TC and LDL-C are negatively correlated with bone mineral density in patients with osteoporosis.

OBJECTIVE: To investigate the relationships of multiple lipid metabolism indicators and bone turnover markers (BTMs) with bone mineral density (BMD) and osteoporosis, in order to identify high-risk populations. METHODS: A total of 380 patients were recruited and their general information was collected. Linear and logistic regression models were used to analyze the correlation of these indicators with BMD and osteoporosis. RESULTS: Lipid metabolism indices and BTMs exhibited varying degrees of positive or negative correlation with BMD. Elevated levels of triglycerides (r = -0.204, P = 0.004), total cholesterol (TC) (r = -0.244, P < 0.001), low-density lipoprotein cholesterol (LDL-C) (r = -0.256, P < 0.001), apoprotein B (r = -0.292, P < 0.001) and lipoprotein-associated phospholipase A2 (Lp-PLA2) (r = -0.221, P = 0.002) in women were associated with a reduction in BMD. This relationship persisted even after adjusting for confounding factors and in the subgroup analysis of elderly women. In males, TC (r = 0.159, P = 0.033), LDL-C (r = 0.187, P = 0.012), apoprotein B (r = 0.157, P = 0.035), and Lp-PLA2 (r = 0.168, P = 0.024) exhibited a positive correlation with BMD, while free fatty acid (FFA) (r = -0.153, P = 0.041) was negatively correlated with BMD. However, after adjusting for confounding factors, only FFA remained negatively correlated with BMD, which was not observed in the age subgroup analysis. Furthermore, elevated levels of TC and LDL-C in elderly women were positively associated with the risk of osteoporosis or low bone mass. CONCLUSION: Elevated levels of TC and LDL-C not only indicate a decrease in BMD in females but also positively correlate with the occurrence of osteoporosis and low bone mass in elderly females.

Therapeutic Effects of Mechanical Stress-Induced C2C12-Derived Exosomes on Glucocorticoid-Induced Osteoporosis Through miR-92a-3p/PTEN/AKT Signaling Pathway.

Introduction: Osteoporosis is a common bone disease in which the bone loses density and strength and is prone to fracture. Bone marrow mesenchymal stem cells (BMSCs) are important in bone-related diseases. Exosomes, as mediators of cell communication, have potential in cell processes. Previous studies have focused on muscle factors' regulation of bone remodeling, but research on exosomes is lacking. Methods:  In order to confirm the therapeutic effect of mechanically stimulated myocytes (C2C12) derived exosomes (Exosome-MS) on the Glucocorticoid-induced osteoporosis(GIOP) compared with unmechanically stimulated myocytes (C2C12) derived exosomes (Exosomes), we established a dexamethasone-induced osteoporosis model in vivo and in vitro. Cell viability and proliferation were assessed using CCK8 and EDU assays. Osteogenic potential was evaluated through Western blotting, real-time PCR, alkaline phosphatase activity assay, and alizarin red staining. Differential expression of miRNAs was determined by high-throughput sequencing. The regulatory mechanism of miR-92a-3p on cell proliferation and osteogenic differentiation via the PTEN/AKT pathway was investigated using real-time PCR, luciferase reporter gene assay, Western blotting, and immunofluorescence. The therapeutic effects of exosomes were evaluated in vivo using microCT, HE staining, Masson staining, and immunohistochemistry. Results:  In this study, we found that exosomes derived from mechanical stress had a positive impact on the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). Importantly, we demonstrated that miR-92a-3p mimics could reverse dexamethasone-induced osteoporosis in vitro and in vivo, indicating that mechanical stress-induced mouse myoblast-derived exosomes could promote osteogenesis and prevent the occurrence and progression of osteoporosis in mice through miR-92a-3p/PTEN/AKT signaling pathway. Conclusion:  Exosomes derived from mechanical stress-induced myoblasts can promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells through miR-92a-3p/PTEN/AKT signaling pathway, and can have a therapeutic effect on glucocorticoid-induced osteoporosis in mice in vivo.

Neuroprotective effect of chlorogenic acid on Parkinson's disease like symptoms through boosting the autophagy in zebrafish.

