Wild type transthyretin amyloidosis,a reason not to be forgotten for heart failure of preserved ejection fraction in the elderly

Amyloidosis is a multisystem disease that is characterized by deposition of fibrils in extracellular tissue,which mainly involves the kidney,heart,and autonomic nervous system.Two types of amyloidosis typically infiltrate the heart,including immunoglobulin light-chain(AL)and amyloid transthyretin(ATTR).ATTR is further subdivided into wild-type ATTR and variant ATTR caused by point mutations in the TTR gene.[1]Wild-type ATTR is considered as not uncommon in older patients with heart failure.Recently,a comprehensive set of consensus recommendations for the suspicion and diagnosis of ATTR was published,with particular focus on the combined application of noninvasive methods.[2]We present here a case of wild-type TTR cardiac amyloidosis(ATTRwt-CA),which was diagnosed by noninvasive modalities,and provide an overview of the recommended diagnostic approach of CA.Furthermore,to the best of our knowledge,this is the first Chinese case of ATTRwt-CA reported to date.

Research progress on the health status and community management service needs of mobile elderly people

The health status of the floating elderly population and the need of community management services are complex and important problems.Owing to the change of living environment and the insecure medical care,the mobile elderly people have more health and psy-chological problems compared to the rest of the elderly population.Therefore,the community should take a variety of measures to meet their needs and help them to better adapt to the new living environment,and maintain their physical and mental health.This paper aims to summarize recent studies on the physical and emotional health problems of mobile elderly people and related community management service needs.

渝东南地区五峰–龙马溪组层序地层特征及地质意义

在野外露头观察与实测的基础上,将钻井、录井及实测资料相结合,运用沉积学与层序地层学相关理论方法,对渝东南地区五峰–龙马溪组沉积特征和层序充填特征进行深入研究,并探索油气地质意义。五峰–龙马溪组页岩野外沉积特征明显,识别度高,厚度60～130m,下部为硅质页岩,向上砂质含量逐渐增加,岩性过渡为泥质粉砂岩。通过测井曲线特征、岩性、沉积构造响应及海平面升降,可将五峰–龙马溪组划分为4个三级层序,海侵体系域和高位体系域对称发育,横向上连续性较好,纵向上特征明显;Sq1海侵体系域在研究区内广泛发育,沉积一套硅质页岩,Sq2上部开始发育高位体系域沉积,对应为泥质粉砂岩沉积,Sq3下部发育海侵体系域泥岩沉积,Sq4以高位体系域为主,发育泥质陆棚相炭质页岩沉积。五峰–龙马溪组在整个层序充填过程中受到构造运动和海平面升降的控制,物源主要来自东南缘雪峰古隆起,早期的火山热事件为页岩中大范围硅化现象提供硅质来源的同时,为该时期浮游生物提供了生存条件,促进了五峰–龙马溪组页岩中有机质的发育,为页岩气富集成藏提供了充足的气源。

钢结构梁柱节点抗火性能研究进展与展望

钢结构不耐火,在一定程度上制约了钢结构的应用和发展。为了探明钢结构在火灾下的工作性能,对钢结构基本构件的抗火性能进行了大量研究,提出了相应的设计理论与方法。梁柱节点是钢结构中十分关键的部位,在火灾下的可靠连接是保证钢结构火灾安全的重要前提。为了全面了解钢结构梁柱节点在火灾下的受力性能和破坏机理,从试验研究、有限元模拟和组件分析法三个方面系统地梳理了国内外关于节点抗火性能的研究进展。比较了高温下不同类型节点的力学性能,探讨了多种关键参数对节点在火灾下工作性能的影响规律,介绍了不同类型节点的高温组件分析模型以及弯矩―转角曲线数学模型。分析表明:节点构造形式对钢框架火灾安全有较大影响;通过设置柱腹板加劲肋、提高螺栓强度等级以及增加连接件的厚度等可以有效地提高节点的抗火性能。最后剖析了钢结构梁柱节点抗火研究中存在的不足,对未来值得关注和重视的关键问题提出展望。

Prevalence and prognostic implications of sarcopenia in older patients with coronary heart disease

