BACKGROUND Type 2 diabetes mellitus(T2DM),a fast-growing issue in public health,is one of the most common chronic metabolic disorders in older individuals.Osteoporosis and sarcopenia are highly prevalent in T2DM patie...BACKGROUND Type 2 diabetes mellitus(T2DM),a fast-growing issue in public health,is one of the most common chronic metabolic disorders in older individuals.Osteoporosis and sarcopenia are highly prevalent in T2DM patients and may result in fractures and disabilities.In people with T2DM,the association between nutrition,sarcopenia,and osteoporosis has rarely been explored.AIM To evaluate the connections among nutrition,bone mineral density(BMD)and body composition in patients with T2DM.METHODS We enrolled 689 patients with T2DM for this cross-sectional study.All patients underwent dual energy X-ray absorptiometry(DXA)examination and were categorized according to baseline Geriatric Nutritional Risk Index(GNRI)values calculated from serum albumin levels and body weight.The GNRI was used to evaluate nutritional status,and DXA was used to investigate BMD and body composition.Multivariate forward linear regression analysis was used to identify the factors associated with BMD and skeletal muscle mass index.RESULTS Of the total patients,394 were men and 295 were women.Compared with patients in tertile 1,those in tertile 3 who had a high GNRI tended to be younger and had lower HbA1c,higher BMD at all bone sites,and higher appendicular skeletal muscle index(ASMI).These important trends persisted even when the patients were divided into younger and older subgroups.The GNRI was positively related to ASMI(men:r=0.644,P<0.001;women:r=0.649,P<0.001),total body fat(men:r=0.453,P<0.001;women:r=0.557,P<0.001),BMD at all bone sites,lumbar spine(L1-L4)BMD(men:r=0.110,P=0.029;women:r=0.256,P<0.001),FN-BMD(men:r=0.293,P<0.001;women:r=0.273,P<0.001),and hip BMD(men:r=0.358,P<0.001;women:r=0.377,P<0.001).After adjustment for other clinical parameters,the GNRI was still significantly associated with BMD at the lumbar spine and femoral neck.Additionally,a low lean mass index and higherβ-collagen special sequence were associated with low BMD at all bone sites.Age was negatively correlated with ASMI,whereas weight was positively correlated with ASMI.CONCLUSION Poor nutrition,as indicated by a low GNRI,was associated with low levels of ASMI and BMD at all bone sites in T2DM patients.Using the GNRI to evaluate nutritional status and using DXA to investigate body composition in patients with T2DM is of value in assessing bone health and physical performance.展开更多
The estimates of global incidence and prevalence of non-alcoholic fatty liver disease(NAFLD) are worrisome, due to the parallel burden of obesity and its metabolic complications. Indeed, excess adiposity and insulin r...The estimates of global incidence and prevalence of non-alcoholic fatty liver disease(NAFLD) are worrisome, due to the parallel burden of obesity and its metabolic complications. Indeed, excess adiposity and insulin resistance represent two of the major risk factors for NAFLD; interestingly, in the last years a growing body of evidence tended to support a novel mechanistic perspective, in which the liver is at the center of a complex interplay involving organs and systems, other than adipose tissue and glucose homeostasis. Bone and the skeletal muscle are fat- free tissues which appeared to be independently associated with NAFLD in several cross-sectional studies. The deterioration of bone mineral density and lean body mass, leading to osteoporosis and sarcopenia, respectively, are age-related processes. The prevalence of NAFLD also increases with age. Beyond physiological aging, the three conditions share some common underlying mechanisms, and their elucidations could be of paramount importance to design more effective treatment strategies for the management of NAFLD. In this review, we provide an overview on epidemiological data as well as on potential contributors to the connections of NAFLD with bone and skeletal muscle.展开更多
