Exploring the biocompatibility and healing activity of actinobacterial-enhanced reduced nano-graphene oxide in in vitro and in vivo model and induce bone regeneration through modulation of OPG/RANKL/RUNX2/ALP pathways.

BACKGROUND: The development of cost-effective, simple, environment-friendly biographene is an area of interest. To accomplish environmentally safe, benign culturing that has advantages over other methods to reduce the graphene oxide (GO), extracellular metabolites from actinobacteria associated with mushrooms were used for the first time. METHODS: Bactericidal effect of GO against methicillin-resistant Staphylococcus aureus, antioxidant activity, and hydroxyapatite-like bone layer formation, gene expression analysis and appropriate biodegradation of the microbe-mediated synthesis of graphene was studied. RESULTS: Isolated extracellular contents Streptomyces achromogenes sub sp rubradiris reduced nano-GO to graphene (rGO), which was further examined by spectrometry and suggested an efficient conversion and significant reduction in the intensity of all oxygen-containing moieties and shifted crystalline peaks. Electron microscopic results also suggested the reduction of GO layer. In addition, absence of significant toxicity in MG-63 cell line, intentional free radical scavenging prowess, liver and kidney histopathology, and Wistar rat bone regeneration through modulation of OPG/RANKL/RUNX2/ALP pathways show the feasibility of the prepared nano GO. CONCLUSIONS: The study demonstrates the successful synthesis of biographene from actinobacterial extracellular metabolites, its potential biomedical applications, and its promising role in addressing health and environmental concerns.

Facilitating Rapid Na+ Storage through MoWSe/C Heterostructure Construction and Synergistic Electrolyte Matching Strategy.

The realization of sodium-ion devices with high-power density and long-cycle capability is challenging due to the difficulties of carrier diffusion and electrode fragmentation in transition metal selenide anodes. Herein, a Mo/W-based metal-organic framework is constructed by a one-step method through rational selection, after which MoWSe/C heterostructures with large angles are synthesized by a facile selenization/carbonization strategy. Through physical characterization and theoretical calculations, the synthesized MoWSe/C electrode delivers obvious structural advantages and excellent electrochemical performance in an ethylene glycol dimethyl ether electrolyte. Furthermore, the electrochemical vehicle mechanism of ions in the electrolyte is systematically revealed through comparative analyses. Resultantly, ether-based electrolytes advantageously construct stable solid electrolyte interfaces and avoid electrolyte decomposition. Based on the above benefits, the Na half-cell assembled with MoWSe/C electrodes demonstrated excellent rate capability and a high specific capacity of 347.3 mA h g-1 even after cycling 2000 cycles at 10 A g-1. Meanwhile, the constructed sodium-ion capacitor maintains ∼80% capacity retention after 11,000 ultralong cycles at a high-power density of 3800 W kg-1. The findings can broaden the mechanistic understanding of conversion anodes in different electrolytes and provide a reference for the structural design of anodes with high capacity, fast kinetics, and long-cycle stability.

Environmentally Friendly Water-Based Reduced Graphene Oxide/Cellulose Nanofiber Ink for Supercapacitor Electrode Applications.

The agglomeration of reduced graphene oxide (rGO) in water makes the development of rGO inks for supercapacitor printing challenging. Cellulose nanofiber (CNF), a biodegradable and renewable nanomaterial, can act as a nanospacer, preventing the agglomeration and restacking of rGO flakes. In this work, rGO/CNF films were fabricated using an environmentally friendly water-based rGO/CNF ink. In the absence of an additional binder/surfactant, the rGO/CNF films demonstrated remarkably enhanced hydrophilicity while retaining good electrical conductivity. The concentration of CNF was varied to observe the variation in the electrochemical performance. At a current density of 1 mA/cm2, the rGO/CNF-15 film exhibited a maximum areal capacitance of 98.61 mF/cm2, closely matching that of pure rGO films. Because of its excellent electrical performance, ease of manufacturing, and environmental friendliness, this water-based rGO/CNF ink may have promising applications in the printing of supercapacitor electrodes.

