Malignant tumors in farmers over 60 years old of the high-risk population in a town in Shanghai / 上海预防医学

Objective To screen for malignant tumors and high-risk factors in rural residents over 60 years old， so as to prevent and control the occurrence and development of tumors in the future. Methods The survey was conducted with reference to part of the questionnaire in the "Urban Cancer Early Diagnosis and Treatment Project and Evaluation of High-risk Populations". Clinical examinations included serum tumor marker detection， CT screening for lung cancer， occult blood （+） plus colonoscopy screening for colorectal cancer， and mammography screening. Individuals who were positive in the abovementioned clinical tests were defined as high-risk subjects. Results A total of 271 high-risk subjects （1.91%） were screened out of 14 161. Among the high-risk subjects， 71 cases of malignant tumors （26.19%） were found， with an incidence rate of 501.38 per 105. The top five tumors （63.38% of all diagnosed） were mainly concentrated in lung， upper digestive tract， blood system， urinary system， and rectum-colon. The proportion of malignant tumors detected by positive indicators was 61.54% of blood； 46.15% of carcinoembryonic antigen and carbohydrate antigen 125； 23.08% of alpha-fetoprotein； 16.66% of lung CT； and 3.09% of prostate PSA. The positive indicators in the high-risk subjects were mainly for the tumors in the prostate， lungs， liver， and CEA/CA125. The subjects with positive test indicators had lower average annual income in the last 5 years than the normal subject group （χ2=3.380， P=0.040）. The subjects with positive test indicators had higher proportion in family history of tumors than the normal group （χ2=2.596， P=0.046）. People in thehigh-risk group had a higher proportion than the normal group in suffering from hypertension， liver disease， gastrointestinal disease， respiratory system disease， and surgical treatment. Patients with high-risk tumors were found to have higher proportion than the normal group in showing pre-tumor clinical symptoms in the last 1 year. Study of the tumor-related risk factors found that the high-risk group had a higher proportion of high-fat/high-cholesterol diet， alcohol drinking， passive smoking， and personality depression. Conclusion High tumor risk factors have been identified in this population. It is necessary to strengthen the corresponding intervention and follow-up treatment of precancerous diseases in the future. We recommend the government to conduct tumor screening among high-risk groups to improve cost-effectiveness.

Trends in cerebrovascular disease mortality in Hangzhou City from 2006 to 2020 / 预防医学

Objective@#To investigate the trends in cerebrovascular disease mortality among residents in Hangzhou City from 2006 to 2020, so as to provide insights into the development of cerebrovascular disease control strategy. @*Methods@#The cerebrovascular disease mortality data were collected from residents in Hangzhou City through household registered death cards recorded in county- and city-level hospitals, township-level health centers and community health service centers in Hangzhou City and standardized by the national population census data in China in 2010. The changes in the crude and standardized mortality of cerebrovascular diseases were estimated using annual percent change (APC) in Hangzhou City from 2006 to 2020.@*Results@#A total of 109 891 deaths were reported due to cerebrovascular diseases among residents in Hangzhou City from 2006 to 2020, accounting for 17.72% of all deaths in Hangzhou City during the 15-year period, and the crude and standardized mortality rates were 105.83/105 and 65.15/105, which both appeared a tendency towards a decline (crude mortality: APC=-0.80%, t=-3.832, P=0.002; standardized mortality: APC=-4.69%, t=-24.084, P<0.001). The crude mortality of hemorrhagic stroke showed a tendency towards a decline among residents in Hangzhou City from 2006 to 2020 (APC=-7.97%, t=-30.704, P<0.001), and the crude mortality of both ischemic stroke (APC=5.76%, t=6.106, P<0.001) and sequelae of cerebrovascular disease (APC=13.20%, t=18.103, P<0.001) appeared a tendency towards a rise. The crude mortality of cerebrovascular diseases was significantly higher in men than in women (110.15/105 vs. 101.51/105; χ2=183.078, P<0.001), which both appeared a tendency towards a decline among residents in Hangzhou City from 2006 to 2020 (men: APC=-0.90%, t=-4.669, P<0.001; women: APC=-0.70%, t=-2.658, P=0.020). The crude mortality of cerebrovascular diseases was 101.95/105 and appeared a tendency towards a decline among urban residents in Hangzhou City from 2006 to 2020 (APC=-1.19%, t=-3.655, P=0.003), and was 112.46/105 and showed no remarkable changes among rural residents in Hangzhou City from 2006 to 2020 (t=-0.719, P=0.485). In addition, the crude mortality of cerebrovascular diseases appeared a tendency towards a rise with age among residents in Hangzhou City from 2006 to 2020 (χ2trend=502 711.682, P<0.001), and the crude mortality of cerebrovascular diseases appeared a tendency towards a decline among residents at ages of 35 years and above in Hangzhou City from 2006 to 2020 (P<0.05). @*Conclusions@#The cerebrovascular disease mortality appeared a tendency towards a decline among residents in Hangzhou City from 2006 to 2020, and a high mortality rate of cerebrovascular diseases was seen among rural residents, men and the elderly.