Parkinson's disease (PD) is characterized by both motor and non-motor symptoms, including hypokinesia, postural instability, dopaminergic (DA) neurons loss, and α-synuclein (α-syn) accumulation. A growing number of patients show negative responses towards the current therapies. Thus, preventative or disease-modifying treatment agents are worth to further research. In recent years, compounds extracted from natural sources become promising candidates to treat PD. Chlorogenic acid (CGA) is a phenolic compound appearing in coffee, honeysuckle, and eucommia that showed their potential as antioxidants and neuroprotectors. In this study, we investigated the anti-PD activity of CGA by testing its effect on 1-methyl-4-phenyl-1-1,2,3,6-tetrahydropyridine (MPTP) zebrafish model of PD. It was shown that CGA relieved MPTP-induced PD-like symptoms including DA neurons and blood vessel loss, locomotion reduction, and apoptosis events in brain. Moreover, CGA modulated the expression of PD- and autophagy-related genes (α-syn, lc3b, p62, atg5, atg7, and ulk1b), showing its ability to promote the autophagy which was interrupted in the PD pathology. The unblocked effect of CGA on autophagy was further verified in 6-hydroxydopamine (6-OHDA)-modeled SHSY5Y cells. Our findings indicated that CGA might relieve PD by boosting the autophagy in neuronal cells that makes CGA a potential candidate for anti-PD treatment.

Sleep disorder, Mediterranean diet, and all-cause and cause-specific mortality: a prospective cohort study.

BACKGROUND: There is a bidirectional effect between sleep disorders and Mediterranean diet (MED), but the joint effect of MED and sleep disorders on mortality is unclear. The aim of this study was to investigate whether there is a synergistic effect of adherence to MED and sleep disorders on all-cause and cause-specific mortality. METHODS: The study included 23,212 individuals in the National Health and Nutrition Examination Survey (NHANES) from 2005 to 2014. A 9-point evaluation score, alternative Mediterranean diet (aMED) index was used to assess adherence to MED. Sleep disorder and hours of sleep were assessed by structured questionnaires. Cox regression models were used to assess the relationship between sleep disorders, aMED and all-cause mortality, cause-specific mortality (cardiovascular-related death, cancer-related death). The interaction effect of sleep disorders with aMED on mortality was further assessed. RESULTS: Results showed that participants with lower aMED and presence of sleep disorders had significantly higher risk of all-cause mortality and cardiovascular-related mortality (HR, 2.16, 95% CI, 1.49-3.13, P < 0.0001; HR, 2.68, 95% CI, 1.58-4.54, P = 0.0003). A significant interaction effect was found between aMED and sleep disorders on cardiovascular mortality (p for interaction = 0.033). No significant interaction existed between aMED and sleep disorders on all-cause mortality (p for interaction = 0.184) and cancer-related mortality (p for interaction = 0.955). CONCLUSIONS: Poorer adherence to MED and sleep disorders synergistically increased long-term all-cause mortality and cardiovascular mortality in NHANES population.

Gut microbiota and cardiac arrhythmia.

One of the most prevalent cardiac diseases is cardiac arrhythmia, however the underlying causes are not entirely understood. There is a lot of proof that gut microbiota (GM) and its metabolites have a significant impact on cardiovascular health. In recent decades, intricate impacts of GM on cardiac arrythmia have been identified as prospective approaches for its prevention, development, treatment, and prognosis. In this review, we discuss about how GM and its metabolites might impact cardiac arrhythmia through a variety of mechanisms. We proposed to explore the relationship between the metabolites produced by GM dysbiosis including short-chain fatty acids(SCFA), Indoxyl sulfate(IS), trimethylamine N-oxide(TMAO), lipopolysaccharides(LPS), phenylacetylglutamine(PAGln), bile acids(BA), and the currently recognized mechanisms of cardiac arrhythmias including structural remodeling, electrophysiological remodeling, abnormal nervous system regulation and other disease associated with cardiac arrythmia, detailing the processes involving immune regulation, inflammation, and different types of programmed cell death etc., which presents a key aspect of the microbial-host cross-talk. In addition, how GM and its metabolites differ and change in atrial arrhythmias and ventricular arrhythmias populations compared with healthy people are also summarized. Then we introduced potential therapeutic strategies including probiotics and prebiotics, fecal microbiota transplantation (FMT) and immunomodulator etc. In conclusion, the GM has a significant impact on cardiac arrhythmia through a variety of mechanisms, offering a wide range of possible treatment options. The discovery of therapeutic interventions that reduce the risk of cardiac arrhythmia by altering GM and metabolites is a real challenge that lies ahead.

