Choice of baseline hazards in joint modeling of longitudinal and time-to-event cancer survival data.

Longitudinal time-to-event analysis is a statistical method to analyze data where covariates are measured repeatedly. In survival studies, the risk for an event is estimated using Cox-proportional hazard model or extended Cox-model for exogenous time-dependent covariates. However, these models are inappropriate for endogenous time-dependent covariates like longitudinally measured biomarkers, Carcinoembryonic Antigen (CEA). Joint models that can simultaneously model the longitudinal covariates and time-to-event data have been proposed as an alternative. The present study highlights the importance of choosing the baseline hazards to get more accurate risk estimation. The study used colon cancer patient data to illustrate and compare four different joint models which differs based on the choice of baseline hazards [piecewise-constant Gauss-Hermite (GH), piecewise-constant pseudo-adaptive GH, Weibull Accelerated Failure time model with GH & B-spline GH]. We conducted simulation study to assess the model consistency with varying sample size (N = 100, 250, 500) and censoring (20 %, 50 %, 70 %) proportions. In colon cancer patient data, based on Akaike information criteria (AIC) and Bayesian information criteria (BIC), piecewise-constant pseudo-adaptive GH was found to be the best fitted model. Despite differences in model fit, the hazards obtained from the four models were similar. The study identified composite stage as a prognostic factor for time-to-event and the longitudinal outcome, CEA as a dynamic predictor for overall survival in colon cancer patients. Based on the simulation study Piecewise-PH-aGH was found to be the best model with least AIC and BIC values, and highest coverage probability(CP). While the Bias, and RMSE for all the models showed a competitive performance. However, Piecewise-PH-aGH has shown least bias and RMSE in most of the combinations and has taken the shortest computation time, which shows its computational efficiency. This study is the first of its kind to discuss on the choice of baseline hazards.

Targeting YAP/TAZ mechanosignaling to ameliorate stiffness-induced Schlemm's canal cell pathobiology.

Pathological alterations in the biomechanical properties of the Schlemm's canal (SC) inner wall endothelium and its immediate vicinity are strongly associated with ocular hypertension in glaucoma due to decreased outflow facility. Specifically, the underlying trabecular meshwork is substantially stiffer in glaucomatous eyes compared with that from normal eyes. This raises the possibility of a critical involvement of mechanotransduction processes in driving SC cell dysfunction. Yes-associated protein (YAP) has emerged as a key contributor to glaucoma pathogenesis. However, the molecular underpinnings of SC cell mechanosignaling via YAP and transcriptional coactivator with PDZ-binding motif (TAZ) in response to glaucomatous extracellular matrix (ECM) stiffening are not well understood. Using a novel biopolymer hydrogel that facilitates dynamic and reversible stiffness tuning, we investigated how ECM stiffening modulates YAP/TAZ activity in primary human SC cells, and whether disruption of YAP/TAZ mechanosignaling attenuates SC cell pathobiology and increases ex vivo outflow facility. We demonstrated that ECM stiffening drives pathologic YAP/TAZ activation and cytoskeletal reorganization in SC cells, which was fully reversible by matrix softening in a distinct time-dependent manner. Furthermore, we showed that pharmacologic or genetic disruption of YAP/TAZ mechanosignaling abrogates stiffness-induced SC cell dysfunction involving altered cytoskeletal and ECM remodeling. Finally, we found that perfusion of the clinically used, small molecule YAP/TAZ inhibitor verteporfin (without light activation) increases ex vivo outflow facility in normal mouse eyes. Collectively, our data provide new evidence for a pathologic role of aberrant YAP/TAZ mechanosignaling in SC cell dysfunction and suggest that YAP/TAZ inhibition has therapeutic value for treating ocular hypertension in glaucoma.NEW & NOTEWORTHY Pathologically altered biomechanical properties of the Schlemm's canal (SC) inner wall microenvironment were recently validated as the cause for increased outflow resistance in ocular hypertensive glaucoma. However, the involvement of specific mechanotransduction pathways in these disease processes is largely unclear. Here, we demonstrate that Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) are central regulators of glaucoma-like SC cell dysfunction in response to extracellular matrix stiffening and that targeted disruption of YAP/TAZ mechanosignaling attenuates SC cell pathobiology and enhances outflow function.

