Antidiabetic effectiveness of Phyllanthus niruri bioactive compounds via targeting DPP-IV.

Phyllanthus niruri Linn. (Euphorbiaceae) is a small herb and is categorised as one of the rich medicinal plants throughout the world. This study aimed to evaluate the P. niruri L. whole plant extract (PNE) for secondary metabolite assay (total phenolic and terpenoid content) followed by the potential antioxidant activity (ABTS diammonium salt radical assay, DPPH· activity, superoxide anion (O2-) radicals' assay, and nitric oxide (NO) radical generation) and antidiabetic activity in vivo and in vitro in streptozotocin (STZ) induced albino mice. PNE showed good scavenging activity with a value of 286.45 ± 6.55 mg TE/g and 194.54 ± 4.64 mg TE/g in ABTS and DPPH assays respectively. In the superoxide anion assay, the PNE caused a dose-dependent inhibition at the lowest IC25 value of 0.17 ± 0.00 mg/mL compared to ascorbic acid (IC25 of 0.25 ± 0.02 mg/mL). The scavenging ability of PNE against nitric oxide showed an IC25 of 1.13 ± 0.04 mg/mL compared to ascorbic acid (IC25 4.78 ± 0.09 mg/mL). Unlike diabetic control mice, the PNE-treated diabetic mice presented significant amelioration of glycaemia and lipid dysmetabolism. Phytochemicals like Astragalin, Gallocatechin, Ellagic acid, Gallic acid, Brevifolin carboxylic acid, Phyllnirurin, and Hypophyllanthin showed significant docking score (> -4) of inhibitory potential with DPP-IV protein. Results indicated that PNE phytochemicals could be a promising antidiabetic agent by targeting DPP-IV.

Synchronization of Mycobacterium life cycle: A possible novel mechanism of antimycobacterial drug resistance evolution and its manipulation.

Mycobacterium Tuberculosis (Mtb) causing Tuberculosis (TB) is a widespread disease infecting millions of people worldwide. Additionally, emergence of drug resistant tuberculosis is a major challenge and concern in high TB burden countries. Most of the drug resistance in mycobacteria is attributed to developing acquired resistance due to spontaneous mutations or intrinsic resistance mechanisms. In this review, we emphasize on the role of bacterial cell cycle synchronization as one of the intrinsic mechanisms used by the bacteria to cope with stress response and perhaps involved in evolution of its drug resistance. The importance of cell cycle synchronization and its function in drug resistance in cancer cells, malarial and viral pathogens is well understood, but its role in bacterial pathogens has yet to be established. From the extensive literature survey, we could collect information regarding how mycobacteria use synchronization to overcome the stress response. Additionally, it has been observed that most of the microbial pathogens including mycobacteria are responsive to drugs predominantly in their logarithmic phase, while they show resistance to antibiotics when they are in the lag or stationary phase. Therefore, we speculate that Mtb might use this novel strategy wherein they regulate their cell cycle upon antibiotic pressure such that they either enter in their low metabolic phase i.e., either the lag or stationary phase to overcome the antibiotic pressure and function as persister cells. Thus, we propose that manipulating the mycobacterial drug resistance could be possible by fine-tuning its cell cycle.

Molecular drilling to combat salmonella typhi biofilm using L-Asparaginase via multiple targeting process.

OBJECTIVES: Salmonella Typhibiofilm condition is showing as a major public health problem due to the development of antibiotic resistance and less available druggable target proteins. Therefore, we aimed to identify some more druggable targets of S. Typhibiofilm using computational drilling at the genome/proteome level so that the target shortage problem could be overcome and more antibiofilm agents could be designed in the future against the disease. METHODS: We performed protein-protein docking and interaction analysis between the homological identified target proteins of S.Typhi biofilm and a therapeutic protein L-Asparaginase. RESULTS: We have identified some druggable targets CsgD, BcsA, OmpR, CsgG, CsgE, and CsgF in S.Typhi. These targets showed high-binding affinity BcsA (-219.8 Kcal/mol) >csgF (-146.52 Kcal/mol) >ompR (-135.68 Kcal/mol) >CsgE (-134.66 Kcal/mol) >CsgG (-113.81 Kcal/mol) >CsgD(-95.39 Kcal/mol) with therapeutic enzyme L-Asparaginase through various hydrogen-bonds and salt-bridge. We found six proteins of S. Typhi biofilm from the Csg family as druggable multiple targets. CONCLUSION: This study provides insight into the idea of identification of new druggable targets and their multiple targeting with L-Asparaginase to overcome target shortage in S. Typhibiofilm-mediated infections. Results further indicated that L-Asparaginase could potentially be utilized as an antibiofilm biotherapeutic agent against S.Typhi.