Severe burn injury triggers the body's nonspecific adaptive responses to acute insult, including the systemic inflammatory and stress responses, as well as the sympathetic response to immobilization. These responses ...Severe burn injury triggers the body's nonspecific adaptive responses to acute insult, including the systemic inflammatory and stress responses, as well as the sympathetic response to immobilization. These responses trigger inflammatory bone resorption followed by glucocorticoid-induced apoptosis of osteoblasts and probably osteocytes. Because these patients are catabolic, they suffer concomitant muscle wasting and negative nitrogen balance. The use of anabolic agents such as recombinant human growth hormone and oxandrolone results in improved bone mineral content and muscle strength after approximately I year. Use of bisphosphonates within the first 10 days of a severe burn completely blocks the resorptive bone loss and has the added advantage of appearing to preserve muscle protein from excessive breakdown. The mechanism for the protective effect on muscle is not currently known. However, if the effect of bisphosphonates on muscle can be confirmed, it raises the possibility that bone communicates with muscle.展开更多
Osteogenesis imperfecta(OI) comprises a group of heritable connective tissue disorders generally defined by recurrent fractures, low bone mass, short stature and skeletal fragility. Beyond the skeletal complications...Osteogenesis imperfecta(OI) comprises a group of heritable connective tissue disorders generally defined by recurrent fractures, low bone mass, short stature and skeletal fragility. Beyond the skeletal complications of OI,many patients also report intolerance to physical activity, fatigue and muscle weakness. Indeed, recent studies have demonstrated that skeletal muscle is also negatively affected by OI, both directly and indirectly. Given the well-established interdependence of bone and skeletal muscle in both physiology and pathophysiology and the observations of skeletal muscle pathology in patients with OI, we investigated the therapeutic potential of simultaneous anabolic targeting of both bone and skeletal muscle using a soluble activin receptor 2B(ACVR2B) in a mouse model of type Ⅲ OI(oim). Treatment of 12-week-old oim mice with ACVR2 B for 4 weeks resulted in significant increases in both bone and muscle that were similar to those observed in healthy,wild-type littermates. This proof of concept study provides encouraging evidence for a holistic approach to treating the deleterious consequences of OI in the musculoskeletal system.展开更多
Autophagy in eukaryotic cells is a constitutive process and functions as a homeostatic mechanism; it is up-regulated in response to specific stress stimuli such as starvation, hypoxia and as oxidative stress. In addit...Autophagy in eukaryotic cells is a constitutive process and functions as a homeostatic mechanism; it is up-regulated in response to specific stress stimuli such as starvation, hypoxia and as oxidative stress. In addition to playing a crucial role in adaptive responses to different stimuli, autophagy is also required for intracellular quality control. This second aspect is important to prevent the activation of pathological processes. Autophagy also plays a central role in cellular development and differentiation because it is involved in the regulation of energetic balance. This final aspect is critical for maintaining proper bone and muscle function as well as to prevent any pathological changes. Therefore, identifying new molecular targets involved in autophagy is critical to assure a good quality of life.展开更多
Objective: To study the muscle injury, inflammatory response and bone metabolism after paravertebral muscle space and minimally invasive percutaneous approach surgeries for thoracolumbar fracture. Methods: A total of ...Objective: To study the muscle injury, inflammatory response and bone metabolism after paravertebral muscle space and minimally invasive percutaneous approach surgeries for thoracolumbar fracture. Methods: A total of 92 patients with thoracolumbar fractures who were treated in Baoji Central Hospital between May 2015 and January 2017 were selected and randomly divided into paravertebral group and minimally invasive percutaneous group who accepted pedicle screw fixation under different approaches. Before operation as well as 1 d and 3 d after operation, serum was collected to determine the contents of creatase, inflammatory reaction molecules and bone metabolism indexes. Results: Serum Myo, CK, LDH, TNF-α, MCP-1, HMGB-1, CRP, IL-1β, OC, OPG, PICP