Background Sarcopenia is a progressive and generalized skeletal muscle disorder that is associated with an increased likelihood of adverse outcomes, including falls, fractures, physical disability, and mortality. However, there have been few systematic studies of the prevalence and prognostic values of sarcopenia in older patients with coronary heart disease(CHD). This study aimed to investigate the prevalence of sarcopenia in hospitalized older patients with CHD, and to prospectively evaluate the effect of sarcopenia on the short-term prognosis of these patients. Methods Patients aged ≥ 65 years, with the diagnosis of CHD from Peking Union Medical College Hospital between December 2017 and November 2018, were included. Sarcopenia was diagnosed according to consensus of the Asian Working Group for Sarcopenia in 2014. Follow-up items included unscheduled return visits, occurrence of major adverse cardiac and cerebral events (MACCE), and all-cause mortality. The MACCE-free survival curve of sarcopenic and non-sarcopenic older patients with CHD was estimated by the Kaplan-Meier method. Cox regression analysis was used to analyze the association between sarcopenia and an unscheduled return visits, MACCE, and all-cause mortality. Results A total of 345 older patients with CHD were enrolled in the study, with a median age of 74 years. Among the patients, 78 (22.6%) were diagnosed with sarcopenia. During the follow-up time, there were significantly more unscheduled return visits in sarcopenic patients than in non-sarcopenic patients (34.2% vs. 21.8%,χ^2 = 4.418, P = 0.036), while there was no significant difference in the occurrence of MACCE (χ^2 = 2.869, P = 0.09) or all-cause mortality (χ^2 = 1.673, P = 0.196) between these patient groups. The Kaplan-Meier curve showed that the MACCE-free survival time of sarcopenic patients was significantly shorter than that in non-sarcopenic patients (χ^2 = 4.102, P = 0.043). After adjusting for sex, age, and the Charlson comorbidity index, sarcopenia was not an independent risk factor of unscheduled return visits (HR = 1.002, 95% CI: 0.556–1.807). However, the complication of anxiety and depression was an independent risk factor (HR = 1.876, 95% CI: 1.012–3.477, P = 0.046) for unscheduled return visits in older patients with CHD. Conclusions There is a high prevalence of sarcopenia among hospitalized older adults with CHD. A shorter MACCE-free survival time and more unscheduled return visits are found in sarcopenic older patients with CHD. Clinicians should pay more attention to the functional status of older patients with CHD, as well as identification and management of geriatric syndromes.

天然膜仿生递药系统在肿瘤治疗中的应用进展

药物递送系统在肿瘤治疗中具有广阔的应用前景,但作为外源物质,易被机体免疫系统所识别与清除,临床应用受到限制。近年来,受自然界生物系统启发,随着仿生技术发展,天然膜仿生递药系统成为研究热点。天然膜仿生药物递送系统主要是利用机体内源性细胞膜对功能性纳米药物载体进行修饰,有效地将细胞膜的天然属性与纳米载体的优势相结合,具有免疫原性低、血液循环时间长的特点。本文将根据天然膜的来源不同,对仿生药物递送系统在肿瘤治疗中的研究进展并对其未来的发展进行综述。

Evolutionary impacts of purine metabolism genes on mammalian oxidative stress adaptation

Many mammals risk damage from oxidative stress stemming from frequent dives(i.e., cycles of ischemia/reperfusion and hypoxia/reoxygenation),high altitude and subterranean environments, or powered flight. Purine metabolism is an essential response to oxidative stress, and an imbalance between purine salvage and de novo biosynthesis pathways can generate damaging reactive oxygen species(ROS). Here, we examined the evolution of 117 purine metabolism-related genes to explore the accompanying molecular mechanisms of enhanced purine metabolism in mammals under high oxidative stress. We found that positively selected genes,convergent changes, and nonparallel amino acid substitutions are possibly associated with adaptation to oxidative stress in mammals. In particular, the evolution of convergent genes with c AMP and c GMP regulation roles may protect mammals from oxidative damage. Additionally, 32 genes were identified as under positive selection in cetaceans, including key purine salvage enzymes(i.e., HPRT1), suggesting improved re-utilization of non-recyclable purines avoid hypoxanthine accumulation and reduce oxidative stress. Most intriguingly, we found that six unique substitutions in cetacean xanthine dehydrogenase(XDH), an enzyme that regulates the generation of the ROS precursor xanthine oxidase(XO) during ischemic/hypoxic conditions, show enhanced enzyme activity and thermal stability and diminished XO conversion activity. These functional adaptations are likely beneficial for cetaceans by reducing radical oxygen species production during diving. In summary, our findings offer insights into the molecular and functional evolution of purine metabolism genes in mammalian oxidative stress adaptations.