Association between ADHD and vision problems. A systematic review and meta-analysis.

AIM: To conduct the first systematic review and meta-analysis assessing whether attention-deficit/hyperactivity disorder (ADHD) is associated with disorders of the eye, and/or altered measures of visual function. METHOD: Based on a pre-registered protocol (PROSPERO: CRD42021256352), we searched PubMed, Web of Knowledge/Science, Ovid Medline, Embase and APA PsycINFO up to 16th November 2021, with no language/type of document restrictions. We included observational studies reporting at least one measure of vision in people of any age meeting DSM/ICD criteria for ADHD and in people without ADHD; or the prevalence of ADHD in people with and without vision disorders. Study quality was assessed with the Appraisal tool for Cross-Sectional Studies (AXIS). Random effects meta-analyses were used for data synthesis. RESULTS: We included 42 studies in the narrative synthesis and 35 studies in the meta-analyses (3,250,905 participants). We found meta-analytic evidence of increased risk of astigmatism (OR = 1.79 [CI: 1.50, 2.14]), hyperopia and hypermetropia (OR = 1.79 [CI: 1.66, 1.94]), strabismus (OR = 1.93 [CI: 1.75, 2.12]), unspecified vision problems (OR = 1.94 [CI: 1.38, 2.73]) and reduced near point of convergence (OR = 5.02 [CI: 1.78, 14.11]); increased lag (Hedge's g = 0.63 [CI: 0.30, 0.96]) and variability (Hedge's g = 0.40 [CI: 0.17, 0.64]) of the accommodative response; and increased self-reported vision problems (Hedge's g = 0.63 [CI: 0.44, 0.82]) in people with ADHD compared to those without ADHD (with no significant heterogeneity). We also found meta-analytic evidence of no differences between people with and without ADHD on retinal nerve fiber layer thickness (Hedge's g = -0.19 [CI: -0.41, 0.02]) and refractive error (Hedge's g = 0.08 [CI: -0.26, 0.42]) (with no significant heterogeneity). DISCUSSION: ADHD is associated with some self-reported and objectively ascertained functional vision problems, but not with structural alterations of the eye. Further studies should clarify the causal relationship, if any, between ADHD and problems of vision. TRIAL REGISTRATION: PROSPERO registration: CRD42021256352.

Association between Autism Spectrum Disorder (ASD) and vision problems. A systematic review and meta-analysis.

AIM: To conduct a systematic review and meta-analysis assessing whether vision and/or eye disorders are associated with Autism Spectrum Disorder (ASD). METHOD: Based on a pre-registered protocol (PROSPERO: CRD42022328485), we searched PubMed, Web of Knowledge/Science, Ovid Medline, Embase and APA PsycINFO up to 5th February 2022, with no language/type of document restrictions. We included observational studies 1) reporting at least one measure of vision in people of any age with a diagnosis of ASD based on DSM or ICD criteria, or ADOS; or 2) reporting the prevalence of ASD in people with and without vision disorders. Study quality was assessed with the Appraisal tool for Cross-Sectional Studies (AXIS). Random-effects meta-analyses were used for data synthesis. RESULTS: We included 49 studies in the narrative synthesis and 46 studies in the meta-analyses (15,629,159 individuals distributed across multiple different measures). We found meta-analytic evidence of increased prevalence of strabismus (OR = 4.72 [95% CI: 4.60, 4.85]) in people with versus those without ASD (non-significant heterogeneity: Q = 1.0545, p = 0.7881). We also found evidence of increased accommodation deficits (Hedge's g = 0.68 [CI: 0.28, 1.08]) (non-significant heterogeneity: Q = 6.9331, p = 0.0741), reduced peripheral vision (-0.82 [CI: -1.32, -0.33]) (non-significant heterogeneity: Q = 4.8075, p = 0.4398), reduced stereoacuity (0.73 [CI: -1.14, -0.31]) (non-significant heterogeneity: Q = 0.8974, p = 0.3435), increased color discrimination difficulties (0.69 [CI: 0.27,1.10]) (non-significant heterogeneity: Q = 9.9928, p = 0.1890), reduced contrast sensitivity (0.45 [CI: -0.60, -0.30]) (non-significant heterogeneity: Q = 9.9928, p = 0.1890) and increased retinal thickness (=0.29 [CI: 0.07, 0.51]) (non-significant heterogeneity: Q = 0.8113, p = 0.9918) in ASD. DISCUSSION: ASD is associated with some self-reported and objectively measured functional vision problems, and structural alterations of the eye, even though we observed several methodological limitations in the individual studies included in our meta-analyses. Further research should clarify the causal relationship, if any, between ASD and problems of vision during early life. PROSPERO REGISTRATION: CRD42022328485.