Mediterranean diet lowers all-cause and cardiovascular mortality for patients with metabolic syndrome.

A Mediterranean-style diet (MED) can promote people lengthen the span of life and avoid atherosclerotic cardiovascular disease (ASCVD) in primary prevention. Metabolic syndrome (MetS) can significantly reduce life expectancy and increase the risk of ASCVD. However, few studies have focused on the role of the Mediterranean diet in patients with MetS. Participants in the National Health and Nutrition Examination Survey (NHANES) with MetS (N = 8301) from 2007 to 2018 were examined. A 9-point evaluation scorewas used to measure the degree of adherence to the MED diet. In order to compare the various levels of adherence to the MED diet and the effects of the specific MED diet components on all-cause and cardiovascular mortality, Cox regression models were utilized. Among the 8301 participants with MetS, about 13.0% (1080 of 8301) died after a median follow-up of 6.3 years. In this study, participants with MetS with adherence to high-quality and moderate-quality Mediterranean diet were significantly associated with lower all-cause mortality as well as cardiovascular mortality during the follow-up period. Futhermore, in joint analysis of the Mediterranean diet and sedentary behavior or depression, we found that high-quality or moderate-quality Mediterranean diet could attenuate, even reverse the adverse effects of sedentary behavior and depression on all-cause and cardiovascular mortality in participants with MetS. Among the components of the MED diet, greater intakes of vegetables, legumes, nuts and high MUFA/SFA ratio were significantly associated with lower all-cause mortality and greater vegetables intake was significantly associated with lower cardiovascular mortality, while more red/processed meat intake was significantly associated with higher cardiovascular mortality in participants with MetS.

Causal association of genetically predicted urinary sodium-potassium ratio and upper urinary calculi.

The causality between the urinary sodium-potassium ratio and upper urinary calculi has not been clarified and easily affected by confounders. We performed two-sample and multivariable Mendelian randomization (MR) analysis to evaluate the potential causal role of the urinary sodium-potassium ratio in upper urinary calculi. Data of the urinary sodium-potassium ratio (N = 326,938), upper urinary calculi (N = 337,199), and confounding factors including BMI (N = 336,107), ever-smoke (N = 461,066), hypertension (N = 218,754), diabetes (N = 218,792), and alcohol intake frequency (N = 462,346) were obtained from the IEU OpenGWAS Project database. The inverse-variance weighted (IVW), weighted median, and MR-Egger methods were used to estimate MR effects. The MR-Egger intercept test, Cochran's Q test, MR-PRESSO, leave-one-out method, and funnel plot were used for sensitivity analysis. A causal relationship was found between the urinary sodium-potassium ratio and upper urinary calculi (OR = 1.008, 95% CI = 1.002-1.013, P = 0.011). FinnGen data supported this conclusion (OR = 2.864, 95% CI = 1.235-6.641, P = 0.014). The multivariable Mendelian randomization analysis result showed that after adjusting for the effects of five confounders, the urinary sodium-potassium ratio was still positively correlated with upper urinary calculi (OR = 1.005, 95% CI = 1.001-1.009, P = 0.012). This study demonstrated a positive causal association between the urinary sodium-potassium ratio and upper urinary calculi using MR analysis. Timely identification of changes in urine composition and dietary regulation of sodium and potassium intake could greatly reduce the incidence of future urinary calculi.

Mechanism of immune infiltration in synovial tissue of osteoarthritis: a gene expression-based study.

