Clinical outcomes and complications of a new high-density polyethylene-based spherical integrated porous orbital implant.

PURPOSE: To describe our experience over 6 years using a new high-density polyethylene-based spherical integrated porous orbital implants (Oculfit). METHODS: This is an observational retrospective case series study analyzing all cases requiring Oculfit implants between February 2015 and September 2021. Clinical information regarding the population included, the characteristics of the implant, and the outcomes and complications during the follow-up were noted. The success of the implant was defined according to anatomical and functional parameters. RESULTS: The study analyzed 90 cases of anophthalmic patients. The main causes for enucleation or evisceration were ocular decompensations (36.7%) and neoplasms (27.8% uveal melanoma and 7.8% retinoblastoma). Anatomical success was identified in 63 (70.0%) cases, functional success in 79 (87.8%) and complete success (anatomical + functional) in 61 (67.7%) cases. Factors associated with the functional success were age and exposure of the primary orbital implant. Complications appeared in 11 (12.2%) cases, which were completely resolved without sequelae in 4 (4.4%). Orbital explant was required in 5 (5.6%) cases. CONCLUSION: In our experience, Oculfit can be considered a useful alternative among the currently available options for orbital implants and has a good efficacy/safety profile.

Enhanced Electrocatalysis on Copper Nanostructures: Role of the Oxidation State in Sulfite Oxidation.

The influence of surface morphology and the oxidation state on the electrocatalytic activity of nanostructured electrodes is well recognized, yet disentangling their individual roles in specific reactions remains challenging. Here, we investigated the electrooxidation of sulfite ions in an alkaline environment using cyclic voltammetry on copper oxide nanostructured electrodes with different oxidation states and morphologies but with similar active areas. To this aim, we synthesized nanostructured Cu films made of nanoparticles or nanorods on top of glassy carbon electrodes. Our findings showed an enhanced sensitivity and a lower detection threshold when utilizing Cu(I) over Cu(II). Density functional theory-based thermochemical analysis revealed the underlying oxidation mechanism, indicating that while the energy gain associated with the process is comparable for both oxide surfaces, the desorption energy barrier for the resulting sulfate molecules is three times higher on Cu(II). This becomes the limiting step of the reaction kinetics and diminishes the overall electrooxidation efficiency. Our proposed mechanism relies on the tautomerization of hydroxyl groups confined on the surface of Cu-based electrodes. This mechanism might be applicable to electrochemical reactions involving other sulfur compounds that hold technological significance.

Therapeutic strategy for spinal muscular atrophy by combining gene supplementation and genome editing.

Defect in the SMN1 gene causes spinal muscular atrophy (SMA), which shows loss of motor neurons, muscle weakness and atrophy. While current treatment strategies, including small molecules or viral vectors, have shown promise in improving motor function and survival, achieving a definitive and long-term correction of SMA's endogenous mutations and phenotypes remains highly challenging. We have previously developed a CRISPR-Cas9 based homology-independent targeted integration (HITI) strategy, enabling unidirectional DNA knock-in in both dividing and non-dividing cells in vivo. In this study, we demonstrated its utility by correcting an SMA mutation in mice. When combined with Smn1 cDNA supplementation, it exhibited long-term therapeutic benefits in SMA mice. Our observations may provide new avenues for the long-term and efficient treatment of inherited diseases.

Literature Review of Deep-Learning-Based Detection of Violence in Video.

Physical aggression is a serious and widespread problem in society, affecting people worldwide. It impacts nearly every aspect of life. While some studies explore the root causes of violent behavior, others focus on urban planning in high-crime areas. Real-time violence detection, powered by artificial intelligence, offers a direct and efficient solution, reducing the need for extensive human supervision and saving lives. This paper is a continuation of a systematic mapping study and its objective is to provide a comprehensive and up-to-date review of AI-based video violence detection, specifically in physical assaults. Regarding violence detection, the following have been grouped and categorized from the review of the selected papers: 21 challenges that remain to be solved, 28 datasets that have been created in recent years, 21 keyframe extraction methods, 16 types of algorithm inputs, as well as a wide variety of algorithm combinations and their corresponding accuracy results. Given the lack of recent reviews dealing with the detection of violence in video, this study is considered necessary and relevant.

Emerging Technologies in Edge Computing and Networking.

