Sleep in the human aging process and circadian sleep rhythm disruptions / Sono no processo de envelhecimento humano e interrupções do ritmo circadiano do sono

The aging process is often related to sleeping difficulties, often due to changes in circadian rhythms. The circadian timing system is centered in the suprachiasmatic nucleus - the master biological clock - which synchronizes the rhythm of oscillators throughout the body, including the sleep-wake cycle. This affects the time, duration and quality of sleep according to the development and aging process, under external and internal influences. This review addresses the human circadian timing system, including endogenous and exogenous influences on circadian rhythms, their age-related particularities, as well as the repercussions of circadian misalignment in neurodegenerative diseases. Circadian rhythms naturally weaken with aging, but there are particularities according to age. Throughout life, sleep and circadian rhythm disorders are strongly bidirectionally related to the pathophysiology of some psychiatric and neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. This knowledge could potentially create valuable opportunities to improve the health of the world's population that is under circadian misalignment and aging.

O processo de envelhecimento está frequentemente relacionado a dificuldades de dormir, muitas vezes decorrentes de alterações nos ritmos circadianos. O sistema de ronometragem circadiana está centrada no núcleo supraquiasmático - o relógio biológico mestre - o qual sincroniza o ritmo dos osciladores em todo o corpo, incluindo o ciclo sono-vigília. Isso afeta o tempo, a duração e a qualidade do sono de acordo com o processo de desenvolvimento e envelhecimento, sob influências externas e internas. Esta revisão aborda o sistema de temporização circadiana humana, incluindo as influências endógenas e exógenas nos ritmos circadianos, suas particularidades relacionadas à idade, bem como as repercussões do desalinhamento circadiano nas doenças neurodegenerativas. Os ritmos circadianos enfraquecem naturalmente com o envelhecimento, mas há particularidades de acordo com a idade. Ao longo da vida, os transtornos do sono e do ritmo circadiano estão fortemente relacionados bidirecionalmente à fisiopatologia de algumas doenças psiquiátricas e neurodegenerativas, como as doenças de Alzheimer e Parkinson. Esse conhecimento pode potencialmente criar oportunidades valiosas para melhorar a saúde da população mundial que está sob desalinhamento circadiano e envelhecimento.

Progress on the neuroprotective effects of ginsenoside Rg1 / 药学学报

Ginsenoside Rg1 is one of the most important saponins in ginseng. It has a wide range of pharmacological activities. It is considered to be a powerful neuroprotective agent. It has neuroprotective effects such as anti-neuroinflammation, anti-oxidative stress, anti-neuronal apoptosis, and enhancing memory. Rg1 shows a good application prospect in the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, stroke, and mental diseases such as depression. This paper reviews the research on the neuroprotective mechanism of Rg1 at home and abroad in recent years, in order to provide new research ideas for the clinical treatment of nervous system diseases.

Neuroprotection of Natural Products Based on Keap1/Nrf2/ARE Signaling Pathway： A Review / 中国实验方剂学杂志

The pathological manifestations of neurodegenerative diseases， such as Alzheimer's disease， Parkinson's disease， and multiple sclerosis， are abnormal protein aggregation and accumulation， microglia activation， and mitochondrial dysfunction， which eventually lead to the gradual loss of neuronal structure or function and deteriorate over time. These pathological processes are related to the production of reactive oxygen species （ROS）， which can cause oxidative stress and damage proteins， lipids， and DNA， leading to cell and tissue injuries. The Kelch-like ECH-associated protein 1 （Keap1）/nuclear factor E2-related factor 2 （Nrf2）/antioxidant response element （ARE） signaling pathway is the main mechanism to maintain the redox balance of the body and defend against oxidative stress injury. Nrf2 activates the expression of a series of antioxidant genes related to ARE through the dissociation of Keap1 and nuclear transfer in the cytoplasm to protect the body from oxidative damage. Therefore， the discovery and study of the Keap1/Nrf2/ARE signaling pathway activator is of great significance for the prevention and treatment of neurodegenerative diseases. Because of the remarkable biological activity and slight side effects， natural products are a treasure trove for new drug research and development. Studies have shown that a variety of natural products can activate the Keap1/Nrf2/ARE signaling pathway and play a neuroprotective role. According to the structural characteristics， natural products can be divided into flavonoids， terpenoids， volatile oils， polyphenols， and phenylpropanoids. This study summarized the underlying mechanism of the Keap1/Nrf2/ARE signaling pathway in regulating diseases and reviewed the research progress on natural products based on this signaling pathway in neuroprotection to provide references for the development of clinical drugs for the prevention and treatment of neurodegenerative diseases.