Pulmonary tuberculosis presenting with aortic pseudoaneurysm, diffuse alveolar hemorrhage, and cardiomyopathy: Is there an intriguing relationship?

A 22-year-old Chinese man presented with fever, cough, hoarseness, neck pain, acute heart failure and hemoptysis. Pulmonary tuberculosis was proved by sputum culture. Chest imaging showed an aortic arch pseudoaneurysm and bilateral ground glass opacities. Echocardiography confirmed cardiomyopathy. With anti-TB drugs, high-dose prednisone and surgery, the life of this patient was successfully prolonged for more than four months. The concomitant disorders of aortic pseudoaneurysm, alveolar hemorrhage and cardiomyopathy in pulmonary tuberculosis are intriguing. We postulate that immune-mediated small vessel vasculitis triggered by pathogens plays an important role in the pathogenesis of this disease, rather than a direct TB infection.

Tyrosine 23 Phosphorylation of Annexin A2 Promotes Proliferation,Invasion,and Stat3 Phosphorylation in the Nucleus of Human Breast Cancer SK-BR-3 Cells

Objective To investigate the role of tyrosine 23 （Tyr23） phosphorylation of Annexin A2 （Anxa2） in regulating the proliferation and invasion of human breast cancer SK-BR-3 cells. Methods A panel of lentivirus plasmids expressing Anxa2-wide type （Ana2-WT）, Anxa2-Y23A, and Anxa2-Y23D was generated and infected with SK-BR-3 cells. The monoclonal strains were screened. The expression of Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D was determined by Western blot analysis. The ability of the cells to proliferate was detected through an MTT [3-（4,5-Dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide] test. Boyden chamber assays were employed to examine migration and invasion abilities. The interaction between Anxa2 and Stat3 was analyzed by immunoprecipitation analyses. Nucleoprotein and cytosolic protein were extracted from SK-BR-3, Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D cells to analyze the expression and localization of Stat3 phosphorylation. Results The monoclonal strains constitutively expressing Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D were screened. Both Anxa2-WT and Anxa2-Y23D enhanced the proliferation, migration and invasion abilities of SK-BR-3 cells （P〈0.05）. Immunoprecipitation analysis revealed that Anxa2 and Stat3 interacted with each other, and the expression of Stat3 phosphorylation in the nucleus was enhanced by Anxa2-Y23D. Conclusions Tyr23 phosphorylation of Anxa2 promotes the proliferation and invasion of human breast cancer SK-BR-3 cells and the phosphorylation of Stat3 in the nucleus.

How to establish precise proprietary Chinese medicine in post-marketing reappraisal?

The clinical efficacy and safety of proprietary Chinese medicine has become a more prevailing public health concern.The China Food and Drug Administration has pushed drug companies to conduct post-marketing reappraisal for proprietary Chinese medicine to provide additional evidence supporting the efficacy and safety of these medicine.In this paper,we propose a reappraisal protocol called“precise proprietary Chinese medicine”to precisely i)define the therapeutic aim;ii)design the protocol;iii)control the quality of proprietary Chinese medicine;iv)implement the protocol in the trial;v)study the mechanism-of-action of the proprietary Chinese medicine and vi)describe the indications of the proprietary Chinese medicine.We hope that these steps facilitate the post-marketing reappraisal of proprietary Chinese medicine.