Enhanced Sodium Ion Batteries' Performance: Optimal Strategies on Electrolytes for Different Carbon-based Anodes.

Carbon-based materials have emerged as promising anodes for sodium-ion batteries (SIBs) due to the merits of cost-effectiveness and renewability. However, the unsatisfactory performance has hindered the commercialization of SIBs. During the past decades, tremendous attention has been put into enhancing the electrochemical performance of carbon-based anodes from the perspective of improving the compatibility of electrolytes and electrodes. Hence, a systematic summary of strategies for optimizing electrolytes between hard carbon, graphite, and other structural carbon anodes of SIBs is provided. The formulations and properties of electrolytes with solvents, salts, and additives added are comprehensively presented, which are closely related to the formation of solid electrolyte interface (SEI) and crucial to the sodium ion storage performance. Cost analysis of commonly used electrolytes has been provided as well. This review is anticipated to provide guidance in future rational tailoring of electrolytes with carbon-based anodes for sodium-ion batteries.

Novel same-metal three electrode system for cyclic voltammetry studies.

Conventional three-electrode systems used in electrochemical measurement demand time-consuming and maintenance intensive procedures to enable accurate and repeatable electrochemical measurements. Traditionally, different metal configurations are used to establish the electrochemical gradient required to acquire the redox activity, and vary between different electrochemical measurement platforms. However, in this work, we report using the same metal (gold) for the counter, working and reference electrodes fabricated on a miniaturized printed circuit board (PCB) for a much simpler design. Potassium ferricyanide, widely used as a redox probe for electrochemical characterization, was utilized to acquire cyclic voltametric profiles using both the printed circuit board-based gold-gold-gold three-electrode and conventional three-electrode systems (glassy carbon electrode or graphite foil as the working electrode, platinum wire as the counter electrode, and Ag/AgCl as the reference electrode). The results show that both types of electrode systems generated similar cyclic voltammograms within the same potential window (-0.5 to +0.7 V). However, the novel PCB-based same-metal three-electrode electrochemical cell only required a few activation cycles and exhibited impressive cyclic voltametric repeatability with higher redox sensitivity and detection window, while using only trace amounts of solutions/analytes.

Industrial functional safety assessment for WSN using QoS metrics.

Wireless Sensor Networks are increasingly getting deployed for the safety use cases in industrial applications. While several research papers discuss about the Quality & Reliability improvement techniques in WSN systems to achieve minimal delay, higher node life, optimal routing etc., very limited work is witnessed on assessment of safety integrity levels of WSN systems. In this paper we tried to bridge this gap by bringing out a QoS metric-based safety integrity assessment for the end-to-end industrial Wireless Sensor Network (WSN) system. To identify relevant QoS metrics for monitoring the safety integrity levels, we also bring out a 4-step mapping methodology to link the QoS metrics and communication defenses/safety mechanisms. This mapping approach is expected to serve the network safety design engineers. Finally, a simulation case example is discussed to illustrate safety integrity assessment and we conclude by bringing out future research opportunities to improve safety integrity levels of industrial WSN systems.

Silver nanoparticles based spectroscopic sensing of eight metal ions in aqueous solutions.

