Target association rule mining to explore novel paediatric illness patterns in emergency settings.

BACKGROUND AND AIMS: To assess the hospitalized sick children admitted to the pediatric emergency department (ED) and to find new patterns of clinical and laboratory attributes using association rule mining (ARM). METHODS: In this observational study, 158 children with median (IQR) age 11 months and a PRISM III score of 5 (2-9) were enrolled. Hotspot data mining method was applied to assess clinical attributes, lab investigations and pre-defined outcome parameters of children and their association in sick hospitalized children aged 1 month to 12 years. RESULTS: We obtained 30 rules with value for outcome as discharge is given attributes as follows: duration of hospitalization > 4 days, lactate > 1.2 mmol/L, platelet = 3.67/µL, dur_ventil = 0 h, serum K = 5.2 mmol/L, SBP = 120 mmHg, pCO2 = 41.9 mmHg, PaO2 = 163 mmHg, age = 92 months, heart rate > 114-159 per minute, temperature > 98 °F, GCS (Glasgow Coma Scale) > 7-14, gas K = 4.14 mmol/L, gas Na = 138.1 mmol/L, BUN (Blood Urea Nitrogen) = 18.69 mg/dL, Diagnosis > 1-718, Creatinine = 1.2 mg/dL, serum Na = 148 mmol/L, shock = 2, Glucose = 144 mg/dL, Mg(i) > 0.23 meq/L, BUN > 6.54 mg/dL. CONCLUSION: ARM is an effective data analysis technique to find meaningful patterns using clinical features with actual numbers in pediatric critical illness. It can prove to be important while analysing the association of clinical attributes with disease pattern, its features, and therapeutic or intervention success patterns.

Urease Inhibitory Kinetic Studies of Various Extracts and Pure Compounds from Cinnamomum Genus.

Urease is an enzyme that plays a significant role in the hydrolysis of urea into carbonic acid and ammonia via the carbamic acid formation. The resultant increase in pH leads to the onset of various pathologies such as gastric cancer, urolithiasis, hepatic coma, hepatic encephalopathy, duodenal ulcers and peptic ulcers. Urease inhibitors can reduce the urea hydrolysis rate and development of various diseases. The Cinnamomum genus is used in a large number of traditional medicines. It is well established that stem bark of Cinnamomum cassia exhibits antiulcerogenic potential. The present study evaluated the inhibitory effect of seven extracts of Cinnamomum camphora, Cinnamomum verum and two pure compounds Camphene and Cuminaldehyde on urease enzyme. Kinetic studies of potential inhibitors were carried out. Methanol extract (IC50 980 µg/mL) of C. camphora and a monoterpene Camphene (IC50 0.147 µg/mL) possess significant inhibitory activity. The Lineweaver Burk plot analysis suggested the competitive inhibition by methanol extract, hexane fraction and Camphene. The Gas Chromatography-Mass Spectroscopy (GC-MS) analysis of hexane fraction revealed the contribution of various terpenes. The present study targets terpenes as a new class of inhibitors that have potential therapeutic value for further development as novel drugs.

Gum xanthan-psyllium-cl-poly(acrylic acid-co-itaconic acid) based adsorbent for effective removal of cationic and anionic dyes: Adsorption isotherms, kinetics and thermodynamic studies.

The present work highlights the synthesis of the adsorbent based on Gum xanthan-psyllium hybrid backbone graft co-polymerized with polyacrylic acid-co-polyitaconic acid chains for the rapid sequestration of auramine-O (Aur-O) and eriochrome black-T (EBT) dyes from the aqueous fluid. The excellent dye removal efficiency of 90.53% for EBT and 95.63% for Aur-O was found at initial dye concentration of 30mgL-1 (EBT) and 15 mgL-1 (Aur-O) 40mL-1 with an adsorbent dose of 600mg within time duration of 5h and 323K temp. The adsorption isotherm data fitted well with Langmuir isotherm and Freundlich isotherm for Aur-O and EBT dyes (R2 ≥ 0.90), respectively. The adsorption kinetics depicted that pseudo-second order kinetics was followed simultaneously with intra-particle diffusion for both the dyes. Thermodynamic parameters such as ΔG°, ΔH° and ΔS° were also calculated and confirmed the spontaneity, randomness and endothermic nature of the adsorption process. Further, the adsorbent exhibited good recyclability efficiency for the capture of Aur-O and EBT from aqueous solution with minimal activity decline after six and three cycles, respectively. So, the synthesized adsorbent could be used successfully by the textile industries for the treatment of dye contaminated water with excellent competency.

Bone-specific alkaline phosphatase - a potential biomarker for skeletal growth assessment.

OBJECTIVE: The present study was aimed to assess levels of serum Bone-specific alkaline phosphatase (BALP) and serum Insulin-like growth factor-1 (IGF-1) and comparing with cervical vertebral maturation index (CVMI) stages. DESIGN: Cross-sectional study. SETTING: Maulana Azad Institute of Dental Sciences, New Delhi, India. PARTICIPANTS: 150 subjects (75 males and 75 females) in the age group of 8-20 years. METHODS: Subjects were divided into six CVMI stages. Enzyme-linked immunosorbant assay was performed for the estimation of serum BALP and serum IGF-1 levels. Mann-Whitney U test was performed to compare mean ranks of serum BALP and serum IGF-1 with different CVMI stages. Spearman correlation between serum BALP and serum IGF-1 was done across 6 CVMI stages. RESULTS: Peak serum IGF-1 levels were found at CVMI stages 4 and 3 for males and females respectively. Peak levels for serum BALP were found at stage 3 for both genders with significant differences from other stages. A statistically significant correlation was seen between serum IGF-1 and serum BALP from CVMI stages 1 to 3 and 4 to 6 (p < .01). CONCLUSIONS: BALP showed promising results and can be employed as a potential biomarker for the estimation of growth status.