A Rare Entanglement: Self-Knotting of a Nasogastric Tube.

While nasogastric intubation is a commonplace procedure characterized by its utility in enteral feeding and gastrointestinal decompression, instances of unexpected complications are relatively infrequent. Herein, we describe an unusual and rare complication, knot formation, that surfaced during routine patient care. This unique case prompts a re-evaluation of the potential complications associated with nasogastric tube insertion and offers insights into the challenges faced in its management. Through this report, we aim to contribute to the understanding of rare complications in enteral feeding practices.

Modulators of Candida albicans Membrane Drug Transporters: A Lucrative Portfolio for the Development of Effective Antifungals.

The escalating prevalence of membrane drug transporters and drug efflux pumps in pathogenic yeast like Candida albicans necessitates a comprehensive understanding of their roles in MDR. The overexpression of drug transporter families, ABC and MFS, implicated in MDR through drug efflux and poses a significant challenge in the diagnosis and treatment of fungal infection. Various mechanisms have been proposed for MDR; however, the upregulation of ABC and MFS superfamily transporters is most noticeable in MDR. The direct inhibition of these transporters seems an efficient strategy to overcome this problem. The goal of the article is to present an overview of the prospect of utilizing these modulators of C. albicans drug transports as effective antifungal molecules against MDR addressing a critical gap in the field. The review tries to address to prevent drug extrusion by modulating the expression of drug transporters of C. albicans. The review discussed the progress in identifying potent, selective, and non-toxic modulators of these transporters to develop some effective antifungals and overcome MDR. We reviewed major studies in this area and found that recent work has shifted toward the exploration of natural compounds as potential modulators to restore drug sensitivity in MDR fungal cells. The focus of this review is to survey and interpret current research information on modulators of C. albicans drug transporters from natural sources emphasizing those compounds that are potent antifungal agents.

Interrogating Salmonella Typhi biofilm formation and dynamics to understand antimicrobial resistance.

AIMS: Salmonella Typhi biofilm-mediated infections are globally rising. Due to the emergence of drug resistance antibiotics did not show effective results against S. Typhi biofilm. Therefore, there is an urgent need for an in-depth interrogation of S. Typhi biofilm to understand its formation kinetics, compositions, and surface charge value. METHODS: This study utilized the S. Typhi MTCC-733 strain from a microbial-type culture collection in India. The S. Typhi biofilm was formed on a glass slide in a biofilm development apparatus. Typhoidal biofilm analysis was done with the help of various assays such as a crystal violet assay, SEM analysis, FTIR analysis, Raman analysis, and zeta potential analysis. KEY FINDING: This article contained a comprehensive assessment of the typhoid biofilm formation kinetics, biofilm compositions, and surface charge which revealed that cellulose was a major molecule in the typhoidal biofilm which can be used as a major biofilm drug target against typhoidal biofilm. SIGNIFICANCE: This study provided interrogations about typhoidal biofilm kinetics which provided ideas about the biofilm composition. The cellulose molecule showed a major component of S. Typhi biofilm and it could potentially involved in drug resistance, and offer a promising avenue for developing a new antibiofilm therapeutic target to conquer the big obstacle of drug resistance. The obtained information can be instrumental in designing novel therapeutic molecules in the future to combat typhoidal biofilm conditions effectively for overcoming antibiotic resistance against bacterial infection Salmonella.

Synergies in stem cell research: Integrating technologies, strategies, and bionanomaterial innovations.