A Mechanistic Review of Methotrexate and Celecoxib as a Potential Metronomic Chemotherapy for Oral Squamous Cell Carcinoma.

The combination of low-dose methotrexate and celecoxib as metronomic chemotherapy (MCT) is a novel therapy, believed to act by modulating the immune response, inhibiting angiogenesis and its cytotoxic action, though the exact mechanism of action is unclear. Clinically, MCT was found to be very effective in delaying tumor progression in patients with head and neck squamous cell carcinoma in both curative and palliative settings. This review was aimed to give a brief insight into the mechanism of action and potential molecular alterations of MCT in the treatment of oral cancers taking into consideration the various in vivo and in vitro studies.

Dioscorea Villosa (Wild Yam) Root Extract.

The Expert Panel for Cosmetic Ingredient Safety reviewed updated information that has become available since their original assessment from 2004, along with updated information regarding product types, and frequency and concentrations of use, and reaffirmed their original conclusion that Dioscorea Villosa (Wild Yam) Root Extract is safe as a cosmetic ingredient in the practices of use and concentration as described in this report.

Methyl Alcohol.

The Expert Panel for Cosmetic Ingredient Safety reviewed updated information that has become available since their original assessment from 2001, along with updated information regarding product types, and frequency and concentrations of use, and reaffirmed their original conclusion that Methyl Alcohol is safe as used to denature alcohol in the practices of use and concentration as described in this report.

Polyamino Sugar Condensate.

The Expert Panel for Cosmetic Ingredient Safety reviewed updated information that has become available since their original assessment from 1982, along with updated information regarding product types, and frequency and concentrations of use, and reaffirmed their original conclusion that Polyamino Sugar Condensate is safe for topical application to humans in the practices of use and concentration as described in this report.

Peanut Glycerides.

The Expert Panel for Cosmetic Ingredient Safety reviewed updated information that has become available since their original assessment from 2001, along with updated information regarding product types, and frequency and concentrations of use, and reaffirmed their original conclusion that Peanut Glycerides is safe as a cosmetic ingredient in the practices of use and concentration as described in this report.

Glyceryl Diesters.

The Expert Panel for Cosmetic Ingredient Safety reviewed newly available studies since their original assessment in 2002, along with updated information regarding product types and concentrations of use, and confirmed that these 17 glyceryl diesters are safe as cosmetic ingredients in the practices of use and concentration as described in this report.

Cottonseed Glyceride and Hydrogenated Cottonseed Glyceride.

The Expert Panel for Cosmetic Ingredient Safety reviewed newly available studies since their original assessment in 2001, along with updated information regarding product types and concentrations of use, and confirmed that Cottonseed Glyceride and Hydrogenated Cottonseed Glyceride are safe as cosmetic ingredients in the practices of use and concentration as described in this report, provided that established and imposed limits on gossypol, heavy metals, and pesticide concentrations are not exceeded.

Polyacrylamide.

The Expert Panel for Cosmetic Ingredient Safety reviewed updated information that has become available since their original assessment from 1991, along with updated information regarding product types, and frequency and concentrations of use, and reaffirmed their original conclusion that Polyacrylamide is safe as a cosmetic ingredient in the practices of use and concentration as described in this report.

Erythorbic Acid and Sodium Erythorbate.

The Expert Panel for Cosmetic Ingredient Safety reviewed newly available studies since their original assessment in 1999, along with updated information regarding product types and concentrations of use, and confirmed that Erythorbic Acid and Sodium Erythorbate are safe as cosmetic ingredients in the practices of use and concentration as described in this report.