中俄教育合作:历史、现状与展望

当今世界,国际合作已成为推动全球社会进步和文明交流的关键。在全球化的背景下,中俄两国致力于加强彼此间的教育合作。这种合作不仅对两国文化交流乃至“民心相通”具有深远影响,更为大国之间的文明互鉴和共同发展树立了典范,也是践行新型大国关系理念的重要措施之一。本文旨在研究中俄教育合作的历史和发展趋势,重点关注双方合作的领域、项目的成果以及对双方教育的影响。同时,文章将进一步分析未来中俄教育合作的潜在机遇,以期为两国教育合作提供更多的启示和建议。

Self-adaptive hydrogel for breast cancer therapy via accurate tumor elimination and on-demand adipose tissue regeneration

The irregular defects and residual tumor tissue after surgery are challenges for effective breast cancer treatment.Herein,a smart hydrogel with self-adaptable size and dual responsive cargos release was fabricated to treat breast cancer via accurate tumor elimination,on-demand adipose tissue regeneration and effective infection inhibition.The hydrogel consisted of thiol groups ended polyethylene glycol(SH-PEG-SH)and doxorubicin encapsulated mesoporous silica nanocarriers(DOX@MSNs)double crosslinked hyaluronic acid(HA)after loading of antibacterial peptides(AP)and adipose-derived stem cells(ADSCs).A pH-cleavable unsaturated amide bond was pre-introduced between MSNs and HA frame to perform the tumor-specific acidic environment dependent DOX@MSNs release,meanwhile an esterase degradable glyceryl dimethacrylate cap was grafted on MSNs,which contributed to the selective chemotherapy in tumor cells with over-expressed esterase.The bond cleavage between MSNs and HA would also cause the swelling of the hydrogel,which not only provide sufficient space for the growth of ADSCs,but allows the hydrogel to fully fill the irregular defects generated by surgery and residual tumor atrophy,resulting in the on-demand regeneration of adipose tissue.Moreover,the sustained release of AP could be simultaneously triggered along with the size change of hydrogel,which further avoided bacterial infection to promote tissue regeneration.

A privacy-preserving vehicle trajectory clustering framework

As one of the essential tools for spatio‒temporal traffic data mining,vehicle trajectory clustering is widely used to mine the behavior patterns of vehicles.However,uploading original vehicle trajectory data to the server and clustering carry the risk of privacy leakage.Therefore,one of the current challenges is determining how to perform vehicle trajectory clustering while protecting user privacy.We propose a privacy-preserving vehicle trajectory clustering framework and construct a vehicle trajectory clustering model(IKV)based on the variational autoencoder(VAE)and an improved K-means algorithm.In the framework,the client calculates the hidden variables of the vehicle trajectory and uploads the variables to the server;the server uses the hidden variables for clustering analysis and delivers the analysis results to the client.The IKV’workflow is as follows:first,we train the VAE with historical vehicle trajectory data(when VAE’s decoder can approximate the original data,the encoder is deployed to the edge computing device);second,the edge device transmits the hidden variables to the server;finally,clustering is performed using improved K-means,which prevents the leakage of the vehicle trajectory.IKV is compared to numerous clustering methods on three datasets.In the nine performance comparison experiments,IKV achieves optimal or sub-optimal performance in six of the experiments.Furthermore,in the nine sensitivity analysis experiments,IKV not only demonstrates significant stability in seven experiments but also shows good robustness to hyperparameter variations.These results validate that the framework proposed in this paper is not only suitable for privacy-conscious production environments,such as carpooling tasks,but also adapts to clustering tasks of different magnitudes due to the low sensitivity to the number of cluster centers.

The spectrum of rare monogenic diseases in patients with premature coronary artery disease

To the Editor:The global burden of premature coronary artery disease(CAD)is increasing among the young population.Premature CAD generally refers to the occurrence of obstructive coronary atherothrombotic lesions in men and women aged<55 years and<65 years,respectively,and particularly in those aged<45 years.[1]The incidence of CAD in the younger population has remained stable or has increased,despite the declining incidence in older adults.Patients with premature CAD have a poor long-term prognosis,experiencing a high rate of recurrent ischemic events,rapid progression to multivessel disease,and frequent premature death.[1]Thus,it is crucial to identify the underlying causes of premature CAD that are distinct from those of age-related CAD and improve patient outcomes through precise diagnosis and treatment.