The global evolution of the Internet is experiencing a notable and inevitable change towards a convergent scenario known as the Internet of Things (IoT), where a large number of devices with heterogeneous characteristics and requirements have to be interconnected to serve different verticals, such as smart cities, intelligent transportation systems, smart grids, (ITS) or e-health [...].

Nutritional composition of quinoa leafy greens: An underutilized plant-based food with the potential of contributing to current dietary trends.

Quinoa (Chenopodium quinoa Willd.) leafy greens (QLGs) are plant-based foods of high nutritional value that have been scarcely studied. In this work, the nutritional and functional composition of three QLGs varieties was evaluated. A protein content higher than 35 g 100 g-1 dw with a well-balanced essential amino acid composition was found making them a good source of vegetable protein. In addition, elevated contents of dietary fibre and minerals, higher than those detected in quinoa seeds and other leafy vegetables, were found. The lipid profile showed higher contents of linoleic (C18:2, ω6) (20.2 %) and linolenic acids (C18:3, ω3) (52.8 %) with low ω6/ ω3 ratios (∼0.4/1). A total sugar content <1 g 100 g-1 dw was found for all varieties tested, lower than that obtained in seeds. The saponin content varied between 0.76 and 0.87 %. Also, high values of total phenolic compounds (969.8-1195.4 mg gallic acid 100 g-1), mainly hydroxycinnamic acids and flavonoids, and great antioxidant activities (7.64-8.90 g Trolox kg-1) were found. Multivariate analysis here used allowed us to classify the samples according to the quinoa variety evaluated, and the sequential stepwise multiple regression applied revealed that the PUFA and sucrose contents negatively influenced the protein content while the palmitic acid content affected positively this parameter. Overall, this study shows that QLGs are promising nutritious and functional plant-based foods supporting the necessity of promoting their cultivation, commercialization, and consumption.

An overview of machine learning and deep learning techniques for predicting epileptic seizures.

Epilepsy is a neurological disorder (the third most common, following stroke and migraines). A key aspect of its diagnosis is the presence of seizures that occur without a known cause and the potential for new seizures to occur. Machine learning has shown potential as a cost-effective alternative for rapid diagnosis. In this study, we review the current state of machine learning in the detection and prediction of epileptic seizures. The objective of this study is to portray the existing machine learning methods for seizure prediction. Internet bibliographical searches were conducted to identify relevant literature on the topic. Through cross-referencing from key articles, additional references were obtained to provide a comprehensive overview of the techniques. As the aim of this paper aims is not a pure bibliographical review of the subject, the publications here cited have been selected among many others based on their number of citations. To implement accurate diagnostic and treatment tools, it is necessary to achieve a balance between prediction time, sensitivity, and specificity. This balance can be achieved using deep learning algorithms. The best performance and results are often achieved by combining multiple techniques and features, but this approach can also increase computational requirements.

Adverse events of antibody–drug conjugates on the ocular surface in cancer therapy

Antibody–drug conjugates consist of a monoclonal antibody attached to a cytotoxic therapeutic molecule by a connector. This association allows a highly specific therapy, which increases their effectiveness and decreases their potential toxicity. This new therapy emerged approximately 20 years ago; since then, numerous combinations have appeared in the field of treatment-related neoplasms as an alternative for patients who do not achieve good results with conventional treatment options. Adverse effects of these drugs on the ocular surface are frequent and varied. Their prevalence ranges from 20 to 90% depending on the drug and administration condition, probably due to multiple receptor-mediated factors or mechanisms not mediated by specific receptors, such as macropinocytosis. These adverse events can greatly limit patients’ comfort; thus, the objectives of this article were, in the first place, to compile the information currently available on different types of adverse effects of antibody–drug conjugates on the ocular surface, including pathophysiology, prevalence, and treatment, and in second place, to contribute to the correct identification and management of these events, which will result in a lower rate of cessation of treatment, which is necessary for the survival of candidate patients (AU)

Adverse events of antibody-drug conjugates on the ocular surface in cancer therapy.

