Computational Exploration of Naturally Occurring Flavonoids as TGF-ß Inhibitors in Breast Cancer: Insights from Docking and Molecular Dynamics Simulations and In-vitro Cytotoxicity Study.

Breast cancer is a global health concern, demanding innovative treatments. Targeting the Transforming Growth Factor-beta (TGF-ß) signaling pathway, pivotal in breast cancer, is a promising approach. TGF-ß inhibits proliferation via G1 phase cell cycle arrest, acting as a suppressor initially, but in later stages, it promotes progression by enhancing motility, invasiveness, and metastasis formation. This study explores naturally occurring flavonoids' interactions with TGF-ß. Using molecular docking against the protein's crystal structure (PDB Id: 1PY5), Gossypin showed the highest docking score and underwent molecular dynamics simulation, revealing complex flexibility and explaining how flavonoids impede TGF-ß signaling in breast cancer. ADMET predictions adhered to Lipinski's rule of Five. Insights into flavonoid-TGF-ß binding offer a novel angle for breast cancer treatment. Flavonoids having a good docking score like gossypin, morin, luteolin and taxifolin shown potent cytotoxic effect on breast cancer cell line, MCF-7. Understanding these interactions could inspire flavonoid-based therapies targeting TGF-ß to halt breast cancer growth. These findings pave the way for personalized, targeted breast cancer therapies, offering hope against this formidable disease.

Emerging green synthetic routes for thiazole and its derivatives: Current perspectives.

This review article provides an overview of the green synthesis of thiazole derivatives, emphasizing sustainable and environmentally friendly methodologies. Thiazole derivatives possess significant value and find diverse applications across various fields. However, conventional synthesis methods often involve hazardous reagents and generate substantial waste, posing environmental concerns. The green synthesis of thiazole derivatives employs renewable starting materials, nontoxic catalysts, and mild reaction conditions to minimize environmental impact. Innovative techniques such as microwave irradiation, ultrasound synthesis, green solvents, a green catalyst-based approach, and mechanochemistry-mediated synthesis are employed, offering advantages in terms of scalability, cost-effectiveness, and purification simplicity. The resulting thiazole derivatives exhibit comparable or enhanced biological activities, showcasing the feasibility and practicality of green synthesis in drug discovery. This review paper underscores the importance of sustainable approaches in functional molecular synthesis and encourages further research in this domain.

Probiotic Formulations: A Patent Landscaping Using the Text Mining Approach.

The outstanding research outcomes and registrations of myriads of probiotic strains have flooded the health market with various innovative probiotic-based products and their patents. The study of patented formulations of probiotics can give an overall insight into its existing application. A landscaping review of patents for probiotic-based preparations is presented in the current work. The patent search was performed over commercially available patent databased and analysis tool-PatSeer Pro®. Search strings containing words "Formulation" and "Composition" resulted in more than 3700 patents. Landscaping review of 400 + patents from the last 20 years (2000-2020) was performed using the Text-Mining approach. Text-Mining helped to identify 19 technological clusters which represent these patents. These clusters include the patents of probiotic preparations on animal feed, human food, cosmetics, antimicrobial, antidiabetic, arthritis, etc. A review of this massive number of patents unveiled many exciting preparations. Probiotic-based innovative products for depression, diabetes, Parkinson's, tumor, acne, and animal husbandry are reviewed comprehensively. The present work also unravels a few new-flanged products like probiotic layered condoms, products for acute alcoholism, and traditional Chinese medicine with probiotics. The patent landscape of probiotic-based preparations has presented a whole scenario of probiotic-based preparations. It has also revealed many unexplored areas where innovation can be excelled.

Predictors of clinically relevant differences between noninvasive versus arterial blood pressure.