and PINP levels of both groups 1 d and 3 d after operation were significantly higher than those before operation while TRACP5b, RANKL, CTX and NTX levels were significantly lower than those before operation, and serum Myo, CK, LDH, TNF-α, MCP-1, HMGB-1, CRP, IL-1β, TRACP5b, RANKL, CTX and NTX levels of paravertebral group 1 d and 3 d after operation were significantly lower than those of minimally invasive percutaneous group while OC, OPG, PICP and PINP levels were significantly higher than those of minimally invasive percutaneous group. Conclusion:Paravertebral muscle space surgery for thoracolumbar fracture is more effective than minimally invasive percutaneous surgery in reducing muscle injury and inflammatory response, and improving bone metabolism.展开更多
Objective: To explore the correlation between muscle strength, muscle mass and bone mineral density (BMD) in Zhuang female population, body composition analysis and grip strength, and to analyze the possible influenci...Objective: To explore the correlation between muscle strength, muscle mass and bone mineral density (BMD) in Zhuang female population, body composition analysis and grip strength, and to analyze the possible influencing factors of BMD. Methods: 182 postmenopausal women were selected from Guangxi Province of China. Broadband ultrasound attenuation (BUA) was used to evaluate BMD. Grip dynamometer to assess muscle strength. Height, weight and muscle mass of each part were measured by body composition measuring instrument. Body mass index (BMI), skeletal muscle mass index (SMI) and limb skeletal muscle mass (SM) were calculated according to the measurement results. Results: BUA, grip strength and SMI in postmenopausal women of Zhuang nationality showed a decreasing trend with age (p p p r = 0.305, p Conclusion: With the increase of age, the decline rate of muscle strength of postmenopausal Zhuang women in Guangxi is slower than that of BMD and muscle mass. SM can better reflect the BMD level of the body than SMI, and the LSM is the main influencing factor of BMD.展开更多
Purpose: Bone marrow and muscle adiposity have been considered to correlate with osteoporosis and Sarcopenia. Proton Density Fat Fraction (PDFF) can be measured by Magnetic Resonance Imaging (MRI). The purpose of the ...Purpose: Bone marrow and muscle adiposity have been considered to correlate with osteoporosis and Sarcopenia. Proton Density Fat Fraction (PDFF) can be measured by Magnetic Resonance Imaging (MRI). The purpose of the present study was to measure PDFF in the lumbar spine, paraspinal muscle and subcutaneous fat tissue. Methods: Participants were comprised of 30 patients (18 males, 12 females;age range, 14 - 87 years) who underwent MRI due to low back symptoms. PDFFs for the body of the fourth lumbar vertebra (L4), paraspinal muscle, and subcutaneous fat were measured. Results: PDFFs of the vertebral body and subcutaneous fat were significantly higher than that of paraspinal muscle (p < 0.001). PDFF was significantly higher for subcutaneous fat than for the vertebral body (p < 0.001). Although no significant differences in PDFF of the vertebral body, paraspinal muscle, and subcutaneous fat between females and males, PDFFs of the vertebral body and paraspinal muscle were significantly higher in older subjects (>63 years) than in younger subjects (Conclusions: PDFF of the vertebral body was significantly higher than that of paraspinal muscle. PDFFs of the vertebral body and paraspinal muscle were significantly lower in younger subjects than in older subjects.展开更多
In order to investigate the origin of neointimal smooth muscle cells in transplant arterio- sclerosis in rat aortic allograft, sex-mismatched bone marrow transplantation was performed from male Wistar rats to female W...In order to investigate the origin of neointimal smooth muscle cells in transplant arterio- sclerosis in rat aortic allograft, sex-mismatched bone marrow transplantation was performed from male Wistar rats to female Wistar rats. Four weeks after transplantation, the aortic transplant model was established by means of micro-surgery in rats. The recipients were divided into 4 groups: female Wistar-female Wistar aortic isografts, female SD-female Wistar aortic allografts, male SD-male Wis- tar aortic allografts, female SD-chimera Wistar aortic