Silica Microparticles from Sugarcane By-Products as an Encapsulation System for Retinoids Aimed at Topical Sustained Release.

The encapsulation of retinol within silica microparticles has emerged as a promising opportunity in the realm of cosmetic and pharmaceutical formulations, driven by the need to reinforce the photoprotection and oxidation stability of retinol. This work examines the process of encapsulating retinol into silica microparticles. The association efficiency, microparticle size, molecular structure, morphology, oxidation, and release profile, as well as biocompatibility and skin sensitization, were evaluated. Results showed that 0.03% of retinol and 9% of emulsifier leads to an association efficiency higher than 99% and a particle size with an average of 5.2 µm. FTIR results indicate that there is an association of retinol with the silica microparticles, and some may be on the surface. Microscopy indicates that when association happens, there is less aggregation of the particles. Oxidation occurs in two different phases, the first related to the retinol on the surface and the second to the associated retinol. In addition, a burst release of up to 3 h (30% free retinol, 17% associated retinol) was observed, as well as a sustained release of 44% of retinol up to 24 h. Encapsulation allowed an increase in the minimal skin cytotoxic concentrations of retinol from 0.04 µg/mL to 1.25 mg/mL without skin sensitization. Overall, retinol is protected when associated with silica microparticles, being safe to use in cosmetics and dermatology.

New Natural and Sustainable Cosmetic Preservative Based on Sugarcane Straw Extract.

Preservative ingredients in cosmetic formulations undertake a necessary role in the prevention of microbial contamination. In this field, there is an unmet need for natural, sustainable, and effective preservatives. Thus, the main goal of this work was to evaluate a sugarcane straw extract-based ingredient and investigate its potential as a preservative for cosmetic applications. Different ingredients were developed using several cosmetic solvents to improve the solubility of the extracted compounds. The antimicrobial activity was assessed against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. The 1,2-hexanediol was the solvent that allowed us to achieve the ingredient (20% dry extract dispersed in 25% 1,2-hexanediol in water) with the best antimicrobial performance, showing a minimum inhibitory concentration of between 5% and 3% (I). The 5% (w/v) concentration of this ingredient complied with the USP51 standards for cosmetic preservatives. Real-time (25 °C, 65% RH) and accelerated stability (40 °C, 75% RH) tests were conducted to determine the ingredient stability, and it was found that one month of storage time at room temperature would be ideal for better ingredient stability and performance in terms of composition, pH, color, and antioxidant activity.

Obtention and Characterization of Microcrystalline Cellulose from Industrial Melon Residues Following a Biorefinery Approach.

Residual melon by-products were explored for the first time as a bioresource of microcrystalline cellulose (MCC) obtention. Two alkaline extraction methods were employed, the traditional (4.5% NaOH, 2 h, 80 °C) and a thermo-alkaline in the autoclave (2% NaOH, 1 h, 100 °C), obtaining a yield of MCC ranging from 4.76 to 9.15% and 2.32 to 3.29%, respectively. The final MCCs were characterized for their chemical groups by Fourier-transform infrared spectroscopy (FTIR), crystallinity with X-ray diffraction, and morphology analyzed by scanning electron microscope (SEM). FTIR spectra showed that the traditional protocol allows for a more effective hemicellulose and lignin removal from the melon residues than the thermo-alkaline process. The degree of crystallinity of MCC ranged from 51.51 to 61.94% and 54.80 to 55.07% for the thermo-alkaline and traditional processes, respectively. The peaks detected in X-ray diffraction patterns indicated the presence of Type I cellulose. SEM analysis revealed microcrystals with rough surfaces and great porosity, which could remark their high-water absorption capacity and drug-carrier capacities. Thus, these findings could respond to the need to valorize industrial melon by-products as raw materials for MCC obtention with potential applications as biodegradable materials.

Healing profiles in patients with a chronic diabetic foot ulcer: An exploratory study with machine learning.