Engineering a 3D hydrogel system to study optic nerve head astrocyte morphology and behavior.

In glaucoma, astrocytes within the optic nerve head (ONH) rearrange their actin cytoskeleton, while becoming reactive and upregulating intermediate filament glial fibrillary acidic protein (GFAP). Increased transforming growth factor beta 2 (TGF ß2) levels have been implicated in glaucomatous ONH dysfunction. A key limitation of using conventional 2D culture to study ONH astrocyte behavior is the inability to faithfully replicate the in vivo ONH microenvironment. Here, we engineer a 3D ONH astrocyte hydrogel to better mimic in vivo mouse ONH astrocyte (MONHA) morphology, and test induction of MONHA reactivity using TGF ß2. Primary MONHAs were isolated from C57BL/6J mice and cell purity confirmed. To engineer 3D cell-laden hydrogels, MONHAs were mixed with photoactive extracellular matrix components (collagen type I, hyaluronic acid) and crosslinked for 5 minutes using a photoinitiator (0.025% riboflavin) and UV light (405-500 nm, 10.3 mW/cm2). MONHA-encapsulated hydrogels were cultured for 3 weeks, and then treated with TGF ß2 (2.5, 5.0 or 10 ng/ml) for 7 days to assess for reactivity. Following encapsulation, MONHAs retained high cell viability in hydrogels and continued to proliferate over 4 weeks as determined by live/dead staining and MTS assays. Sholl analysis demonstrated that MONHAs within hydrogels developed increasing process complexity with increasing process length over time. Cell processes connected with neighboring cells, coinciding with Connexin43 expression within astrocytic processes. Treatment with TGF ß2 induced reactivity in MONHA-encapsulated hydrogels as determined by altered F-actin cytoskeletal morphology, increased GFAP expression, and elevated fibronectin and collagen IV deposition. Our data sets the stage for future use of this 3D biomimetic ONH astrocyte-encapsulated hydrogel to investigate astrocyte behavior in response to injury.

Review on NAD(P)H dehydrogenase quinone 1 (NQO1) pathway.

NQO1 is an enzyme present in humans which is encoded by NQO1 gene. It is a protective antioxidant agent, versatile cytoprotective agent and regulates the oxidative stresses of chromatin binding proteins for DNA damage in cancer cells. The oxidization of cellular pyridine nucleotides causes structural alterations to NQO1 and changes in its capacity to binding of proteins. A strategy based on NQO1 to have protective effect against cancer was developed by organic components to enhance NQO1 expression. The quinone derivative compounds like mitomycin C, RH1, E09 (Apaziquone) and ß-lapachone causes cell death by NQO1 reduction of two electrons. It was also known to be overexpressed in various tumor cells of breast, lung, cervix, pancreas and colon when it was compared with normal cells in humans. The mechanism of NQO1 by the reduction of FAD by NADPH to form FADH2 is by two ways to inhibit cancer cell development such as suppression of carcinogenic metabolic activation and prevention of carcinogen formation. The NQO1 exhibit suppression of chemical-mediated carcinogenesis by various properties of NQO1 which includes, detoxification of quinone scavenger of superoxide anion radical, antioxidant enzyme, protein stabilizer. This review outlines the NQO1 structure, mechanism of action to inhibit the cancer cell, functions of NQO1 against oxidative stress, drugs acting on NQO1 pathways, clinical significance.

Signet ring cell cancer of stomach and gastro-esophageal junction: molecular alterations, stage-stratified treatment approaches, and future challenges.