allografts. Eight weeks after transplantation, aortic grafts were removed at autopsy and processed for histological evaluation and immunohisto- chemistry. The results indicated that excessive accumulation of α-SMA-positive smooth muscle cells resulted in significant neointima formation and vascular lumen stricture in rat aortic allografts. Neointima assay revealed that the neointimal area and NIA/MA ratio of transplanted artery were sig- nificantly increased in all of aortic allograft groups as compared with those in aortic isograft group (P<0.01). Neointimal smooth muscle cells were harvested from cryostat sections of aortic allograft by microdissection method. The Sry gene-specific PCR was performed, and the result showed that a dis- tinct DNA band of 225 bp emerged in the male-male aortic allograft group and chimera aortic al- lograft group respectively, but not in the female-female aortic allograft group. It was suggested that recipient bone-marrow cells, as the origin of neointimal smooth muscle cells, contributed to the pathological neointimal hyperplasia of aortic allograft and transplant arteriosclerosis.展开更多
目的浅析椎动脉型颈椎病(cervical spondylotic vertebral arteriopathy,CSA)患者实施基于“筋骨平衡”理论针刺推拿疗法对其中医症状积分、颈性眩晕症状与功能评估量表(cervical vertigo symptom and function assessment scale,ESCV)...目的浅析椎动脉型颈椎病(cervical spondylotic vertebral arteriopathy,CSA)患者实施基于“筋骨平衡”理论针刺推拿疗法对其中医症状积分、颈性眩晕症状与功能评估量表(cervical vertigo symptom and function assessment scale,ESCV)评分、颈椎曲度及脑血流参数的影响。方法选择该院2019年12月—2021年12月就诊的CSA患者86例实施研究,随机数字表法分作观察组与对照组,各43例;其中对照组脱落2例,剔除1例,共完成40例;观察组脱落1例,剔除1例,共完成41例;对照组予以常规牵引疗法,观察组予以基于“筋骨平衡”理论针刺推拿疗法;比较两组治疗前与治疗后中医症状积分、ESCV评分、颈椎曲度及脑血流参数的变化,并对比两组疗效。结果治疗后,观察组中医症状积分皆显著低于对照组(P<0.05)。治疗后,观察组ESCV评分和颈椎曲度皆明显高于对照组(P<0.05)。治疗后,观察组椎底动脉及左、右椎动脉的Vs与Vd水平均显著高于对照组(P<0.05)。观察组总有效率显著高于对照组[95.12%(39/41)vs 80.00%(32/40),P<0.05]。结论基于“筋骨平衡”理论针刺推拿疗法对CSA患者疗效显著,有效改善中医症状积分、ESCV评分以及颈椎曲度,增强脑血流参数。展开更多
基金Supported by Social Development Projects of Nantong,No.MS22021008 and No.QNZ2022005.
文摘BACKGROUND Type 2 diabetes mellitus(T2DM),a fast-growing issue in public health,is one of the most common chronic metabolic disorders in older individuals.Osteoporosis and sarcopenia are highly prevalent in T2DM patients and may result in fractures and disabilities.In people with T2DM,the association between nutrition,sarcopenia,and osteoporosis has rarely been explored.AIM To evaluate the connections among nutrition,bone mineral density(BMD)and body composition in patients with T2DM.METHODS We enrolled 689 patients with T2DM for this cross-sectional study.All patients underwent dual energy X-ray absorptiometry(DXA)examination and were categorized according to baseline Geriatric Nutritional Risk Index(GNRI)values calculated from serum albumin levels and body weight.The GNRI was used to evaluate nutritional status,and DXA was used to investigate BMD and body composition.Multivariate forward linear regression analysis was used to identify the factors associated with BMD and skeletal muscle mass index.RESULTS Of the total patients,394 were men and 295 were women.Compared with patients in tertile 1,those in tertile 3 who had a high GNRI tended to be younger and had lower HbA1c,higher BMD at all bone sites,and higher appendicular skeletal muscle index(ASMI).These important trends persisted even when the patients were divided into younger and older subgroups.The GNRI was positively related to ASMI(men:r=0.644,P<0.001;women:r=0.649,P<0.001),total body fat(men:r=0.453,P<0.001;women:r=0.557,P<0.001),BMD at all bone sites,lumbar spine(L1-L4)BMD(men:r=0.110,P=0.029;women:r=0.256,P<0.001),FN-BMD(men:r=0.293,P<0.001;women:r=0.273,P<0.001),and hip BMD(men:r=0.358,P<0.001;women:r=0.377,P<0.001).After adjustment for other clinical parameters,the GNRI was still significantly associated with BMD at the lumbar spine and femoral neck.Additionally,a low lean mass index and higherβ-collagen special sequence were associated with low BMD at all bone sites.Age was negatively correlated with ASMI,whereas weight was positively correlated with ASMI.CONCLUSION Poor nutrition,as indicated by a low GNRI,was associated with low levels of ASMI and BMD at all bone sites in T2DM patients.Using the GNRI to evaluate nutritional status and using DXA to investigate body composition in patients with T2DM is of value in assessing bone health and physical performance.