Anthropogenic releases from different outlets of industry, municipal sewage and the road traffic can give rise to higher concentrations of heavy metals in food commodities which imposes a threat to human health and environment. A simple silver nanoparticle (Ag NPs) used for the sensing of heavy metal ions, Cd2+, Cu2+, Fe2+, Hg2+, Mn2+, Ni2+, Pb2+ and Zn2+ in aqueous solution is described qualitatively and quantitatively using spectroscopic tool. FE-SEM and TEM images confirmed that the particles are spherical in shape with an average diameter of 23.4 nm. Presence of heavy metal ions with Ag NPs gives, new peak at around 925, 898, 643, 665, 688, and 838 nm for Cd2+, Cu2+, Fe2+, Hg2+, Mn2+ and Zn2+ in addition to the peak found at 410 nm for Ag NPs. Further, the addition of Ni2+ and Pb2+ metal ion solution with Ag NPs increased the SPR band from 410 nm to 436 and 462 nm respectively. Citrate functionalized Ag NPs aggregate in solution in the presence of divalent metal ions by ions-template chelating process and are easily measurable changes in the UV-vis absorption spectrum of the particles. Further, studies also confirmed the interaction of Ag NPs with metal ions using FT-IR spectroscopy. The proposed method was found to be useful for simple UV-vis spectroscopic sensing of metal ions in aqueous solutions and real contaminated samples.

Overexpression of IGF-1 During Early Development Expands the Number of Mammary Stem Cells and Primes them for Transformation.

Insulin-like growth factor I (IGF-1) has been implicated in breast cancer due to its mitogenic and anti-apoptotic effects. Despite substantial research on the role of IGF-1 in tumor progression, the relationship of IGF-1 to tissue stem cells, particularly in mammary tissue, and the resulting tumor susceptibility has not been elucidated. Previous studies with the BK5.IGF-1 transgenic (Tg) mouse model reveals that IGF-1 does not act as a classical, post-carcinogen tumor promoter in the mammary gland. Pre-pubertal Tg mammary glands display increased numbers and enlarged sizes of terminal end buds, a niche for mammary stem cells (MaSCs). Here we show that MaSCs from both wild-type (WT) and Tg mice expressed IGF-1R and that overexpression of Tg IGF-1 increased numbers of MaSCs by undergoing symmetric division, resulting in an expansion of the MaSC and luminal progenitor (LP) compartments in pre-pubertal female mice. This expansion was maintained post-pubertally and validated by mammosphere assays in vitro and transplantation assays in vivo. The addition of recombinant IGF-1 promoted, and IGF-1R downstream inhibitors decreased mammosphere formation. Single-cell transcriptomic profiles generated from 2 related platforms reveal that IGF-1 stimulated quiescent MaSCs to enter the cell cycle and increased their expression of genes involved in proliferation, plasticity, tumorigenesis, invasion, and metastasis. This study identifies a novel, pro-tumorigenic mechanism, where IGF-1 increases the number of transformation-susceptible carcinogen targets during the early stages of mammary tissue development, and "primes" their gene expression profiles for transformation.

The impacts of COVID-19 lockdown on wildlife in Deccan Plateau, India.

The outbreak of the COVID-19 pandemic brought unprecedented changes in human activity via extensive lockdowns worldwide. Large-scale shifts in human activities bestowed both positive and negative impacts on wildlife. Unforeseen reduction in the activities of people allowed wildlife to venture outside of forested areas to exploit newfound habitats and increase their diurnal activities. While on a negative note, a reduction in forest-related law enforcement led to substantial increase in illegal activities such as poaching. We conducted mammal surveys in forested and nearby farmland of a fragmented landscape under two distinct scenarios: pre-lockdown and lockdown. An increase in poaching activities observed during the lockdown period in our study area provided us an opportunity to investigate the impact of the lockdown on wildlife. Camera trapping data of four highly poached mammalian species, namely black-naped hare Lepus nigricollis, wild pig Sus scrofa, four-horned antelope Tetracerus quadricornis and leopard Panthera pardus were considered to investigate activity patterns and habitat use, to understand the effect of lockdown. The pre-lockdown period was used as a baseline to compare any changes in trends of activity patterns, habitat use and detection probabilities of targeted species. Species-specific changes in activity patterns of study species were observed, with an increment in daytime activity during lockdown. The results showed species-specific increase in the habitat use of study species during lockdown. Reduction in the detection probability of all study species was witnessed. This is the first study to highlight the effect of the COVID-19 lockdown on the responses of wildlife by considering the changes in their temporal and spatial use before and during lockdown. The knowledge gained on wildlife during reduced human mobility because of the pandemic aid in understanding the effect of human disturbances and developing future conservation strategies in the shared space, to manage both wildlife and humans.