BACKGROUND: Osteoarthritis is a chronic degenerative joint disease, and increasing evidences suggest that the pathogenic mechanism involves immune system and inflammation. AIMS: The aim of current study was to uncover hub genes linked to immune infiltration in osteoarthritis synovial tissue using comprehensive bioinformatics analysis and experimental confirmation. METHODS: Multiple microarray datasets (GSE55457, GSE55235, GSE12021 and GSE1919) for osteoarthritis in Gene Expression Omnibus database were downloaded for analysis. Differentially expressed genes (DEGs) were identified using Limma package in R software, and immune infiltration was evaluated by CIBERSORT algorithm. Then weighted gene co-expression network analysis (WGCNA) was performed to uncover immune infiltration-associated gene modules. Protein-protein interaction (PPI) network was constructed to select the hub genes, and the tissue distribution of these genes was analyzed using BioGPS database. Finally, the expression pattern of these genes was confirmed by RT-qPCR using clinical samples. RESULTS: Totally 181 DEGs between osteoarthritis and normal control were screened. Macrophages, mast cells, memory CD4 T cells and B cells accounted for the majority of immune cell composition in synovial tissue. Osteoarthritis synovial showed high abundance of infiltrating resting mast cells, B cells memory and plasma cells. WGCNA screened 93 DEGs related to osteoarthritis immune infiltration. These genes were involved in TNF signaling pathway, IL-17 signaling pathway, response to steroid hormone, glucocorticoid and corticosteroid. Ten hub genes including MYC, JUN, DUSP1, NFKBIA, VEGFA, ATF3, IL-6, PTGS2, IL1B and SOCS3 were selected by using PPI network. Among them, four genes (MYC, JUN, DUSP1 and NFKBIA) specifically expressed in immune system were identified and clinical samples revealed consistent change of these four genes in synovial tissue retrieved from patients with osteoarthritis. CONCLUSION: A 4-gene-based diagnostic model was developed, which had well predictive performance in osteoarthritis. MYC, JUN, DUSP1 and NFKBIA might be biomarkers and potential therapeutic targets in osteoarthritis.

The therapeutic effect of capsaicin on oropharyngeal dysphagia: A systematic review and meta-analysis.

Objectives: Capsaicin is a specific agonist of TRPV1 (multimodal sensory receptor), which improves oropharyngeal dysphagia by increasing sensory input from the oropharynx and hypopharynx and by increasing repetitive stimulation of the cerebral cortex. The aim of this systematic review was to evaluate the therapeutic effect of capsaicin on swallowing disorders in stroke patients and the elderly. Method: We searched Medline, Embase, PubMed, and Cochrane Library databases. We used the Mesh terms search database to screen all clinical trials that complied with the inclusion criteria. Studies were subjected to literature screening, quality assessment, and data extraction to remove studies that did not meet the inclusion criteria. After literature screening, quality assessment, and data extraction, a systematic review and meta-analysis of the included study were performed. Results: This systematic review and meta-analysis were prospectively registered on PROSPERO under registration number CRD42022313958. Five high-quality randomized controlled trials were ultimately included. The results of our meta-analysis showed a more significant reduction in swallowing function score change in the capsaicin group compared to the control group [SMD = -1.30, 95% CI: (-2.35, -0.25), P = 0.01] and on the Water swallowing test the improvement was significantly higher in the capsaicin group [RR = 2.46, 95% CI: (1.73, 3.50), P < 0.0001]. Conclusions: Although the results of our meta-analysis showed that capsaicin improved swallowing function, most studies had an unclear bias and included few studies. More studies are needed to support this in the future. Systematic review registration: www.crd.york.ac.uk/prospero/display_record.php?RecordID=304061, identifier: 304061.

The Circular RNA circSKA3 Facilitates the Malignant Biological Behaviors of Medulloblastoma via miR-520 h/CDK6 Pathway.

Circular RNAs (circRNAs) are reported to participate in the development of diverse human malignancies. This work investigated the mechanism of circSKA3 in modulating medulloblastoma progression. A total of 15 cases of medulloblastoma were collected in this work. Daoy cells were used to construct cell models. The expression level of circSKA3, microRNA-520 h (miR-520 h), and cyclin-dependent kinase 6 (CDK6) mRNA in tissues or cells was examined by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was employed to detect CDK6 protein expression. CCK-8 experiment, Transwell assay, and flow cytometry were applied to detect the regulatory effects of circSKA3 on cell proliferation, migration, invasion, and cell cycle. Dual-luciferase reporter gene experiment was executed to determine the relationship between circSKA3 and miR-520 h, and between miR-520 h and CDK6. circSKA3 was remarkably up-modulated in medulloblastoma tissues. CircSKA3 depletion markedly suppressed Daoy cell viability, migration, invasion, and cell cycle progression. CircSKA3 overexpression induced the opposite effects. circSKA3 could decoyed miR-520 h, which targeted the 3' UTR of CDK6. circSKA3 expression in medulloblastoma tissues was negatively correlated with miR-520 h expression and positively correlated with CDK6 expression. "Rescue" experiments revealed that miR-520 h down-modulation or CDK6 overexpression remarkably counteracted the inhibitory effect of circSKA3 knockdown on Daoy cells. circSKA3 facilitates medulloblastoma progression through miR-520 h/CDK6.