Antibody-drug conjugates consist of a monoclonal antibody attached to a cytotoxic therapeutic molecule by a connector. This association allows a highly specific therapy, which increases their effectiveness and decreases their potential toxicity. This new therapy emerged approximately 20 years ago; since then, numerous combinations have appeared in the field of treatment-related neoplasms as an alternative for patients who do not achieve good results with conventional treatment options. Adverse effects of these drugs on the ocular surface are frequent and varied. Their prevalence ranges from 20 to 90% depending on the drug and administration condition, probably due to multiple receptor-mediated factors or mechanisms not mediated by specific receptors, such as macropinocytosis. These adverse events can greatly limit patients' comfort; thus, the objectives of this article were, in the first place, to compile the information currently available on different types of adverse effects of antibody-drug conjugates on the ocular surface, including pathophysiology, prevalence, and treatment, and in second place, to contribute to the correct identification and management of these events, which will result in a lower rate of cessation of treatment, which is necessary for the survival of candidate patients.

Rapid degeneration of iPSC-derived motor neurons lacking Gdap1 engages a mitochondrial-sustained innate immune response.

Charcot-Marie-Tooth disease is a chronic hereditary motor and sensory polyneuropathy targeting Schwann cells and/or motor neurons. Its multifactorial and polygenic origin portrays a complex clinical phenotype of the disease with a wide range of genetic inheritance patterns. The disease-associated gene GDAP1 encodes for a mitochondrial outer membrane protein. Mouse and insect models with mutations in Gdap1 have reproduced several traits of the human disease. However, the precise function in the cell types affected by the disease remains unknown. Here, we use induced-pluripotent stem cells derived from a Gdap1 knockout mouse model to better understand the molecular and cellular phenotypes of the disease caused by the loss-of-function of this gene. Gdap1-null motor neurons display a fragile cell phenotype prone to early degeneration showing (1) altered mitochondrial morphology, with an increase in the fragmentation of these organelles, (2) activation of autophagy and mitophagy, (3) abnormal metabolism, characterized by a downregulation of Hexokinase 2 and ATP5b proteins, (4) increased reactive oxygen species and elevated mitochondrial membrane potential, and (5) increased innate immune response and p38 MAP kinase activation. Our data reveals the existence of an underlying Redox-inflammatory axis fueled by altered mitochondrial metabolism in the absence of Gdap1. As this biochemical axis encompasses a wide variety of druggable targets, our results may have implications for developing therapies using combinatorial pharmacological approaches and improving therefore human welfare. A Redox-immune axis underlying motor neuron degeneration caused by the absence of Gdap1. Our results show that Gdap1-/- motor neurons have a fragile cellular phenotype that is prone to degeneration. Gdap1-/- iPSCs differentiated into motor neurons showed an altered metabolic state: decreased glycolysis and increased OXPHOS. These alterations may lead to hyperpolarization of mitochondria and increased ROS levels. Excessive amounts of ROS might be the cause of increased mitophagy, p38 activation and inflammation as a cellular response to oxidative stress. The p38 MAPK pathway and the immune response may, in turn, have feedback mechanisms, leading to the induction of apoptosis and senescence, respectively. CAC, citric acid cycle; ETC, electronic transport chain; Glc, glucose; Lac, lactate; Pyr, pyruvate.

Unique nutritional features that distinguish Amaranthus cruentus L. and Chenopodium quinoa Willd seeds.

Univariate (Analysis of Variance_ANOVA) and multivariate (Principal Component Analysis (PCA) and Canonical Discriminant Analysis (CDA)) analyses were performed in order to classify and authenticate the seeds from different varieties of quinoa (Chenopodium quinoa Will.), and amaranth (Amaranthus cruentus L.). The univariate analysis showed differences between species for sucrose, K, Ca, unsaturated fatty acids, and the ω6/ω3 ratio. Nevertheless, to strengthen this classification, a PCA was applied separating the samples in 2 groups; group 1, formed by quinoa seeds, presented higher contents of margaroleic, eicosadienoic, behenic, erucic, linolenic, linoleic, and gadoleic acids, proteins, sucrose, and total sugars. Group 2, formed by amaranth seeds, showed positive values for Mn, Mg, Fe, P, Zn, Ca, fiber, glucose, and ω6/ω3 ratio. Furthermore, the CDA models developed resulted in a probability of event of 100% when classifying the samples in the groups quinoa or amaranth, highlighting the good sensitivity of the models used.

Peripheral Ulcerative Keratopathy following Systemic Treatment with Bortezomib: A Case Report.

