LRE), and aerobic exercise (AE). beta-stiffness index was measured as an index of arterial stiffness, and global cerebral blood flow was measured as an index of cerebral blood flow. These were measured at rest, immediately after exercise, and 10 minutes post-exercise.RESULTS: Global cerebral blood flow was significantly increased immediately after and 10 minutes after AE. In contrast, a significant decrease of global cerebral blood flow was observed immediately after and 10 minutes following HRE. However, no changes were observed in LRE. With HRE, beta-stiffness index was significantly increased immediately after and 10 minutes post-exercise. In contrast, no differences were observed in arterial stiffness associated with aerobic exercise and LRE. A negative correlation was found between global cerebral blood flow and arterial stiffness at rest and immediately after and 10 minutes post-exercise.CONCLUSIONS: These results suggested that HRE increased arterial stiffness and decreased cerebral blood flow.

The aim of this study was to investigate whether accommodating elastic bands with barbell back squats (BSQ) increase muscular force during the deceleration subphase. Ten healthy men (mean ± standard deviation: Age: 23 ± 2 years; height: 170.5 ± 3.7 cm; mass: 66.7 ± 5.4 kg; and BSQ one repetition maximum (RM): 105 ± 23.1 kg; BSQ 1RM/body mass: 1.6 ± 0.3) were recruited for this study. The subjects performed band-resisted parallel BSQ (accommodating elastic bands each sides of barbell) with five band conditions in random order. The duration of the deceleration subphase, mean mechanical power, and the force and velocity during the acceleration and deceleration subphases were calculated. BSQ with elastic bands elicited greater mechanical power output, velocity, and force during the deceleration subphase, in contrast to that elicited with traditional free weight (p < 0.05). BSQ with elastic bands also elicited greater mechanical power output and velocity during the acceleration subphase. However, the force output during the acceleration subphase using an elastic band was lesser than that using a traditional free weight (p < 0.05). This study suggests that BSQ with elastic band elicit greater power output during the acceleration and deceleration subphases.

The deceleration sub-phase during the back squat (BSQ) makes it difficult to stimulate the muscles throughout the full range of motion, and it has only been reported for one load during BSQ. The purpose of this study was to investigate whether a deceleration sub-phase occurs during BSQ with different loads and to assess the influence of load on the deceleration sub-phase duration and negative impulse during the deceleration sub-phase. Sixteen healthy men (mean ± standard deviation: age: 25 ± 3 years; height: 1.73 ± 0.07 m; mass: 83.2 ± 16.1 kg; BSQ one repetition maximum (1RM): 163.8 ± 36.6 kg; BSQ 1RM/body weight: 2.0 ± 0.4) who had performed resistance training for at least 1 year were recruited for this study. The subjects performed parallel BSQ with 0%, 12%, 27%, 42%, 56%, 71%, and 85% of each 1RM on a force plate in a random order. The deceleration sub-phase duration and negative impulse during the deceleration sub-phase were calculated from force-time data. The absolute durations of the deceleration sub-phase were not significantly different according to load except for 27% 1RM and 85% 1RM (p = 0.01). However, as the load increased from 12 to 85% 1RM, the relative duration of the deceleration sub-phase decreased (p < 0.05). The negative impulse during the deceleration sub-phase also increased from 0 to 42% 1RM (p < 0.05). A deceleration sub-phase occurs regardless of the load (0%-85% 1RM), and a large portion of the deceleration sub-phase occupied the concentric phase, with low-moderate loads, and a large amount of negative impulse occurred during the short deceleration sub-phase with a high load.

BACKGROUND: Increased central arterial stiffness and/or decreased compliance reduces buffer function and increases cerebral blood flow (CBF) pulsatility, which leads to increased cerebral microvascular damage, resulting in the augmentation of the risk of cerebrovascular diseases. Resistance-trained men showed higher central arterial stiffness and lower arterial compliance than age-matched, sedentary men. This study examined the effect of increased central arterial stiffness and/or decreased arterial compliance on CBF pulsatility. METHODS: The study participants included 31 young healthy men (15 resistance-trained men, aged 21 ± 1 years; and 16 controls, aged 23 ± 1 years). β-Stiffness index and arterial compliance were measured in the right carotid artery as index of central arterial stiffness and compliance, respectively. The pulsatility index (PI) was measured in the middle cerebral artery as index of CBF pulsatility. RESULTS: β-Stiffness index and PI were significantly higher in the resistance-trained group than in the control group (β-stiffness index: 5.3 ± 0.3 vs. 3.5 ± 0.3 a.u., P < 0.05, PI: 0.80 ± 0.02 vs. 0.70 ± 0.02, P < 0.05). The resistance-trained group showed significantly lower arterial compliance than the control group (0.16 ± 0.01 vs. 0.23 ± 0.01 mm2/mm Hg, P < 0.05). Positive and negative correlations were observed between β-stiffness index and PI (r = 0.39, P < 0.05), and between arterial compliance and PI (r = -0.59, P < 0.05), respectively. CONCLUSIONS: The resistance-trained group showed higher central arterial stiffness and PI and lower arterial compliance. Central arterial stiffness and arterial compliance were associated with PI. Increased arterial stiffness and decreased arterial compliance with resistance training impair buffer function, resulting in increased CBF pulsatility. CLINICAL TRIAL REGISTRATION: Trial Number UMIN000023816 URL: http://www.umin.ac.jp/icdr/index.html Official scientific title of the study: effect of increase arterial stiffness by resistance training on cerebral hemodynamic.

PURPOSE: It remains unclear whether rehydration restores retinal blood flow reduced by exhaustive exercise. We investigated the effect of fluid intake on retinal blood flow after exhaustive exercise. METHODS: Blood flow in the inferior (ITRA) and superior temporal retinal arterioles (STRA) was measured before and after incremental cycling exercise until exhaustion in 13 healthy males. After the exercise, the subjects rested without drinking (control condition: CON) or with drinking an electrolyte containing water (rehydrate condition: REH) and were followed up for a period of 120 min. To assess the hydration state, the body mass was measured, and venous blood samples were collected and plasma volume (PV) was calculated. RESULTS: Body mass decreased in CON after the trial [- 1.1 ± 0.1% (mean ± SE), p < 0.05]. PV was lower in CON than in REH during recovery. The ITRA and STRA blood flows decreased immediately after exercise from the resting baseline (ITRA; - 23 ± 4% in REH and - 30 ± 4% in CON, p < 0.05). The ITRA blood flow recovered baseline level at 15 min of recovery in REH (- 9 ± 3%, p = 0.5), but it remained reduced in CON (-14 ± 3%, p < 0.05). The STRA blood flow was higher in REH than in CON at 15 min (2 ± 3 vs. - 5 ± 3%, p < 0.05). CONCLUSIONS: The results of this study suggest that the reduction in retinal blood flow induced by exhaustive exercise can be recovered early by rehydration.