Influence of quercetin on aging of bone marrow mesenchymal stem cells induced by microgravity / 中国药理学通报

Aim To investigate the effect of quercetin on the aging model of bone marrow mesenchymal stem cells established under microgravity. Methods Using 3D gyroscope, a aging model of bone marrow mesenchymal stem cells was constructed, and after receiving quercetin and microgravity treatment, the anti-aging effect of the quercetin was evaluated by detecting related proteins and oxidation indexes. Results Compared to the control group, the expressions of age-related proteins p21, pi6, p53 and RB in the microgravity group significantly increased, while the expressions of cyclin D1 and lamin B1 significantly decreased, with statistical significance (P<0.05). In the microgravity group, mitochondrial membrane potential significantly decreased (P<0.05), ROS accumulation significantly increased (P <0.05), SOD content significantly decreased and MDA content significantly increased (P<0.05). Compared to the microgravity group, the expressions of age-related proteins p21, pi6, p53 and RB in the quercetin group significantly decreased, while the expressions of cyclin D1 and lamin B1 significantly increased, with statistical significance (P<0.05). In the quercetin group, mitochondrial membrane potential significantly increased (P<0.05), ROS accumulation significantly decreased (P<0.05), SOD content significantly increased and MDA content significantly decreased (P<0.05). Conclusions Quercetin can resist oxidation, protect mitochondrial function and normal cell cycle, thus delaying the aging of bone marrow mesenchymal stem cells induced by microgravity.

Síndrome de Progeria Hutchinson-Gilford

El Síndrome de Progeria de Hutchinson- Gilford es una enfermedad que se caracteriza por el envejecimiento prematuro en niños, debido a una mutación en el gen de Lámina tipo A involucrado en la mitosis celular. En el presente trabajo, con el objetivo de dar difusión al conocimiento de esta enfermedad, se señalan los procesos involucrados en su desarrollo, así como los avances científicos y el alcance de nuevas ventanas terapéuticas. La revisión se realizó consultando artículos en español e inglés empleando los motores de búsqueda Pubmed y Google Académico. La actualización del personal de salud sobre las enfermedades genéticas congénitas es de vital importancia para mejorar su detección, atención y manejo.

Hutchinson-Gilford Progeria Syndrome is a disease characterized by premature aging in children, due to a mutation in the Lamina type A, gene involved in cellular mitosis. In the present work, with the aim of spreading the knowledge of this disease, the processes involved in its development, the scientific advances, and the scope of new therapeutic treatments were summarized. The review was carried out by consulting articles in Spanish and English using the Pubmed and Google Academic search engines. The updating of health personnel on congenital genetic diseases is of vital importance to improve their detection, care and management.

A Síndrome de Hutchinson-Gilford Progeria é uma doença caracterizada pelo envelhecimento prematuro em crianças, devido a uma mutação no gene lamina tipo A envolvido na mitose celular. No presente trabalho, como objetivo de divulgar o conhecimento desta doença, são indicados os processos envolvidos no seu desenvolvimento, bem como os avanços científicos e o âmbito de novas janelas terapêuticas. A análise foi realizada através da consulta de artigos em espanhol e inglês utilizando os motores de busca pubmed e Google Scholar. A atualização do pessoal de saúde sobre doenças genéticas congénitas é de importância vital para melhorar a sua deteção, cuidados e gestão.

Isolated Left Ventricular Apical Hypoplasia without Lamin A/C Gene Mutation

Abstract Isolated left ventricular apical hypoplasia is a rare cardiomyopathy, with a broad range of clinical presentations. Since this entity was already described in association with osteomuscular diseases, mutation in the Lamin A/C gene has been regarded as a possible cause of this disease. This study describes the case of an asymptomatic teenager with isolated left ventricular apical hypoplasia and arthrogriposis but with no mutations in the entire Lamin A/C gene.

