Oxidized Low-density Lipoproteins and Lipopolysaccharides Augment Carotid Artery Plaque Vulnerability in Hypercholesterolemic Microswine.

Atherosclerosis is a chronic inflammatory disease and hypercholesterolemia is a risk factor. This study aims to compare the potency of lipopolysaccharide (LPS) and oxidized low-density lipoproteins (oxLDL) to induce plaque formation and increase plaque vulnerability in the carotid artery of hypercholesterolemic Yucatan microswine. Atherosclerotic lesions at the common carotid artery junction and ascending pharyngeal artery were induced in hypercholesterolemic Yucatan microswine at 5-6 months of age with balloon angioplasty. LPS or oxLDL were administered intraluminally at the site of injury after occluding the arterial flow temporarily. Pre-intervention ultrasound (US), angiography, and optical coherence tomography (OCT) were done at baseline and just before euthanasia to assess post-op parameters. The images from the US, OCT, and angiography in the LPS and the oxLDL-treated group showed increased plaque formation with features suggestive of unstable plaque, including necrotic core, thin fibrous caps, and a signal poor region more with oxLDL compared to LPS. Histomorphology of the carotid artery tissue near the injury corroborated the presence of severe lesions in both LPS and oxLDL-treated pigs but more in the oxLDL group. Vascular smooth muscle and endothelial cells treated with LPS and oxLDL showed increased folds changes in mRNA transcripts of the biomarkers of inflammation and plaque vulnerability compared to untreated cells. Collectively, the results suggest that angioplasty-mediated intimal injury of the carotid arteries in atherosclerotic swine with local administration of LPS or ox-LDL induces vulnerable plaques compared to angioplasty alone and oxLDL is relatively more potent than LPS in inducing vulnerable plaque.

Pyridine coupled pyrazole analogues as lethal weapon against MRSA: An in-vitro and in-silico approach.

The treatment of Methicillin-resistant staphylococcus aureus (MRSA) infections has become challenging due to the growth of multidrug resistance in the bacteria. Here we report the synthesis of pyridine-coupled pyrazoles as an antimicrobial agent against MRSA. A series of pyridine coupled pyrazoles were synthesized and synthesized compounds were characterized using FT-IR, 1H NMR, and Mass spectroscopy. The ADMET results of all the 14 active compounds are interpreted. To identify the potent compound the synthesized compounds screened for minimum inhibitory concentrations against MRSA and compared with standard drug vancomycin. Among the synthesized compounds 6d exhibited good antibacterial activity with MIC value 21 µg/mL, bacterial cell membrane damage study was studied potassium efflux, and cellular content leakage assay. Anticoagulant study for the potent compound also studied and validated by molecular docking and molecular dynamics simulation studies. The docking study of the synthesized compound was carried out and the study depicted that the pyridine ring of all the analogues binds with the various amino acids in the binding pocket of the active site of the Staphylocoagulase and PBP2a protein of MRSA.

Design, synthesis of pyridine coupled pyrimidinone/pyrimidinthione as anti-MRSA agent: Validation by molecular docking and dynamics simulation.

Methicillin Resistant Staphylococcus aureus (MRSA) is a major cause of severe hospital and infections acquired by the population and related morbidity and mortality. In this unique situation, there is a need of dynamic strong drug candidates to control MRSA diseases. Thus, the present work focuses on the synthesis and characterization of pyrimidinones and pyrimidinthiones coupled pyridine derivatives as anti-MRSA agent. The synthesized compounds were characterized by different spectroscopic techniques and evaluated against MRSA strain. Among them, 4e and 4 g possessed better antibacterial activity with MIC values of 10 µg and 8 µg respectively. The key determinant of the wide range beta-lactam resistance in MRSA strains is the Penicillin-Binding Protein 2a (PBP2a) but the gene encodes PBP2a which has a low affinity towards ß-lactam antibiotics. Because of this, the present investigation focused on the mechanism of PBP2a protein binding studies by in-silico studies. The synthesized compounds showed very good interactions with PBP2A compared with standard drug Vancomycin, among them compound 4 g showed better interaction with the binding score of -9.8 kcal/mol. Antibacterial activity was validated with molecular docking and molecular dynamic simulation. Simulation results revealed that protein-ligand interactions of 4 g compound stably sustained up to 20,000ps.Communicated by Ramaswamy H. Sarma.