A Promising Glycolysis- and Immune-Related Prognostic Signature for Glioblastoma.

BACKGROUND: Glioblastoma multiforme (GBM) is a malignant brain tumor with a poor prognosis. Aerobic glycolysis and an immunosuppressive microenvironment are potentially correlated with progression of GBM. However, the prognostic value of glycolysis-immune-related genes has not been studied in GBM. METHODS: Using GBM-related data downloaded from Chinese Glioma Genome Atlas database, the overlapped differentially expressed genes were identified between the GBM patients with a different glycolysis status and immune score, which had also undergone functional enrichment. Univariate Cox regression analysis and LASSO (least absolute shrinkage and selection operator) Cox regression analysis were used for risk score construction. Multivariate Cox regression analysis and survival analysis determined the independent prognostic factors. RESULTS: We found 277 overlapped differentially expressed genes between high glycolysis and low glycolysis, a high immune score and low immune score, and a combination of low glycolysis status and a low immune score and high glycolysis status and a high immune score. These were significantly enriched in 301 gene otology terms and 25 Kyoto Encyclopedia of Genes and Genomes pathways. Of these, 8 genes were found to be optimal for building a risk score. The risk score was an independent prognostic factor for GBM patients, and patients with a high score had a worse prognosis. Moreover, between the high- and low-risk GBM patients, 17 types of immune cells were differentially infiltrated, and 5 immune checkpoints were differentially expressed. CONCLUSIONS: The glycolysis-immune-related risk score using CACNG2, CSMD3, GABRA3, KCNIP2, KSR2, PTPRT, TNFRSF12A, and TNR was able to predict the prognosis of GBM patients relatively reliably.

Elevated stress hyperglycemia and the presence of intracranial artery stenosis increase the risk of recurrent stroke.

Background: Stress hyperglycemia has served as a reliable biomarker to predict poor outcomes after ischemic stroke. However, recent studies have reported some contrary conclusions. Different stroke subtypes may respond inconsistently to stress hyperglycemia. The progression of intracranial atherosclerotic stenosis (ICAS) is tightly related to hyperglycemia. Thus, this study aims to determine the relationship between stress hyperglycemia and recurrent stroke in ischemic stroke patients with or without intracranial atherosclerotic stenosis. Methods: This is a multicenter retrospective observational cohort study. Patients with acute minor ischemic stroke and eligible computed tomography and magnetic resonance imaging data were enrolled. The severity of stress hyperglycemia is measured by the stress hyperglycemia ratio (SHR). SHR was calculated based on fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) levels. The primary outcome was stroke recurrence during hospitalization. The interaction of SHR levels with the presence of ICAS on the primary outcome was investigated using univariable and multivariable Cox proportional hazards models. Restricted cubic splines were applied to determine the nonlinear relationship between SHR and primary outcome. A two-piecewise linear regression model was used to identify the threshold of SHR. Results: A total of 610 participants were included in the study. The average age of the patients was 61.4 ± 12.9 years old, and approximately 70% of participants were males. A total of 189 (30.98%) patients had ICAS. The patients were categorized into 3 groups based on the tertiles of SHR. Compared with the group with a lower SHR, a higher SHR was significantly associated with the risk of stroke recurrence in the ICAS group (hazard ratio [HR], 8.52, 95% confidence interval [CI], 3.16-22.96, P<0.001). When SHR was treated as a continuous variable, each 0.1-unit increase in SHR in the ICAS group was associated with a 1.63-fold increase in the risk of recurrence (HR, 1.63, 95% CI, 1.39-1.9, P<0.001) with a threshold of 0.75. FPG but not HbA1c was associated with stroke recurrence in ICAS patients (HR, 1.17, 95% CI, 1.08-1.26, P<0.001). Sensitive analyses showed consistent results after adjusting for previous diabetes mellitus, oral hypoglycemic agents and insulin injection. Conclusions: SHR represents a better biomarker to predict the risk of stroke recurrence in patients with ICAS than FPG and HbA1c regardless of previous diabetes mellitus. Trial registration: https://www.chictr.org.cn/showproj.aspx?proj=125817; Identifier, [ChiCTR2100046958].

