AIM: To detect the expression of PTEN encoding productin normal mucosa, intestinal metaplasia (IM), dysplasia andcarcinoma of the stomach, and to investigate its clinicalimplication in tumorigenesis and progression of...AIM: To detect the expression of PTEN encoding productin normal mucosa, intestinal metaplasia (IM), dysplasia andcarcinoma of the stomach, and to investigate its clinicalimplication in tumorigenesis and progression of gastriccarcinoma.METHODS: Formalin-fixed paraffin embedded specimens from184 cases of gastric carcinoma, their adjacent normal mucosa,IM and dysplasia were evaluated for PTEN protein expressionby SABC immunohistochemistry. PTEN expression wascompared with tumor stage, lymph node metastasis, Lauren'sand WHO's histological classification of gastric carcinoma.Expression of VEGF was also detected in 60 cases of gastriccarcinoma and its correlation with PTEN was concerned.RESULTS: The positive rates of PTEN protein were 100 %(102/102), 98.5 %(65/66), 66.7 % (4/6) and 47.8 %(88/184)in normal mucosa, IM, dysplasia and carcinoma of the stomach,respectively. The positive rates in dysplasia and carcinomawere lower than in normal mucosa and IM (P<0.01).Advanced gastric cancers expressed less frequent PTEN thanearly gastric cancer (42.9 % v567.6 %, P<0.01). The positiverate of PTEN protein was lower in gastric cancer with thanwithout lymph node metastasis (40.3 % v563.3 %, P<0.01).PTEN was less expressed in diffuse-type than in intestinal-type gastric cancer (41.5 % v557.8 %,P<0.05). Signet ringcell carcinoma showed the expression of PTEN at the lowestlevel (25.0 %, 7/28); less than well and moderatelydifferentiated ones (P<0.01). Expression of PTEN was notcorrelated with expression of VEGF (P>0.05).CONCLUSION: Loss or reduced expression of PTEN proteinoccures commonly in tumorigenesis and progression of gastriccarcinoma. It is suggested that PTEN can be an objective markerfor pathologically biological behaviors of gastric carcinoma.展开更多
Background: We estimated the incidence and mortality of nasopharyngeal carcinoma(NPC) in China in 2010 according to the data of 145 domestic population-based cancer registries in 2014, and no such reports since then.H...Background: We estimated the incidence and mortality of nasopharyngeal carcinoma(NPC) in China in 2010 according to the data of 145 domestic population-based cancer registries in 2014, and no such reports since then.Hence, to further and better understand its epidemiology in China and to provide more precise scientific information for its control and prevention in China, we analyzed the NPC incidence and mortality of 255 domestic populationbased cancer registries, and estimated the national rates in 2013 again.Methods: NPC incidence and mortality data of 255 domestic cancer registries in 2013, accepted by the 2016 National Cancer Registry Annual Report, were collected and collated, and the indices of NPC such as the numbers of new cases and deaths, crude rates, age-standardized rates, and truncated rates of incidence and mortality were calculated and analyzed. The incidence and mortality in China and its constituent areas were estimated according to the national population in 2013.Results: An estimated 42,100 new cases and 21,320 deaths were attributed to NPC in China in 2013, accounting for1.14% of all new cancer cases and 0.96% of all cancer-related deaths that year in China. Crude incidence and mortality of NPC were 3.09/100,000 and 1.57/100,000, respectively. World age-standardized incidence and mortality were2.17/100,000 and 1.08/100,000, respectively. The incidence and mortality of males were obviously higher than those of females and slightly higher in urban areas than in rural areas. Among seven Chinese administrative regions, NPC incidence and mortality were obviously higher in South China than in other regions and lowest in North China. Top3 incidence and mortality provinces and registering areas all located in South China. The age-specific incidence and mortality rose quickly from age 25-29 and 35 to 39 years, respectively, peaked at different ages and varied by location.Conclusions: These results demonstrated that NPC incidence and mortality in China in 2013 were also at high levels worldwide, which s展开更多