PURPOSE: There has been an increase in the incidence of signet ring cell cancer (SRCC) of the stomach and gastro-esophageal junction (GEJ). The multistage carcinogenesis involving genetic and epigenetic aberrations may have a major role in the increasing incidence of SRCC. Although there are numerous studies on the prognostic value of SRCC, they are markedly inconsistent in their results, making it impossible to draw any meaningful conclusions. We aimed to examine the available evidences on molecular alterations and stage-stratified treatment approaches in SRCC of the stomach and GEJ. METHODS: A systematic search was carried out in PubMed. Studies available in English related to SRCC of stomach and gastro-esophageal junction were identified and evaluated. RESULTS: This study reviewed the current evidence and provided an insight into the molecular alterations, stage-stratified treatment approaches, and future challenges in the management of SRCC of the stomach and GEJ. Specific therapeutic strategies and personalized multimodal treatment have been recommended based on the tumor characteristics of SRCC. CONCLUSION: Multistage carcinogenesis involving genetic and epigenetic aberrations in SRCC is interlinked with stage-dependent prognosis. Specific therapeutic strategy and personalized multimodal treatment should be followed based on the tumor characteristics of SRCC. Endoscopic resection, radical surgery, and perioperative chemotherapy should be offered in carefully selected patients based on stage and prognostic stratification. Future studies in genetic and molecular analysis, histopathological classification, and options of multimodality treatment will improve the prognosis and oncological outcomes in SRCC of gastric and GEJ.

Synergistic effect of Betulinic acid, Apigenin and Skimmianine (BASk) in high cholesterol diet rabbit: Involvement of CD36-TLR2 signaling pathway.

BACKGROUND: Progression of chronic inflammatory disease, atherosclerosis is a multifactorial process. Cluster of differentiation 36 (CD36) mediated downstream activation of Toll like receptor 2 (TLR2) and NLRP3 (Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome signaling pathway actively participates during chronic inflammation. Nowadays, synergistic combinations of bioactive compounds attained priority in the field of drug discovery and development as therapeutic agents. An investigation regarding the anti-inflammatory potential of a novel drug formulation, BASk which is a combination of three bioactive compounds Betulinic acid (B):Apigenin (A):Skimmianine (Sk) remains the focus area of this research study. We also elucidate the molecular mechanism behind the therapeutic potential of BASk through CD36 mediated activation TLR2-NLRP3 signaling pathway. METHODS: OxLDL induced hPBMCs used to screen out a suitable combination of BASk via MTT, COX, LOX, NOS and MPO assays. Hypercholesterolemia is induced in rabbits by supplementing with 1% cholesterol + 0.5% cholic acid and treated with BASk (2:2:1) (5 mg/Kg) and atorvastatin (10 mg/Kg) for 60 days. CD36, TLR2, NLRP3, NFκB, cytokines, endothelial damage were quantified by reverse transcription, real time PCR, ELISA, flow cytometry and histopathology. RESULTS: hPBMCs pretreated with BASk at 2:2:1 ratio significantly decreased the activities of COX, 15-LOX, NOS and MPO on OxLDL induction than quercetin. Down regulation of CD36, TLR2, MyD88, TRAF6 by BASk further buttressed NLRP3 inflammasome activation mediated by the transcription factor NFκB. This is in correlation with the effect of BASk by balancing pro (IL-1ß, IL-18) and anti-inflammatory (TGF-ß) mediators in the aortic endothelial cells. CONCLUSION: BASk exerted its anti-inflammatory potential by reducing pro-inflammatory mediators during cholesterol supplementation via down regulating CD36 mediated TLR2 - NLRP3 inflammasome cascade. This deciphers a synergistic combination named BASk (2:2:1) as a novel drug formulation against chronic inflammatory disease, atherosclerosis.

Mechanosensitive channel inhibition attenuates TGFß2-induced actin cytoskeletal remodeling and reactivity in mouse optic nerve head astrocytes.

Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, which coincides with astrocyte reactivity and extracellular matrix (ECM) deposition. Elevated transforming growth factor beta 2 (TGFß2) levels within astrocytes have been described in glaucoma, and TGFß signaling induces actin cytoskeletal remodeling and ECM deposition in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGFß2-mediated optic nerve head astrocyte actin cytoskeletal remodeling, reactivity, and ECM deposition. Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with vehicle control, TGFß2 (5 ng/ml), GsMTx4 (a mechanosensitive channel inhibitor; 500 nM), or TGFß2 (5 ng/ml) + GsMTx4 (500 nM) for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining and immunoblotting for GFAP. Levels of fibronectin and collagen IV deposition were also quantified. Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (F4/80) and oligodendrocytes (OSP1). Significantly increased %CLAN-positive cells were observed after 48-h treatment with TGFß2 vs. control in a dose-dependent manner. Co-treatment with GsMTx4 significantly decreased %CLAN-positive cells vs. TGFß2 treatment and the presence of f-actin stress fibers. TGFß2 treatment significantly increased GFAP, fibronectin, and collagen IV levels, and GsMTx4 co-treatment ameliorated GFAP immunoreactivity. Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic strategy to modulate actin cytoskeletal remodeling within the optic nerve head in glaucoma.