OBJECTIVE: Blood pressure (BP) measurements are important for managing patients with hypertensive emergencies (HE). Previous studies showed that there was significant difference between IABP and NIBP but no information whether these differences changed management. Our study investigated the factors associated with the differences affecting BP management of patients with HE. METHODS: This was a retrospective study involving adult patients admitted to a resuscitation unit. We screened all patients who received IABP upon admission between 06/01/2017 and 12/31/2017 as sample size calculation recommended 64 patients. Primary outcome was the clinical relevance of the difference of IABP vs. NIBP, which was defined as having both: a) difference of 10 mm of mercury (mmHg), and b) resulting in possible change of blood pressure managements according to treatment guidelines. We performed backward stepwise multivariable logistic regression to measure associations. RESULTS: We analyzed 147 patients whose mean age was 69 (±16) years and included 69 (47%) patients with spontaneous intracerebral hemorrhage (sICH). Mean difference between IABP and NIBP was 21 (±16) mmHg while 41 (28%) patients who had difference affecting managements. In multivariable regression, sICH (Odd Ratios 13.5, 95%CI 2.3-79.5, p-value < 0.001) was significantly associated with clinically relevant difference between the two modalities of BP monitoring. CONCLUSIONS: There was a large difference between IABP and NIBP among patients with hypertensive emergencies. Up to 30% of patients had clinically relevant differences. Patients with sICH were more likely to have differences affecting BP management. Further studies are needed to confirm our observation.

Neuroticism in temporal lobe epilepsy is associated with altered limbic-frontal lobe resting-state functional connectivity.

Neuroticism, a core personality trait characterized by a tendency towards experiencing negative affect, has been reported to be higher in people with temporal lobe epilepsy (TLE) compared with healthy individuals. Neuroticism is a known predictor of depression and anxiety, which also occur more frequently in people with TLE. The purpose of this study was to identify abnormalities in whole-brain resting-state functional connectivity in relation to neuroticism in people with TLE and to determine the degree of unique versus shared patterns of abnormal connectivity in relation to elevated symptoms of depression and anxiety. Ninety-three individuals with TLE (55 females) and 40 healthy controls (18 females) from the Epilepsy Connectome Project (ECP) completed measures of neuroticism, depression, and anxiety, which were all significantly higher in people with TLE compared with controls. Resting-state functional connectivity was compared between controls and groups with TLE with high and low neuroticism using analysis of variance (ANOVA) and t-test. In secondary analyses, the same analytics were performed using measures of depression and anxiety and the unique variance in resting-state connectivity associated with neuroticism independent of symptoms of depression and anxiety identified. Increased neuroticism was significantly associated with hyposynchrony between the right hippocampus and Brodmann area (BA) 9 (region of prefrontal cortex (PFC)) (p < 0.005), representing a unique relationship independent of symptoms of depression and anxiety. Hyposynchrony of connection between the right hippocampus and BA47 (anterior frontal operculum) was associated with high neuroticism and with higher depression and anxiety scores (p < 0.05), making it a shared abnormal connection for the three measures. In conclusion, increased neuroticism exhibits both unique and shared patterns of abnormal functional connectivity with depression and anxiety symptoms between regions of the mesial temporal and frontal lobe.

Survival outcome and prognostic model of patients with colorectal cancer on phase 1 trials.

Background Patients with metastatic colorectal cancer (mCRC) who progress on standard therapies may be eligible for phase I trials. To better delineate the risk-benefit ratio, we assessed toxicities, clinical outcomes and prognostic factors. Methods Records of mCRC patients on phase I trials at our institution over 18 years were reviewed. Univariable (UVA) and multivariable analyses (MVA) were undertaken and a prognostic model developed. Results There were 187 enrollments on 37 phase I trials. Median age was: 59 (29-83) years and number of prior therapies: 3 (0-8). The clinical benefit rate (CBR): response (5.6%) + stable disease, was 43.1%. Median progression free survival (PFS) and overall survival (OS) was 7.7 weeks and 43.7 weeks, respectively. The MVA identified age > 60 years (HR 1.63, p < 0.004), albumin<3.5 g/dL (HR 3.69, p < 0.001), direct bilirubin>ULN (HR1.69, p < 0.01), and WBC ≥ 5.2 k/uL (HR 1.97, p < 0.001) as negative prognostic factors. A risk score based on the MVA revealed that patients with a score of 0-1 had an improved OS (58.7 weeks) compared to a score of 2 (49.9 weeks, p < 0.01) and 3 (14.1 weeks, p < 0.001). Conclusions Phase 1 trials may offer similar or better clinical outcome for mCRC patients than standard third line therapies; the prognostic model could assist in selecting appropriate patients.

Neuroanatomical correlates of personality traits in temporal lobe epilepsy: Findings from the Epilepsy Connectome Project.