Nasopharyngeal carcinoma(NPC) is common in South China. Although regional epidemiological data on NPC in China is available, national epidemiological data have been unavailable up to now. The goal of this study was to...Nasopharyngeal carcinoma(NPC) is common in South China. Although regional epidemiological data on NPC in China is available, national epidemiological data have been unavailable up to now. The goal of this study was to analyze the NPC incidence and mortality data in some domestic cancer registries, estimate these rates in China in 2010, and provide scientific information that can be harnessed for NPC control and prevention. To accomplish this goal, NPC incidence and mortality data for 2010 were collected from 145 Chinese cancer registries from which data were included in the 2013 National Cancer Registry Annual Report. Such indices as its incident and death numbers, crude rates, age-standardized rates and truncated rates were calculated and analyzed. The incidence and mortality in China and constituent areas were estimated according to the national population in 2010. An estimated 41,503 new cases and 20,058 deaths were attributed to NPC in China in 2010, accounting for 1.34% of all new cancer cases and 1.03% of all cancer-related deaths that year in China. Crude incidence and mortality were 3.16/100,000 and 1.53/100,000, respectively. World age-standardized incidence and mortality were 2.44/100,000 and 1.18/100,000, respectively. Incidence and mortality were higher among males than among females and slightly higher in urban areas than in rural areas. Among seven Chinese administrative regions, NPC incidence and mortality were obviously higher in South China than in other regions and lowest in North China. The male and female age-specific incidence and mortality both rose quickly from age 25-29 years, but peaked at different ages and varied by location. These results demonstrated that NPC incidence and mortality in China especially in South China were at high levels in the world, and suggested that control and prevention efforts should be enhanced.展开更多
Liver cancer is a common malignant tumor in China and a major health concern. We aimed to estimate the liver cancer incidence and mortality in China in 2010 using liver cancer data from some Chinese cancer registries ...Liver cancer is a common malignant tumor in China and a major health concern. We aimed to estimate the liver cancer incidence and mortality in China in 2010 using liver cancer data from some Chinese cancer registries and provide reference for liver cancer prevention and treatment. We collected and evaluated the incidence and mortality data of liver cancer in 2010 from 145 cancer registries, which were included in the 2013 Chinese Cancer Registry Annual Report, calculated crude, standardized, and truncated incidences and mortalities, and estimated new liver cancer cases and deaths from liver cancer throughout China and in different regions in 2010 from Chinese practical population. The estimates of new liver cancer cases and deaths were 358,840 and 312,432, respectively, in China in 2010. The crude incidence, age-standardized rate by Chinese standard population(ASR China), and age-standardized rate by world standard population(ASR world) were 27.29/100,000, 21.35/100,000, and 20.87/100,000, respectively; the crude, ASR China, and ASR world mortalities were 23.76/100,000, 18.43/100,000, and 18.04/100,000, respectively. The incidence and mortality were the highest in western regions, higher in rural areas than in urban areas, and higher in males than in females. The age-specific incidence and mortality of liver cancer showed a rapid increase from age 30 and peaked at age 80-84 or 85+. Our results indicated that the 2010 incidence and mortality of liver cancer in China, especially in undeveloped rural areas and western regions, were among high levels worldwide. The strategy for liver cancer prevention and treatment should be strengthened.展开更多
功能性运动障碍(functional movement disorders,FMDs)是一类常见的分离/转换性障碍,已成为运动障碍疾病的重要分支,但多数临床医师对此疾病的认知不足。基于此,本文综述了FMDs的临床表现、辅助检查、诊断及鉴别诊断、治疗及预后,以期...功能性运动障碍(functional movement disorders,FMDs)是一类常见的分离/转换性障碍,已成为运动障碍疾病的重要分支,但多数临床医师对此疾病的认知不足。基于此,本文综述了FMDs的临床表现、辅助检查、诊断及鉴别诊断、治疗及预后,以期提高临床医生对FMDs的认识,提高临床诊断正确率,为准确治疗提供可能性。展开更多
Identification of a suitable nonhuman primate(NHP)model of COVID-19 remains challenging.Here,we characterized severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection in three NHP species:Old World monkeys...Identification of a suitable nonhuman primate(NHP)model of COVID-19 remains challenging.Here,we characterized severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection in three NHP species:Old World monkeys Macaca mulatta(M.mulatta)and Macaca fascicularis(M.fascicularis)and New World monkey Callithrix jacchus(C.jacchus).Infected M.mulatta and M.fascicularis showed abnormal chest radiographs,an increased body temperature and a decreased body weight.Viral genomes were detected in swab and blood samples from all animals.Viral load was detected in the pulmonary tissues of M.mulatta and M.fascicularis but not C.jacchus.Furthermore,among the three animal species,M.mulatta showed the strongest response to SARS-CoV-2,including increased inflammatory cytokine expression and pathological changes in the pulmonary tissues.Collectively,these data revealed the different susceptibilities of Old World and New World monkeys to SARS-CoV-2 and identified M.mulatta as the most suitable for modeling COVID-19.展开更多