A tissue-engineered human trabecular meshwork hydrogel for advanced glaucoma disease modeling.

Abnormal human trabecular meshwork (HTM) cell function and extracellular matrix (ECM) remodeling contribute to HTM stiffening in primary open-angle glaucoma (POAG). Most current cellular HTM model systems do not sufficiently replicate the complex native three dimensional (3D) cell-ECM interface, limiting their use for investigating POAG pathology. Tissue-engineered hydrogels are ideally positioned to overcome shortcomings of current models. Here, we report a novel biomimetic HTM hydrogel and test its utility as a POAG disease model. HTM hydrogels were engineered by mixing normal donor-derived HTM cells with collagen type I, elastin-like polypeptide and hyaluronic acid, each containing photoactive functional groups, followed by UV crosslinking. Glaucomatous conditions were induced with dexamethasone (DEX), and effects of the Rho-associated kinase (ROCK) inhibitor Y27632 on cytoskeletal organization and tissue-level function, contingent on HTM cell-ECM interactions, were assessed. DEX exposure increased HTM hydrogel contractility, f-actin and alpha smooth muscle actin abundance and rearrangement, ECM remodeling, and fibronectin deposition - all contributing to HTM hydrogel condensation and stiffening consistent with glaucomatous HTM tissue behavior. Y27632 treatment produced precisely the opposite effects and attenuated the DEX-induced pathologic changes, resulting in HTM hydrogel relaxation and softening. For model validation, confirmed glaucomatous HTM (GTM) cells were encapsulated; GTM hydrogels showed increased contractility, fibronectin deposition, and stiffening vs. normal HTM hydrogels despite reduced GTM cell proliferation. We have developed a biomimetic HTM hydrogel model for detailed investigation of 3D cell-ECM interactions under normal and simulated glaucomatous conditions. Its bidirectional responsiveness to pharmacological challenge and rescue suggests promising potential to serve as screening platform for new POAG treatments with focus on HTM biomechanics.

Dietary amaranths modulate the immune response via balancing Th1/Th2 and Th17/Treg response in collagen-induced arthritis.

Imbalance between Th1/Th2 and Th17/Treg is crucial in RA progression. Various dietary factors can modulate the disease severity by restoring the balance in differentiation of CD4+ T cell subsets. Dietary amaranths hold an important part of diet as vegetables, where commonly consumed species includes Amaranthus cruentus (Ac), Amaranthus viridis (Av), and Amaranthus hybridus (Ah). The present study focuses on to evaluate whether these dietary amaranths can modulate the immune activation in collagen-induced arthritis. For in vivo study, Female Wistar rats were immunized with type II collagen and after immunization period, rats were separately supplemented with cooked Ac, Av, and Ah at 500 mg/100 g bwt concentration mixed with standard rat feed for 60 days. HPTLC fingerprint analysis identified peaks for compounds in these three amaranths. The results showed a protective role of immunomodulation in Th1/Th2 response of the three dietary amaranths, by significantly augmenting lymphocyte activation with increased IL-4 secretion, but decreased IFN-γ by cultured spleen lymphocytes subjected to collagen-induced inflammation. Moreover, Th17/Treg imbalance created by increase in IL-17 and decrease in IL-10 was significantly balanced by the three dietary supplemented groups. Furthermore, Th1/Th2 status reflected from Tbet/GATA3 ratio and Th17/Treg status reflected from RORγt/FOXP3 ratio was significantly decreased in the three dietary amaranth supplemented groups. Thus, dietary amaranths provide an immune-modulating role by keeping the balance between Th1/Th2 and Th17/Treg response in collagen-induced inflammation.