Effect of Aqueous Extract of Trametes robiniophila on Proliferation of Human Prostate Cancer VCaP Cells / 中国实验方剂学杂志

ObjectiveTo investigate effect of aqueous extract of Trametes robiniophila （TRM，Huaier） on autophagy of human prostate cancer VCaP cells and Lamin B1 expression， so as to uncover its role in the proliferation of VCaP cells. MethodThe inhibitory effect of 0， 2， 4， 6， 8， 10 g·L-1 TRM aqueous extract on the proliferation of human prostate cancer VCaP cells at different time points were determined by cell counting kit-8 （CCK-8） assay. Acridine orange staining was conducted for analyzing the effect of TRM aqueous extract on the formation of autolysosomes in VCaP cells. After medication， the expression of microtubule-associated protein Ⅰ light chain 3 （LC3）， autophagy-related protein 3 （Atg3）， autophagy-related protein 5 （Atg5）， and autophagy-related protein 7 （Atg7） in VCaP cells were detected by Western blot. The effect of TRM aqueous extract alone and its combination with autophagy inhibitor bafilomycin A1 on the proliferation of VCaP cells were assayed by CCK-8 assay. RNA interference technology was used to explore the role of Lamin B1 in anti-proliferation of VCaP cells by TRM. ResultCompared with the blank group， TRM aqueous extract inhibited the proliferation of human prostate cancer VCaP cells in a time- and concentration-dependent manner （P<0.01）. Acridine orange staining showed that TRM aqueous extract promoted the formation of autolysosomes in VCaP cells. As revealed by Western blotting， TRM aqueous extract up-regulated the expression levels of LC3-Ⅱ， Atg3， Atg5， and Atg7 in contrast to those in the blank group （P<0.05）. All these indicated that TRM aqueous extract induced the autophagy of VCaP cells. In addition， autophagy inhibition impaired the sensitivity of VCaP cells to TRM aqueous extract （P<0.05）. The comparison with the blank group showed that TRM aqueous extract inhibited Lamin B1 protein expression in VCaP cells （P<0.01）， which in turns weakened the sensitivity of VCaP cells to TRM aqueous extract. ConclusionTRM aqueous extract inhibited the proliferation of human prostate cancer VCaP cells possibly by inducing autography and down-regulating Lamin B1 expression. This study has provided a theoretical basis for the clinical application of TRM.

Nuclear peripheral chromatin-lamin B1 interaction is required for global integrity of chromatin architecture and dynamics in human cells

The eukaryotic genome is folded into higher-order conformation accompanied with constrained dynamics for coordinated genome functions. However, the molecular machinery underlying these hierarchically organized three-dimensional (3D) chromatin architecture and dynamics remains poorly understood. Here by combining imaging and sequencing, we studied the role of lamin B1 in chromatin architecture and dynamics. We found that lamin B1 depletion leads to detachment of lamina-associated domains (LADs) from the nuclear periphery accompanied with global chromatin redistribution and decompaction. Consequently, the inter-chromosomal as well as inter-compartment interactions are increased, but the structure of topologically associating domains (TADs) is not affected. Using live-cell genomic loci tracking, we further proved that depletion of lamin B1 leads to increased chromatin dynamics, owing to chromatin decompaction and redistribution toward nucleoplasm. Taken together, our data suggest that lamin B1 and chromatin interactions at the nuclear periphery promote LAD maintenance, chromatin compaction, genomic compartmentalization into chromosome territories and A/B compartments and confine chromatin dynamics, supporting their crucial roles in chromatin higher-order structure and chromatin dynamics.

Research progress on lamin and Hutchinson-Gilford progeria syndrome / 中华老年医学杂志

Many studies have shown that abnormal expression and modification of lamin are closely related to aging.Hutchinson-Gilford progeria syndrome(HGPS)is a rare and severe premature aging disease caused by mutations in the gene encoding nuclear envelope proteins of A-type lamins (LMNA).The pathogenesis of HGPS is similar to the aging process of normal individuals,thus research on HGPS will be helpful for understanding the mechanisms of senescence and developing antiaging drugs.This paper reviews recent advances in lamin and the pathogenesis and treatment of HGPS,in order to provide a reference for further basic and clinical research on HGPS.

Regulation of lamin a/c in epigenetic pharmacology of stem cells / 中国药理学通报

Lamin A/C is I he main component of nuclear lamina, ami its important role of cpigenetie pharmacology in si em cells has been recognizcd in recent years, lmin A/C hus been reported to maintain the nuclear homeostasis, protect the chromatin, anil act as a reactor for mcchanical stimulation of the nucleus to transmit mechanical signals. Besides, Lamin A/C has been discovered to participate directly or indirectly in the transcriptional regulation of the genes. This article aims to review the regulation of Lamin A/C in stem cells by mechanical signals of microenvi- ronment, and live effect of Lamin A/C on chromosome modification and genes regulation.