The capability of electrocatalytic reducti on of carbon dioxide(CO2)using nitrogen(N)-doped carb on strongly depe nds on the N-dopi ng level and their types.In this work,we developed a strategy to generate mesoporous ...The capability of electrocatalytic reducti on of carbon dioxide(CO2)using nitrogen(N)-doped carb on strongly depe nds on the N-dopi ng level and their types.In this work,we developed a strategy to generate mesoporous N-doped carb on frameworks with tun able configurati ons and contents of N dopants,by using a secondary doping process via the treatment of N,N-dimethylformamide(DMF)solvent.The obtained mesoporous N-doped carbon(denoted as MNC-D)served as an efficient electrocatalyst for electroreduction of CO2 to CO.A high Faradaic efficiency of^92%and a partial current density for CO of-6.8 mA·cm^-2 were achieved at a potential of-0.58 V vs.RHE.Electrochemical analyses further revealed that the active sites within the N-doped carb on catalysts were the pyridinic N and defects gen erated by the DMF treatme nt,which enhan ced the activati on and adsorpti on CO2 molecules.Our study suggests a new approach to develop efficie nt carb on-based catalysts for potential scalable CO2 reduction reaction(CO2RR)to fuels and chemicals.展开更多
Osteoporosis is a common disease that affects patient quality of life, especially among the elderly population. Although inflammation contributes significantly to osteoporosis, the underlying mechanism is unclear. In ...Osteoporosis is a common disease that affects patient quality of life, especially among the elderly population. Although inflammation contributes significantly to osteoporosis, the underlying mechanism is unclear. In this study, we found that tumor necrosis factor (TNF)-a, an inflammatory environment mimic, inhibits osteogenesis of bone mesenchymal stem ceils (BMSCs), induces miR-146a and decreases Smad4. Moreover, overexpression of miR-146a inhibited the osteogenic ability of BMSCs, whereas blocking miR-146a partially rescued the osteogenesis deficiency under TNF-a treatment. Molecularly, miR-146a decreased Smad4 expression at the protein level by binding to an element located in the Smad4 3'-untranslated region, and restoration of Smad4 reversed the inhibitory effects of miR-146a on osteogenesis. Together, our results showed that the inflammatory environment mimic TNF-ol inhibits osteogenesis via upregulation of miR-146a and subsequent downregulation of Smad4, thus suggesting that therapeutic manipulation of miR-146a maybe a potential strategy to improve osteogenesis in the context of osteoporosis.展开更多
Macrophages (Mφ) are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. We have recently found that tumors can alter the normal developmental process of Mφ to trig...Macrophages (Mφ) are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. We have recently found that tumors can alter the normal developmental process of Mφ to trigger transient activation of monocytes, but the underlying regulatory mechanisms are incompletely understood. Here, we showed that the protein expression of transcription factor C/EBPβ was markedly elevated in tumor-associated Mφ both in vitro and human tumors in situ. The expression of C/EBPβ protein correlated with cytokine production in tumor-activated monocytes. Moreover, we found that C/EBPβ expression was regulated at the post-transcriptional level and correlated with sustained reduction of microRNA-155 (miR-155) in tumor-activated monocytes. Bioinformatic analysis revealed that C/EBPβ is a potential target of miR-155 and luciferase assay confirmed that C/EBPβ translation is suppressed by miR-155 through interaction with the 3'UTR of C/EBPβ mRNA. Further analysis showed that induction of miR-155 suppressed C/EBPβ protein expression as well as cytokine production in tumor-activated monocytes, an effect which could be mimicked by silencing of C/EBPβ. These results indicate that tumor environment causes a sustained reduction of miR-155 in monocytes/Mφ, which in turn regulates the functional activities of monocytes/Mφ by releasing the translational inhibition of transcription factor C/EBPβ.展开更多
基金the National Natural Science Foundation of China No.30070845"Outstanding Research Training Program",Ministry of Education No.[1999] 2Science Foundation of Liaoning Education Bureau No.20121031
