Case report: A rare case of cyclodialysis after cataract surgery managed exclusively with medical therapy.

We present the case of a 68-year-old female who presented with a substantial cyclodialysis after phacoemulsification identified by anterior segment optical coherence tomography and ultrasound biomicroscopy. Gonioscopy is challenging because of its shallow anterior chamber. Due to lack of response to topical steroids and refusal by the patient for surgical treatment, topical steroids were tapered, and cycloplegic was initiated. After two weeks, the cyclodialysis cleft was closed on optical coherence tomography persisting residual subchoroidal fluid, topical treatment was mantained. Despite of this, visual acuity and hypotony were restored. Finally at 2 months visit subchoroidal fluid was reabsorbed and patient was discharged. Clinics should consider the possibility of cyclodialysis in cases of unjustified hypotony during postoperative period. In addition, we recommend performing anterior segment imaging which includes ultrasound biomicroscopy and optical coherence tomography, when the evaluation of angular structures is unfeasible. Finally, we propose medical management for severe cyclodialysis as the first option considering anti-inflammatory drugs could perpetrate the separation between scleral spur and ciliary muscle.

Development of functional consortia for the pretreatment of compostable lignocellulosic waste: A simple and effective solution to a large-scale problem.

Microorganisms drive the degradation of organic matter thanks to their enzymatic versatility. However, the structure of lignocellulose poses a great challenge for the microbiota inhabiting a compost pile. Our purpose was to increase the biodegradability of vegetable waste in the early stages of the composting process by applying a microbial consortium with lignocelllulolytic capacity. For this, a previous screening was performed among the culturable microbiota from different composting processes to find inoculants with ligninocellulolytic activity. Selected strains were applied as a pure culture and as a microbial consortium. The starting material was composed of tomato plant and pruning remains mixed in a ratio (50:50 v/v), whose humidity was adjusted to around 65%. To determine the ability of both treatments to activate the biodegradation of the mixtures, moisture, organic matter, ash, C/N ratio, 4-day cumulative respirometric index (AT4) and degradation rates of cellulose, hemicellulose and lignin were evaluated. Subsequently, a real composting process was developed in which the performance of the microbial consortium was compared with the composting process without inoculum (control). According to our tests, three microbial strains (Bacillus safensis, Bacillus licheniformis and Fusarium oxysporum) were selected. The results showed that the application of the bacteria strains at low doses (104 CFU g-1 on the complete residual material of the pile) resulted in higher rates of lignocelullose degradation after 10 days of treatment compared to that observed after application of the fungus in pure culture or untreated controls. The implementation of the strategy described in this work resulted in obtaining compost with better agronomic quality than the uninoculated controls. Therefore, the application of this consortium could be considered as an interesting tool for bioactivation of lignocellulosic waste prior to the composting process.

Hereditary Optic Neuropathies: A Systematic Review on the Interplay between Biomaterials and Induced Pluripotent Stem Cells.

Hereditary optic neuropathies (HONs) such as dominant optic atrophy (DOA) and Leber Hereditary Optic Neuropathy (LHON) are mitochondrial diseases characterized by a degenerative loss of retinal ganglion cells (RGCs) and are a cause of blindness worldwide. To date, there are only limited disease-modifying treatments for these disorders. The discovery of induced pluripotent stem cell (iPSC) technology has opened several promising opportunities in the field of HON research and the search for therapeutic approaches. This systematic review is focused on the two most frequent HONs (LHON and DOA) and on the recent studies related to the application of human iPSC technology in combination with biomaterials technology for their potential use in the development of RGC replacement therapies with the final aim of the improvement or even the restoration of the vision of HON patients. To this purpose, the combination of natural and synthetic biomaterials modified with peptides, neurotrophic factors, and other low- to medium-molecular weight compounds, mimicking the ocular extracellular matrices, with human iPSC or iPSC-derived cell retinal progenitors holds enormous potential to be exploited in the near future for the generation of transplantable RGC populations.

A multidisciplinary challenge: Therapy with phosphorus-32 for locally advanced pancreatic cancer.