Corynoxine B targets at HMGB1/2 to enhance autophagy for α-synuclein clearance in fly and rodent models of Parkinson's disease

Parkinson's disease (PD) is the most common neurodegenerative movement disease. It is featured by abnormal alpha-synuclein (α-syn) aggregation in dopaminergic neurons in the substantia nigra. Macroautophagy (autophagy) is an evolutionarily conserved cellular process for degradation of cellular contents, including protein aggregates, to maintain cellular homeostasis. Corynoxine B (Cory B), a natural alkaloid isolated from Uncaria rhynchophylla (Miq.) Jacks., has been reported to promote the clearance of α-syn in cell models by inducing autophagy. However, the molecular mechanism by which Cory B induces autophagy is not known, and the α-syn-lowering activity of Cory B has not been verified in animal models. Here, we report that Cory B enhanced the activity of Beclin 1/VPS34 complex and increased autophagy by promoting the interaction between Beclin 1 and HMGB1/2. Depletion of HMGB1/2 impaired Cory B-induced autophagy. We showed for the first time that, similar to HMGB1, HMGB2 is also required for autophagy and depletion of HMGB2 decreased autophagy levels and phosphatidylinositol 3-kinase III activity both under basal and stimulated conditions. By applying cellular thermal shift assay, surface plasmon resonance, and molecular docking, we confirmed that Cory B directly binds to HMGB1/2 near the C106 site. Furthermore, in vivo studies with a wild-type α-syn transgenic drosophila model of PD and an A53T α-syn transgenic mouse model of PD, Cory B enhanced autophagy, promoted α-syn clearance and improved behavioral abnormalities. Taken together, the results of this study reveal that Cory B enhances phosphatidylinositol 3-kinase III activity/autophagy by binding to HMGB1/2 and that this enhancement is neuroprotective against PD.

Prevention and alleviation of neurodegenerative diseases： time restricted feeding / 上海预防医学

With the rapid development of aging around the world， the incidence and prevalence of age-related dementia have increased significantly， leading to a huge burden on patients， families and society. So far， there are no drugs that can completely reverse degeneration of the nervous system. The core pathological mechanisms of dementia are not clear， and the occurrence and progression of degenerative diseases in the nervous system appears to be only rely on the prevention through lifestyle interventions. Among lifestyle interventions， a large amount of laboratory evidence and a small amount of epidemiological evidence suggest that time restricted feeding have excellent effects on preventing the occurrence and progression of degenerative diseases in the nervous system. However， due to the lack of evidence in human population， the application of time restricted feeding lacks scientific basis. By synthesizing the existing evidence， this review discussed the pathway network of time restricted feeding that antagonizes the nervous system degradation， suggesting that future studies should focus on population evidence and the combined effects of multiple effect pathways.

Tualang and Kelulut Honey Reduced Lipopolysaccharidesstimulated Inflammatory Responses of Mic

@#Microglial activity is crucial in maintaining the central nervous system (CNS) homeostasis. However, prolonged microglial activation have been implicated in the pathology of neurodegenerative diseases. Activated microglia will increase the production of inflammatory cytokines, reactive oxygen species (ROS) and alter their surface marker expression levels. This study used Malaysian honey, Tualang honey (TH), and Kelulut honey (KH) to determine lipopolysaccharide (LPS)-stimulated inflammatory responses of microglia. TH and KH at 0.1% were used in the current study as our findings showed no significant difference in the cell viability between BV2 cells treated with 0.1 % of TH and KH and control group. TH and KH reduced the ROS level significantly by 41.62±1.06% and 49.16±0.63%, respectively, and slightly reduced the expression of co-stimulatory molecules, CD40 and CD11b in LPS-activated BV2 cells. Our preliminary findings proposed an in-depth future study on the anti-inflammatory effect of TH and KH on microglial activation.