Molecular mechanisms of adaptive evolution in wild animals and plants

Wild animals and plants have developed a variety of adaptive traits driven by adaptive evolution,an important strategy for species survival and persistence.Uncovering the molecular mechanisms of adaptive evolution is the key to understanding species diversification,phenotypic convergence,and inter-species interaction.As the genome sequences of more and more non-model organisms are becoming available,the focus of studies on molecular mechanisms of adaptive evolution has shifted from the candidate gene method to genetic mapping based on genome-wide scanning.In this study,we reviewed the latest research advances in wild animals and plants,focusing on adaptive traits,convergent evolution,and coevolution.Firstly,we focused on the adaptive evolution of morphological,behavioral,and physiological traits.Secondly,we reviewed the phenotypic convergences of life history traits and responding to environmental pressures,and the underlying molecular convergence mechanisms.Thirdly,we summarized the advances of coevolution,including the four main types:mutualism,parasitism,predation and competition.Overall,these latest advances greatly increase our understanding of the underlying molecular mechanisms for diverse adaptive traits and species interaction,demonstrating that the development of evolutionary biology has been greatly accelerated by multi-omics technologies.Finally,we highlighted the emerging trends and future prospects around the above three aspects of adaptive evolution.

长链非编码RNA LINC00342在头颈鳞癌中的生物学功能及临床意义

目的明确长链非编码RNA LINC00342表达水平与头颈鳞状细胞癌(简称鳞癌)患者临床病理参数的关系,研究LINC00342在头颈鳞癌细胞中的生物学功能。方法分析癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库头颈鳞癌转录组中LINC00342的表达,利用转录组测序技术检测山西医科大学第一医院27例喉鳞癌组织中该基因的表达;利用实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,qPCR)检测人胚肺二倍体细胞2BS和头颈鳞癌细胞系FD-LSC-1、CAL-27、Detroit562中LINC00342的表达水平;使用RNA干扰(RNA interference,RNAi)技术敲降头颈鳞癌细胞中该基因的表达,利用细胞增殖检测试剂盒(cell counting kit-8,CCK-8)、克隆形成、流式细胞术、Transwell迁移和侵袭等实验检测肿瘤细胞恶性表型的变化;生物信息学方法构建以LINC00342为核心的竞争性内源RNA(competing endogenous RNA,ceRNA)调控网络,并进行基因本体论(gene ontology,GO)富集分析。使用SPSS 25.0软件和GraphPad Prism 6软件进行统计学分析及作图。结果TCGA数据库收录的头颈鳞癌组织与数据库对应的正常组织相比,LINC00342的相对表达量差异无统计学意义(P=0.522);但其表达水平与患者颈淋巴结转移、病理学分级呈正相关,男性患者的表达水平高于女性患者(P值均<0.05)。通过转录组测序发现,LINC00342在我院27例喉鳞癌中的相对表达量高于癌旁正常黏膜组织(t=1.56,P=0.036)。LINC00342在头颈鳞癌细胞系FD-LSC-1、CAL-27和Detroit562中表达均显著上调(t值分别为-12.17、-23.26和-388.57,P值均<0.001)。分别转染si-LINC00342-1和si-LINC00342-2敲降LINC00342,可抑制头颈鳞癌细胞FD-LSC-1、CAL-27和Detroit562增殖(t值分别为8.95和4.84,2.70和5.55,2.02和3.70)、克隆形成(t值分别为6.66和6.17,7.38和11.65,4.90和5.79)、迁移(t值分别为8.21和7.19,5.76和6.46,6.28和9.92)和侵袭能力(t值分别为9.29和10.25,11.30和11.36,8.02和8.66),同时促进FD-LSC-1及CAL-27细胞凋亡(t值分别为-2.21和-5.83,-3.05和-5.25),差异有统计学意义(P值均<0.05)。以LINC00342为中心的ceRNA调控网络包括10个下调的微核糖核酸(microRNA)节点和647个上调的mRNA节点,GO分析表明这些mRNA主要聚类在22个生物过程、32个分子功能以及12个细胞组分方面。结论LINC00342高表达与头颈鳞癌恶性进展相关,具有促进头颈鳞癌细胞增殖、迁移、侵袭以及拮抗凋亡的重要生物学功能,是头颈鳞癌潜在的重要分子标志物。

LDformer:a parallel neural network model for long-term power forecasting

Accurate long-term power forecasting is important in the decision-making operation of the power grid and power consumption management of customers to ensure the power system’s reliable power supply and the grid economy’s reliable operation.However,most time-series forecasting models do not perform well in dealing with long-time-series prediction tasks with a large amount of data.To address this challenge,we propose a parallel time-series prediction model called LDformer.First,we combine Informer with long short-term memory(LSTM)to obtain deep representation abilities in the time series.Then,we propose a parallel encoder module to improve the robustness of the model and combine convolutional layers with an attention mechanism to avoid value redundancy in the attention mechanism.Finally,we propose a probabilistic sparse(ProbSparse)self-attention mechanism combined with UniDrop to reduce the computational overhead and mitigate the risk of losing some key connections in the sequence.Experimental results on five datasets show that LDformer outperforms the state-of-the-art methods for most of the cases when handling the different long-time-series prediction tasks.