Since the 1960 s, there has been a substantial amount of research directed towards investigating the biology of several types of stem cells, including embryonic stem cells, brain cells, and mesenchymal stem cells. In contemporary times, a wide array of stem cells has been utilized to treat several disorders, including bone marrow transplantation. In recent years, stem cell treatment has developed as a very promising and advanced field of scientific research. The progress of therapeutic methodologies has resulted in significant amounts of anticipation and expectation. Recently, there has been a notable proliferation of experimental methodologies aimed at isolating and developing stem cells, which have emerged concurrently. Stem cells possess significant vitality and exhibit vigorous proliferation, making them suitable candidates for in vitro modification. This article examines the progress made in stem cell isolation and explores several methodologies employed to promote the differentiation of stem cells. This study also explores the method of isolating bio-nanomaterials and discusses their viewpoint in the context of stem cell research. It also covers the potential for investigating stem cell applications in bioprinting and the usage of bionanomaterial in stem cell-related technologies and research. In conclusion, the review article concludes by highlighting the importance of incorporating state-of-the-art methods and technological breakthroughs into the future of stem cell research. Putting such an emphasis on constant innovation highlights the ever-changing character of science and the never-ending drive toward unlocking the maximum therapeutic potential of stem cells. This review would be a useful resource for researchers, clinicians, and policymakers in the stem cell research area, guiding the next steps in this fast-developing scientific concern.

Candida die-off: Adverse effect and neutralization with phytotherapy approaches.

Candida albicans is the main species that causes 3rd most common bloodstream infection candidiasis in hospitalization. Once it has been diagnosed and treated with antifungal medications accurately, large amounts of Candida cells are killed off rapidly known as Candida die-off or Jarisch-Herxheimer reactions. When Candida cells are killed off quickly, a large no. of toxic substances are released simultaneously. This flood of endotoxins is noxious (harmful) and causes the kidneys and liver to work overtime to try and remove them which causes worsening of symptoms in patients. As a complementary and holistic approach to addressing Candida die-off and its associated symptoms, plant-based remedies i.e., phytotherapy have been gaining increased attention. In this review paper, we have discussed major factors involved in provoking Candida die-off, their management by phytotherapy, challenges associated with the toxic effects due to die-off, and neutralization of Candida die-off through phytotherapy to manage this problem and challenges. In conclusion, this article serves as a meticulous compilation of knowledge on the intriguing subject of Candida die-off, presenting a distinct and informative perspective that has the potential to pave the way for new insights in the realm of plant-based antifungal therapeutics.

microRNA as biomarkers in tuberculosis: a new emerging molecular diagnostic solution.

Tuberculosis (TB) caused by Mycobacterium tuberculosis is a lethal infectious disease that is prevalent worldwide. During TB infection, host microRNAs change their expression in the form of up/down-regulation. The identification of unique host microRNAs during TB could serve as potential biomarkers in the early detection of TB. microRNAs fulfill the required criteria for being an ideal biomarker, such as sensitivity, high specificity, and accessibility. Therefore, the recognition of potential host microRNAs can be valuable for the diagnosis of TB. The field of miRNA biomarkers in TB requires more extensive research to identify potential biomarkers. This review provides an overview of the biogenesis and biological functions of microRNAs and presents the findings of various studies on the identification of potential biomarkers for TB. Research momentum is gaining in this field and we anticipate that miRNAs will become a routine approach in the development of reliable diagnostic and specific therapeutic interventions in future.

Articulate Chemotherapeutic Strategies for the Development of Effective Drugs against a Fatal Disease, Visceral Leishmaniasis.

Visceral Leishmaniasis (VL) control relies mainly on chemotherapy in the absence of no effective vaccines. However, available anti-VL drugs are limited in number, having toxicity issues, adverse reactions, low efficacy, and resistance observed against antileishmanial. A significant decrease in efficacy (~tenfold increase in dosage and duration) was reported against the usual treatment with Pentavalent antimonials (the most recommended antileishmanial drug discovered 90 years ago). Amphotericin B is the second line of treatment but limits wider use due to its high cost. Pentamidine is another anti-VL drug, but its therapeutic efficacy has decreased significantly in different areas. These conventional therapeutics for VL have become almost outdated due to a significant increase in therapeutic failure in terms of percentage. Due to this, the search for an effective future anti-VL drug spans several decades, and now it is in high demand in the current situation. Some conventional therapeutics are modified, but they are also not satisfactory. Therefore, this article aimed to discuss conventional and modified therapeutics while emphasizing innovative chemotherapeutic measures against VL that could speed up the slow pace of antileishmanial drugs and overcome the drug resistance problem in the future.