Magnetic Resonance Imaging Studies of Neurodegenerative Disease: From Methods to Translational Research / 神经科学通报·英文版

Neurodegenerative diseases (NDs) have become a significant threat to an aging human society. Numerous studies have been conducted in the past decades to clarify their pathologic mechanisms and search for reliable biomarkers. Magnetic resonance imaging (MRI) is a powerful tool for investigating structural and functional brain alterations in NDs. With the advantages of being non-invasive and non-radioactive, it has been frequently used in both animal research and large-scale clinical investigations. MRI may serve as a bridge connecting micro- and macro-level analysis and promoting bench-to-bed translational research. Nevertheless, due to the abundance and complexity of MRI techniques, exploiting their potential is not always straightforward. This review aims to briefly introduce research progress in clinical imaging studies and discuss possible strategies for applying MRI in translational ND research.

Analysis of SOD1 and C9orf72 mutations in patients with amyotrophic lateral sclerosis in Antioquia, Colombia / Análisis de mutaciones en los genes SOD1 y C9orf72 en pacientes con esclerosis lateral amiotrófica, Antioquia, Colombia

Introduction: Amyotrophic lateral sclerosis is a neurodegenerative disease with a possible multifactorial origin characterized by the progressive degeneration of motor neurons. There is a relatively high prevalence of this disease in Antioquia; however, there is no published genetic study to date in Colombia. Despite its unknown etiopathogenesis, more genetic risk factors possibly involved in the development of this disease are constantly found. Objetives: To evaluate G93A and D90A mutations in SOD1 gene and a short tandem repeat in C9orf72 within a cohort of amyotrophic lateral sclerosis patients from Antioquia, Colombia. Materials y methods: Thirty-four patients previously diagnosed with amyotrophic lateral sclerosis were included in the study. Peripheral blood samples were used for DNA extraction and genotyping. Results: No mutations were found in SOD1 (G93A and D90A) in any of the patients, while C9orf72 exhibited an allele with a statistically significant high prevalence in the study sample (8 hexanucleotide repeats of CAGCAG). Conclusions: These results suggest an association between this short tandem repeat (STR) in C9orf72 and the presence of amyotrophic lateral sclerosis in the studied population. However, this association should be established in a larger sample size and with controls from the same population. In addition, there also seems to be a genetic anticipation effect for the disease regarding this locus, since patients with this genotype present an earlier onset.

Introducción. La esclerosis lateral amiotrófica es una enfermedad neurodegenerativa con un posible origen multifactorial, caracterizado por una degeneración progresiva de las neuronas motoras. Hay una gran prevalencia relativa de esta enfermedad en Antioquia; sin embargo, no hay publicaciones de estudios genéticos en Colombia. A pesar de su etiopatogénesis desconocida, hay varios factores de riesgo genético que se encuentran constantemente en el desarrollo de esta enfermedad. Objetivo. Evaluar las mutaciones G93A y D90A del gen SOD1 y una repetición corta en tándem (Short Tandem Repeat, STR) en el locus C9orf72, en una cohorte de pacientes con esclerosis lateral amiotrófica en Antioquia, Colombia. Materiales y métodos. Se incluyeron 34 pacientes previamente diagnosticados en el estudio. Una muestra de sangre periférica se usó para extraer el ADN y, posteriormente, genotipificarlo. Resultados. No se encontraron mutaciones en el gen SOD1 (G93A y D90A), mientras que el C9orf72 exhibe un alelo con una significativa prevalencia en los pacientes del estudio (8 repeticiones del hexanucleótido G4C2). Conclusiones. Se sugiere una asociación entre la repetición en tándem en C9orf72 y la presencia de la esclerosis lateral amiotrófica en la población estudiada. Sin embargo, se sugiere hacer estudios adicionales e incluir un grupo control de la misma población. Además, se detecta un fenómeno de anticipación genética de la enfermedad, dado que los pacientes con el alelo de 8 repeticiones en C9orf72 presentan una edad temprana de aparición de los síntomas.