Behavioral and personality disorders in temporal lobe epilepsy (TLE) have been a topic of interest and controversy for decades, with less attention paid to alterations in normal personality structure and traits. In this investigation, core personality traits (the Big 5) and their neurobiological correlates in TLE were explored using the Neuroticism Extraversion Openness-Five Factor Inventory (NEO-FFI) and structural magnetic resonance imaging (MRI) through the Epilepsy Connectome Project (ECP). NEO-FFI scores from 67 individuals with TLE (34.6 ±â€¯9.5 years; 67% women) were compared to 31 healthy controls (32.8 ±â€¯8.9 years; 41% women) to assess differences in the Big 5 traits (agreeableness, openness, conscientiousness, neuroticism, and extraversion). Individuals with TLE showed significantly higher neuroticism, with no significant differences on the other traits. Neural correlates of neuroticism were then determined in participants with TLE including cortical and subcortical volumes. Distributed reductions in cortical gray matter volumes were associated with increased neuroticism. Subcortically, hippocampal and amygdala volumes were negatively associated with neuroticism. These results offer insight into alterations in the Big 5 personality traits in TLE and their brain-related correlates.

A study on the wear of enamel caused by monolithic zirconia and the subsequent phase transformation compared to two other ceramic systems.

PURPOSE: The purpose of this in vitro research project was to evaluate and compare the wear behavior of human tooth enamel opposing monolithic zirconia and other different ceramic restorative materials and also to observe the tetragonal to monoclinic phase transformation in zirconia-based ceramics that may occur while simulating wear occurring at room temperature in a wet environment. MATERIALS AND METHODS: A total of sixty samples were prepared for this study. Fifteen discs of glazed zirconia, 15 discs of polished zirconia without glaze, 15 discs of metal ceramic, and 15 discs of lithium disilicate were fabricated. Sixty extracted premolars were collected and randomly divided into four groups of 15 each. The discs and extracted human premolars were placed onto holders on a two-body wear machine under a constant load of 5 kg to simulate the oral wear cycle. A diffractometer was used to analyze phase transformation. One-way analysis of variance and Tukey's post hoc tests was used. RESULTS: The mean loss of height of tooth samples and its standard deviation for Group I (monolithic zirconia with glaze), Group II (mechanically polished monolithic zirconia without glaze), Group III (porcelain fused to metal), and Group IV (glazed monolithic lithium disilicate) was obtained as 0.2716 ± 0.1409, 0.1240 ± 0.0625, 0.1567 ± 0.0996, and 0.2377 ± 0.1350, respectively. The highest mean loss in height was observed in Group I and the least was observed in Group II. CONCLUSION: Mechanically polished zirconia showed the least amount of enamel wear followed by porcelain fused to metal and glazed monolithic lithium disilicate, whereas glazed monolithic zirconia showed the highest enamel wear.

Decoupling the contribution of surface energy and surface area on the cohesion of pharmaceutical powders.

PURPOSE: Surface area and surface energy of pharmaceutical powders are affected by milling and may influence formulation, performance and handling. This study aims to decouple the contribution of surface area and surface energy, and to quantify each of these factors, on cohesion. METHODS: Mefenamic acid was processed by cryogenic milling. Surface energy heterogeneity was determined using a Surface Energy Analyser (SEA) and cohesion measured using a uniaxial compression test. To decouple the surface area and surface energy contributions, milled mefenamic acid was "normalised" by silanisation with methyl groups, confirmed using X-ray Photoelectron Spectroscopy. RESULTS: Both dispersive and acid-base surface energies were found to increase with increasing milling time. Cohesion was also found to increase with increasing milling time. Silanised mefenamic acid possessed a homogenous surface with a surface energy of 33.1 ± 1.4 mJ/m(2) , for all milled samples. The cohesion for silanised mefenamic acid was greatly reduced, and the difference in the cohesion can be attributed solely to the increase in surface area. For mefenamic acid, the contribution from surface energy and surface area on cohesion was quantified to be 57% and 43%, respectively. CONCLUSIONS: Here, we report an approach for decoupling and quantifying the contribution from surface area and surface energy on powder cohesion.

Eribulin in Cancer Treatment.