Pancreatic cancer is a disease with a poor prognosis, and overall survival has improved the least in the last 40 years of all cancers. Locally advanced pancreatic adenocarcinoma, without distant metastasis but with limiting vascular involvement, constitutes almost one third of these patients. This group is the focus of most research efforts to introduce treatments to increase surgical salvage rates and/or survival, with two main objectives: local control and prevention of systemic progression. Intratumoural treatment with phosphorus-32 microparticles, guided by echoendoscopy and combined with standard chemotherapy may have significant and clinically relevant benefits in these patients, and therefore a valuable treatment option in a disease where there is an urgent need to develop new therapies to help improve outcomes.

Sustainable approach to the control of airborne phytopathogenic fungi by application of compost extracts.

The search for new sustainable alternatives for plant disease control has gained interest in the last decades. Compost extracts are nowadays considered a remarkable alternative to agrochemicals due to their biopesticidal properties. However, these properties could be affected by the different variables of extraction protocols and by starting compost. This work focused on the physicochemical and biological characterization of compost extracts obtained from a wide range of composted materials and different extraction protocols (CEP). CEP-1 and CEP-4 involved incubation at 20 °C for 48 h and 14 days, respectively; CEP-2 incubation for 24 h at 40 °C; while CEP-3 were incubated for 12 h at 70 °C. Electrical conductivity, pH, total organic carbon (TOC) and phenolic content were determined as well as the actinobacterial count and enzyme profiles related to plant pathogen suppression. Additionally, the influence of the different materials and protocols on the in vitro growth inhibition of Alternaria alternata and Botrytis cinerea was determined. The starting materials and extraction protocols significantly influenced the physicochemical and biological characteristics of extracts. Treatments based on long incubation times at 20 °C, as well as those based on short incubation times at 40 °C, resulted in extracts with increased suppressive properties. However, extracts derived from CEP-3 protocol were characterized by high phenolic and TOC content, low functional biodiversity, and a more discreet antagonistic capacity. Therefore, the development and optimization of suitable extraction protocols could lead to compost extracts with increased phytoprotective capacities, thus becoming an effective and sustainable alternative to chemical inputs.

Creation of an iPSC-Based Skeletal Muscle Model of McArdle Disease Harbouring the Mutation c.2392T>C (p.Trp798Arg) in the PYGM Gene.

McArdle disease is a rare autosomal recessive condition caused by mutations in the PYGM gene. This gene encodes the skeletal muscle isoform of glycogen phosphorylase or myophosphorylase. Patients with McArdle disease have an inability to obtain energy from their muscle glycogen stores, which manifests as a marked exercise intolerance. Nowadays, there is no cure for this disorder and recommendations are intended to prevent and mitigate symptoms. There is great heterogeneity among the pathogenic variants found in the PYGM gene, and there is no obvious correlation between genotypes and phenotypes. Here, we present the generation of the first human iPSC-based skeletal muscle model harbouring the second most frequent mutation in PYGM in the Spanish population: NM_005609.4: c.2392T>C (p.Trp798Arg). To this end, iPSCs derived from a McArdle patient and a healthy control were both successfully differentiated into skeletal muscle cells using a small molecule-based protocol. The created McArdle skeletal muscle model was validated by confirming distinctive biochemical aspects of the disease such as the absence of myophosphorylase, the most typical biochemical feature of these patients. This model will be very valuable for use in future high-throughput pharmacological screenings.

Epidemiology, patterns of care and prognosis of acute kidney injury in critically ill patients: A multicenter study in Argentina (The EPIRA study).

BACKGROUND: Acute kidney injury (AKI) is associated with high morbidity and mortality rates in the intensive care unit (ICU). In low- and middle-income countries (LMICs), epidemiological information about this condition is still scarce. Our main objective was to characterize its epidemiology, prognosis, and its treatment. METHODS: This multicenter prospective cohort study included 1466 patients from 35 ICUs during 6 months in Argentina in 2018. Risk factors and outcomes in patients with and without AKI, and between AKI on admission (AKIadm) and that developed during hospitalization (AKIhosp) were analyzed. RESULTS: AKI occurred in 61.3% of patients (900/1466); 72.6% were AKIadm and 27.3% AKIhosp. Risk factors were age, BMI, arterial hypertension, cardiovascular diseases, diabetes, SOFA, APACHE II, dehydration, sepsis, vasopressor use, radiocontrast, diuresis/h and mechanical ventilation. Independent predictors for AKI were sepsis, diabetes, dehydration, vasopressors on admission, APACHE II and radiocontrast use. Renal replacement therapies (RRT) requirement in AKI patients was 14.8%. Hospital mortality in AKI vs. non-AKI was 38.7% and 23.3% (p < 0.001); and in AKIadm vs. AKIhosp, 41.2% and 37.8% (p = 0.53). CONCLUSIONS: ICU-acquired AKI has high incidence, complications and mortality. Risk factors for AKI and RRT utilization were similar to those described in other epidemiological studies. AKIadm was more frequent than AKIhosp, but had equal prognosis.