Bioinformatics study on role of IL-17 signaling in manganese poisoning and manganese-related neurodegenerative diseases / 环境与职业医学

Background Manganese (Mn) is one of the environmental factors of Parkinson's disease (PD), and long-term exposure to Mn can cause nerve damage. It is important to explore the common mechanism of neurotoxic effects of Mn and neurodegenerative diseases (NDD), especially PD, for early diagnosis of the disease. Objective To comprehensively analyze the core messenger RNA (mRNA)-microRNAs (miRNAs) co-expressed in frontal cortex of NDD patients and neuronal cells exposed to Mn via bioinformatics, and to reveal the potential common mechanism between Mn-induced neurotoxicity and NDD, especially PD. Methods Difference of the mRNAs from frontal cortex of NDD patients (GSE150696) and human neuroblastoma (SH-SY5Y) cells exposed to Mn were analyzed by R software; Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed on the overlapping differentially expressed genes (DEGs). The miRNAs were predicted using the miRNet database, mRNA-miRNA interactions were identified by the starBase and miRTarBase databases, and mRNA-miRNA regulatory networks were constructed with Cytoscape software. The core miRNAs associated with PD (GSE77667) were incorporated into Weighted Gene Co-Expression Network Analysis (WGCNA) and the mRNA-miRNA regulatory network was comparatively analyzed. Results A total of 34 overlapping DEGs were identified in the frontal cortical of NDD patients and the neuronal cells exposed to Mn, mainly enriched in interleukin-17 (IL-17) signaling pathway, cyclic adenosine monophosphate (cAMP) signaling pathway, and primary immunodeficiency. Based on the results of database prediction, 52 miRNAs with 71 pairs of interaction relationships were finally included to construct the miRNA-mRNA regulatory network. Six core miRNAs were screened by WGCNA: hsa-let-7i-5p, hsa-mir-155-5p, hsa-mir-219-2-3p, hsa-mir-221-3p, hsa-mir-485-3p, and hsa-mir-509-3-5p, among which hsa-let-7i-5p interacted with the target gene FBXW2 and hsa-mir-155-5p interacted with the target gene CCL2. The results of the KEGG analysis indicated that CCL2 was closely related to the IL-17 signaling pathway. Conclusion There are similar molecular regulatory mechanisms involved in the neurotoxicity of Mn and NDD, and the IL-17 signaling pathway may play a role in Mn-related NDD through CCL2 and hsa-mir-155-5p.

The importance of promoting physical activity and exercise training as adjuvant therapy for people with multiple sclerosis

Abstract Aim: This review aimed to provide evidence and highlight the importance of including physical activity (PA) and/or exercise training (ET) as part of the comprehensive multiple sclerosis (MS) care. Methods: Using the current literature on the subject, we provide a brief overview of MS incorporating its definition, common symptoms, prevalence, and potential disease consequences. We further succinctly describe MS as the first line of treatment, as well as the role of PA and ET in the disease. We end the commentary highlighting important recommendations from an international initiative to improve MS-related physical activity research that we believe will help not only improve the area of study but also best practices within this population. Results: There is compelling evidence for the beneficial effects of PA and/or ET on MS-related symptoms and consequently health and quality of life. There is preliminary evidence suggesting the potential for a disease-modifying effect. Conclusion: Fomenting this discussion is timely due to the increased prevalence of MS in different regions of the globe, and people with MS report low levels of PA participation and high amounts of sedentary time. The consequences of inactivity in this population can be drastic. The current body of evidence supports the notion that PA and ET are safe health behaviors that should be adopted as an adjuvant treatment option within the comprehensive and complex MS care due to its benefits on a variety of disease-related symptoms and its potential for improving health and quality of life in this population.