文摘AIM: To detect the expression of PTEN encoding productin normal mucosa, intestinal metaplasia (IM), dysplasia andcarcinoma of the stomach, and to investigate its clinicalimplication in tumorigenesis and progression of gastriccarcinoma.METHODS: Formalin-fixed paraffin embedded specimens from184 cases of gastric carcinoma, their adjacent normal mucosa,IM and dysplasia were evaluated for PTEN protein expressionby SABC immunohistochemistry. PTEN expression wascompared with tumor stage, lymph node metastasis, Lauren'sand WHO's histological classification of gastric carcinoma.Expression of VEGF was also detected in 60 cases of gastriccarcinoma and its correlation with PTEN was concerned.RESULTS: The positive rates of PTEN protein were 100 %(102/102), 98.5 %(65/66), 66.7 % (4/6) and 47.8 %(88/184)in normal mucosa, IM, dysplasia and carcinoma of the stomach,respectively. The positive rates in dysplasia and carcinomawere lower than in normal mucosa and IM (P<0.01).Advanced gastric cancers expressed less frequent PTEN thanearly gastric cancer (42.9 % v567.6 %, P<0.01). The positiverate of PTEN protein was lower in gastric cancer with thanwithout lymph node metastasis (40.3 % v563.3 %, P<0.01).PTEN was less expressed in diffuse-type than in intestinal-type gastric cancer (41.5 % v557.8 %,P<0.05). Signet ringcell carcinoma showed the expression of PTEN at the lowestlevel (25.0 %, 7/28); less than well and moderatelydifferentiated ones (P<0.01). Expression of PTEN was notcorrelated with expression of VEGF (P>0.05).CONCLUSION: Loss or reduced expression of PTEN proteinoccures commonly in tumorigenesis and progression of gastriccarcinoma. It is suggested that PTEN can be an objective markerfor pathologically biological behaviors of gastric carcinoma.
文摘Background: We estimated the incidence and mortality of nasopharyngeal carcinoma(NPC) in China in 2010 according to the data of 145 domestic population-based cancer registries in 2014, and no such reports since then.Hence, to further and better understand its epidemiology in China and to provide more precise scientific information for its control and prevention in China, we analyzed the NPC incidence and mortality of 255 domestic populationbased cancer registries, and estimated the national rates in 2013 again.Methods: NPC incidence and mortality data of 255 domestic cancer registries in 2013, accepted by the 2016 National Cancer Registry Annual Report, were collected and collated, and the indices of NPC such as the numbers of new cases and deaths, crude rates, age-standardized rates, and truncated rates of incidence and mortality were calculated and analyzed. The incidence and mortality in China and its constituent areas were estimated according to the national population in 2013.Results: An estimated 42,100 new cases and 21,320 deaths were attributed to NPC in China in 2013, accounting for1.14% of all new cancer cases and 0.96% of all cancer-related deaths that year in China. Crude incidence and mortality of NPC were 3.09/100,000 and 1.57/100,000, respectively. World age-standardized incidence and mortality were2.17/100,000 and 1.08/100,000, respectively. The incidence and mortality of males were obviously higher than those of females and slightly higher in urban areas than in rural areas. Among seven Chinese administrative regions, NPC incidence and mortality were obviously higher in South China than in other regions and lowest in North China. Top3 incidence and mortality provinces and registering areas all located in South China. The age-specific incidence and mortality rose quickly from age 25-29 and 35 to 39 years, respectively, peaked at different ages and varied by location.Conclusions: These results demonstrated that NPC incidence and mortality in China in 2013 were also at high levels worldwide, which s
文摘Nasopharyngeal carcinoma(NPC) is common in South China. Although regional epidemiological data on NPC in China is available, national epidemiological data have been unavailable up to now. The goal of this study was to analyze the NPC incidence and mortality data in some domestic cancer registries, estimate these rates in China in 2010, and provide scientific information that can be harnessed for NPC control and prevention. To accomplish this goal, NPC incidence and mortality data for 2010 were collected from 145 Chinese cancer registries from which data were included in the 2013 National Cancer Registry Annual Report. Such indices as its incident and death numbers, crude rates, age-standardized rates and truncated rates were calculated and analyzed. The incidence and mortality in China and constituent areas were estimated according to the national population in 2010. An estimated 41,503 new cases and 20,058 deaths were attributed to NPC in China in 2010, accounting for 1.34% of all new cancer cases and 1.03% of all cancer-related deaths that year in China. Crude incidence and mortality were 3.16/100,000 and 1.53/100,000, respectively. World age-standardized incidence and mortality were 2.44/100,000 and 1.18/100,000, respectively. Incidence and mortality were higher among males than among females and slightly higher in urban areas than in rural areas. Among seven Chinese administrative regions, NPC incidence and mortality were obviously higher in South China than in other regions and lowest in North China. The male and female age-specific incidence and mortality both rose quickly from age 25-29 years, but peaked at different ages and varied by location. These results demonstrated that NPC incidence and mortality in China especially in South China were at high levels in the world, and suggested that control and prevention efforts should be enhanced.