The role of ketamine in major depressive disorders: Effects on parvalbumin-positive interneurons in hippocampus.

Major depressive disorder (MDD) is a complex illness that is arising as a growing public health concern. Although several brain areas are related to this type of disorders, at the cellular level, the parvalbumin-positive cells of the hippocampus interplay a very relevant role. They control pyramidal cell bursts, neuronal networks, basic microcircuit functions, and other complex neuronal tasks involved in mood disorders. In resistant depressions, the efficacy of current antidepressant treatments drops dramatically, so the new rapid-acting antidepressants (RAADs) are being postulated as novel treatments. Ketamine at subanesthetic doses and its derivative metabolites have been proposed as RAADs due to their rapid and sustained action by blocking N-methyl-d-aspartate (NMDA) receptors, which in turn lead to the release of brain-derived neurotrophic factor (BDNF). This mechanism produces a rapid plasticity activation mediated by neurotransmitter homeostasis, synapse recovery, and increased dendritic spines and therefore, it is a promising therapeutic approach to improve cognitive symptoms in MDD.

Mitigation of phytotoxic effect of compost by application of optimized aqueous extraction protocols.

The abuse of chemical fertilizers in recent decades has led the promotion of less harmful alternatives, such as compost or aqueous extracts obtained from it. Therefore, it is essential to develop liquid biofertilizers, which in addition of being stable and useful for fertigation and foliar application in intensive agriculture had a remarkable phytostimulant extracts. For this purpose, a collection of aqueous extracts was obtained by applying four different Compost Extraction Protocols (CEP1, CEP2, CEP3, CEP4) in terms of incubation time, temperature and agitation of compost samples from agri-food waste, olive mill waste, sewage sludge and vegetable waste. Subsequently, a physicochemical characterization of the obtained set was performed in which pH, electrical conductivity and Total Organic Carbon (TOC) were measured. In addition, a biological characterization was also carried out by calculating the Germination Index (GI) and determining the Biological Oxygen Demand (BOD5). Furthermore, functional diversity was studied using the Biolog EcoPlates technique. The results obtained confirmed the great heterogeneity of the selected raw materials. However, it was observed that the less aggressive treatments in terms of temperature and incubation time, such as CEP1 (48 h, room temperature (RT)) or CEP4 (14 days, RT), provided aqueous compost extracts with better phytostimulant characteristics than the starting composts. It was even possible to find a compost extraction protocol that maximize the beneficial effects of compost. This was the case of CEP1, which improved the GI and reduced the phytotoxicity in most of the raw materials analyzed. Therefore, the use of this type of liquid organic amendment could mitigate the phytotoxic effect of several composts being a good alternative to the use of chemical fertilizers.

Biorecovery of olive mill wastewater sludge from evaporation ponds.

Olive mill wastewater (OMW) resulting from the olive oil extraction process is usually disposed of in evaporation ponds where it concentrates generating a sludge that pollutes the ponds nearby area. In this study, four bio-treatments were applied for the in-situ bioremediation and valorization of OMW sludge: Landfarming, phytoremediation, composting and vermicomposting. In all cases, the OMW sludge was added with organic residues (mushroom compost, rabbit manure, and chicken manure). The bio-treatments were carried out in duplicate, inoculated and non-inoculated, to determine the effect of a specialized fungal consortium (Aspergillus ochraceus H2 and Scedosporium apiospermum H16) on the efficacy of the bio-treatments. The evaluation of chemical parameters, toxicity, and functional microbial biodiversity revealed that the four techniques depleted the toxicity and favored the stimulation of functional microbiota. Landfarming and phytoremediation allowed the decontamination and improvement of soils. Composting and vermicomposting also offered high-quality products of agronomic interest. Inoculation improved the bioremediation effectiveness. Biological treatments are effective for the safe recovery of contaminated OMW sludge into high-quality services and products.