A 60-year-old patient was diagnosed with multiple myeloma in November 2020, and started treatment in December 2020 with bortezomib, dexamethasone, and thalidomide. Four months later, he presented to the ophthalmology emergency department with inflammation and pain in the left eye. Examination of the anterior segment revealed severe, mixed anterior squamous blepharitis with significant bilateral palpebral inflammation, dysfunction of the meibomian glands, and several styes on both eyelids bilaterally. A peripheral ulcerative keratopathy was detected while examining the left eye cornea, with an inferior infiltrate and significant thinning, approximately 5 mm in length, at the limbal margin (Figure 1). Tear break-up time was shortened bilaterally. In addition to palpebral hygiene and oral treatment with doxycycline 100 mg every 24 hours, the patient was prescribed the following topical treatment without preservatives: artificial tears with lipid emulsion, netilmicin 0,3% 0,4 ml eye drops every 6 hours and dexamethasone 1 mg/ml every 8 hours.

Myc Supports Self-Renewal of Basal Cells in the Esophageal Epithelium.

It is widely believed that cellular senescence plays a critical role in both aging and cancer, and that senescence is a fundamental, permanent growth arrest that somatic cells cannot avoid. Here we show that Myc plays an important role in self-renewal of esophageal epithelial cells, contributing to their resistance to cellular senescence. Myc is homogeneously expressed in basal cells of the esophageal epithelium and Myc positively regulates their self-renewal by maintaining their undifferentiated state. Indeed, Myc knockout induced a loss of the undifferentiated state of esophageal epithelial cells resulting in cellular senescence while forced MYC expression promoted oncogenic cell proliferation. A superoxide scavenger counteracted Myc knockout-induced senescence, therefore suggesting that a mitochondrial superoxide takes part in inducing senescence. Taken together, these analyses reveal extremely low levels of cellular senescence and senescence-associated phenotypes in the esophageal epithelium, as well as a critical role for Myc in self-renewal of basal cells in this organ. This provides new avenues for studying and understanding the links between stemness and resistance to cellular senescence.

In vivo partial reprogramming alters age-associated molecular changes during physiological aging in mice.

Partial reprogramming by expression of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) for short periods of time restores a youthful epigenetic signature to aging cells and extends the life span of a premature aging mouse model. However, the effects of longer-term partial reprogramming in physiologically aging wild-type mice are unknown. Here, we performed various long-term partial reprogramming regimens, including different onset timings, during physiological aging. Long-term partial reprogramming lead to rejuvenating effects in different tissues, such as the kidney and skin, and at the organismal level; duration of the treatment determined the extent of the beneficial effects. The rejuvenating effects were associated with a reversion of the epigenetic clock and metabolic and transcriptomic changes, including reduced expression of genes involved in the inflammation, senescence and stress response pathways. Overall, our observations indicate that partial reprogramming protocols can be designed to be safe and effective in preventing age-related physiological changes. We further conclude that longer-term partial reprogramming regimens are more effective in delaying aging phenotypes than short-term reprogramming.

Correlation Between Retrograde Trans-Synaptic Degeneration of Ganglion Cells and Optical Coherence Tomography Angiography Following Ischemic Stroke.

Objective Following nerve injury, the projection of posterior visual pathway lesions into the macular ganglion cell layer (GCL) region indicates retrograde trans-synaptic degeneration (RTSD) as a mechanism of functional damage. Our purpose is to assess GCL damage and the impacts of ischemic brain lesions affecting the visual pathway on macular microvascularization in patients with stroke. Methods In a case-control study, we examined 15 ischemic stroke patients who showed visual field defects and 50 healthy controls using the high-resolution optical coherence tomography (OCT) techniques such as spectral domain-OCT (SD-OCT) to measure retinal nerve fiber layer (RNFL) and GCL thicknesses, and OCT angiography (OCTA) to assess damage to the macular microvasculature. Results In the cases, the correlation was detected among the site of vascular damage, visual field defect, retinal GCL thinning, and normal RNFL thickness. Further observations were significant reductions in macular thickness, GCL thickness, outer retinal layer vascular density, and vascular area in deeper retinal layers (p < 0.05). Conclusion Our findings suggest that ocular microvasculature abnormalities could serve as diagnostic and/or prognostic markers in patients with stroke and support the described use of GCL thickness as an image marker of visual pathway RTSD after brain injury.

c-MYC Triggers Lipid Remodelling During Early Somatic Cell Reprogramming to Pluripotency.