Blood homocysteine level in Shanghai rural residents with high risk of stroke / 上海预防医学

Objective:To determine the blood level of homocysteine （Hcy） and its influencing factors among Shanghai rural residents with high risk of stroke and to verify if hyperhomocysteinemia （HHcy） is a main biomarker of stroke. Methods:With a clustered random sampling method， questionnaire survey and physical examination were conducted among 4 073 rural residents， aged 55 years and above， in Luojing community， Shanghai， in 2018. A total of 470 residents were at high-risk for stroke based on screening of plasma Hcy and other blood indicators. Multivariate logistic regression method was performed for data analysis. Results:The overall level of Hcy was （18.92±6.37）μmol/L， with （20.40±5.89）μmol/L for men and （17.87±2.12）μmol/L for women （t=5.431，P<0.001）. HHcy was detected in 78.94%（371/470） of the participants， in which 85.77%（235/274） were men and 69.39%（136/196） were women （χ2=12.400，P=0.001）. In the high-risk male group， subjects with smoking history， overweight or obesity， exercise frequency <1 h/d， and hypertension has a higher detection rate of HHcy than those without smoking history， normal body mass index， exercise frequency ≥1 h/d and without hypertension （χ2=11.340，8.170，8.200， and 12.400， respectively， all P<0.01）. However， there was no significant difference in HHcy detection rate in different age groups and between the patients with or without diabetes， and dyslipidemia（χ2=3.120，2.311， and 0.984， respectively， all P>0.05）. In the high-risk women group， HHcy detection rate increased with age （χ2=13.874，P<0.01）， and it was higher in participants with overweight or obesity， exercise frequency < 1 h/d， hypertension， and dyslipidemia（χ2=10.278， 13.840， 14.100， and 12.330， respectively， all P<0.01）. Unconditional logistic regression analysis showed that the factors affecting HHcy in the high-risk population of stroke include being male， smoking， hypertension， overweight and obesity. Conclusion:Screening of high-risk populations for stroke should include blood level of Hcy.

Role of connexin 43 in odontoblastic differentiation and structural maintenance in pulp damage repair / 国际口腔科学杂志·英文版

Dental pulp can initiate its damage repair after an injury of the pulp-dentin complex by rearrangement of odontoblasts and formation of newly differentiated odontoblast-like cells. Connexin 43 (Cx43) is one of the gap junction proteins that participates in multiple tissue repair processes. However, the role of Cx43 in the repair of the dental pulp remains unclear. This study aimed to determine the function of Cx43 in the odontoblast arrangement patterns and odontoblastic differentiation. Human teeth for in vitro experiments were acquired, and a pulp injury model in Sprague-Dawley rats was used for in vivo analysis. The odontoblast arrangement pattern and the expression of Cx43 and dentin sialophosphoprotein (DSPP) were assessed. To investigate the function of Cx43 in odontoblastic differentiation, we overexpressed or inhibited Cx43. The results indicated that polarized odontoblasts were arranged along the pulp-dentin interface and had high levels of Cx43 expression in the healthy teeth; however, the odontoblast arrangement pattern was slightly changed concomitant to an increase in the Cx43 expression in the carious teeth. Regularly arranged odontoblast-like cells had high levels of the Cx43 expression during the formation of mature dentin, but the odontoblast-like cells were not regularly arranged beneath immature osteodentin in the pulp injury models. Subsequent in vitro experiments demonstrated that Cx43 is upregulated during odontoblastic differentiation of the dental pulp cells, and inhibition or overexpression of Cx43 influence the odontoblastic differentiation. Thus, Cx43 may be involved in the maintenance of odontoblast arrangement patterns, and influence the pulp repair outcomes by the regulation of odontoblastic differentiation.