基金supported by the Chinese National Twelveth Five-year Program for Science and Technology Development (No. 2012ZX10002010)
文摘Liver cancer is a common malignant tumor in China and a major health concern. We aimed to estimate the liver cancer incidence and mortality in China in 2010 using liver cancer data from some Chinese cancer registries and provide reference for liver cancer prevention and treatment. We collected and evaluated the incidence and mortality data of liver cancer in 2010 from 145 cancer registries, which were included in the 2013 Chinese Cancer Registry Annual Report, calculated crude, standardized, and truncated incidences and mortalities, and estimated new liver cancer cases and deaths from liver cancer throughout China and in different regions in 2010 from Chinese practical population. The estimates of new liver cancer cases and deaths were 358,840 and 312,432, respectively, in China in 2010. The crude incidence, age-standardized rate by Chinese standard population(ASR China), and age-standardized rate by world standard population(ASR world) were 27.29/100,000, 21.35/100,000, and 20.87/100,000, respectively; the crude, ASR China, and ASR world mortalities were 23.76/100,000, 18.43/100,000, and 18.04/100,000, respectively. The incidence and mortality were the highest in western regions, higher in rural areas than in urban areas, and higher in males than in females. The age-specific incidence and mortality of liver cancer showed a rapid increase from age 30 and peaked at age 80-84 or 85+. Our results indicated that the 2010 incidence and mortality of liver cancer in China, especially in undeveloped rural areas and western regions, were among high levels worldwide. The strategy for liver cancer prevention and treatment should be strengthened.
文摘功能性运动障碍(functional movement disorders,FMDs)是一类常见的分离/转换性障碍,已成为运动障碍疾病的重要分支,但多数临床医师对此疾病的认知不足。基于此,本文综述了FMDs的临床表现、辅助检查、诊断及鉴别诊断、治疗及预后,以期提高临床医生对FMDs的认识,提高临床诊断正确率,为准确治疗提供可能性。
基金supported by the National Research and Development Project of China(2020YFC0841100,2020YFC0846400,and 2020YFA0707600)CAMS Innovation Fund for Medical Sciences(2016-I2M-2-006 and 2020-I2M-CoV19-012)+1 种基金the Talents Project of Yunnan Province(2017HB068)the Special Funds for High-level Health and Family Planning Technical Personnel training of Yunnan Province(D-201653).
文摘Identification of a suitable nonhuman primate(NHP)model of COVID-19 remains challenging.Here,we characterized severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection in three NHP species:Old World monkeys Macaca mulatta(M.mulatta)and Macaca fascicularis(M.fascicularis)and New World monkey Callithrix jacchus(C.jacchus).Infected M.mulatta and M.fascicularis showed abnormal chest radiographs,an increased body temperature and a decreased body weight.Viral genomes were detected in swab and blood samples from all animals.Viral load was detected in the pulmonary tissues of M.mulatta and M.fascicularis but not C.jacchus.Furthermore,among the three animal species,M.mulatta showed the strongest response to SARS-CoV-2,including increased inflammatory cytokine expression and pathological changes in the pulmonary tissues.Collectively,these data revealed the different susceptibilities of Old World and New World monkeys to SARS-CoV-2 and identified M.mulatta as the most suitable for modeling COVID-19.