Deciphering Target Protein Cascade in Salmonella typhi Biofilm using Genomic Data Mining, and Protein-protein Interaction.

Background: Salmonella typhi biofilm confers a serious public health issue for lengthy periods and the rise in antibiotic resistance and death rate. Biofilm generation has rendered even the most potent antibiotics ineffective in controlling the illness, and the S. typhi outbreak has turned into a fatal disease typhoid. S. typhi infection has also been connected to other deadly illnesses, such as a gall bladder cancer. The virulence of this disease is due to the interaction of numerous genes and proteins of S. typhi. Objective: The study aimed to identify a cascade of target proteins in S. typhi biofilm condition with the help of genomic data mining and protein-protein interaction analysis. Methods: The goal of this study was to notice some important pharmacological targets in S. typhi. using genomic data mining, and protein-protein interaction approaches were used so that new drugs could be developed to combat the disease. Results: In this study, we identified 15 potential target proteins that are critical for S. typhi biofilm growth and maturation. Three proteins, CsgD, AdrA, and BcsA, were deciphered with their significant role in the synthesis of cellulose, a critical component of biofilm's extracellular matrix. The CsgD protein was also shown to have high interconnectedness and strong interactions with other important target proteins of S. typhi. As a result, it has been concluded that CsgD is involved in a range of activities, including cellulose synthesis, bacterial pathogenicity, quorum sensing, and bacterial virulence. Conclusion: All identified targets in this study possess hydrophobic properties, and their cellular localization offered proof of a potent therapeutic target. Overall results of this study, drug target shortage in S. typhi is also spotlighted, and we believe that obtained result could be useful for the design and development of some potent anti-salmonella agents for typhoid fever in the future.

Effects of extended-release 7-nitroindazole gel formulation treatment on the behavior of Shank3 mouse model of autism.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by behavioral deficits such as abnormalities in communication, social interaction, anxiety, and repetitive behavior. We have recently shown that the Shank3 mutation in mice representing a model of ASD causes excessive nitric oxide (NO) levels and aberrant protein S-nitrosylation. Further, 10-day daily injections of 7-NI, a neuronal nitric oxide synthase inhibitor, into Shank3Δ4-22 and Cntnap2(-/-) mutant mice (models of ASD) at a dose of 80 mg/kg reversed the manifestations of ASD phenotype. In this study, we proposed an extended release of 7-NI using a novel drug system. Importantly, unlike the intraperitoneal injections, our new preparation of poly (sebacic acid-co-ricinoleic acid) (PSARA) gel containing 7-NI was injected subcutaneously into the mutant mice only once. The animals underwent behavioral testing starting from day 3 post-injection. It should be noted that the developed PSARA gel formulation allowed a slow release of 7-NI maintaining the plasma level of the drug at ∼45 µg/ml/day. Further, we observed improved memory and social interaction and reduced anxiety-like behavior in Shank3 mutant mice. This was accompanied by a reduction in 3-nitrotyrosine levels (an indicator of nitrative/nitrosative stress) in plasma. Overall, we suggest that our single-dose formulation of PSARA gel is very efficient in rendering a therapeutic effect of 7-NI for at least 10 days. This approach may provide in the future a rational design of an effective ASD treatment using 7-NI and its clinical translation.

Molecular Determinants Involved in Candida albicans Biofilm Formation and Regulation.

Candida albicans is known for its pathogenicity, although it lives within the human body as a commensal member. The commensal nature of C. albicans is well controlled and regulated by the host's immune system as they live in the harmonized microenvironment. However, the development of certain unusual microhabitat conditions (change in pH, co-inhabiting microorganisms' population ratio, debilitated host-immune system) pokes this commensal fungus to transform into a pathogen in such a way that it starts to propagate very rapidly and tries to breach the epithelial barrier to enter the host's systemic circulations. In addition, Candida is infamous as a major nosocomial (hospital-acquired infection) agent because it enters the human body through venous catheters or medical prostheses. The hysterical mode of C. albicans growth builds its microcolony or biofilm, which is pathogenic for the host. Biofilms propose additional resistance mechanisms from host immunity or extracellular chemicals to aid their survival. Differential gene expressions and regulations within the biofilms cause altered morphology and metabolism. The genes associated with adhesiveness, hyphal/pseudo-hyphal growth, persister cell transformation, and biofilm formation by C. albicans are controlled by myriads of cell-signaling regulators. These genes' transcription is controlled by different molecular determinants like transcription factors and regulators. Therefore, this review has focused discussion on host-immune-sensing molecular determinants of Candida during biofilm formation, regulatory descriptors (secondary messengers, regulatory RNAs, transcription factors) of Candida involved in biofilm formation that could enable small-molecule drug discovery against these molecular determinants, and lead to disrupt the well-structured Candida biofilms effectively.