The effects of altered BMP4 signaling in first branchial-arch-derived murine embryonic orofacial tissues / 国际口腔科学杂志·英文版

The first branchial arch (BA1), which is derived from cranial neural crest (CNC) cells, gives rise to various orofacial tissues. Cre mice are widely used for the determination of CNC and exploration of gene functions in orofacial development. However, there is a lack of Cre mice specifically marked BA1's cells. Pax2-Cre allele was previously generated and has been widely used in the field of inner ear development. Here, by compounding Pax2-Cre and R26R-mTmG mice, we found a specific expression pattern of Pax2

Evaluation of the effect of self-management model in diabetic patients in community under the guidance of general practitioners / 中华健康管理学杂志

Objective:To evaluate the effect of self-management model of community diabetes patients under the guidance of general practitioners.Methods:A total of 22 communities in Hangzhou were selected as intervention pilot from 2016 to 2017, and 10-15 diabetic patients were recruited to form a self-management team in each community. Relying on the construction of medical alliance and there sources of contracted service of family doctors, intervention teams were set up to conduct regular, personalized and continuous self-management interventions on patients. Intervention continued for 4-6 months. Before, after and 6 months after the intervention, the basic information of patients, knowledge of diabetes, faith in managing disease, self-management behavior, self-management ability of patients, and quality of life were collected through questionnaire survey, body mass index (BMI), 2-hour postprandial blood glucose and glycosylated hemoglobin were detected uniformly, and statistical analysis was conducted.Results:A total of 292 patients were involved in the self-management group, the average age was (66.38±7.90) years old, most of them were female (64.73%). Six months after the intervention, the awareness rate of basic knowledge, risk factors and prevention and treatment knowledge about diabetes in patients (68.64%, 78.75%, 63.41%) were significantly higher than those before the intervention (28.08%, 48.29%, 39.38%), the proportion of patients with reasonable diet, moderate exercise, active problem-solving, management plan making and action according to the plan increased (from 65.41%, 28.76%, 39.04%, 31.85%, 27.74% to 86.76%, 60.28%, 65.16%, 78.05%, 73.17%)(all P<0.05).In terms of time effect, the scores of physical functioning, role-physical, bodily pain, general health, vitality, social functioning, role-emotional and mental health in SF-36 after intervention and 6 months after intervention were all increased step by step (all P<0.05). Before the intervention, BMI, 2-hour postprandial blood glucose and glycosylated hemoglobin were (24.20±3.11) kg/m 2, (9.29±2.23) mmol/L, (6.81±1.01)% respectively. After the intervention, the three indexes all showed a downward trend, which were (22.89±2.98) kg/m 2, (8.81±1.61) mmol/L, (6.57±0.76)% respectively ( F=14.832, 6.828, 9.667; all P<0.05). Conclusion:Under the guidance of general practitioners, the self-management model of community diabetes patients can effectively improve the level of recognition, belief and behavior of patients, improve their self-management ability and quality of life, and improve BMI and blood glucose control level.

Epidemiological characteristics of mumps in mainland China from 2004 to 2018 and key population for prevention and control / 中国当代儿科杂志

OBJECTIVE@#To investigate the epidemiological characteristics of mumps in mainland China from 2004 to 2018, and to provide data for the key population for prevention and control of mumps.@*METHODS@#The epidemiological characteristics of mumps were analyzed with reference to the data of the cases of mumps reported in the National Scientific Data Sharing Platform for Population and Health and Disease Prevention and Control Bureau of National Health Commission of the People's Republic of China. Descriptive epidemiology was used to analyze the epidemiological characteristics of mumps.@*RESULTS@#A total of 4 272 368 cases of mumps were reported in China during 2004-2018, with an average annual reported incidence rate of 21.44/100 000. A single dose of mumps-containing vaccine was added to the national Expanded Program of Immunization in 2008, but the annual incidence rate ranged from 12.84/100 000 to 35.59/100 000. The second dose of measles, mumps and rubella combined attenuated live vaccine was included in the routine immunization in Beijing, Tianjin and Shanghai, and then the average incidence rate of mumps reported in these three regions dropped to about 10/100 000. From 2004 to 2016, the population aged 3-14 years accounted for 81.16% of all patients with mumps. The children aged 6 years had the highest incidence rate of mumps during 2004-2013.@*CONCLUSIONS@#A single dose of mumps-containing vaccine has no obvious effect on the incidence rate of mumps. Children aged 6 years have the highest incidence rate of mumps. A booster dose of mumps-containing vaccine should be given to preschool children.