Design and validation of cyanobacteria-rhizobacteria consortia for tomato seedlings growth promotion.

The use of rhizobacteria provide great benefits in terms of nitrogen supply, suppression of plant diseases, or production of vitamins and phytohormones that stimulate the plant growth. At the same time, cyanobacteria can photosynthesize, fix nitrogen, synthesize substances that stimulate rhizogenesis, plant aerial growth, or even suppose an extra supply of carbon usable by heterotrophic bacteria, as well as act as biological control agents, give them an enormous value as plant growth promoters. The present study focused on the in vitro establishment of consortia using heterotrophic bacteria and cyanobacteria and the determination of their effectiveness in the development of tomato seedlings. Microbial collection was composed of 3 cyanobacteria (SAB-M612 and SAB-B866 belonging to Nostocaceae Family) and GS (unidentified cyanobacterium) and two phosphate and potassium solubilizing heterotrophic bacteria (Pseudomonas putida-BIO175 and Pantoea cypripedii-BIO175). The results revealed the influence of the culture medium, incubation time and the microbial components of each consortium in determining their success as biofertilizers. In this work, the most compatible consortia were obtained by combining the SAB-B866 and GS cyanobacteria with either of the two heterotrophic bacteria. Cyanobacteria GS promoted the growth of both rhizobacteria in vitro (increasing logarithmic units when they grew together). While Cyanobacteria SAB-B866 together with both rhizobacteria stimulated the growth of tomato seedlings in planta, leading to greater aerial development of the treated seedlings. Parameters such as fresh weight and stem diameter stood out in the plants treated with the consortia (SAB-B866 and both bacteria) compared to the untreated plants, where the values doubled. However, the increase was more discrete for the parameters stem length and number of leaves. These results suggest that the artificial formulation of microbial consortia can have positive synergistic effects on plant growth, which is of enormous agro-biotechnological interest.

Usefulness of chest X-rays for evaluating prognosis in patients with COVID-19.

BACKGROUND AND AIMS: The pandemia caused by SARS-CoV-2 (COVID-19) has been a diagnostic challenge in which chest X-rays have had a key role. This study aimed to determine whether the Radiological Scale for Evaluating Hospital Admission (RSEHA) applied to chest X-rays of patients with COVID-19 when they present at the emergency department is related with the severity of COVID-19 in terms of the need for admission to the hospital, the need for admission to the intensive care unit (ICU), and/or mortality. MATERIAL AND METHODS: This retrospective study included 292 patients with COVID-19 who presented at the emergency department between March 16, 2020 and April 30, 2020. To standardize the radiologic patterns, we used the RSEHA, categorizing the radiologic pattern as mild, moderate, or severe. We analyzed the relationship between radiologic severity according to the RSEHA with the need for admission to the hospital, admission to the ICU, and mortality. RESULTS: Hospital admission was necessary in 91.4% of the patients. The RSEHA was significantly associated with the need for hospital admission (p = 0.03) and with the need for ICU admission (p < 0.001). A total of 51 (17.5%) patients died; of these, 57% had the severe pattern on the RSEHA. When we analyzed mortality by grouping patients according to their results on the RSEHA and their age range, the percentage of patients who died increased after age 70 years in patients classified as moderate or severe on the RSEHA. CONCLUSIONS: Chest X-rays in patients with COVID-19 obtained in the emergency department are useful for determining the prognosis in terms of admission to the hospital, admission to the ICU, and mortality; radiologic patterns categorized as severe on the RSEHA are associated with greater mortality and admission to the ICU.

Usefulness of chest X-rays for evaluating prognosis in patients with COVID-19. / Utilidad de la radiografía de tórax para evaluar el pronóstico de pacientes con COVID-19.