A soft-computation hybrid method for search of the antibiotic-resistant gene in Mycobacterium tuberculosis for promising drug target identification and antimycobacterial lead discovery.

Tuberculosis (TB) control programs were already piloted before the COVID-19 pandemic commenced and the global TB response was amplified by the pandemic. To combat the global TB epidemic, drug repurposing, novel drug discovery, identification and targeting of the antimicrobial resistance (AMR) genes, and addressing social determinants of TB are required. The study aimed to identify AMR genes in Mycobacterium tuberculosis (MTB) and a new anti-mycobacterial drug candidate. In this research, we used a few software to explore some AMR genes as a target protein in MTB and identified some potent antimycobacterial agents. We used Maestro v12.8 software, along with STRING v11.0, KEGG and Pass Server databases to gain a deeper understanding of MTB AMR genes as drug targets. Computer-aided analysis was used to identify mtrA and katG AMR genes as potential drug targets to depict some antimycobacterial drug candidates. Based on docking scores of -4.218 and -6.161, carvacrol was identified as a potent inhibitor against both drug targets. This research offers drug target identification and discovery of antimycobacterial leads, a unique and promising approach to combating the challenge of antibiotic resistance in Mycobacterium, and contributes to the development of a potential futuristic solution.

Paclitaxel Delivery to the Brain for Glioblastoma Treatment.

The development of paclitaxel-loaded polymeric nanoparticles for the treatment of brain tumors was investigated. Poly(lactide-glycolide) (PLGA) nanoparticles containing 10% w/w paclitaxel with a particle size of 216 nm were administered through intranasal and intravenous routes to male Sprague-Dawley rats at a dose of 5 mg/kg. Both routes of administration showed appreciable accumulation of paclitaxel in brain tissue, liver, and kidney without any sign of toxicity. The anti-proliferative effect of the nanoparticles on glioblastoma tumor cells was comparable to that of free paclitaxel.

Cellular Attributes of Candida albicans Biofilm-Associated in Resistance Against Multidrug and Host Immune System.

One of the ubiquitous hospital-acquired infections is associated with Candida albicans fungus. Usually, this commensal fungus causes no harm to its human host, as it lives mutually with mucosal/epithelial tissue surface cells. Nevertheless, due to the activity of various immune weakening factors, this commensal starts reinforcing its virulence attributes with filamentation/hyphal growth and building an absolute microcolony composed of yeast, hyphal, and pseudohyphal cells, which is suspended in an extracellular gel-like polymeric substance (EPS) called biofilms. This polymeric substance is the mixture of the secreted compounds from C. albicans as well as several host cell proteins. Indeed, the presence of these host factors makes their identification and differentiation process difficult by host immune components. The gel-like texture of the EPS makes it sticky, which adsorbs most of the extracolonial compounds traversing through it that aid in penetration hindrance. All these factors further contribute to the multidrug resistance phenotype of C. albicans biofilm that is spotlighted in this article. The mechanisms it employs to escape the host immune system are also addressed effectively. The article focuses on cellular and molecular determinants involved in the resistance of C. albicans biofilm against multidrug and the host immune system.

Secondary metabolites from endophytic fungi: Production, methods of analysis, and diverse pharmaceutical potential.