Metabolic rewiring and mitochondrial dynamics remodelling are hallmarks of cell reprogramming, but the roles of the reprogramming factors in these changes are not fully understood. Here we show that c-MYC induces biosynthesis of fatty acids and increases the rate of pentose phosphate pathway. Time-course profiling of fatty acids and complex lipids during cell reprogramming using lipidomics revealed a profound remodelling of the lipid content, as well as the saturation and length of their acyl chains, in a c-MYC-dependent manner. Pluripotent cells displayed abundant cardiolipins and scarce phosphatidylcholines, with a prevalence of monounsaturated acyl chains. Cells undergoing cell reprogramming showed an increase in mitochondrial membrane potential that paralleled that of mitochondrial-specific cardiolipins. We conclude that c-MYC controls the rewiring of somatic cell metabolism early in cell reprogramming by orchestrating cell proliferation, synthesis of macromolecular components and lipid remodelling, all necessary processes for a successful phenotypic transition to pluripotency. c-MYC promotes anabolic metabolism, mitochondrial fitness and lipid remodelling early in cell reprogramming. A high rate of aerobic glycolysis is crucial to provide intermediaries for biosynthetic pathways. To ensure the availability of nucleotides, amino acids and lipids for cell proliferation, cells must provide with a constant flux of the elemental building blocks for macromolecule assembly and fulfil the anabolic demands to reach the critical cellular mass levels to satisfactorily undergo cell division. A high rate of aerobic glycolysis is induced by c-MYC, increasing the amounts of intracellular Glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and glyceraldehyde-3-phosphate (GA3P), which can all enter pentose phosphate pathway (PPP) to produce Ribose-5-Phosphate (R5P) and NADPH, which are necessary for the biosynthesis of biomolecules such as proteins, nucleic acids, or lipids. C-MYC-dependent activation of glucose-6-phosphate dehydrogenase (G6PD) may play a critical role in the shunting of G6P to PPP and generation of NADPH. High glycolytic flux increases the amounts of dihydroxyacetone phosphate (DHAP), which is crucial for biosynthesis of phospholipids and triacylglycerols, and pyruvate (Pyr), which can be converted to citrate (Cit) in the mitochondria and enter the biosynthesis of fatty acids (FA). During cell reprogramming, c-MYC-dependent lipid remodelling leads to Polyunsaturated Fatty Acid (PUFA) downregulation and Monounsaturated Fatty Acid (MUFA) upregulation, which may play critical roles in cytoarchitectural remodelling of cell membrane or non-canonical autophagy, respectively. Cardiolipin (pink dots) rise early in cell reprogramming correlates with an increase in mitochondrial fitness, suggesting that c-MYC may restore proper levels of cardiolipins and antioxidant proteins, such as UCP2, to guarantee an optimal mitochondrial function while upholding ROS levels, reinforcing the idea of cell rejuvenation early in cell reprogramming.

Concordancia entre termografía con teléfono inteligente y angio-TAC para detección de perforantes cutáneas en colgajos DIEP / Correlation between smartphone thermography and angio-CT to detect perforators in DIEP flaps

INTRODUCCIÓN Y OBJETIVO: Existe una gran variedad de técnicas para valorar e identificar perforantes. De todas ellas, la angio-TAC es la prueba considerada como modelo de referencia. La termografía detecta las radiaciones infrarrojas emitidas por los objetos. Esta técnica ha sufrido grandes avances y en la actualidad, está disponible una cámara termal que se puede conectar a los teléfonos inteligentes. El objetivo del presente estudio es analizar la correlación que existe a la hora de determinar la localización exacta de la salida de las perforantes en colgajos libres DIEP entre la termografía para móviles, en comparación con angio-TAC y entre la termografía y el Doppler manual para la localización de perforantes. MATERIAL Y MÉTODO: Diseñamos un estudio de tipo observacional descriptivo de evaluación de pruebas diagnósticas con prueba de referencia. Incluimos un total de 14 pacientes, sometidas a reconstrucción mamaria con colgajo libre DIEP en el Servicio de Cirugía Plástica y Reparadora del Hospital Universitario Río Hortega de Valladolid (España), en el periodo comprendido entre septiembre de 2018 y mayo de 2019. RESULTADOS: El coeficiente de correlación de Pearson de la angio-TAC con la termografía fue de 0.866 (p < 0.01), y el del Doppler con la termografía de 0.952 (p < 0.01). El análisis de regresión lineal arrojó los siguientes datos: angio-TAC = 0.804 X termografía. Doppler = 0.982 X termografía, siendo ambas variables dependientes significativas (p < 0.01). CONCLUSIONES: Presentamos un estudio para la realización de un mapeo de perforantes de forma rápida, que necesita un material de bajo coste ya que solo emplea una cámara infrarroja y un teléfono inteligente con conexión mini USB, que requiere un entrenamiento mínimo, y proporciona una buena fiabilidad en comparación con la angio-TAC, considerada como la prueba de referencia en la actualidad