Relationship between the after-school schedule and bone mineral density in middle-school students in Shanghai / 中国学校卫生

Objective@#To investigate the relationship between after-school schedule and bone mineral density in middle-school students in Shanghai, to provide a reference for taking targeted measures.@*Methods@#From November 2017 to April 2018，eighteen classes of six middle schools in Shanghai (from urban districts, urban-suburb combined districts and suburban districts, respectively) were selected based on cluster random sampling. In each school, 2-4 classes were further randomly chosen in the 6th grade. Questionnaires were completed by 518 students and their parents, as well as physical examination and bone mineral density assessment.@*Results@#The average time spent on after-school academic learning during weekends was 4.0 (IQR: 2.0, 6.0) h. The Z-score of bone density was 0.3 (IQR: -0.7, 1.0). The smooth curve fit model showed a non-linear relationship between after-school academic learning time during weekends and the Z-scores of bone mineral densities. A two-stage multiple linear regression analysis was further applied according to the fit results, and the results showed that when total afterschool academic learning time <4.5 hours during weekends, the learning time was inversely correlated with the bone density Z-scores (β=-0.11,P=0.01), and when the learning time ≥4.5 hours, there was no significant correlation between the learning time and bone density Z-scores (β=0.02, P=0.65). Parent and student questionnaires showed that there was a non-linear relationship between students’ daily time spent on outdoor sports-related activities and bone density Z-scores. When time spent on the outdoor activities ≥45 minutes per day, outdoor activities were positively correlated with bone density Z-scores (P<0.05). However, when the time spent on outdoor activities <45 minutes per day, there was no significant relationship between outdoor activities and bone density Z-scores (P>0.05).@*Conclusion@#Too much time on after-school academic learning during weekends or limited time on outdoor activities are both related to impaired bone mineral densities. Therefore, a reasonable after-school schedule for middle-school students is important for physical development, especially during weekends.

Changes and clinical significance of CD4 + CD25 + Foxp3 + regulatory T cell and relative cell factors in peripheral blood of steroid-resistant allergic rhinitis patients / 中国医师杂志

Objective To investigate the changes of CD4 + CD25 + Foxp3 + regulatory T cell (Treg) and relative cell factors in peripheral blood of steroid-resistant allergic rhinitis (AR) patients,and to study their functions in occurrence of steroid-resistant AR.Methods The CD4 + CD25 + Foxp3 + Treg cell in 30 cases general physical examination (The control group),30 cases steroid-sensitive AR patients (steroidsensitive group) and 30 cases steroid-resistant AR patients (steroid-resistant group) were detected by flow cytometry.The levels of serum interleukin 10 (IL-10) and transforming growth factor β1 (TGF-β1) in the three groups were detected by enzyme-linked immunoadsorbent assay (ELISA).Results Compare to the control group,proportion of CD4 + CD25 + Foxp3 + Treg cell and the levels of serum IL-10,TGF-β1 in steroid-sensitive group and steroid-resistant group were significantly reduced (P ＜ 0.01).The proportion of CD4 + CD25 + Foxp3 + Treg cell and the levels of serum IL-10,TGF-β1 in steroid-resistant group were decreased significantly than steroid-sensitive group (P ＜ 0.01).The proportion of CD4 + CD25 + Foxp3 + Treg cell were positively correlated with the serum IL-10,and TGF-β1 (P ＜ 0.01).Conclusions The reduced proportion of CD4 + CD25 + Foxp3 + Treg cell and lower serum IL-10,TGF-β1 play an important role in the occurrence of steroid-resistant AR patients.