Halichondrin B is a complex, natural, polyether macrolide derived from marine sponges. Eribulin is a structurally-simplified, synthetic, macrocyclic ketone analogue of Halichondrin B. Eribulin was approved by United States Food and Drug Administration in 2010 as a third-line therapy for metastatic breast cancer patients who have previously been treated with an anthracycline and a taxane. It has a unique microtubule dynamics inhibitory action. Phase III studies have either been completed or are currently ongoing in breast cancer, soft tissue sarcoma, and non-small cell lung cancer. Phase I and II studies in multiple cancers and various combinations are currently ongoing. This article reviews the available information on eribulin with respect to its clinical pharmacology, pharmacokinetics, pharmacodynamics, mechanism of action, metabolism, preclinical studies, and with special focus on clinical trials.

Investigating the Antioxidant Capacity of Newly Synthesized Flavonoids via DPPH Assay.

BACKGROUND: Numerous naturally occurring and artificially synthesized flavonoids have garnered attention for their impressive ability to combat oxidative stress and scavenge free radicals when evaluated in laboratory settings. The core aim of our investigation revolved around assessing the antioxidant potential of a diverse range of synthesized flavonoids through in vitro experiments. METHOD: We crafted 29 distinct flavonoids using the aldol condensation mechanism via a chalcone intermediate to accomplish this. We meticulously characterized these newly formed compounds using a variety of spectroscopic techniques. We employed the widely recognized DPPH free radical method for the crucial antioxidant evaluation, a benchmark in such studies Result: The radical scavenging efficacy of our synthesized flavonoids was then meticulously compared to that of the positive control, ascorbic acid, renowned for its antioxidant prowess, and the IC50 values for each compound were calculated and examined. Surprisingly, our results showed that the flavonoids we tested had a wide range of antioxidant activity, with IC50 values that ranged from 75.8 ± 8.30 to 397 ± 25.10 µg/mL. CONCLUSION: Intriguingly, compounds US5, US13, US16, US17, US18, and US21 outshone even ascorbic acid in their antioxidant potential, displaying remarkable scavenging abilities against free radicals. This discovery holds promise for further exploration of these compounds as potential antioxidants with potential applications in health and wellness.

Decrypting the multifaceted peripheral neuropathy based on molecular pathology and therapeutics: a comprehensive review.

CONTEXT: Peripheral neuropathy (PN) is a multifaceted complication characterized by nerve damage due to oxidative stress, inflammatory mediators, and dysregulated metabolic processes. Early PN manifests as sensory changes that develop progressively in a "stocking and glove" pattern. METHODS AND MECHANISMS: A thorough review of literature has been done to find the molecular pathology, clinical trials that have been conducted to screen the effects of different drugs, current treatments and novel approaches used in PN therapy. Diabetic neuropathy occurs due to altered protein kinase C activity, elevated polyol pathway activity in neurons, and Schwann cells-induced hyperglycemia. Other causes involve chemotherapy exposure, autoimmune ailments, and chronic ethanol intake. CONCLUSION: Symptomatic treatments for neuropathic pain include use of tricyclic antidepressants, anticonvulsants, and acetyl-L-carnitine. Patients will have new hope if clinicians focus on novel therapies including gene therapy, neuromodulation techniques, and cannabidiol as an alternative to traditional medications, as management is still not ideal.

Structural Insights of PD-1/PD-L1 Axis: An In silico Approach.

BACKGROUND: Interaction of PD-1 protein (present on immune T-cell) with its ligand PD-L1 (over-expressed on cancerous cell) makes the cancerous cell survive and thrive. The association of PD-1/PD-L1 represents a classical protein-protein interaction (PPI), where receptor and ligand binding through a large flat surface. Blocking the PD-1/PDL-1 complex formation can restore the normal immune mechanism, thereby destroying cancerous cells. However, the PD-1/PDL1 interactions are only partially characterized. OBJECTIVE: We aim to comprehend the time-dependent behavior of PD-1 upon its binding with PD-L1. METHOD: The current work focuses on a molecular dynamics simulation (MDs) simulation study of apo and ligand bound PD-1. RESULTS: Our simulation reveals the flexible nature of the PD-1, both in apo and bound form. Moreover, the current study also differentiates the type of strong and weak interactions which could be targeted to overcome the complex formation. CONCLUSION: The current article could provide a valuable structural insight about the target protein (PD-1) and its ligand (PD-L1) which could open new opportunities in developing small molecule inhibitors (SMIs) targeting either PD-1 or PD-L1.