Functions of lamin B1 and the new progress of its roles in neurological diseases and tumors / 生物工程学报

Lamin B1 is one of the essential members of the nuclear lamina protein family. Its main function is to maintain the integrity of nuclear skeleton, as well as to participate in the cell proliferation and aging by affecting the chromosome distribution. gene expression, and DNA damage repair. The abnormal expression of lamin B1 is related to certain diseases, including neurological diseases [e.g. neural tube defects (NDTs), adult-onset autosomal dominant leukodystrophy (ADLD)] and tumors (e.g. pancreatic cancer). It is also a potential tumor marker as well as drug target. Further research on lamin B1 will help people understand the molecular mechanism of the emergence and development of neural system diseases and tumors, and define a new future in drug target.

Differential stem cell aging kinetics in Hutchinson-Gilford progeria syndrome and Werner syndrome

Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) are two of the best characterized human progeroid syndromes. HGPS is caused by a point mutation in lamin A (LMNA) gene, resulting in the production of a truncated protein product-progerin. WS is caused by mutations in WRN gene, encoding a loss-of-function RecQ DNA helicase. Here, by gene editing we created isogenic human embryonic stem cells (ESCs) with heterozygous (G608G/+) or homozygous (G608G/G608G) LMNA mutation and biallelic WRN knockout, for modeling HGPS and WS pathogenesis, respectively. While ESCs and endothelial cells (ECs) did not present any features of premature senescence, HGPS- and WS-mesenchymal stem cells (MSCs) showed aging-associated phenotypes with different kinetics. WS-MSCs had early-onset mild premature aging phenotypes while HGPS-MSCs exhibited late-onset acute premature aging characterisitcs. Taken together, our study compares and contrasts the distinct pathologies underpinning the two premature aging disorders, and provides reliable stem-cell based models to identify new therapeutic strategies for pathological and physiological aging.

Early-Onset LMNA-Associated Muscular Dystrophy with Later Involvement of Contracture

BACKGROUND AND PURPOSE: The early diagnosis of LMNA-associated muscular dystrophy is important for preventing sudden arrest related to cardiac conduction block. However, diagnosing early-onset Emery-Dreifuss muscular dystrophy (EDMD) with later involvement of contracture and limb-girdle muscular dystrophy type 1B is often delayed due to heterogeneous clinical presentations. We aimed to determine the clinical features that contribute to a delayed diagnosis. METHODS: We reviewed four patients who were recently diagnosed with LMNA-associated muscular dystrophy by targeted exome sequencing and who were initially diagnosed with nonspecific or other types of muscular dystrophy. RESULTS: Certain clinical features such as delayed contracture involvement and calf hypertrophy were found to contribute to a delayed diagnosis. Muscle biopsies were not informative for the diagnosis in these patients. CONCLUSIONS: Genetic testing of single or multiple genes is useful for confirming a diagnosis of LMNA-associated muscular dystrophy. Even EDMD patients could experience the later involvement of contracture, so clinicians should consider early genetic testing for patients with undiagnosed muscular dystrophy or laminopathy.

Time-dependent proteomic and genomic alterations in Toll-like receptor-4-activated human chondrocytes: increased expression of lamin A/C and annexins

Activation of Toll-like receptor-4 (TLR-4) in articular chondrocytes increases the catabolic compartment and leads to matrix degradation during the development of osteoarthritis. In this study, we determined the proteomic and genomic alterations in human chondrocytes during lipopolysaccharide (LPS)-induced inflammation to elucidate the underlying mechanisms and consequences of TLR-4 activation. Human chondrocytes were cultured with LPS for 12, 24, and 36 h to induce TLR-4 activation. The TLR-4-induced inflammatory response was confirmed by real-time PCR analysis of increased interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) expression levels. In TLR-4-activated chondrocytes, proteomic changes were determined by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-mass spectroscopy analysis, and genomic changes were determined by microarray and gene ontology analyses. Proteomics analysis identified 26 proteins with significantly altered expression levels; these proteins were related to the cytoskeleton and oxidative stress responses. Gene ontology analysis indicated that LPS treatment altered specific functional pathways including ‘chemotaxis’, ‘hematopoietic organ development’, ‘positive regulation of cell proliferation’, and ‘regulation of cytokine biosynthetic process’. Nine of the 26 identified proteins displayed the same increased expression patterns in both proteomics and genomics analyses. Western blot analysis confirmed the LPS-induced increases in expression levels of lamin A/C and annexins 4/5/6. In conclusion, this study identified the time-dependent genomic, proteomic, and functional pathway alterations that occur in chondrocytes during LPS-induced TLR-4 activation. These results provide valuable new insights into the underlying mechanisms that control the development and progression of osteoarthritis.