The synthesis of secondary metabolites is a constantly functioning metabolic pathway in all living systems. Secondary metabolites can be broken down into numerous classes, including alkaloids, coumarins, flavonoids, lignans, saponins, terpenes, quinones, xanthones, and others. However, animals lack the routes of synthesis of these compounds, while plants, fungi, and bacteria all synthesize them. The primary function of bioactive metabolites (BM) synthesized from endophytic fungi (EF) is to make the host plants resistant to pathogens. EF is a group of fungal communities that colonize host tissues' intracellular or intercellular spaces. EF serves as a storehouse of the above-mentioned bioactive metabolites, providing beneficial effects to their hosts. BM of EF could be promising candidates for anti-cancer, anti-malarial, anti-tuberculosis, antiviral, anti-inflammatory, etc. because EF is regarded as an unexploited and untapped source of novel BM for effective drug candidates. Due to the emergence of drug resistance, there is an urgent need to search for new bioactive compounds that combat resistance. This article summarizes the production of BM from EF, high throughput methods for analysis, and their pharmaceutical application. The emphasis is on the diversity of metabolic products from EF, yield, method of purification/characterization, and various functions/activities of EF. Discussed information led to the development of new drugs and food additives that were more effective in the treatment of disease. This review shed light on the pharmacological potential of the fungal bioactive metabolites and emphasizes to exploit them in the future for therapeutic purposes.

Phospholipase C: underrated players in microbial infections.

During bacterial infections, one or more virulence factors are required to support the survival, growth, and colonization of the pathogen within the host, leading to the symptomatic characteristic of the disease. The outcome of bacterial infections is determined by several factors from both host as well as pathogen origin. Proteins and enzymes involved in cellular signaling are important players in determining the outcome of host-pathogen interactions. phospholipase C (PLCs) participate in cellular signaling and regulation by virtue of their ability to hydrolyze membrane phospholipids into di-acyl-glycerol (DAG) and inositol triphosphate (IP3), which further causes the activation of other signaling pathways involved in various processes, including immune response. A total of 13 PLC isoforms are known so far, differing in their structure, regulation, and tissue-specific distribution. Different PLC isoforms have been implicated in various diseases, including cancer and infectious diseases; however, their roles in infectious diseases are not clearly understood. Many studies have suggested the prominent roles of both host and pathogen-derived PLCs during infections. PLCs have also been shown to contribute towards disease pathogenesis and the onset of disease symptoms. In this review, we have discussed the contribution of PLCs as a determinant of the outcome of host-pathogen interaction and pathogenesis during bacterial infections of human importance.

Effect of toxicological interaction of chlorpyrifos, cypermethrin, and arsenic on soil dehydrogenase activity in the terrestrial environment.

Soil is the most widespread area for the co-occurrence of two or more numbers of contaminants. Therefore, toxicity assessments based on contaminants mixture are urgently required to assess their combined impacts on soil enzymes. In the present study, the median effect plot and the combination index isobologram were studied to evaluate the dose-response curve for individual and interactive impacts of chlorpyrifos (Chl), cypermethrin (Cyp), and arsenic (As) on soil dehydrogenase, a potential marker of soil health. Along with these methods, a two-way ANOVA was also tested and the results showed significant changes with respect to different treatments. The results also showed that the Dm value increases in the order of As0.25 fa level. However, Chl + Cyp unveiled a synergistic impact over soil dehydrogenase on day 30th. The overall impact of applied chemicals on dehydrogenase activity was contributed by bioavailability and the nature of toxicological interactions between them. This study would be one of the exclusive studies for the agricultural sector to predict the potential risk associated with the co-existence of these or similar contaminants in the terrestrial environment.

Sodium lignosulfonate causes cell membrane perturbation in the human fungal pathogen Candida albicans.

Underestimating fungal infections led to a gap in the development of antifungal medication. However, rising rates of morbidity and mortality with fungal infection have revealed an alarming rise in antifungal resistance also. Due to the eukaryotic properties of fungi and the close evolutionary similarity between fungal cells and human hosts, therapeutic targeting of Candida infections is troublesome, along with the development of resistance. The discovery of new antifungals is so far behind schedule that the antifungal pipeline is nearly empty. Previously, we have reported the activity and susceptibility of Sodium lignosulfonate (LIG) against C. albicans. In this work, we have established the mechanistic actions of LIG's activity. We performed flow cytometric analysis for membrane integrity, ergosterol binding assay, crystal violet assay, and membrane leakage assay to analyze quantitatively that the C. albicans membrane is being disrupted in response to LIG. Electron microscopic analysis with SEM and TEM confirmed changes in Candida cellular morphology and membrane perturbation respectively. These findings indicated that LIG causes cell membrane damage in C. albicans. This knowledge about LIG's mechanism of action against C. albicans could be used to explore it further as a lead antifungal molecule to develop it as a potent candidate for antifungal therapeutics in the future.