4-acetamido-3-nitrobenzoic acid - structural, quantum chemical studies, ADMET and molecular docking studies of SARS-CoV2.

In December 2019, a new type of SARS corona virus emerged from China and caused a globally pandemic corona virus disease (COVID-19). This highly infectious virus has been named as SARS-CoV-2 by the International Committee of the Taxonomy of Viruses. It has severely affected a large population and economy worldwide. Globally various scientific communities have been involved in studying this newly emerged virus and is lifecycle. Multiple diverse studies are in progress to design novel therapeutic agents, in which understanding of interactions between the target and drug ligand is a significant key for this challenge. Structures of proteins involved in the life cycle of the virus have been revealed in RCSB PDB by researchers. In this study, we employed molecular docking study of 4-Acetamido-3-nitrobenzoic acid (ANBA) with corona virus proteins (spike protein, spike binding domain with ACE2 receptor and Main protease, RNA-dependent RNA polymerase). Single crystal X-ray analysis and density functional theory calculations were carried out for ANBA to explore the structural and chemical-reactive parameters. Intermolecular interactions which are involved in the ligand-protein binding process are validated by Hirshfeld surface analysis. To study the behaviour of ANBA in a living organism and to calculate the physicochemical parameters, ADMET analysis was done using SwissADME and Osiris data warrior tools. Further, Toxicity of ANBA was predicted using pkCSM online software. Based on the molecular docking analysis, we introduce here a potent drug molecule that binds to the COVID-19 proteins.Communicated by Ramaswamy H. Sarma.

Molecular profiling and anti-infective potency of endophytic actinomycetes inhabiting Madhuca insignis Radlk., from Western Ghats of India.

BACKGROUND: Endophytic actinomycetes are well known for their diverse bioactive entities and considered as an important source for drug development research. RESULTS: We isolated and identified four potential endophytic Streptomyces species, i.e., Streptomyces misionensis MI22, Streptomyces roietensis MI24, Streptomyces glaucescens MI29, and Streptomyces sp. MI04 inhabiting Madhuca insignis by its characteristic morphological features and 16S rRNA gene sequence analysis. S. misionensis MI22 exhibits a broad spectrum of anti-microbial activity against methicillin-resistant Staphylococcus aureus (25.00 ± 1.00 mm) followed by Bacillus subtilis (23.66 ± 0.57 mm), Escherichia coli (22.00 ± 0.00 mm), and Candida albicans (18.00 ± 0.00 mm). Minimum inhibitory concentrations of the ethyl acetate fraction of S. misionensis MI22 against test pathogens were ranged from 25 to 100 µg/mL. Indeed, strain MI22 also exhibited significant anti-proliferative activity against HeLa cell line with IC50 value 98 µg/mL and showed no cytotoxicity effect to the normal human embryonic kidney cell line in the MTT assay. The anti-microbial metabolites from strain MI22 were detected at Rf 0.55 as depicted by the inhibition zone on the intensive band in TLC-bioautography assay. CONCLUSION: The study indicates that, anti-microbial metabolites of these endophytic Streptomyces species, especially S. misionensis MI22 as a prolific source to discover novel bioactive metabolites to combat multidrug-resistant pathogens.

The whole-genome sequence analysis of Enterobacter cloacae strain Ghats1: insights into endophytic lifestyle-associated genomic adaptations.

Enterobacter cloacae is normally considered to be an opportunistic human pathogen. Here, we report on the whole-genome sequence of an endophytic E. cloacae, strain "Ghats1", isolated from leaves of the medicinal plant Coscinium fenestratum Gaertn. Functional analysis of the Ghats1 genome revealed an enrichment for genes involved in the uptake and exchange of nutrients, for chemotaxis and for plant colonization. Unexpectedly though, there were no ORFs belonging to the "virulence factors and antibiotic resistance". Moreover, the presence of hydrolytic enzymes and motility functions reveals the characteristics of an endophyte lifestyle of a bacterium that can colonize and adapt to plant environment. These results provide a better understanding of an endophytic lifestyle through plant-microbe interaction, which can be further exploited as a biocontrol agent.

Isolation and characterization of bioactive compounds with antibacterial, antioxidant and enzyme inhibitory activities from marine-derived rare actinobacteria, Nocardiopsis sp. SCA21.