A novel c.563 T>G, p.L189R lamin A/C mutation in identical twins with dilated cardiomyopathy

No abstract available.

Progeria: A rare genetic premature ageing disorder.

Progeria is characterized by clinical features that mimic premature ageing. Although the mutation responsible for this syndrome has been deciphered, the mechanism of its action remains elusive. Progeria research has gained momentum particularly in the last two decades because of the possibility of revealing evidences about the ageing process in normal and other pathophysiological conditions. Various experimental models, both in vivo and in vitro, have been developed in an effort to understand the cellular and molecular basis of a number of clinically heterogeneous rare genetic disorders that come under the umbrella of progeroid syndromes (PSs). As per the latest clinical trial reports, Lonafarnib, a farnesyltranferase inhibitor, is a potent ‘drug of hope’ for Hutchinson-Gilford progeria syndrome (HGPS) and has been successful in facilitating weight gain and improving cardiovascular and skeletal pathologies in progeroid children. This can be considered as the dawn of a new era in progeria research and thus, an apt time to review the research developments in this area highlighting the molecular aspects, experimental models, promising drugs in trial and their implications to gain a better understanding of PSs.

Avaliação bioquímica, hormonal e genética das famílias de duas pacientes brasileiras portadoras de lipodistrofia parcial familiar tipo 2 / Biochemical, hormonal and genetic evaluation of the families of two Brazilian patients with type 2 familial partial lipodystrophy

OBJETIVO: Avaliar características clínicas, bioquímicas, hormonais e genéticas de familiares de duas pacientes portadoras de lipodistrofia parcial familiar (FPLD) tipo 2. MATERIAIS E MÉTODOS: Foram avaliados 50 indivíduos de duas famílias brasileiras não relacionadas a partir de dois propósitos com fenótipo de FPLD. Foi confirmada a mutação no éxon 8 do gene LMNA em 18 destes e identificada a substituição em heterozigose no códon 482, resultando na mutação p.R482W. Com base na presença ou não da mutação, os indivíduos foram separados em afetados e não afetados, e comparados quanto a parâmetros clínicos, bioquímicos e hormonais. RESULTADOS: Indivíduos afetados tiveram 2,8 vezes mais chance de manifestar diabetes e síndrome dos ovários policísticos (SOP), maiores índices HOMA-IR, níveis de insulina e de triglicérides e menores níveis de leptina. Essas alterações precedem o início do diabetes, pois foram evidenciadas nos afetados diabéticos e não diabéticos. Foi constatada heterogeneidade fenotípica entre os portadores da mutação. CONCLUSÃO: A mutação no gene da LMNA é determinante de alterações clínicas, bioquímicas e hormonais que implicam deterioração metabólica nos portadores da mutação.

OBJECTIVE: To evaluate clinical, biochemical, hormonal and genetic characteristics of relatives of two patients with familial partial lipodystrophy (FPLD) type 2. MATERIALS AND METHODS: Fifty subjects, members of two non-related Brazilian families from two different probands with FPLD phenotype, were evaluated. A mutation in exon 8 of LMNA gene was confirmed in 18 of them, and a heterozygous substitution at codon 482 was identified, predicting a p.R482W mutation. Based on the presence or absence of the mutation, subjects were classified in affected and unaffected, and compared in terms of clinical, biochemical and hormonal parameters. RESULTS: Affected subjects were 2.8 times more likely to manifest diabetes and PCOS, higher HOMA-IR, insulin and triglyceride levels, and lower levels of leptin. These changes preceded the onset of diabetes, because they were observed in diabetic and non-diabetic affected patients. A phenotypic heterogeneity was found among mutation carriers. CONCLUSION: A mutation in the LMNA gene is a determinant of clinical, biochemical and hormonal changes that imply in metabolic deterioration in mutation carriers.