Exosomes derived from platelet-rich plasma present a novel potential in alleviating knee osteoarthritis by promoting proliferation and inhibiting apoptosis of chondrocyte via Wnt/ß-catenin signaling pathway.

BACKGROUND: Platelet-rich plasma (PRP) provides a nonsurgical approach for treating osteoarthritis (OA). Exosomes that play vital roles in intercellular communication have been studied extensively. Here, we investigated the therapeutic potential and molecular mechanism of exosomes derived from PRP (PRP-Exos) in alleviating OA. METHODS: Exosomes derived from PRP(PRP-Exos) were isolated and purified using the exoEasy Maxi Kit and then identified and analyzed. Primary rabbit chondrocytes were isolated and treated with interleukin 1 beta (IL-1ß) to establish the OA model in vitro. Proliferation, migration, and apoptosis assays were measured and compared between PRP-Exos and activated PRP (PRP-As) to evaluate the therapeutic effects on OA. The mechanism involving the Wnt/ß-catenin signaling pathway was investigated by Western blot analysis. In vivo, we established animal knee OA model by surgery to compare the therapeutic effect of PRP-Exos and PRP-As. RESULTS: We successfully isolated and purified exosomes from PRP using the exoEasy Maxi Kit. We also isolated and identified chondrocytes from the New Zealand white rabbit and established the IL-1ß-induced OA model; meanwhile, PRP-Exos and PRP-As both inhibited the release of tumor necrosis factor-α(TNF-α) and there was no statistically significant difference between the two. In proliferation, migration, scratch assay, the promoting effect of PRP-Exos was significantly more better than PRP-As. Furthermore, PRP-Exos could significantly decreased apoptotic rate of OA chondrocyte compared with PRP-As. In Western blot analysis, the expression of ß-catenin, and RUNX2, Wnt5a were increased in IL-1ß-treated chondrocytes, but PRP-Exos and PRP-As could both reverse these changes, and the reversal effect of the former was better than the latter. In vivo, we found that both PRP-Exos and PRP-As displayed the progression of OA, and the effect of PRP-Exos was obviously better than PRP-As by chondrocyte count and Osteoarthritis Research Society International (OARSI) scoring system. CONCLUSION: The therapeutic effects of PRP-Exos on OA were similar or better compared with those of PRP-As in vitro or in vivo. PRP-Exos acting as carriers containing growth factors derived from PRP present a novel therapy for OA by activating the Wnt/ß-catenin signaling pathway.

MicroRNA-520b affects the proliferation of human glioblastoma cells by directly targeting cyclin D1.

Glioblastoma (GBM) represents one of most common tumors in humans. However, the biological processes and molecular mechanisms of GBM are still unclear. It is known that microRNA-520b (miR-520b) participates in the development of various tumor progressions. The present study was to evaluate the level of miR-520b in GBM tissues and cells. We further investigated the molecular mechanisms of miR-520b in U87 and U251 cell lines. Here, our data showed that the expression levels of miR-520b were significantly reduced in clinical GBM tissues and cell lines. Accordingly, the expression levels of cyclin D1 were significantly increased in clinical GBM tissues and cell lines. Ectopic expression of miR-520b in U87 and U251 cells resulted in decreased cell proliferation and enhanced cell apoptosis. Further study characterized the 3' untranslated region (3'-UTR) of cyclin D1 gene as a direct target of miR-520b in U87 and U251 cells as determined by luciferase reporter assays. In addition, ectopic expression of miR-520b led to the down-regulation of phosphorylated retinoblastoma (p-Rb, a downstream effector of cyclin D1), while the overexpression of cyclin D1 reversed the miR-520b-induced inhibition of p-Rb expression. In conclusion, this study highlights the importance of miR-520b in regulating the proliferation and apoptosis of GBM by directly targeting cyclin D1, and miR-520b may represent a potential therapeutic strategy for GBM.