Nanomaterials as drug delivery agents for overcoming the blood-brain barrier: A comprehensive review.

Background and Purpose: The blood-brain barrier (BBB), a critical interface of specialized endothelial cells, plays a pivotal role in regulating molecular and ion transport between the central nervous system (CNS) and systemic circulation. Experimental Approach: This review aims to delve into the intricate architecture and functions of the BBB while addressing challenges associated with delivering therapeutics to the brain. Historical milestones and contemporary insights underscore the BBB's significance in protecting the CNS. Key Results: Innovative approaches for enhanced drug transport include intranasal delivery exploiting olfactory and trigeminal pathways, as well as techniques like temporary BBB opening through chemicals, receptors, or focused ultrasound. These avenues hold the potential to reshape conventional drug delivery paradigms and address the limitations posed by the BBB's selectivity. Conclusion: This review underscores the vital role of the BBB in maintaining CNS health and emphasizes the importance of effective drug delivery through this barrier. Nanoparticles emerge as promising candidates to overcome BBB limitations and potentially revolutionize the treatment of CNS disorders. As research progresses, the application of nanomaterials shows immense potential for advancing neurological therapeutics, albeit with careful consideration of safety aspects.

Clinical outcome and prognostic markers for patients with gynecologic malignancies in phase 1 clinical trials: a single institution experience from 1999 to 2010.

OBJECTIVES: There is a scarcity of outcome data regarding phase 1 trials for patients with gynecologic malignancy. The objective of this study was to assess toxicity, clinical benefit and prognosticators in gynecologic oncology patients participating in phase 1 trials. METHODS: All phase 1 oncology trials conducted at Albert Einstein Cancer Center from 1999 to 2010 were reviewed and extracted for relevant demographic and clinical data concerning patients with gynecologic malignancy. Cox-proportional and logistic regression modeling were used for multivariate analysis. RESULTS: 120 distinct patients with gynecologic malignancy participated in 41 trials, constituting 30.6% of all phase 1 patients enrolled in the same time period. The median age is 59 years. Out of the 184 patients enrolled, 160 individual responses were evaluable. Seventeen DLT events (9.2%) occurred, including 1 (0.5%) treatment-related mortality. There were 27.2%≥ grade 3 hematologic and 24.4% non-hematologic toxicity. Eighty patients had stable disease (SD, 50%), including 21.9% with SD ≥ 4 months, 11 (6.3%) with partial response (PR), and 3 (1.9%) achieving complete response (CR). The clinical benefit rate (CBR=SD+CR+PR) was 58.1%. Albumin (Alb)≤ 3.5 g/dL and abnormal ANC were independent negative prognosticators of survival. We also found a continuous correlation between changes in Albumin (p=0.02) and LDH (p=0.02) and odds of achieving CBR≥4month. CONCLUSIONS: Our clinical outcome and safety data suggested that phase 1 trials may be a reasonable option for patients with advanced and recurrent gynecologic cancer. The potential prognosticators identified should be further validated in larger trials.

A review of the symptomatic management of malignant gliomas in adults.

Malignant brain tumors are aggressive tumors with a very poor prognosis. Survival is on average 12 to 18 months. Patients with malignant gliomas are subject to multiple medical problems that can significantly impact their overall survival and quality of life, including seizures, cerebral edema, venous thromboembolism, cognitive and psychiatric disorders, and side effects of chemotherapy, such as nausea, vomiting, myelosuppression, constipation, and diarrhea. This article examines the evidence for managing many of these issues to reduce symptoms and improve quality of life.

Molecular pathology and therapeutics of the diabetic foot ulcer; comprehensive reviews.

Diabetes mellitus (DM) is a chronic metabolic condition linked to high blood sugar levels. Diabetes causes complications like neuropathy, nephropathy, and retinopathy. Diabetes foot ulcer (DFU) is a significant and serious wound healing issue resulting from uncontrolled DM. The main causes of the development of the DFU are oxidative stress brought on by the NO moiety, release of pro-inflammatory cytokines like tumour necrosis factor (TNF)-α and interleukin (IL-1), cellular dysfunction, and pathogenic microorganisms including staphylococcus and streptococcus species. The two main types of wounds that are prevalent in DFU patients are neuropathic and neuroischemic. If this wound is not properly treated or cared for, a lower limb may have to be amputated. There are several therapy options for DFU, including antibiotics, debridement, dressings, nano formulations, and growth factor preparations like PDGF-BB, to help the wound heal and prevent amputation. Other novel approaches involved the use of nerve taps, microneedle patches, nanotechnology-based formulations and stem cell applications to promote healing. There are possibilities of drug repurposing for the DFU treatment based on targeting specific enzymes. This article summarises the current pathophysiological aspects of DFU and its probable future targets.