Liposuction in body-shaping treatment for Kobberling-Dunnigan syndrome / Lipoaspiração no tratamento do contorno corporal na síndrome de Kobberling-Dunnigan

INTRODUCTION: Kobberling-Dunnigan syndrome is characterized by a series of alterations in metabolism and body fat distribution. Body dysmorphism and the Cushingoid appearance may have negative social and psychological impacts on the patient, including difficulty with social adaptation. In such cases, liposuction is used with the aim of improving body contour, with consequent improvements in self-esteem. The results are long lasting when body weight is maintained. CASE REPORT: Liposuction of the back, abdomen, and cervical regions in a woman with Kobberling-Dunnigan syndrome led to significant improvement in her body shape, and the results were maintained nine months postoperatively.

INTRODUÇÃO: A síndrome de Kobberling-Dunnigan caracteriza-se por um conjunto de alterações metabólicas e de distribuição da gordura corporal. O dismorfismo corporal e o aspecto cushingoide trazem repercussões psicológicas e sociais, com dificuldade de ajuste social. Nesses casos, a lipoaspiração é empregada com o objetivo de melhorar o contorno corporal e, consecutivamente, as alterações de autoestima. Os resultados apresentam-se duradouros quando há manutenção do peso. RELATO DO CASO: Paciente do sexo feminino, portadora de síndrome de Kobberling-Dunnigan, submetida a lipoaspiração de dorso, abdome e região cervical, com melhora significativa do contorno corporal e manutenção dos resultados nove meses após a operação.

Lamins, laminopathies and disease mechanisms: Possible role for proteasomal degradation of key regulatory proteins.

Lamins are major structural proteins of the nucleus and are essential for nuclear integrity and organization of nuclear functions. Mutations in the human lamin genes lead to highly degenerative genetic diseases that affect a number of different tissues such as muscle, adipose or neuronal tissues, or cause premature ageing syndromes. New findings on the role of lamins in cellular signalling pathways, as well as in ubiquitin-mediated proteasomal degradation, have given important insights into possible mechanisms of pathogenesis.

Pleiotropic consequences of misexpression of the developmentally active and stress-inducible non-coding hsrω gene in Drosophila.

The non-coding hsrω gene of Drosophila melanogaster is expressed in nearly all cell types and developmental stages. However, in the absence of conventional mutant alleles of this gene, its developmental functions remain largely unknown. In the present study, we used a variety of GAL4 drivers to overexpress or ablate this gene’s transcripts in specific tissues and examined the developmental consequences thereof. Our results show that a balanced expression of these non-coding transcripts is critical for survival and normal development in all the tissue types tested, since any change in cellular levels of these transcripts in a given cell type generally has detrimental effects, with extreme cases resulting in organismal lethality, although in a few cases the misexpression of these transcripts also suppresses the mutant phenotype due to other genetic conditions. Evidence is also presented for existence of a new spliced variant of the hsrω-n nuclear transcript. Following the RNAi-mediated down-regulation of hsrω transcripts, the omega speckles disappear so that the nucleoplasmic hnRNPs get diffusely distributed, while upregulation of these transcripts results in greater sequestration of these proteins into omega speckle clusters; either of these conditions would affect activities of the hnRNPs and other hsrω-RNA interacting proteins, which is likely to have cascading consequences. The present findings, together with our earlier observations on effects of altered levels of the hsrω transcripts on induced apoptosis and expanded polyQ-mediated neurodegeneration, further confirm that ncRNA species like the hsrω, far from being evolutionary hangovers, provide critical information for important functions in normal cells.