TGF-ß-induced DACT1 biomolecular condensates repress Wnt signalling to promote bone metastasis.

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid-liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGFß and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2-a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis.

Intense bloom of the diatom Hemidiscus hardmanianus (Greville) in relation to water quality and plankton communities in Tuticorin coast, Gulf of Mannar, India.

The present study reports a dense bloom of the marine-diatom Hemidiscus hardmanianus observed off the Tuticorin coast in the Gulf of Mannar (GoM), India. The surface water discoloration (pale green) was observed during a coastal survey conducted in the initial period of the northeast monsoon (October 2018). The bloom extended over an area of approximately 5 km2 around the Tuticorin harbor. Distribution and relative abundance of the phytoplankton and zooplankton species together with the water quality and Chlorophyll-a were studied in the area of bloom. H. hardmanianus density was maximum (10.57 × 104 cells L-1) in the bloom site, which was almost 97% of the total phytoplankton population. The present report is the first record of H. hardmanianus bloom in the Gulf of Mannar. The chain-forming diatom Biddulphia biddulphiana was also observed in strong numbers (802 and 432 cells L-1), which has been rarely reported from the Indian coastal waters.

Printed-Circuit-Board-Based Two-Electrode System for Electronic Characterization of Proteins.

Proteins have been increasingly suggested as suitable candidates for the fabrication of biological computers and other biomolecular-based electronic devices mainly due to their interesting structure-related intrinsic electrical properties. These natural biopolymers are environmentally friendly substitutes for conventional inorganic materials and find numerous applications in bioelectronics. Effective manipulation of protein biomolecules allows for accurate fabrication of nanoscaled device dimensions for miniaturized electronics. The prerequisite, however, demands an interrogation of its various electronic properties prior to understanding the complex charge transfer mechanisms in protein molecules, the knowledge of which will be crucial toward development of such nanodevices. One significantly preferred method in recent times involves the utilization of solid-state sensors where interactions of proteins could be investigated upon contact with metals such as gold. Therefore, in this work, proteins (hemoglobin and collagen) were integrated within a two-electrode system, and the resulting electronic profiles were investigated. Interestingly, structure-related electronic profiles representing semiconductive-like behaviors were observed. These characteristic electronic profiles arise from the metal (Au)-semiconductor (protein) junction, clearly demonstrating the formation of a Schottky junction. Further interpretation of the electronic behavior of proteins was done by the calculation of selected solid-state parameters. For example, the turn-on voltage of hemoglobin was measured to occur at a lower turn-on voltage, indicating the possible influence of the hem group present as a cofactor in each subunit of this tetrameric protein.

Enhancing the Efficiency of a Dye-Sensitized Solar Cell Based on a Metal Oxide Nanocomposite Gel Polymer Electrolyte.