Diabetic foot ulcers (DFU) are one of the most frequent and debilitating complications of diabetes. DFU wound healing is a highly complex process, resulting in significant medical, economic and social challenges. Therefore, early identification of patients with a high-risk profile would be important to adequate treatment and more successful health outcomes. This study explores risk assessment profiles for DFU healing and healing prognosis, using machine learning predictive approaches and decision tree algorithms. Patients were evaluated at baseline (T0; N = 158) and 2 months later (T1; N = 108) on sociodemographic, clinical, biochemical and psychological variables. The performance evaluation of the models comprised F1-score, accuracy, precision and recall. Only profiles with F1-score >0.7 were selected for analysis. According to the two profiles generated for DFU healing, the most important predictive factors were illness representations on T1 IPQ-B (IPQ-B ≤ 9.5 and < 10.5) and the DFU duration (≤ 13 weeks). The two predictive models for DFU healing prognosis suggest that biochemical factors are the best predictors of a favorable healing prognosis, namely IL-6, microRNA-146a-5p and PECAM-1 at T0 and angiopoietin-2 at T1. Illness perception at T0 (IPQ-B ≤ 39.5) also emerged as a relevant predictor for healing prognosis. The results emphasize the importance of DFU duration, illness perception and biochemical markers as predictors of  healing in chronic DFUs. Future research is needed to confirm and test the obtained predictive models.

Skin Microbiota and the Cosmetic Industry.

Skin harbors an important microbial ecosystem - the skin microbiota that is in homeostasis with its host and is beneficial for human health. Cosmetic products have the potential to interfere with this microbial community; therefore their impact should be assessed. The aim of this review is to highlight the importance of skin microbiota in the cosmetic industry. Several studies determined that cosmetic ingredients have the potential to disrupt the skin microbiota equilibrium leading to the development of skin diseases and dysregulation of immune response. These studies led their investigation by using different methodologies and models, concluding that methods must be chosen according to the aim of the study, the skin site to be evaluated, and the target population of the cosmetics. Overall, it is crucial to test the impact of cosmetics in the skin microbiota and to stablish standard procedures, as well as specific criteria that allow to classify a cosmetic product as skin microbiota friendly.

Anti-Aging Potential of a Novel Ingredient Derived from Sugarcane Straw Extract (SSE).

Natural and sustainable anti-aging ingredients have gained attention from the cosmetic industry. This study evaluated the anti-aging potential of a sugarcane straw extract-based (SSE) cosmetic ingredient. First, cytotoxicity tests were assessed in keratinocytes and fibroblast cell lines, and sensitization was carried out through the direct peptide reactivity assay. Subsequently, various anti-aging properties were investigated, including inhibiting skin aging-related enzymes, promoting elastin and hyaluronic acid synthesis, and anti-pollution activity. Finally, a permeability assay using a synthetic membrane resembling skin was conducted. The results demonstrated that the SSE ingredient effectively inhibited elastase (55%), collagenase (25%), and tyrosinase (47%) while promoting hyaluronic acid production at non-cytotoxic and low-sensitizer concentrations. Moreover, it reduced the inflammatory response provoked by urban pollution, as evidenced by decreased levels of IL1-α and IL-6. However, it was observed that the phenolic compounds predominantly reached the skin's surface, indicating a limited ability to penetrate deeper layers of the skin. Therefore, it can be concluded that the SSE ingredient holds anti-aging properties, albeit with limited penetration into deeper skin layers. Further research and formulation advancements are needed to optimize the ingredient's ability to reach and exert its effects in deeper skin layers.

Biocontamination and diversity of epilithic bacteria and fungi colonising outdoor stone and mortar sculptures.