Association of progerin-interactive partner proteins with lamina proteins:Mel18 is associated with emerin in HGPS / 北京大学学报(医学版)

Objective :The Hutchinson-Gilford progeria syndrome (HGPS or progeria) is a childhood disorder with features of premature aging and is caused by mutations in the lamin A gene resulting in the production of an abnormal protein, termed progerin. To investigate the underlying pathogenic mecha-nism, we studied the nuclear co-localization and association of progerin interactive partner proteins (PIPPs) with lamina proteins. Methods:Both wild-type (WT) and progeria fibroblasts were studied by various methods including eonfocal microscopy, immunopreeipitation and Western blot. Results:All PIPPs discovered so-far co-localized with lamin A/C. In addition, the PIPPs were selectively associated with lamina proteins. An increased immunofluorescent staining signal was found for Mel18 in HGPS as com-pared to WT cells. An association of Me118 with emerin was observed in HGPS, but not in WT cells.Conclusion: Based on these findings, we propose that PIPPs, along with associated lamina proteins may form a pathogenic progerin-containing protein complex.

Atypical generalized lipoatrophy and severe insulin resistance due to a heterozygous LMNA p. T10I mutation / Lipoatrofia generalizada atípica e resistência insulínica grave devido à mutação p. T10I em heterozigose no gene LMNA

Lipodystrophies are a group of heterogeneous disorders characterized by the loss of adipose tissue and metabolic complications. The main familial forms of lipodystrophy are Congenital Generalized Lipodystrophy and Familial Partial Lipodystrophy (FPLD). FPLD may result from mutations in the LMNA gene. Besides FPLD, mutations in LMNA have been shown to be responsible for other inherited diseases called laminopathies. Here we describe the case of a 15-year-old girl who was referred to our service due to diabetes mellitus and severe hypertriglyceridemia. Physical examination revealed generalized loss of subcutaneous fat, confirmed by DEXA (total body fat 8.6 percent). As the patient presented with pubertal-onset of generalized lipodystrophy and insulin resistance, molecular analysis of the LMNA gene was performed. We identified a heterozygous substitution in exon 1 (c.29C>T) predicting a p.T10I mutation. In summary, we describe an atypical phenotype of lipodistrophy associated with a de novo appearance of the p.T10I mutation in LMNA gene.

As lipodistrofias são um grupo heterogêneo de doenças caracterizadas por perda de tecido adiposo e complicações metabólicas. As formas hereditárias mais importantes de lipodistrofias são: lipodistrofia congênita generalizada e lipodistrofia parcial familiar (LDPF). LDPF resulta de mutações no gene LMNA que codificam as lâminas tipo A. Além da LDPF, mutações no gene LMNA são responsáveis por outras doenças hereditárias, denominadas laminopatias. Descrevemos o caso de uma paciente de 15 anos de idade encaminhada por diabetes melito e hipertrigliceridemia grave. Ao exame físico, apresentava perda generalizada de gordura subcutânea que foi confirmada por DEXA (gordura corporal total 8,6 por cento). Como a paciente apresentava perda de gordura de início na puberdade e resistência insulínica, foi realizada análise molecular do gene LMNA. Identificamos uma substituição em heterozigose no éxon 1 (c.29C>T), resultando na mutação p.T10I. Em sumário, um caso de fenótipo atípico de lipodistrofia generalizada devido à mutação de novo p.T10I no gene LMNA é descrito.

The Pathogenic Mechanisms and Therapeutic Strategies of Hutchinson-Gilford Progeria Syndrome / 生物化学与生物物理进展

Hutchinson-Gilford progeria syndrome (HGPS) is an early onset severe premature aging disorder due to a point mutation in LMNA gene which encodes nuclear lamin A/C. The mutation activates a cryptic splice site within exon 11 of LMNA, resulting in a 50-amino acid in-frame deletion in prelamin A. However, it is not clear how the mutation in a structural protein under the nuclear envelope could give rise to premature aging phenotypes. Recent studies showed that various abnormalities have been found in nuclear structures and functions of HGPS cells, mainly including progerin accumulation and nuclear morphology abnormalities, altered nuclear mechanical properties, changes of histone methylation patterns and epigenetic control, gene misregulation, p53 signalling activation, and increased genomic instability. Two hypotheses recently emerged in the explanation of the pathogenic mechanisms contributing to HGPS. No effective clinical intervention has been developed so far for HGPS. Several fascinating therapeutic strategies have recently been provided, such as farnesyltransferase inhibitors, antisense oligonucleotides and RNA interference. HGPS has been considered to be a model for studying the mechanisms responsible for normal aging. This study will help to elucidate the physiological functions of lamin A and nuclear envelope, together with their roles in normal aging process and diseases.