To overcome the critical limitations of liquid-electrolyte-based dye-sensitized solar cells, quasi-solid-state electrolytes have been explored as a means of addressing long-term device stability, albeit with comparatively low ionic conductivities and device performances. Although metal oxide additives have been shown to augment ionic conductivity, their propensity to aggregate into large crystalline particles upon high-heat annealing hinders their full potential in quasi-solid-state electrolytes. In this work, sonochemical processing has been successfully applied to generate fine Co3O4 nanoparticles that are highly dispersible in a PAN:P(VP-co-VAc) polymer-blended gel electrolyte, even after calcination. An optimized nanocomposite gel polymer electrolyte containing 3 wt % sonicated Co3O4 nanoparticles (PVVA-3) delivers the highest ionic conductivity (4.62 × 10-3 S cm-1) of the series. This property is accompanied by a 51% enhancement in the apparent diffusion coefficient of triiodide versus both unmodified and unsonicated electrolyte samples. The dye-sensitized solar cell based on PVVA-3 displays a power conversion efficiency of 6.46% under AM1.5 G, 100 mW cm-2. By identifying the optimal loading of sonochemically processed nanoparticles, we are able to generate a homogenous extended particle network that effectively mobilizes redox-active species through a highly amorphous host matrix. This effect is manifested in a selective 51% enhancement in photocurrent density (JSC = 16.2 mA cm-2) and a lowered barrier to N719 dye regeneration (RCT = 193 Ω) versus an unmodified solar cell. To the best of our knowledge, this work represents the highest known efficiency to date for dye-sensitized solar cells based on a sonicated Co3O4-modified gel polymer electrolyte. Sonochemical processing, when applied in this manner, has the potential to make meaningful contributions toward the ongoing mission to achieve the widespread exploitation of stable and low-cost dye-sensitized solar cells.

Surface Modification Techniques for Zirconia-Based Bioceramics: A Review.

Zirconia is gaining interest as a ceramic biomaterial for implant applications due to its biocompatibility and desirable mechanical properties. At present, zirconia-based bioceramics is often seen in the applications of hip replacement and dental implants. This article briefly reviews different surface modification techniques that have been applied to zirconia such as polishing, sandblasting, acid etching, biofunctionalization, coating, laser treatment, and ultraviolet light treatment. The potential of surface modification to make zirconia a successful implant material in the future is highly dependent on the establishment of successful in vitro and in vivo studies. Hence, further effort should be made in order to deepen the understanding of tissue response to implant and tissue regeneration process.

Dynamic role of the codon 72 p53 single-nucleotide polymorphism in mammary tumorigenesis in a humanized mouse model.

Female breast cancer (BrCa) is the most common noncutaneous cancer among women in the United States. Human epidemiological studies reveal that a p53 single-nucleotide polymorphism (SNP) at codon 72, encoding proline (P72) or arginine (R72), is associated with differential risk of several cancers, including BrCa. However, the molecular mechanisms by which these variants affect mammary tumorigenesis remain unresolved. To investigate the effects of this polymorphism on susceptibility to mammary cancer, we used a humanized p53 mouse model, homozygous for either P72 or R72. Our studies revealed that R72 mice had a significantly higher mammary tumor incidence and reduced latency in both DMBA-induced and MMTV-Erbb2/Neu mouse mammary tumor models compared to P72 mice. Analyses showed that susceptible mammary glands from E-R72 (R72 x MMTV-Erbb2/Neu) mice developed a senescence-associated secretory phenotype (SASP) with influx of proinflammatory macrophages, ultimately resulting in chronic, protumorigenic inflammation. Mammary tumors arising in E-R72 mice also had an increased influx of tumor-associated macrophages, contributing to angiogenesis and elevated tumor growth rates. These results demonstrate that the p53 R72 variant increased susceptibility to mammary tumorigenesis through chronic inflammation.

Hierarchical Porous Carbon Microfibers Derived from Tamarind Seed Coat for High-Energy Supercapacitor Application.

The overwhelming interest in supercapacitors has led to the search for various carbonaceous materials, leading to hierarchical porous carbons. Herein, we report a natural biomass (tamarind seed)-based hierarchical porous carbon without any template and activated by a facile scheme. The tamarind seed coat-based hierarchical porous carbon possessed a unique configuration, making the material exhibit superior supercapacitor properties. A single carbon fiber hosting a distinctive micro- and mesoporous structure formed a connecting thread between the pores. This unique structure enabled high surface area and high capacitance. The highest surface area obtained by this method was 1702 m2 g-1, whereas the capacitance was 157 F g-1 in 6 M KOH. Further, an ionic liquid-based electrolyte revealed 78 F g-1 at a current density of 0.5 A g-1. Outstanding capacity retentions of 96 and 93% were obtained over 1000 cycles at a current density of 2 A g-1 for aqueous (6 M KOH) and ionic liquid (1-butyl 3-methyl imidazoliumbistrifluorosulfonylimide) electrolytes, respectively. The high charge-storage ability of the porous carbon microfibers (PCMFs) can be ascribed to the coexistence of micro- and mesopores. The power characteristics and the cyclic stability of PCMF materials were appealing in both electrolytes. The synthesis process described is amenable for large-scale applications with less complexity.