Atorvastatin's Reduction of Alzheimer's Disease and Possible Alteration of Cognitive Function in Midlife as well as its Treatment.

Over the past 20 years, advances in the field of pathogenesis have inspired researchers to look into novel pharmacological therapeutics that are more focused on the pathophysiological events of the disease (AD). This review article discussed the prior use of statins for the prevention of Alzheimer's disease, which can help prevent the disease. Other drugs, such as memantine and donepezil, are available, but they cannot prevent the onset of AD in middle age. Based on available clinical data, the valuable effects of statins are mediated by alteration of ß-amyloid (Aß) and tau metabolism, genetic and lifestyle risk factors, along with other clinical aspects of AD. These findings suggested that using statins in middle age may help to prevent Alzheimer's disease by modifying genetic and non-genetic risk factors in later stages of life. In the present review, we elaborated upon the modification of risk factors and amyloid metabolism in the development and progression of AD and their modulation through atorvastatin. Future directions in the research and treatment of Alzheimer's disease patients include the use of antisense oligonucleotides (ASO) to change target expression, and researchers discovered decreased markers of oxidative stress in tissues affected by tau pathology in response to RNA interference treatment.

A Review of the Therapeutic Importance of Indole Scaffold in Drug Discovery.

Indole is known as a versatile heterocyclic building block for its multiple pharmacological activities and has a high probability of success in the race for drug candidates. Many natural products, alkaloids, and bioactive heterocycles contain indole as the active principle pharmacophore. These encourage the researchers to explore it as a lead in the drug development process. The current manuscript will serve as a torchbearer for understanding the structurally diverse class of indole derivatives with extensive pharmacological activity. The current manuscript describes the intermediates and their functional groups responsible for superior biological activity compared to the standard. The review is written to help researchers to choose leads against their target but also to provide crucial insight into the design of a hybrid pharmacophore-based approach in drug design with enhanced potential. The present reviews on the indole derivatives correlate the structures with biological activities as well as essential pharmacophores, which were highlighted. The discussion was explored under challenging targets like dengue, chikungunya (anti-viral), antihypertensive, diuretic, immunomodulator, CNS stimulant, antihyperlipidemic, antiarrhythmic, anti-Alzheimer's, and neuroprotective, along with anticancer, antitubercular, antimicrobial, anti-HIV, antimalarial, anti-inflammatory, antileishmanial, antianthelmintic, and enzyme inhibitors. So, this review includes a discussion of 19 different pharmacological targets for indole derivatives that could be utilized to derive extensive information needed for ligand-based drug design. The article will guide the researchers in the selection, design of lead and pharmacophore, and ligand-based drug design using indole moiety.

Xenobots: Applications in Drug Discovery.

This review work discusses the applications of xenobots in drug discovery. These are the world's first tiny robots that are living. Robots are built of metals and other things that benefit humans to solve various issues; however, in this case, small xenobots were built utilizing Xenopus laevis, frog embryonic stem cells in the blastocyte stage. Xenobots were created by combining bioscience, artificial intelligence, and computer science. Artificial intelligence constructs several forms of design in an in vitro, In-silico model, after which software analyzes the structure; the most substantial and most noticeable forms are filtered out. Later in vivo development create the design of the Petri plate using the MMR solution and makes the same form as the in silico approach. Ultimately evaluation done based on the behavior, movement, function, and features of xenobots. Xenobots are employed in medical research, pharmaceutical research to evaluate novel dosage forms, also useful for biotechnological and environmental research. Xenobots can be utilized to cure neurodegenerative disorders such as Alzheimer's, Parkinson's disease, and cancer-related issues because of their selfrepairing properties, which allow them to repair normal damaged cells, and convey drugs to their specific target, and reduce cytotoxicity in mostly malignancy circumstances. In the future, new approaches will be employed to treat chronic illnesses and their complications.