Microbial communities colonising outdoor sculptures form intricate and dynamic ecosystems, which can accelerate the deterioration processes of the artworks and pose challenges to their conservation. In this study, the bacterial and fungal communities colonising the surfaces of five contemporary outdoor sculptures were characterised by high-throughput sequencing. The sculptures, made of marble, granite, Ançã limestone and mortar, are in urban parks and squares in the district of Porto, Portugal. The analysis of the microbial populations revealed great taxonomic diversity and species richness, including in well-preserved sculptures showing few visible traces of contamination. Proteobacteria, namely the genera Pseudomonas and Sphingomonas, were the core taxa common to all the sculptures, while Massilia and Aureobasidium were dominant only in granite. An abundance of pigment-producing microorganisms, such as Deinococcus, Methylobacterium, Rhodotorula and Sporobolomyces, was also found in granite. These are relevant taxonomic groups that can negatively impact stone and mortar artworks. The study was complemented with colourimetric analyses and bioluminescence assays to measure the adenosine triphosphate (ATP) content of samples collected from specific contaminated areas of the sculptures. The characterisation of the microbiomes of sculptures can provide further knowledge on the deterioration risks of this type of artwork in the region and help outline future targeted conservation strategies. KEY POINTS: • Rich and abundant microbiomes expose sculptures' vulnerability to deterioration. • Well-preserved sculptures are at risk of deterioration by pigment-producing taxa. • ATP and colourimetry quickly identified the most relevant contaminated areas.

Current postbiotics in the cosmetic market-an update and development opportunities.

Postbiotics are a new class of health-promoting molecules that derive from probiotics. These new cosmetic and dermatological ingredients are defined as a 'preparation of inanimate microorganisms and/or their components that confers a health benefit on the host'. This review focuses on what is presently known of these compounds, the benefits of using them, the main postbiotics products available in the market and players, the production key trends and available production methods. The main advantages identified for the use of postbiotics are related to their higher specificity of action on resident microbiota as of interaction with cells of the host compared to probiotics. Postbiotics can be produced/obtained especially through fermentative processes, but most of companies industrial processes are patented. Most of these compounds are usually derived from lactic acid bacteria, Lactobacillus genera and/or yeasts, especially Saccharomyces cerevisiae. Postbiotics go from metabolites like teichoic acids to polysaccharides among others and exhibit important biological properties such as antioxidant, anti-inflammatory, anti-proliferative and immunomodulatory-the reason why their use in cosmetic formulations must be considered. Besides that, when compared to probiotics, postbiotics have longer shelf life and greater safety and do not require viability in the topical formulation which turns them into an innovative approach within the cosmetic ingredients market. The main players are companies that operate in several areas, such as the chemical industry, food innovation, pharmaceutical and cosmetic industries, and the critical trends for production of these compounds include energy efficiency, emission-free mobility, conservation of finite resources and renewable raw material utilization. KEY POINTS: • Postbiotics are mainly derived from lactic acid bacteria and S. cerevisiae. • Postbiotics exhibit several biological properties. • Postbiotics present several advantages over probiotics.

Effects of cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid supplementation on maternal reproductive parameters.

Conjugated linoleic acid (CLA) is a mixture of positional isomers of linoleic acid found in ruminant products and meat. The diet supplementing with CLA is an emerging area, requiring studies to elucidate its effects on animals and human reproduction, as well as its side effects. Therefore, the aim of this study was to evaluate the effects of CLA gastric administration, during the pregestational and gestational period in biometric and reproductive parameters, as well as in ovarian morphophysiology. Animals were distributed in three groups: (1) control (n = 10); (2) fish oil (n = 10); and (3) CLA (n = 10), that daily received, by gavage, phosphate-buffered saline, fish oil and CLA, respectively, carried out over 50 days (before mating, mating and pregnancy). There was an increment in the nasoanal distance and Lee index of the CLA and fish oil-treated groups during the first weeks (P > 0.05). CLA administration did not affect the ovarian follicle mobilization (P > 0.05), the number of follicles (P > 0.05) and the integrated density of lipid content of oocytes included in antral follicles (P > 0.05). There was no effect of CLA administration on the litter weight (P > 0.05; F2 and F3), however, an increment (P < 0.05) in the number of pups per litter (F2) was observed. Overall, this study demonstrated the absence of side effects of the CLA gastric administration on mice reproductive performance and suggests that this treatment would transgenerationally enhance fertility in this species.

Deep Neural Networks Applied to Stock Market Sentiment Analysis.