Erythrocyte sphingolipid species as biomarkers of Alzheimer's disease / 药物分析学报

Diagnosing Alzheimer's disease(AD)in the early stage is challenging.Informative biomarkers can be of great value for population-based screening.Metabolomics studies have been used to find potential biomarkers,but commonly used tissue sources can be difficult to obtain.The objective of this study was to determine the potential utility of erythrocyte metabolite profiles in screening for AD.Unlike some commonly-used sources such as cerebrospinal fluid and brain tissue,erythrocytes are plentiful and easily accessed.Moreover,erythrocytes are metabolically active,a feature that distinguishes this sample source from other bodily fluids like plasma and urine.In this preliminary pilot study,the erythrocyte metab-olomes of 10 histopathologically confirmed AD patients and 10 patients without AD(control(CTRL))were compared.Whole blood was collected post-mortem and erythrocytes were analyzed using ultra-performance liquid chromatography tandem mass spectrometry.Over 750 metabolites were identified in AD and CTRL erythrocytes.Seven were increased in AD while 24 were decreased(P＜0.05).The ma-jority of the metabolites increased in AD were associated with amino acid metabolism and all of the decreased metabolites were associated with lipid metabolism.Prominent among the potential bio-markers were 10 sphingolipid or sphingolipid-related species that were consistently decreased in AD patients.Sphingolipids have been previously implicated in AD and other neurological conditions.Furthermore,previous studies have shown that erythrocyte sphingolipid concentrations vary widely in normal,healthy adults.Together,these observations suggest that certain erythrocyte lipid phenotypes could be markers of risk for development of AD.

Research progress in neurodegenerative langerhans cell histiocytosis / 中华实用儿科临床杂志

Langerhans cell histiocytosis (LCH) is a rare myeloid tumor disease, which is characterized by CD1a + CD 207+ dendritic cell proliferation.The clinical manifestations of LCH vary greatly due to the different locations and different involved organs.Among them, neurodegenerative disease (ND) is one of the manifestations of central nervous system involvement in LCH.The pathogenesis of LCH-ND is unclear and it is mainly characterized by neurological disorders and progressive imaging changes.Due to its unclear etiologies and long progress of LCH-ND, the treatment of LCH-ND remains very challenging.Presently, the main modalities of treatment include intravenous immunoglobulin, chemotherapy and targeted therapy.Early treatment and timely intervention may be the key to halt the progression of LCH-ND, to stabilize the central nervous system function and to improve the quality of life.The pathogenesis, clinical manifestations, diagnosis, treatment and clinical evaluation of LCH-ND are briefly reviewed.

Research progress of small molecule proteasome activators / 药学学报

Proteasome controls the degradation of proteins closely related to life activities and plays a key role in the maintenance of protein homeostasis. Proteasome activities decrease with aging, followed by the overwhelming production of damaged proteins which far exceed the protein consumption. Accumulation of these proteins leads to various diseases including neurodegenerative diseases. Therefore, inducing toxic protein degradation is considered as a promising solution for the treatment of these diseases, while increasing the activity of proteasome is considered as an important strategy. However, the research in this field is still in the preliminary stage, and this review will focus on the discussion of the research progress of various small molecule proteasome activators, including research methods, pharmacological effects, structure-activity relationships and the existing problems.

Misoplegia and dementia: a case study / Misoplegia e demência: relato de caso

ABSTRACT. Brain-damaged patients can develop abnormal attitudes towards their deficits. Misoplegia is one such example, involving exaggerated aversion to an impaired limb, sometimes associated with hatred of paresis and verbal or physical abuse directed at the paretic limb. Few studies or reports on this disorder are available in the literature, prompting the present case report of a patient with misoplegia and vascular dementia.

RESUMO. Pacientes com lesões cerebrais podem apresentar atitudes anormais em relação a seus déficits. Um exemplo é a misoplegia, uma aversão excessiva em relação ao membro com déficit, podendo estar associado a ódio à paralisia e maus tratos verbais ou físicos contra os membros paralisados. Ainda há poucos estudos e relatos sobre esse distúrbio na literatura, evidenciando a importância de um relato de caso de um paciente portador de misoplegia e demência vascular.

Recent advance of aquaporin-4 in neurodegenerative diseases / 中华神经科杂志

Neurodegenerative disease is a type of disease characterized by the progressive loss of neurons, the cause of which is not clear. Aquaporin-4 (AQP4) is a member of the aquaporin family, which plays an important role in maintaining water homeostasis in the brain. In recent years, researchers found that AQP4 has the functions of draining brain metabolic wastes and participating in material exchange through the glmphatic system. This review aims to summarize the current researches of AQP4 in the pathogenesis and progression of neurodegenerative diseases such as Parkinson′s disease and Alzheimer′s disease, and to propose future research directions.