Correlation between fractional exhaled nitric oxide and allergic rhinitis / 中国耳鼻咽喉头颈外科

OBJECTIVETo study the relationship between allergic rhinitis and fractional exhaled nitric oxide (FeNO).METHODS FeNO was measured in 66 allergic rhinitis patients before and after treatment. Visual analogue scale (VAS) score was used to describe the patient's symptoms. RESULTSFeNO and VAS score were significantly decreased after treatment (t=4.25,P=0.0000;t=3.34, P=0.0008), and the decrease proportion were (14.56±27.23)% and (8.68±53.17)% respectively. Correlation analysis showed FeNO and VAS score was significantly correlated before (r=0.43,P=0.0003) and after (r=0.45,P=0.0002) treatment. After treatment, the decreased proportion of FeNO and VAS score were significantly correlated (r=0.25,P=0.0437). CONCLUSIONFeNO can provide objective evidence for the treatment efficacy of allergic rhinitis.

Application of poisson distribution in early warning of HFMD / 预防医学

Objective To understand the value of poisson distribution in monitoring and early warning of hand-foot-mouth disease( HFMD). Methods Alert data based on infectious disease reports from 2009 to 2011 were used to establish a database. Moving average method was employed to set the number of expected cases each week. The probability of outbreak was calculated by poisson's distribution. If the probability was smaller than a standard,the signal of early warning was sent out. Epidemiological investigation was used to verify it. Results Using this method,44 abnormal signals of HFMD were probed,and 33 signals were verified. The positive rate,sensitivity,specificity and Youden 's index were 75. 00%,94. 29%,35. 29% and 29. 58% respectively. Compared to the moving average control chart and the automatic early-warning information system of infectious diseases,it was more efficient. Conclusions The poisson distribution with the moving average method can be used as an effective method in early warning system of HFMD.

Spatial autocorrelation analysis on hand-foot-mouth disease using Moran′s I index / 预防医学

Objective To explore and analyze the spatial distribution characteristics of hand - foot - mouth disease in Hangzhou city. Methods Based on the incidence of hand - foot - mouth disease, the whole and local spatial autocorrelation analysis was used to test the spatial heterogeneity by the software GeoDa095i. Results A total of 5534 cases of hand-foot-mouth disease were reported in Hangzhou city in 2011 with the reported incidence of 63. 61/100000. The wholel Moran’s I index was 0. 33(P<0. 05)indicating that the disease had high spatial cluster occurrence. Local spatial autocorrelation analysis showed that several districts including Gongshu,Jianggan,Xihu and Xiaoshan had high cluster occurrence(LISA=0. 4135,0. 4600,0. 1785 and 0. 3508 respectively,all P<0. 05)while Chun′an county and Tonglu county had the low cluster occurrence(LISA=0. 6923 and 0. 6762 respectively,both P<0. 05). Conclusion The spatial distribution of hand - foot - mouth disease in Hangzhou city is not random distribution and has aggregation phenomenon.

Expression of human angiotensin converting enzyme-C domain in Pichia pastoris / 生物工程学报

Angiotensin I-converting enzyme (ACE, EC3.4.15.1) plays an important role in regulating blood pressure. The C-domain of ACE has been identified as the main catalytic site of angiotensin I cleavage in vivo. The ACE gene fragment of the C-domain was amplified by PCR and cloned into the pPIC9K secretory expression plasmid. The recombinant plasmid was transformed into Pichia pastoris strain GS115. Positive clones were selected and subject to electroporation. Antibiotic G418 was used for the screening of multicopy inserts. After optimization of the expression system, the protein yield reached 0.5 g/L by flask-shaking culture fermentation, and enzyme activity reached 7.178 U/mL in the fermentation supernatant. The purity of the target protein obtained was 97% after Ni+ affinity chromatography. Enzyme inhibitory activity assay using Captopril showed that it is promising to use ACE-C domain as new generation of target for screening ACE inhibitor antihypertensive drugs.

Slow rise of intracellular Ca(2+) concentration in rat primary sensory neurons triggered by loureirin B / 生理学报