The volume of data is growing exponentially and becoming more valuable to organizations that collect it, from e-commerce data, shipping, audio and video logs, text messages, internet search queries, stock market activity, financial transactions, the Internet of Things, and various other sources. The major challenges are related with the way to extract insights from such a rich data environment and whether Deep Learning can be successful with Big Data. To get some insight on these topics, social network data are employed as a case study on how sentiments can affect decisions in stock market environments. In this paper, we propose a generalized Deep Learning-based classification framework for Stock Market Sentiment Analysis. This work comprises the study, the development, and implementation of an automatic classification system based on Deep Learning and the validation of its adequacy and efficiency in any scenario, particularly Stock Market Sentiment Analysis. Distinct datasets and several Deep Learning approaches with different layers and embedded techniques are used, and their performances are evaluated. These developments show how Deep Learning reacts to distinct contexts. The results also give context on how different techniques with different parameter combinations react to certain types of data. Convolution obtained the best results when dealing with complex data inputs, and long short-term layers kept a memory of data, allowing inputs which are not as common to still be considered for decisions. The models that resulted from Stock Market Sentiment Analysis datasets were applied with some success to real-life problems. The best models reached accuracies of 73% in training and 69% in certain test datasets. In a simulation, a model was able to provide a Return on Investment of 4.4%. The results contribute to understanding how to process Big Data efficiently using Deep Learning and specialized hardware techniques.

Valorisation of food agro-industrial by-products: From the past to the present and perspectives.

Food agro-industrial by-products mainly include peels, seeds, stems, bagasse, kernels, and husk, derived during food processing. Due to their overproduction and the lack of sustainable management, such by-products have been conventionally rejected and wasted in landfills, being the principal strategy for their treatment, but nowadays, this strategy has been associated with several environmental, social and economic issues. Hence, we focused on the use of different consolidated biotechnological processes and methodologies as suitable strategies for food by-products management and valorisation, highlighting them as potential bioresources because they still gather high compositional and nutritional value, owing to their richness in functional and bioactive molecules with human health benefits. Food by-products could be utilised for the development of new food ingredients or products for human consumption, promoting their integral valorisation and reincorporation to the food supply chain within the circular bioeconomy concept, creating revenue streams, business and job opportunities. In this review, the main goal was to provide a general overview of the food agro-industrial by-products utilised throughout the years, improving global sustainability and human nutrition, emphasising the importance of biowaste valorisation as well as the methodologies employed for the recovery of value-added molecules.

The potential of insects as food sources - a review.

Entomophagy is a long-time practice and a food source for many cultures. Still, many societies have abandoned it a long time ago, and regard it as a primal behavior. However, nowadays, the challenge for food demand, with the urge of new nutritional sources, and the problems of undernourishment, mainly on underdeveloped countries, has reached a point where a new perspective is demanded. This review gathers some of the most recent studies regarding the potential benefits and concerns of entomophagy, trying to show the potential of insects as food source and possible ways to introduce them in cultures that have disregarded entomophagy. Entomophagy is taking its place, showing the grand potential of insects as feed and food source. As neophobia and disgust are the main western cultures barriers to accept entomophagy, today's comprehension of this practice and processing capabilities can take that source, to any dish in any form. A simple but nutritive insect powder can create a path to a widely, sustainable, rich food source-insects.

Management of Fruit Industrial By-Products-A Case Study on Circular Economy Approach.

The management of industrial fruit by-products is important not only to decrease the volume of food waste accumulated in the landfills but also to develop strategies through reuse with the purpose to valorise and add economic value. The disposal of food waste leads to different global issues in different sectors, such as social, environmental and economical. These by-products represent a rich source of valuable compounds (polyphenols) with high antioxidant activity, which can be extracted through biotechnological methodologies for future industrial applications. In this context, the management of fruit by-products is challenged to move from a linear economy to a circular economy. Therefore, the purpose of this review is to provide a critical view of an integrated valorisation of fruit by-products to overcome a global issue, via the production of antioxidant extracts with high economic value. A case study of pineapple processing industrialization in a circular economy is explored and discussed.

Bioactive compounds from the African medicinal plant Cleistochlamys kirkii as resistance modifiers in bacteria.