Research progress of thioredoxin system / 国际生物医学工程杂志

The thioredoxin system is composed of thioredoxin (Trx), thioredoxin reductase (TR) and reduced nicotinamide adenine dinucleotide phosphate. Trx is an important antioxidant molecule that can resist cell death caused by various stresses and plays a prominent role in redox reactions. TR is a protein containing selenium (selenocysteine), mainly in three forms, i.e. TR1, TR2 and TR3. TR1 mainly distributed in the cytoplasm, TR2 mainly distributed in the mitochondria, and TR3 mainly distributed in the testes. TR can regulate cell growth and apoptosis. After the cell becomes cancerous, the expression of TR increases to promote cell growth and metastasis. Trx system is closely related to neurodegenerative diseases, parasitic infections, acquired immunodeficiency syndrome, rheumatoid arthritis, hypertension, myocarditis and so on. The Trx system can remove the reactive oxygen species (ROS) in the body, keep the inside and outside of the cell in a balanced state, and it interacts with the thioredoxin interacting protein (TXNIP), which plays an important role in the regulation of glucose metabolism and tumor treatment. The Trx system is an important target for drug treatment of many diseases. In this paper, the research progress of the thioredoxin system was reviewed.

Research Progress of CD4 / 中国医学科学院学报

Neurodegenerative diseases are associated with neuroinflammation,oxidative stress,and aging,which can lead to cognitive and motor dysfunctions.Recent studies suggest that the development of neurodegenerative diseases is related to adaptive immunity,in which CD4

Nucleocytoplasmic transport and neurodegenerative diseases / 药学学报

Nucleocytoplasmic transport is the basic cellular activity of eukaryotic cells, which plays a role in cell physiological and pathological processes. A large amount of evidences indicate that impaired nucleocytoplasmic trafficking has emerged as a mechanism contributing to the pathology of neurodegenerative diseases. The regulation of nucleocytoplasmic transport is crucial to elucidate the pathogenesis and intervention in the neurodegenerative diseases. This article summarizes the evidences in disturbed nucleocytoplasmic transport of neurodegenerative diseases in the past two decades, further explores the directions and provides a theoretical basis for the pathogenesis and drug targets in neurodegenerative diseases.

Phospholipid peroxidation: a key factor in "susceptibility" to neurodegenerative diseases / 药学学报

The biochemical integrity of the brain is necessary to maintain normal function. Oxidative damage is one of the mortal important reasons leading to the destruction of this integrity. The nervous system is enriched in phospholipid and polyunsaturated fatty acids (PUFAs). Due to the nature of high oxygen-consumption and rich lipids, brain is particularly vulnerable to oxidative damages. Phospholipid peroxidation is one of the results of imbalance in oxidation-antioxidant system. Once the antioxidant system is insufficient to resist oxidative damage, membrane phospholipids will be prone to free radical attack. Phospholipid peroxidation leads to a variety of toxic oxidation products, including membrane damage, mitochondrial dysfunction, rapid accumulation of amyloid, etc. Multiple proteins and nucleic acids can be covalently modified by peroxidation products, resulting in the loss of the protein functions, which eventually triggers programmed cell death and general neuroinflammation in brain, and ends up with an increased susceptibility to neurodegenerative diseases. Based on the knowledge of mechanisms of phospholipid peroxidation, this review focuses on the characteristics of phospholipid peroxidation as a key factor in the development of neurodegenerative diseases, in order to provide theoretical basis for targeted intervention of phospholipid peroxidation as a potential strategy to prevent neurodegenerative diseases.

Study on blood apolipoprotein as a potential biomarker of neurodegenerative diseases / 药学学报

Neurodegenerative diseases (ND) mainly include Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, ataxia, and other diseases. The number of patients with ND is increasing, but the proportion of patients who can be diagnosed and treated early is less than 30% and the cause of ND is still unclear. In order to intervene in the disease as early as possible, researchers are committed to finding biomarkers that facilitate the early diagnosis of ND. Among them, cerebrospinal fluid (CSF) closely reflects the composition of the extracellular space of the brain, and may be the most sensitive biomarker for evaluating ND. However, the method of taking cerebrospinal fluid is more complicated, and it is not a common method in primary care or elderly medical institutions for the treatment of ND patients. Imaging examinations are expensive and difficult to spread among the community. The peripheral blood collection is convenient and less traumatic, which is a potential early screening and follow-up method. There are many components in the blood for analysis and research. This article reviews the research progress of the changes of apolipoprotein in the blood of ND patients as markers.