A rare actinobacteria strain designated SCA21, producing bioactive metabolites was isolated from marine sediment of Havelock Island, Andaman and Nicobar Islands, India. Analysis of 16S rRNA sequences suggested that the strain SCA21 belonged to the genus Nocardiopsis. Chemical investigation of the fermentation broth led to the isolation of two pure bioactive compounds (1-2). Compound 1: 4-bromophenol, a bromophenol derivative; Compound 2: Bis (2-ethylhexyl) phthalate, a phthalate ester. The structure of compound 1 and 2 were elucidated by the detailed analysis of FT-IR, HR-ESI-MS, 1D and 2D NMR, along with literature data analysis. The isolated metabolites were evaluated for enzyme inhibition activity against α-glucosidase and α-amylase, free radical scavenging activity against DPPH and ABTS radicals, metal chelating and antibacterial activity against clinical pathogens. 1 and 2 exhibited remarkable enzyme inhibitory activities against α-glucosidase. However, Compound 2 was found less active against α-amylase. They showed significant free radical scavenging activity against DPPH and ABTS radicals. In addition, except the strain Salmonella typhi ATCC 25241 and Listeria cytogens ATCC 13932, 1 and 2 showed broad spectrum inhibitory activity against MRSA ATCC NR-46171, MRSA ATCC-46071, Klebsiella pneumonia ATCC 13883, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 12600. In conclusion, to best of our knowledge these findings are the first report of isolation of 4-bromophenol and Bis (2-ethylhexyl) phthalate from genus Nocardiopsis, thus suggesting that rare actinomycetes are promising source of therapeutically important bioactive metabolites.

Potent activity of bioconjugated peptide and selenium nanoparticles against colorectal adenocarcinoma cells.

Nanomaterial based anticancer treatment is promising nowadays because of their small size that can penetrate and interact both inside and outside the cell surface. In this study, a simple protocol was followed for the conjugation of the biologically synthesized selenium nanoparticles (SeNPs) and short chain synthetic peptide. SeNPs was synthesized by using the culture supernatant of Streptomyces griseoruber, actinomycetes isolated from the soil. The short chain peptide Boc-L-F-OMe was synthesized by the conventional solution phase chemistry using a racemization-free fragment condensation strategy. Peptide interaction with different anticancer receptors was preliminarily studied by docking studies. Biosynthesized SeNPs was conjugated with short chain synthetic peptides by means of cysteine conjugation. Characterization of SeNPs with peptide was done by UV-visible spectroscopy and DLS that showed the red shift in the peak and increase in average particle size and zeta potential, respectively. Bioconjugated SeNPs- peptide was tested for its cytotoxicity against the colon cancer cell line HT-29. Bioconjugated SeNPs-peptide showed enhanced cytotoxic activity when compared to the peptide and nanoparticle alone that was tested at 10-50 µg/ml concentration. Further apoptotic studies were done by AO/PI staining and DNA fragmentation assay that confirms the cytotoxicity of the conjugates. Novel peptide-SeNPs conjugates tested in our study has a significant anticancer activity that can be potentially used for targeting the cancer cells.

Effect of peptide-conjugated nanoparticles on cell lines.

Metal nanoparticles are widely used for the delivery and targeting of pharmaceutical, therapeutic and diagnostic agents in cancer therapy in recent years. The multifunctional nanoparticles constructed currently are supposed to show superior effects on cancer cells. This study was conducted to observe the difference between the effect of a biologically important peptide, silver (AgNPs) and gold (AuNPs) nanoparticles and their conjugates on two different cancer cells. Peptide (Boc-L-DP-L-OMe) was acquired from different sources and subjected to conjugation with biosynthesized gold and silver nanoparticles under standard conditions. These conjugates were tested against the colon cancer (HT-29) and breast cancer (MDA MB-231) cell lines. The results clearly depicted the improved activity of nanoparticles in the form of conjugates. Fluorescent dye microscopy and DNA fragmentation assay substantiate the fact that the conjugated nanoparticles cause higher level of disintegration of DNA in cells that consecutively damages and causes apoptosis due to lethality.