基金We thank the following funding agencies for supporting this work:the National Key Research and Development Program of China(Nos.2017YFA0206901 and 2018YFA0209401)the National Natural Science Foundation of China(No.21773036)+1 种基金the Science and Technology Commission of Shanghai Municipality(Nos.17JC1402000 and 19XD1420400)the Innovation Program of Shanghai Municipal Education Commission(No.2019-01-07-00-07-E00045).
文摘The capability of electrocatalytic reducti on of carbon dioxide(CO2)using nitrogen(N)-doped carb on strongly depe nds on the N-dopi ng level and their types.In this work,we developed a strategy to generate mesoporous N-doped carb on frameworks with tun able configurati ons and contents of N dopants,by using a secondary doping process via the treatment of N,N-dimethylformamide(DMF)solvent.The obtained mesoporous N-doped carbon(denoted as MNC-D)served as an efficient electrocatalyst for electroreduction of CO2 to CO.A high Faradaic efficiency of^92%and a partial current density for CO of-6.8 mA·cm^-2 were achieved at a potential of-0.58 V vs.RHE.Electrochemical analyses further revealed that the active sites within the N-doped carb on catalysts were the pyridinic N and defects gen erated by the DMF treatme nt,which enhan ced the activati on and adsorpti on CO2 molecules.Our study suggests a new approach to develop efficie nt carb on-based catalysts for potential scalable CO2 reduction reaction(CO2RR)to fuels and chemicals.
基金supported by the National Natural Science Foundation of China (81570803)Guangzhou Foundation for Science and Technology Planning Project, China (201704030083)+1 种基金Science and Technology Planning Project of Guangdong Province, China (2017A050501013)the Fundamental Research Funds for the Central Universities (17ykjc21)
文摘Osteoporosis is a common disease that affects patient quality of life, especially among the elderly population. Although inflammation contributes significantly to osteoporosis, the underlying mechanism is unclear. In this study, we found that tumor necrosis factor (TNF)-a, an inflammatory environment mimic, inhibits osteogenesis of bone mesenchymal stem ceils (BMSCs), induces miR-146a and decreases Smad4. Moreover, overexpression of miR-146a inhibited the osteogenic ability of BMSCs, whereas blocking miR-146a partially rescued the osteogenesis deficiency under TNF-a treatment. Molecularly, miR-146a decreased Smad4 expression at the protein level by binding to an element located in the Smad4 3'-untranslated region, and restoration of Smad4 reversed the inhibitory effects of miR-146a on osteogenesis. Together, our results showed that the inflammatory environment mimic TNF-ol inhibits osteogenesis via upregulation of miR-146a and subsequent downregulation of Smad4, thus suggesting that therapeutic manipulation of miR-146a maybe a potential strategy to improve osteogenesis in the context of osteoporosis.
文摘Macrophages (Mφ) are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. We have recently found that tumors can alter the normal developmental process of Mφ to trigger transient activation of monocytes, but the underlying regulatory mechanisms are incompletely understood. Here, we showed that the protein expression of transcription factor C/EBPβ was markedly elevated in tumor-associated Mφ both in vitro and human tumors in situ. The expression of C/EBPβ protein correlated with cytokine production in tumor-activated monocytes. Moreover, we found that C/EBPβ expression was regulated at the post-transcriptional level and correlated with sustained reduction of microRNA-155 (miR-155) in tumor-activated monocytes. Bioinformatic analysis revealed that C/EBPβ is a potential target of miR-155 and luciferase assay confirmed that C/EBPβ translation is suppressed by miR-155 through interaction with the 3'UTR of C/EBPβ mRNA. Further analysis showed that induction of miR-155 suppressed C/EBPβ protein expression as well as cytokine production in tumor-activated monocytes, an effect which could be mimicked by silencing of C/EBPβ. These results indicate that tumor environment causes a sustained reduction of miR-155 in monocytes/Mφ, which in turn regulates the functional activities of monocytes/Mφ by releasing the translational inhibition of transcription factor C/EBPβ.