文摘The estimates of global incidence and prevalence of non-alcoholic fatty liver disease(NAFLD) are worrisome, due to the parallel burden of obesity and its metabolic complications. Indeed, excess adiposity and insulin resistance represent two of the major risk factors for NAFLD; interestingly, in the last years a growing body of evidence tended to support a novel mechanistic perspective, in which the liver is at the center of a complex interplay involving organs and systems, other than adipose tissue and glucose homeostasis. Bone and the skeletal muscle are fat- free tissues which appeared to be independently associated with NAFLD in several cross-sectional studies. The deterioration of bone mineral density and lean body mass, leading to osteoporosis and sarcopenia, respectively, are age-related processes. The prevalence of NAFLD also increases with age. Beyond physiological aging, the three conditions share some common underlying mechanisms, and their elucidations could be of paramount importance to design more effective treatment strategies for the management of NAFLD. In this review, we provide an overview on epidemiological data as well as on potential contributors to the connections of NAFLD with bone and skeletal muscle.
基金partial support from P50 GM60338 from the National Institutes of Health and several grants from Shriners Hospitals for Children
文摘Severe burn injury triggers the body's nonspecific adaptive responses to acute insult, including the systemic inflammatory and stress responses, as well as the sympathetic response to immobilization. These responses trigger inflammatory bone resorption followed by glucocorticoid-induced apoptosis of osteoblasts and probably osteocytes. Because these patients are catabolic, they suffer concomitant muscle wasting and negative nitrogen balance. The use of anabolic agents such as recombinant human growth hormone and oxandrolone results in improved bone mineral content and muscle strength after approximately I year. Use of bisphosphonates within the first 10 days of a severe burn completely blocks the resorptive bone loss and has the added advantage of appearing to preserve muscle protein from excessive breakdown. The mechanism for the protective effect on muscle is not currently known. However, if the effect of bisphosphonates on muscle can be confirmed, it raises the possibility that bone communicates with muscle.
基金supported by NIAMS,of the National Institutes of Health,under award numbers R01AR062074 (to DJD) and R01AR060636 (to S-JL)the Harry Headley Charitable and Research Foundation,Punta Gorda,FL(to ELG-L)
文摘Osteogenesis imperfecta(OI) comprises a group of heritable connective tissue disorders generally defined by recurrent fractures, low bone mass, short stature and skeletal fragility. Beyond the skeletal complications of OI,many patients also report intolerance to physical activity, fatigue and muscle weakness. Indeed, recent studies have demonstrated that skeletal muscle is also negatively affected by OI, both directly and indirectly. Given the well-established interdependence of bone and skeletal muscle in both physiology and pathophysiology and the observations of skeletal muscle pathology in patients with OI, we investigated the therapeutic potential of simultaneous anabolic targeting of both bone and skeletal muscle using a soluble activin receptor 2B(ACVR2B) in a mouse model of type Ⅲ OI(oim). Treatment of 12-week-old oim mice with ACVR2 B for 4 weeks resulted in significant increases in both bone and muscle that were similar to those observed in healthy,wild-type littermates. This proof of concept study provides encouraging evidence for a holistic approach to treating the deleterious consequences of OI in the musculoskeletal system.
文摘Autophagy in eukaryotic cells is a constitutive process and functions as a homeostatic mechanism; it is up-regulated in response to specific stress stimuli such as starvation, hypoxia and as oxidative stress. In addition to playing a crucial role in adaptive responses to different stimuli, autophagy is also required for intracellular quality control. This second aspect is important to prevent the activation of pathological processes. Autophagy also plays a central role in cellular development and differentiation because it is involved in the regulation of energetic balance. This final aspect is critical for maintaining proper bone and muscle function as well as to prevent any pathological changes. Therefore, identifying new molecular targets involved in autophagy is critical to assure a good quality of life.
文摘Objective: To study the muscle injury, inflammatory response and bone metabolism after paravertebral muscle space and minimally invasive percutaneous approach surgeries for thoracolumbar fracture. Methods: A total of 92 patients with thoracolumbar fractures who were treated in Baoji Central Hospital between May 2015 and January 2017 were selected and randomly divided into paravertebral group and minimally invasive percutaneous group who accepted pedicle screw fixation under different approaches. Before operation as well as 1 d and 3 d after operation, serum was collected to determine the contents of creatase, inflammatory reaction molecules and bone metabolism indexes. Results: Serum Myo, CK, LDH, TNF-α, MCP-1, HMGB-1, CRP, IL-1β, OC, OPG, PICP and PINP levels of both groups 1 d and 3 d after operation were significantly higher than those before operation while TRACP5b, RANKL, CTX and NTX levels were significantly lower than those before operation, and serum Myo, CK, LDH, TNF-α, MCP-1, HMGB-1, CRP, IL-1β, TRACP5b, RANKL, CTX and NTX levels of paravertebral group 1 d and 3 d after operation were significantly lower than those of minimally invasive percutaneous group while OC, OPG, PICP and PINP levels were significantly higher than those of minimally invasive percutaneous group. Conclusion:Paravertebral muscle space surgery for thoracolumbar fracture is more effective than minimally invasive percutaneous surgery in reducing muscle injury and inflammatory response, and improving bone metabolism.