Attenuation of quorum-sensing-dependent virulence factors and biofilm formation by medicinal plants against antibiotic resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa use small signaling molecules such as acyl homoserine lactones (AHLs), which play an important role in release virulence factors and toxin for further establishment of host infection. Thus, involving with the QS system would provide alternative ways of preventing the pathogenicity. In the present study, totally six medicinal plants (Terminalia bellerica, Celastrus paniculatus, Kingiodendron pinnatum, Schleichera oleosa, Melastoma malabathricum, Garcinia gummi-gutta) were screened for anti-QS activity using biomonitor strain of Chromobacterium violaceum CV12472. The primary screening of antimicrobial activity of all the plant extracts have inhibited the growth of tested bacterial species. Of these at the sub-minimum inhibitory concentration the methanol extract of T. bellerica (0.0625-0.5 mg/ml) has significantly inhibited violacein production (20.07-66.22%) in C. violaceum (CV12472). Consequently, the extract of T. bellerica has reduced the production of pyocyanin, exopolysaccharide and biofilm formation in P. aeruginosa strains. Fluorescence and scanning electron microscopy analysis confirmed the reduction of biofilm formation in P. aeruginosa strains when treated with T. bellerica. GC-MS analysis showed the active compounds inhibited the production of virulence factors of P. aeruginosa. The results suggest the possible use of this T. bellerica as an anti-QS and anti-biofilm agent to control Pseudomonas infection. Interference of QS provides an important means for the inhibition of bacterial virulence and thus aids in treatment strategies.

Prevalence of COMT Val158Met polymorphism in Eastern UP population.

Catechol-O-methyltransferase (COMT) is an abundant S-adenosylmethionine (SAM-)-dependent methyltransferase that methylates catechol compounds, including catecholamines and catecholestrogens.COMT  gene located at chromosome 22q11.2 contains a functional polymorphism at codon 158(Val158Met), which has been related to psychiatric diseases and different types of cancer. COMT might affect tHcy levels because as a by-product it converts SAM to S-adenosylhomocysteine (SAH), which is reversibly converted to homocysteine. The aim of the present study was to determine the frequency of COMT Val158Met polymorphism in scheduled caste (SC) population of Jaunpur district. Total 100 healthy unrelated subjects belonging to SC, between the age group of 18 to70 years were randomly selected for the present study. 3 ml blood samples were collected from each subject. The inclusion criteria of subjects for present study are that they should be domicile of Uttar Pradesh, and healthy without any individual/ family history of genetic or metabolic disorders. COMT Val158Met polymorphism analysis was done by PCR-RFLP method. The Val/Val genotype was found in 48 subjects, Val/Met in 40 subjects and Met/Met genotype in 12 subjects. Genotype frequencies of Val/Val, Val/Met and Met/Met were 0.48, 0.40 and 0.12 respectively. The allele frequency of Val allele was found to be 0.68 and Met allele frequency was 0.32.

Association of C677T polymorphism (rs1801133) in MTHFR gene with depression.

Depression is one of the mental disorders with a state of low mood and aversion to activities that exerts a negative effect on a person's thoughts and behavior. Genetic association studies on MTHFR C677T polymorphism and depression have been repeatedly performed over the last two decades, but results are inconsistent. The aim of the present study was to assess the relationship between MTHFR C677T polymorphism and depression by literature review and meta-analysis. Four electronic databases, PubMed, Google Scholar, Science direct and Springer Link were searched for case control articles focusing on MTHFR C677T polymorphism and the risk of depression. A total of 30 studies including 4,802 cases and 17,362 controls were involved in present meta-analysis. When all the eligible studies were pooled into this meta-analysis,  significant association between depression risk and MTHFR C677T polymorphism was found in three genetic models (Additive model: OR T vs C= 1.20, 95 % CI= 1.00-1.34, p=0.0004; homozygote model: OR TT vs.CC=1.37, 95% CI= 1.13-1.65, p=0.0004; dominant model: OR TT+CT vs CC=1.13, 95 % CI= 0.99-1.28, p=0.04), while meta-analysis with other two genetic models did not show association with other two genetic models ( recessive model: OR TT vs CT+CC= 1.36, 95 % CI = 0.91-2.04, p=0.13; co-dominant model: OR CT vs CC=1.00, 95 % CI=0.93-1.08,p=0.84). Present meta-analysis supports that there is a meager significant association between MTHFR C677T polymorphism and depression risk.