Cleistochlamys kirkii (Benth) Oliv. (Annonaceae) is a medicinal plant traditionally used in Mozambique to treat infectious diseases. The aim of this study was to find resistance modifiers in C. kirkii for Gram-positive and Gram-negative model bacterial strains. One of the most important resistance mechanisms in bacteria is the efflux pump-related multidrug resistance. Therefore, polycarpol (1), three C-benzylated flavanones (2-4), and acetylmelodorinol (5) were evaluated for their multidrug resistance-reverting activity on methicillin-susceptible and methicillin-resistant Staphylococcus aureus and Escherichia coli AG100 and AG100 A strains overexpressing and lacking the AcrAB-TolC efflux pump system. The combined effects of antibiotics and compounds (2 and 4) were also assessed by using the checkerboard microdilution method in both S. aureus strains. The relative gene expression of the efflux pump genes was determined by real-time reverse transcriptase quantitative polymerase chain reaction. The inhibition of quorum sensing was also investigated. The combined effect of the antibiotics and compound 2 or 4 on the methicillin-sensitive S. aureus resulted in synergism. The most active compounds 2 and 4 increased the expression of the efflux pump genes. These results suggested that C. kirkii constituents could be effective adjuvants in the antibiotic treatment of infections.

Solid lipid nanoparticles as oral delivery systems of phenolic compounds: Overcoming pharmacokinetic limitations for nutraceutical applications.

Drug delivery systems, accompanied by nanoparticle technology, have recently emerged as prominent solutions to improve the pharmacokinetic properties, namely bioavailability, of therapeutic and nutraceutical agents. Solid lipid nanoparticles (SLNs) have received much attention from researchers due to their potential to protect or improve drug properties. SLNs have been reported to be an alternative system to traditional carriers, such as emulsions, liposomes, and polymeric nanoparticles. Phenolic compounds are widespread in plant-derived foodstuffs and therefore abundant in our diet. Over the last decades, phenolic compounds have received considerable attention due to several health promoting properties, mostly related to their antioxidant activity, which can have important implications for health. However, most of these compounds have been associated with poor bioavailability being poorly absorbed, rapidly metabolized and eliminated, which compromises its biological and pharmacological benefits. This paper provides a systematic review of the use of SLNs as oral delivery systems of phenolic compounds, in order to overcome pharmacokinetic limitations of these compounds and improved nutraceutical potential. In vitro studies, as well as works describing topical and oral treatments will be revisited and discussed. The classification, synthesis, and clinical application of these nanomaterials will be also considered in this review article.

Therapeutic and nutraceutical potential of rosmarinic acid-Cytoprotective properties and pharmacokinetic profile.

Rosmarinic acid (RA) is a natural polyphenolic antioxidant derived from many common herbal plants. This compound displays several important biological properties, including anti-inflammatory, antiviral, antibacterial, antidepressant, anticarcionogenic, and chemopreventive properties. The importance of its activities and its possible application in processed foods as a natural antioxidant has reached a new interest levels in recent years. The health effects of this polyphenol depend greatly on both its intakes and bioavailability. This review focuses on the importance of RA as a dietary supplement, and summarizes its pharmacokinetics and metabolism, including the factors that limit its oral bioavailability which leads to a lower therapeutic action. Further experimental investigations are needed to optimize and enhance the oral bioavailability of this natural compound which consequently will help increasing therapeutic efficacy of RA in vivo.

Duration of spermatogenesis and daily sperm production in the rodent Proechimys guyannensis.