BACKGROUND AND AIMS: The pandemia caused by SARS-CoV-2 (COVID-19) has been a diagnostic challenge in which chest X-rays have had a key role. This study aimed to determine whether the Radiological Scale for Evaluating Hospital Admission (RSEHA) applied to chest X-rays of patients with COVID-19 when they present at the emergency department is related with the severity of COVID-19 in terms of the need for admission to the hospital, the need for admission to the intensive care unit (ICU), and/or mortality. MATERIAL AND METHODS: This retrospective study included 292 patients with COVID-19 who presented at the emergency department between March 16, 2020 and April 30, 2020. To standardize the radiologic patterns, we used the RSEHA, categorizing the radiologic pattern as mild, moderate, or severe. We analyzed the relationship between radiologic severity according to the RSEHA with the need for admission to the hospital, admission to the ICU, and mortality. RESULTS: Hospital admission was necessary in 91.4% of the patients. The RSEHA was significantly associated with the need for hospital admission (p=0.03) and with the need for ICU admission (p<0.001). A total of 51 (17.5%) patients died; of these, 57% had the severe pattern on the RSEHA. When we analyzed mortality by grouping patients according to their results on the RSEHA and their age range, the percentage of patients who died increased after age 70 years in patients classified as moderate or severe on the RSEHA. CONCLUSIONS: Chest X-rays in patients with COVID-19 obtained in the emergency department are useful for determining the prognosis in terms of admission to the hospital, admission to the ICU, and mortality; radiologic patterns categorized as severe on the RSEHA are associated with greater mortality and admission to the ICU.

Hereditary Optic Neuropathies: Induced Pluripotent Stem Cell-Based 2D/3D Approaches.

Inherited optic neuropathies share visual impairment due to the degeneration of retinal ganglion cells (RGCs) as the hallmark of the disease. This group of genetic disorders are caused by mutations in nuclear genes or in the mitochondrial DNA (mtDNA). An impaired mitochondrial function is the underlying mechanism of these diseases. Currently, optic neuropathies lack an effective treatment, and the implementation of induced pluripotent stem cell (iPSC) technology would entail a huge step forward. The generation of iPSC-derived RGCs would allow faithfully modeling these disorders, and these RGCs would represent an appealing platform for drug screening as well, paving the way for a proper therapy. Here, we review the ongoing two-dimensional (2D) and three-dimensional (3D) approaches based on iPSCs and their applications, taking into account the more innovative technologies, which include tissue engineering or microfluidics.

Olive mill wastewater-evaporation ponds long term stored: Integrated assessment of in situ bioremediation strategies based on composting and vermicomposting.

During the last two decades, the method most widely used to manage olive mill wastewater (OMW) derived from olive oil production has been its disposal in evaporation ponds. Long-term storage of OMW leads to the accumulation of toxic sediments (OMWS) rich in recalcitrant compounds with phytotoxic and antimicrobial properties, which limit their use for agronomic purpose. The aim of this study was to compare the effect of two in situ bioremediation strategies (composting and a combination of composting followed by vermicomposting) to remove the potential toxicity of the sediments derived from long-term stored OMW. The results obtained showed that the composting method assisted with the earthworms enhanced the depletion of phenolic compounds and OMWS ecotoxicity more than composting, especially during the maturation stage. Moreover, vermicomposting was more effective in the reduction of the OMWS salinity. However, a pre-composting process to the OMWS is necessary prior to vermicomposting to provide the suitable conditions for earthworms survival and activity. Furthermore, the final compost showed a phytostimulating effect. Therefore, these in situ bioremediation strategies can be considered potential tools for decontamination and recovery of long-term stored OMWS in evaporation ponds, which currently poses an unsolved environmental problem.

Generation of the iPSC line IISHDOi007-A from peripheral blood mononuclear cells from a patient with McArdle disease harbouring the mutation c.2392 T > C; p.Trp798Arg.

Peripheral blood mononuclear cells (PBMCs) from a McArdle patient carrying a homozygous mutation in the PYGM gene: c.2392 T > C; p.Trp798Arg were used for the generation of the human iPSC line, IISHDOi007-A. For the delivery of the reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc, a non-integrative methodology that implies the use of Sendai virus has been applied.

RELICT HUMID TROPICAL FOREST IN MEXICO PROMOTES DIFFERENTIATION IN BARRED WOODCREEPERS Dendrocolaptes (AVES: FURNARIIDAE).