Actinomycetes mediated synthesis of gold nanoparticles from the culture supernatant of Streptomyces griseoruber with special reference to catalytic activity.

Biogenic synthesis of nanoparticles has received a tremendous attention from the past few decades. The significant progress in the field of nanotechnology has resulted in a cost-effective and eco-friendly process for nanoparticle synthesis. In the present study, the extracellular synthesis of gold nanoparticles was carried out using culture supernatant of Streptomyces griseoruber, actinomycetes isolated from the soil. Bioreduction of gold nanoparticles was confirmed by UV-visible spectrophotometer that showed the peak between 520 and 550 nm. The crystalline nature and mean size of the GNPs were confirmed using XRD. FTIR revealed the possible functional group that could be useful in immobilisation and stabilisation of GNPs. Size and distribution of the biosynthesized GNPs were analysed by HR-TEM that showed the formation of GNPs in the range of 5-50 nm. The synthesised GNPs showed good catalytic activity for the degradation of methylene blue. The study shows the rapid and eco-friendly synthesis of GNPs from Streptomyces griseoruber, and this is the first report on the catalytic activity of GNPs from actinomycetes so far.

Visible upconversion in Er3+/Yb3+ co-doped LaAlO3 phosphors.

The Er3+ doped and Er3+/Yb3+ co-doped LaAlO3 phosphors have been synthesized by the combustion method and characterized their structural, morphological, elemental, vibrational and optical properties. The optical absorption and upconversion properties of the synthesized phosphors have been studied. Upon co-doping Yb3+ ions into Er3+:LaAlO3, the blue, green and red upconversion emissions of Er3+ ions have been enhanced about ~20, ~54 and ~22 times, under 978nm laser excitation. The observed upconversion emissions could be due to excited state absorption in Er3+:LaAlO3, whereas energy transfer is dominant mechanism in Er3+/Yb3+:LaAlO3 phosphors. The tuning in the color emitted from the synthesized phosphors towards the green region has been found due to incorporation of the Yb3+ ions. With increase in the pump power, the color emitted from the co-doped phosphor is not tuned significantly, showing its applicability in making the green display devices.

Dualistic temperature sensing in Er3+/Yb3+ doped CaMoO4 upconversion phosphor.

Temperature sensing performance of Er3+/Yb3+ doped CaMoO4 phosphor prepared via polyol method is reported herein. The X-ray diffraction, Fourier transform infrared spectroscopy and field emission scanning electron microscopy are done to confirm the phase, structure and purity of the synthesized phosphor. The infrared to green upconversion emission is investigated using 980nm diode laser excitation along with its dependence on input pump power and external temperature. The temperature dependent fluorescence intensity ratio of two upconversion emission bands assigned to 2H11/2→4I15/2 (530nm) and 4S3/2→4I15/2 (552nm) transitions has shown two distinct slopes in the studied temperature range - 300 to 760K and therefore, dual nature of temperature sensitivity is observed in this phosphor. This phenomenon in rare earth doped materials is either scarcely reported or overlooked. The material has shown higher sensitivity in the high temperature region (535K

Effect of gamma irradiation on X-ray absorption and photoelectron spectroscopy of Nd-doped phosphate glass.

X-ray absorption near-edge structure (XANES) and X-ray photoelectron spectroscopy (XPS) of Nd-doped phosphate glasses have been studied before and after gamma irradiation. The intensity and the location of the white line peak of the L3-edge XANES of Nd are found to be dependent on the ratio O/Nd in the glass matrix. Gamma irradiation changes the elemental concentration of atoms in the glass matrix, which affects the peak intensity of the white line due to changes in the covalence of the chemical bonds with Nd atoms in the glass (structural changes). Sharpening of the Nd 3d5/2 peak profile in XPS spectra indicates a deficiency of oxygen in the glasses after gamma irradiation, which is supported by energy-dispersive X-ray spectroscopy measurements. The ratio of non-bridging oxygen to total oxygen in the glass after gamma radiation has been found to be correlated to the concentration of defects in the glass samples, which are responsible for its radiation resistance as well as for its coloration.

Structural and optical properties of thermal decomposition assisted Gd2O3:Ho(3+)/Yb(3+) upconversion phosphor annealed at different temperatures.