文摘Objective: To explore the correlation between muscle strength, muscle mass and bone mineral density (BMD) in Zhuang female population, body composition analysis and grip strength, and to analyze the possible influencing factors of BMD. Methods: 182 postmenopausal women were selected from Guangxi Province of China. Broadband ultrasound attenuation (BUA) was used to evaluate BMD. Grip dynamometer to assess muscle strength. Height, weight and muscle mass of each part were measured by body composition measuring instrument. Body mass index (BMI), skeletal muscle mass index (SMI) and limb skeletal muscle mass (SM) were calculated according to the measurement results. Results: BUA, grip strength and SMI in postmenopausal women of Zhuang nationality showed a decreasing trend with age (p p p r = 0.305, p Conclusion: With the increase of age, the decline rate of muscle strength of postmenopausal Zhuang women in Guangxi is slower than that of BMD and muscle mass. SM can better reflect the BMD level of the body than SMI, and the LSM is the main influencing factor of BMD.
文摘Purpose: Bone marrow and muscle adiposity have been considered to correlate with osteoporosis and Sarcopenia. Proton Density Fat Fraction (PDFF) can be measured by Magnetic Resonance Imaging (MRI). The purpose of the present study was to measure PDFF in the lumbar spine, paraspinal muscle and subcutaneous fat tissue. Methods: Participants were comprised of 30 patients (18 males, 12 females;age range, 14 - 87 years) who underwent MRI due to low back symptoms. PDFFs for the body of the fourth lumbar vertebra (L4), paraspinal muscle, and subcutaneous fat were measured. Results: PDFFs of the vertebral body and subcutaneous fat were significantly higher than that of paraspinal muscle (p < 0.001). PDFF was significantly higher for subcutaneous fat than for the vertebral body (p < 0.001). Although no significant differences in PDFF of the vertebral body, paraspinal muscle, and subcutaneous fat between females and males, PDFFs of the vertebral body and paraspinal muscle were significantly higher in older subjects (>63 years) than in younger subjects (Conclusions: PDFF of the vertebral body was significantly higher than that of paraspinal muscle. PDFFs of the vertebral body and paraspinal muscle were significantly lower in younger subjects than in older subjects.
基金grants from the National Natural Sciences Foundation of China (No. 30271242, 30371396)
文摘In order to investigate the origin of neointimal smooth muscle cells in transplant arterio- sclerosis in rat aortic allograft, sex-mismatched bone marrow transplantation was performed from male Wistar rats to female Wistar rats. Four weeks after transplantation, the aortic transplant model was established by means of micro-surgery in rats. The recipients were divided into 4 groups: female Wistar-female Wistar aortic isografts, female SD-female Wistar aortic allografts, male SD-male Wis- tar aortic allografts, female SD-chimera Wistar aortic allografts. Eight weeks after transplantation, aortic grafts were removed at autopsy and processed for histological evaluation and immunohisto- chemistry. The results indicated that excessive accumulation of α-SMA-positive smooth muscle cells resulted in significant neointima formation and vascular lumen stricture in rat aortic allografts. Neointima assay revealed that the neointimal area and NIA/MA ratio of transplanted artery were sig- nificantly increased in all of aortic allograft groups as compared with those in aortic isograft group (P<0.01). Neointimal smooth muscle cells were harvested from cryostat sections of aortic allograft by microdissection method. The Sry gene-specific PCR was performed, and the result showed that a dis- tinct DNA band of 225 bp emerged in the male-male aortic allograft group and chimera aortic al- lograft group respectively, but not in the female-female aortic allograft group. It was suggested that recipient bone-marrow cells, as the origin of neointimal smooth muscle cells, contributed to the pathological neointimal hyperplasia of aortic allograft and transplant arteriosclerosis.