Cognition:Accurate and Consistent Linear Log Parsing Using Template Correction

Logs contain runtime information for both systems and users.As many of them use natural language,a typical log-based analysis needs to parse logs into the structured format first.Existing parsing approaches often take two steps.The first step is to find similar words(tokens)or sentences.Second,parsers extract log templates by replacing different tokens with variable placeholders.However,we observe that most parsers concentrate on precisely grouping similar tokens or logs.But they do not have a well-designed template extraction process,which leads to inconsistent accuracy on particular datasets.The root cause is the ambiguous definition of variable placeholders and similar templates.The consequences include abuse of variable placeholders,incorrectly divided templates,and an excessive number of templates over time.In this paper,we propose our online log parsing approach Cognition.It redefines variable placeholders via a strict lower bound to avoid ambiguity first.Then,it applies our template correction technique to merge and absorb similar templates.It eliminates the interference of commonly used parameters and thus isolates template quantity.Evaluation through 16 public datasets shows that Cognition has better accuracy and consistency than the state-of-the-art approaches.It also saves up to 52.1%of time cost on average than the others.

Screening beneficial bacteriostatic lactic acid bacteria in the intestine and studies of bacteriostatic substances

Lactic acid bacteria(LAB)are a representative probiotic.As the dominant flora in the human intestinal tract,LAB can regulate the balance of human intestinal flora and improve host health.The purpose of this study was to isolate and screen LAB that are well suited to the intestinal characteristics of the Chinese population,with excellent probiotics and high antibacterial activity.After 16S ribosomal RNA(rRNA)homology and phylogenetic tree analysis,potential probiotics were tested for their antibacterial activity,resistance to artificial gastrointestinal fluid and drugs,surface hydrophobicity,and safety.Three strains of LAB with acid resistance,bile salt resistance,epithelial cell adhesion,and no multidrug resistance were selected:Lactobacillus salivarius,Leuconostoc lactis,and Lactobacillus paracasei.Analysis of the antibacterial active substances in the three strains and their fermentation broths revealed that the main antibacterial substances of L.lactis were organic acids,whereas those of L.salivarius and L.paracasei were organic acids and bacteriocins with broad-spectrum antibacterial activity.These three strains of probiotic LAB with high antibacterial activity were identified as bacterial resources that could potentially be used to develop probiotic preparations for the prevention and treatment of intestinal diseases caused by intestinal pathogens.

Engineered neutrophil apoptotic bodies ameliorate myocardial infarction by promoting macrophage efferocytosis and inflammation resolution

Inflammatory response plays a critical role in myocardial infarction(MI)repair.The neutrophil apoptosis and subsequent macrophage ingestion can result in inflammation resolution and initiate regeneration,while the therapeutic strategy that simulates and enhances this natural process has not been established.Here,we constructed engineered neutrophil apoptotic bodies(eNABs)to simulate natural neutrophil apoptosis,which regulated inflammation response and enhanced MI repair.The eNABs were fabricated by combining natural neutrophil apoptotic body membrane which has excellent inflammation-tropism and immunoregulatory properties,and mesoporous silica nanoparticles loaded with hexyl 5-aminolevulinate hydrochloride(HAL).The eNABs actively targeted to macrophages and the encapsulated HAL simultaneously initiated the biosynthesis pathway of heme to produce anti-inflammatory bilirubin after intracellular release,thereby further enhancing the anti-inflammation effects.In in vivo studies,the eNABs efficiently modulated inflammation responses in the infarcted region to ameliorate cardiac function.This study demonstrates an effective biomimetic construction strategy to regulate macrophage functions for MI repair.