Ophthalmic manifestations in Rothmund-Thomson syndrome: Case report and review of literature.

A 24-year-old male patient presented to us with diminution of vision in both eyes with watering and photophobia for the past 8 years. General physical examination showed short stature and poikiloderma. Ocular findings include photophobia with reflex tearing, dry eye, cicatricial ectropion, symblepharon approaching pupillary area of cornea, and multiple superficial punctuate erosions on the cornea. Both eyelids showed scanty meibomian glands on infrared meibography. The rest of the anterior and posterior segment was normal. The patient was treated with topical lubricants which reduced photophobia and corneal erosions. He then underwent symblepharon release with buccal mucosal grafting, which improved ectropion. Patient improved symptomatically with reduction of photophobia and improvement in vision as well.

Parental preferences with regards to disclosure following adverse events occurring in relation to medication use or diagnosis in the care of their children - perspectives from Malaysia.

INTRODUCTION: Open disclosure is poorly understood in Malaysia but is an ethical and professional responsibility. The objectives of this study were to determine: (1) the perception of parents regarding the severity of medical error in relation to medication use or diagnosis; (2) the preference of parents for information following the medical error and its relation to severity; and (3) the preference of parents with regards to disciplinary action, reporting, and legal action. METHODS: We translated and contextualised a questionnaire developed from a previous study. The questionnaire consisted of four case vignettes that described the following: medication error with a lifelong complication; diagnostic error with a lifelong complication; diagnostic error without lifelong effect; and medication error without lifelong effect. Each case vignette was followed by a series of questions examining the subject's perception on the above areas. We also determined the content validity of the questionnaire. We invited parents of Malaysian children admitted to the paediatric wards of Tuanku Jaafar Hospital to participate in the study. RESULTS: One hundred and twenty-three parents participated in the study. The majority of parents wanted to be told regarding the event. As the severity of the case vignettes increased, the desire for information, remedial action, acknowledgement of responsibility, compensation, punishment, legal action, and reporting to a higher agency also increased. The findings did not have strong evidence of a relationship with subject's demographics. CONCLUSION: This study gives insights into previously unexplored perspectives and preferences of parents in Malaysia regarding open disclosure. It also highlights the opportunity for more research in this area with potentially broad applications.

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta.

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.

Comparative analysis of contextual bias around the translation initiation sites in plant genomes.

Nucleotide distribution around translation initiation site (TIS) is thought to play an important role in determining translation efficiency. Kozak in vertebrates and later Joshi et al. in plants identified context sequence having a key role in translation efficiency, but a great variation regarding this context sequence has been observed among different taxa. The present study aims to refine the context sequence around initiation codon in plants and addresses the sampling error problem by using complete genomes of 7 monocots and 7 dicots separately. Besides positions -3 and +4, significant conservation at -2 and +5 positions was also found and nucleotide bias at the latter two positions was shown to directly influence translation efficiency in the taxon studied. About 1.8% (monocots) and 2.4% (dicots) of the total sequences fit the context sequence from positions -3 to +5, which might be indicative of lower number of housekeeping genes in the transcriptome. A three base periodicity was observed in 5' UTR and CDS of monocots and only in CDS of dicots as confirmed against random occurrence and annotation errors. Deterministic enrichment of GCNAUGGC in monocots, AANAUGGC in dicots and GCNAUGGC in plants around TIS was also established (where AUG denotes the start codon), which can serve as an arbiter of putative TIS with efficient translation in plants.