The spiny rat (Proechimys guyannensis) is a neotropical rodent that is used in biomedical research, particularly research related to chronic resistance to epilepsy and infectious diseases. To our knowledge, there are few reports concerning the reproductive biology of this species. Therefore, besides providing basic biometric and morphometric data, in the present study we investigated testis function and spermatogenesis in adult spiny rats. The mean testis weight and gonadosomatic index obtained were 1.63 ± 0.2 g and 1.15 ± 0.1% respectively. Based on the development of the acrosomic system, 12 stages of the seminiferous epithelium cycle were characterized. Stages VI and VII presented the highest frequencies (~17-19%), whilst stages II to V showed the lowest frequencies (~2-4%). The most advanced germ cell types labelled at 1 h or 20 days after BrdU injections were respectively preleptotene/leptotene spermatocytes at stage VII and elongated spermatids at stage III. The mean duration of one cycle was 7.5 ± 0.01 days and the entire spermatogenic process lasted 33.7 ± 0.06 days (~4.5 cycles). The seminiferous tubules (ST) occupied ~96 ± 1% of the testis parenchyma, whereas Leydig cells comprised only 1.5 ± 0.4%. The number of Sertoli cells (SC) per testis gram and the SC efficiency (spermatids/SC) were respectively 78 × 106 ± 11 × 106 and 7.9 ± 1. The daily sperm production per testis gram (spermatogenic efficiency; daily sperm production (DSP)/g/testis) was 78 × 106 ± 8 × 106. To our knowledge, this spermatogenic efficiency is among the highest found for mammals investigated to date and is probably related to the very short duration of spermatogenesis and the very high ST percentage and SC number obtained for this species.

Improving the MDR reversal activity of 6,17-epoxylathyrane diterpenes.

Aiming to optimize macrocyclic lathyrane-type diterpenes as effective Pgp modulators, the phytochemical study of the methanolic extract of Euphorbia boetica aerial parts was carried out. Two new macrocyclic 6,17-epoxylathyrane-type diterpenes, named epoxyboetiranes A (1) and B (2), along with three known analogues (3-5) were isolated. Epoxyboetirane A (1), a triacetate isolated in large amounts, was hydrolyzed to give epoxylathyrol (6). In order to study the effect of the substitution pattern of the macrocyclic scaffold on MDR reversal, 6 was acylated with aroyl, phenylacetyl, cinnamoyl and alkanoyl chlorides/anhydrides, yielding eight new esters, epoxyboetiranes C-J (7-14). The ability of compounds 1-14 as P-glycoprotein (Pgp, ABCB1) modulators was evaluated through combination of transport and chemosensitivity assays, using L5178Y mouse T lymphoma cell line transfected with the human MDR1 gene. In the transport assay, excepting 1, 3 and 6, the compounds, at non-cytotoxic concentrations, displayed strong MDR reversing activity in a dose-dependent mode, exhibiting all the new acyl derivatives (7-14) a many fold increase in the activity when compared with 1. Apart from 11 and 12, all compounds exhibited remarkable synergistic effects in combination with doxorubicin. An ATPase assay, using membrane vesicles from mammalian cells overexpressing Pgp, was also performed with two representatives of the modulators (4 and 5). The results suggest that both compounds compete with substrates for the Pgp drug-binding sites.

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.

Exploring the interplay between stress mediators and skin microbiota in shaping age-related hallmarks: A review.

Psychological stress is a major contributing factor to several health problems (e.g., depression, cardiovascular disease). Around 35 % of the world's population suffers from it, including younger generations. Physiologically, stress manifests through neuroendocrine pathways (Hypothalamic-Pituitary-Adrenal (HPA) axis and Sympathetic-Adrenal-Medullary (SAM) system) which culminate in the production of stress mediators like cortisol, epinephrine and norepinephrine. Stress and its mediators have been associated to body aging, through molecular mechanisms such as telomere attrition, mitochondrial dysfunction, cellular senescence, chronic inflammation, and dysbiosis, among others. Regarding its impact in the skin, stress impacts its structural integrity and physiological function. Despite this review focusing on several hallmarks of aging, emphasis was placed on skin microbiota dysbiosis. In this line, several studies, comprising different age groups, demographic contexts and body sites, have reported skin microbiota alterations associated with aging, and some effects of stress mediators on skin microbiota have also been reviewed in this paper. From a different perspective, since it is not a "traditional" stress mediator, oxytocin, a cortisol antagonist, has been related to glucorticoids inhibition and to display positive effects on cellular aging. This hormone dysregulation has been associated to psychological issues such as depression, whereas its upregulation has been linked to positive social interaction.