BACKGROUND AND OBJECTIVE: A wide range of techniques allow us to assess and identify cutaneous perforators. Out of all of them, angio-CT is the test considered to be gold standard. Thermography detects infrared light from objects. This technique has undergone an enormous advance and is available nowadays as a thermal imager that can be connected to smartphones. Our aim is is to analyze the correlation that exists when determining the exact location of the perforations in DIEP free flaps between mobile thermography compared to angio-CT and between thermography and manual doppler, for the location of perforators Methods: We have carried out a descriptive observational study to assess diagnostic tests with gold standard tests. A total of 14 patients have been included in this study, all of them had undergone breast reconstruction with DIEP technique by the Plastic and Reconstructive Surgery team of Rio Hortega University Hospital in Valladolid (Spain) from September 2018 to May 2019. RESULTS: Pearson correlation coefficient between angio-CT and thermography was 0.866 (p < 0.01) and between doppler and thermography was 0.952 (p < 0.01). Lineal regression analysis showed: angio-CT = 0.804 X thermography (p < 0.01). Doppler = 0.982 X thermograpy (p < 0.01). CONCLUSION: We present a study to map cutaneous perforators in an affordable and quick way, using just an infrared camera and a smartphone. This technique doesn't require special training and has a Good correspondence with angio-CT, which is considered the gold standard technique

Controlled Covalent Functionalization of 2 H-MoS2 with Molecular or Polymeric Adlayers.

Most air-stable 2D materials are relatively inert, which makes their chemical modification difficult. In particular, in the case of MoS2 , the semiconducting 2 H-MoS2 is much less reactive than its metallic counterpart, 1T-MoS2 . As a consequence, there are hardly any reliable methods for the covalent modification of 2 H-MoS2 . An ideal method for the chemical functionalization of such materials should be both mild, not requiring the introduction of a large number of defects, and versatile, allowing for the decoration with as many different functional groups as possible. Herein, a comprehensive study on the covalent functionalization of 2 H-MoS2 with maleimides is presented. The use of a base (Et3 N) leads to the in situ formation of a succinimide polymer layer, covalently connected to MoS2 . In contrast, in the absence of base, functionalization stops at the molecular level. Moreover, the functionalization protocol is mild (occurs at room temperature), fast (nearly complete in 1 h), and very flexible (11 different solvents and 10 different maleimides tested). In practical terms, the procedures described here allow for the chemist to manipulate 2 H-MoS2 in a very flexible way, decorating it with polymers or molecules, and with a wide range of functional groups for subsequent modification. Conceptually, the spurious formation of an organic polymer might be general to other methods of functionalization of 2D materials, where a large excess of molecular reagents is typically used.

Mitochondrial dynamics and metabolism in induced pluripotency.

Somatic cells can be reprogrammed to pluripotency by either ectopic expression of defined factors or exposure to chemical cocktails. During reprogramming, somatic cells undergo dramatic changes in a wide range of cellular processes, such as metabolism, mitochondrial morphology and function, cell signaling pathways or immortalization. Regulation of these processes during cell reprograming lead to the acquisition of a pluripotent state, which enables indefinite propagation by symmetrical self-renewal without losing the ability of reprogrammed cells to differentiate into all cell types of the adult. In this review, recent data from different laboratories showing how these processes are controlled during the phenotypic transformation of a somatic cell into a pluripotent stem cell will be discussed.