The infrared to visible upconversion fluorescent nanoparticles of Ho(3+)/Yb(3+) codoped Gd2O3 phosphor is synthesized via thermal decomposition route. The as-synthesized sample was annealed at 800, 1000 and 1200°C for 3h and then structural and optical properties were studied. The Rietveld refinement of X-ray diffraction (XRD) data was analyzed to probe the effect of Ho(3+)/Yb(3+) dopant on the structural parameters of Gd2O3 host. The upconversion emission spectra of as-synthesized and annealed samples are compared using 980nm diode laser excitation and five emission bands noticed at 490, 539, 550, 667 and 757nm corresponding to the (5)F3→(5)I8, (5)F4→(5)I8, (5)S2→(5)I8,(5)F5→(5)I8 and (5)I4→(5)I8 manifolds, respectively. The local temperature induced by laser light is also calculated. The fluorescence intensity ratio (FIR) of two thermally coupled transitions (5)F4→(5)I8 and (5)S2→(5)I8 is plotted against the sample temperature and sensor sensitivity of sample is calculated.

Genetics of Fusarium Wilt Resistance in Pigeonpea (Cajanus cajan) and Efficacy of Associated SSR Markers.

Inheritance of resistance to Fusarium wilt (FW) disease caused by Fusarium udum was investigated in pigeonpea using four different long duration FW resistant genotypes viz., BDN-2004-1, BDN-2001-9, BWR-133 and IPA-234. Based on the F2 segregation pattern, FW resistance has been reported to be governed by one dominant gene in BDN-2004-1 and BDN-2001-9, two duplicate dominant genes in BWR-133 and two dominant complimentary genes in resistance source IPA-234. Further, the efficacy of six simple sequence repeat (SSR) markers namely, ASSR-1, ASSR-23, ASSR-148, ASSR-229, ASSR-363 and ASSR-366 reported to be associated with FW resistance were also tested and concluded that markers ASSR-1, ASSR-23, ASSR-148 will be used for screening of parental genotypes in pigeonpea FW resistance breeding programs. The information on genetics of FW resistance generated from this study would be used, to introgress FW resistance into susceptible but highly adopted cultivars through marker-assisted backcross breeding and in conventional breeding programs.

Inhibition of a sulfate reducing bacterium, Desulfovibrio marinisediminis GSR3, by biosynthesized copper oxide nanoparticles.

To control the severe problem of microbiologically influenced corrosion, industries require highly potent antibacterial agent which can inhibit the growth of bacteria on man-made surfaces. This need drove the research towards the synthesis of nanoscale antimicrobial compounds. We, therefore, screened several bacteria for the biosynthesis of copper/copper compound nanoparticles which could inhibit the growth of Desulfovibrio marinisediminis, a sulfate reducing bacterium. Supernatant of thirty bacteria isolated from the biofilm formed on ship hull was mixed with 1 mM CuCl2 solution at room temperature. Eight bacterial strains, whose mixtures exhibited colour change, were selected for antimicrobial test. One nanoparticle which has been biosynthesized by Shewanella indica inhibited the growth of D. marinisediminis. Characterization of this particle by UV-visible spectrophotometer, XRD, TEM, DLS and FTIR showed that the particle is polydisperse CuO nanoparticle with average size of 400 nm.

Identification of the traditional and non-traditional sulfate-reducing bacteria associated with corroded ship hull.

Pitting corrosion due to microbial activity is the most severe type of corrosion that occurs in ship hull. Since biogenic sulfide produced by sulfate-reducing bacteria (SRB) is involved in the acceleration of pitting corrosion of marine vessels, so it is important to collect information about SRB community involved in maritime vessel failure. We investigated the SRB community on corroded hull portion of the ship. With the use of common cultural method and 16S rDNA sequencing, ten bacteria with sulfate reduction ability were isolated and identified. They belonged to both traditional (Desulfovibrio, Desulfotomaculum) and non-traditional (Citrobacter) sulfate-reducing bacteria. All the isolates were able to produce a high amount of sulfide. However, only traditional isolates were showing the amplification for the SRB-specific gene, dsrAB. Further studies on corrosion potential of these two groups of bacteria showed that in spite of high sulfide and biofilm production by non-traditional SRB, they are less aggressive towards the mild steel compare to the traditional group.