In the present study, the intracellular free calcium concentration ([Ca(2+)](i)) in acutely isolated rat dorsal root ganglia (DRG) neurons modulated by loureirin B, an active component of "dragon's blood" which is a kind of Chinese herbal medicine, was determined by the means of Fura-2 based microfluorimetry. It was found that loureirin B could evoke the elevation of [Ca(2+)](i) in a dose-dependent manner. However, the elevation of [Ca(2+)](i) evoked in the calcium free solution was much smaller than that in the standard external cell solution, suggesting that most change of [Ca(2+)](i) was generated by the influx of extracellular Ca(2+), not by the activities of intracellular organelles like Ca(2+) stores and mitochondria. In addition, the mixture of loureirin B and caffeine also induced [Ca(2+)](i) rise, but the peak of [Ca(2+)](i) rise induced by the mixture was significantly lower than that by caffeine alone, which means the triggering pathway and the targets of caffeine are probably involved in loureirin B-induced [Ca(2+)](i) rise. Moreover, compared to the transients induced by caffeine, KCl and capsaicin, the loureirin B-induced [Ca(2+)](i) rise is much slower and more stable. These results indicate that the capability of loureirin B of inducing the [Ca(2+)](i) rise is solid and unique.

Expression of hepatitis B virus e gene synthesized in Pichia pastoris and the immunological characterization of the product / 中华检验医学杂志

Objectives To synthesize hepatitis B virus e gene which was suitable for yeast protein expression and express hepatitis B virus e antigen (HBeAg) in Pichia pastoris.Methods Using synonymous codons preferred by yeast usage on protein expression to replace some native codons of wild-type HBV e gene,the synthetic e gene (syneAg-gene) was achieved by a recursive PCR (rPCR).The syneAg- gene was inserted into the yeast expression vector pPICZ?A.The recombinant plasmid was transformed into GS115 yeast by electroporation.The yeast transformant induced by methanol expressed the HBeAg.Results The restriction analysis and DNA sequencing confirmed that the syneAg-gene was inserted to yeast pPICZ?A in correct orientation.SDS-PAGE,immunoblot and ELISA indicated that the secreted form of HBeAg was expressed by the yeast transformant.The expression level of HBeAg in the strain containing syneAg-gene was 63 mg/L.The titer of the recombinant HBeAg in culture superuatant was 1 :81 920 and the maximum OD (optical density) value of absorbance was 2.8 by ELISA.No cross reactivity between Pichia pastoris-derived HBeAg and anti-HBc antibody was found.Conclusion The recombinant HBeAg with high degree of specificity and immunoreactivity is expressed efficiently in Pichia pastoris.

Detection of three kinds of cytokines in patients with viral myocarditis / 中国实用内科杂志

Objective To investigate the changes of TNF-?,IL-2 and IL-1? level in serum of patients with viral myocarditis and its clinical meaning.Methods The level of serum TNF-?,IL-2 and IL-1? in 45 cases of viral myocarditis was detected by ELISA,and the data were compared with control group.Results Serum TNF-?,IL-2 and IL-1? levels in viral myocarditis patients were significantly higher than control group and related to severity of the disease.Conclusion It suggests that immune dysfunction exists in patients with viral myocarditis.Higher level of TNF-?,IL-2 and IL-1? may be associated with development of the viral myocarditis,and it can be a new indicator for viral myocarditis.

Effect of different sex hormones on testicular descent and their relation to expression of INSL3 mRNA in rats / 中华内分泌代谢杂志

Objective To observe the effects of estradiol benzoate (E_2),human cberionic gonadotropin (hCG) and luteinizing hormone-releasing hormone (LHRH) on testicular descent and their relation to the expression of insulin-like factor-3 (INSL3) mBNA in rats.Methods At birth,40 SD male rats were assigned to 4 groups,and each group consisted of 10 rats.Each group received injection of one of the following drugs:normal saline(NS),E_3,hCG+E_2,and LHRH+E_2 for 29 clays after birth.Total RNA was extracted from the testis on day 30 after birth.INSL3 was synthesized and further amplified by RT-PCR.Results (1) Testicular descent rate on clay 30 after birth was 100% (20/20) in the group of NS,0% (0/20) in the group of E_2,85% (17/20) of hCG + E_2,and 70% (14/20) of LHBH + E_2.There was no significant difference between the last two groups ( P = 0.45).(2) Weight of testis differed significantly among the groups ( F = 56.67,P＜0.01 ),being lightest in the group of E_2.(3) E_2 could damage the germ cells of testis,however hCG and LHRH ameliorated this situation. (4) The expression of INSL3 mBNA was absent in the group of E_2,while the other groups showed normal expression.Conclusion E_2 inhibits testicular descent in rats,while hCG and LHRH promote the progress.The mechanism seems to be related to the expression of INSL3 mRNA.