Humid tropical forests in Mesoamerica are distributed along the Atlantic slope and, in scattered locations, along the Mexican Pacific slope. These poorly explored Mexican forests include microendemic bird species. Two species in the genus Dendrocolaptes occur in lowland and foothill humid tropical forests of Mesoamerica. One of these, D. sanctithomae, is comprised of four subspecies, of which the two that occur in Mexico, D. s. sanctithomae and D. s. sheffleri, are distinctly different morphologically, and the latter is a poorly known microendemic taxon of the Mexican Pacific humid tropical forest in the Sierra Madre del Sur. We used both nuclear (nDNA) and mitochondrial DNA (mtDNA) to evaluate the genetic variation of D. sanctithomae in Mexico, and complemented this with a quantitative analysis of phenotypic traits. We also conducted analyses of environmental niche models to test the hypothesis of niche differentiation of D. s. sheffleri from other taxa of D. sanctithomae. Our phylogenetic reconstructions of mtDNA consistently recovered D. s. sheffleri and D. s. sanctithomae as reciprocally monophyletic, while they shared alleles of nDNA. These mtDNA differences are comparable with differences reported between other Dendrocolaptes sister-taxa pairs. Our analysis of phenotypic traits also indicated that the taxa differ in measurements of hallux and feather barring. In contrast, niche differentiation tests suggest that the niches of both taxa are more similar than expected by chance. Our evidence leads us to propose species status for D. sheffleri. This is an additional example of recent speciation in Mexico that indicates active and peripatric evolutionary differentiation in the northern Neotropics.

Mitochondrial Dysfunction and Calcium Dysregulation in Leigh Syndrome Induced Pluripotent Stem Cell Derived Neurons.

Leigh syndrome (LS) is the most frequent infantile mitochondrial disorder (MD) and is characterized by neurodegeneration and astrogliosis in the basal ganglia or the brain stem. At present, there is no cure or treatment for this disease, partly due to scarcity of LS models. Current models generally fail to recapitulate important traits of the disease. Therefore, there is an urgent need to develop new human in vitro models. Establishment of induced pluripotent stem cells (iPSCs) followed by differentiation into neurons is a powerful tool to obtain an in vitro model for LS. Here, we describe the generation and characterization of iPSCs, neural stem cells (NSCs) and iPSC-derived neurons harboring the mtDNA mutation m.13513G>A in heteroplasmy. We have performed mitochondrial characterization, analysis of electrophysiological properties and calcium imaging of LS neurons. Here, we show a clearly compromised oxidative phosphorylation (OXPHOS) function in LS patient neurons. This is also the first report of electrophysiological studies performed on iPSC-derived neurons harboring an mtDNA mutation, which revealed that, in spite of having identical electrical properties, diseased neurons manifested mitochondrial dysfunction together with a diminished calcium buffering capacity. This could lead to an overload of cytoplasmic calcium concentration and the consequent cell death observed in patients. Importantly, our results highlight the importance of calcium homeostasis in LS pathology.

Evaluation of myopic cornea lenticules. A histochemical and clinical correlation.

In this work, we have analyzed the main clinical and corneal histological parameters that may be associated to the spherical equivalent (SE), age and gender of individuals with myopic refractive errors. For this purpose, 108 cornea stroma lenticules were obtained from patients subjected to ReLEx-SMILE myopia correction. Histological analyses were carried out and histochemistry and immunohistochemistry were used to quantify key histological components of the cornea stroma, including mature collagen fibers, reticular and elastic fibers, glycoproteins, proteoglycans, type-V collagen and several crystallins. Clinical and histological data were analyzed to determine their association with SE, age and gender. Results showed a significant correlation between the age range of the patients and the expression of crystallins CRY-α-A, CRY-λ1 and type-V collagen and between CRY-λ1 and corneal thickness, spherical diopters (D) and SE, although correlation between CRY-λ1 and SE was non-significant when age was controlled. Comparison of cases with low myopia and high/moderate myopia found statistical differences for D and lenticule thickness and diameter. The binary logistic regression analysis allowed us to construct a model using two clinical parameters (D and lenticule thickness). Parameters showing significant correlation with the age were the corneal radius, keratometry reading (K), OZ, CRY-α-A and type-V collagen, whereas SE, lenticule thickness, OZ, CRY-λ1 and type-V collagen showed statistically significant differences between the youngest and the oldest patients. A binary logistic regression analysis model was generated including 3 variables (D, cornea radius and OZ). No gender differences were found. The specific clinical and histological modifications found to be associated to the SE and age could be useful for a better understanding of the mechanisms involved in the genesis or progression of myopia and could establish the basement for future therapeutic options.