Sequestration of dyes from artificially prepared textile effluent using RSM-CCD optimized hybrid backbone based adsorbent-kinetic and equilibrium studies.

Present work reports the synthesis of semi-Interpenetrating Network Polymer (semi-IPN) using Gelatin-Gum xanthan hybrid backbone and polyvinyl alcohol in presence of l-tartaric acid and ammonium persulphate as the crosslinker-initiator system. Reaction parameters were optimized with Response Surface Methodology (RSM) in order to maximize the percent gel fraction of the synthesized sample. Polyvinyl alcohol, l-Tartaric acid, ammonium persulphate, reaction temperature, time and pH of the reaction medium were found to make an impact on the percentage gel fraction obtained. Incorporation of polyvinyl alcohol chains onto hybrid backbone and crosslinking between the different polymer chains were confirmed through techniques like FTIR, SEM-EDX and XRD. Semi-IPN was found to be very efficient in the removal of cationic dyes rhodamine-B (70%) and auramine-O (63%) from a mixture with an adsorbent dose of 700 mg, initial concentration of rhodamine-B 6 mgL-1 and auramine-O 26 mgL-1, at an time interval of 22-25 h and 30 °C temp. Further to determine the nature of adsorption Langmuir and Freundlich adsorption isotherm models were studied and it was found that Langmuir adsorption isotherm was the best fit model for the removal of mixture of dyes. Kinetic studies for the sorption of dyes favored the reaction mechanism to occur via a pseudo second order pathway with R2 value about 0.99.

Case of Hallervorden-Spatz Syndrome: A Tale of Twin Sisters.

Hallervorden-Spatz syndrome, now known as pantothenate kinase-associated neurodegeneration (PKAN), is a rare autosomal recessive disorder that is characterized by cerebral iron deposition and leads to progressive extrapyramidal dysfunction and dementia. Most commonly seen in the first two decades of a person's life, it is a differential for patients presenting with atypical progressive extrapyramidal disorder and cognitive impairment. It is characterized by progressive degeneration of the basal ganglia, globus pallidus, and the reticular part of the substantia nigra due to iron accumulation. The characteristic MRI brain pattern of the disease shows the eye-of-the-tiger sign. We report cases of early onset PKAN in two sisters of the same family, in which diagnosis was based on clinical features, lab parameters, and MRI imaging findings. This report aims to differentiate PKAN from other static and progressive neurological illnesses.

Insights on antioxidant therapeutic strategies in type 2 diabetes mellitus: A narrative review of randomized control trials.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a metabolic disease of impaired glucose utilization. Imbalance in generation and elimination of free radicals generate oxidative stress which modulates glucose metabolism and insulin regulation, resulting in the occurrence and progression of diabetes and associated complications. Antioxidant supplements in T2DM can be seen as a potential preventive and effective therapeutic strategy. AIM: To compare randomized controlled trials (RCTs) in which antioxidants have been shown to have a therapeutic effect in T2DM patients. METHODS: We systematically searched the electronic database PubMed by keywords. RCTs evaluating the effect of antioxidant therapy on glycaemic control as well as oxidant and antioxidant status as primary outcomes were included. The outcomes considered were: A reduction in blood glucose; changes in oxidative stress and antioxidant markers. Full-length papers of the shortlisted articles were assessed for the eligibility criteria and 17 RCTs were included. RESULTS: The administration of fixed-dose antioxidants significantly reduces fasting blood sugar and glycated hemoglobin and is associated with decreased malondialdehyde, advanced oxidation protein products, and increased total antioxidant capacity. CONCLUSION: Antioxidant supplements can be a beneficial approach for the treatment of T2DM.

Assessment of Biograft-HT with I-PRF Graft in Immediate Dental Implant Placement.

Objectives: This research was done to assess the efficacy of I-PRF and bone graft in immediate dental implant placement. Materials and Method: Twenty patients were selected randomly into 2 groups with 10 samples in each as Group I- using I-PRF and Group II with synthetic bone replacement alloplast (biograft-HT) after immediate implant placement. Postoperative clinical assessment after graft placement was done based on visual analog scale for pain, modified gingival index and modified plaque index at 2nd, 4th, and 6th months. A radiographic assessment of bone density was performed two and six months after the placement of the implant. Result: There was a statistically considerable variation between the change in modified plaque index and modified gingival index. There was a statistically insignificant divergence in the mean visual analog scale between the two groups at 1, 3, and 6 days. Group I demonstrated a higher change in bone density than group II, with a statistically significant difference (P = .002). Conclusion: An innovative, safe, and efficient method for controlling the healing process around immediate dental implants is provided by the use of I-PRF in conjunction with immediate dental implant placement.

Application of Thermostable Xylanase of Bacillus pumilus in Textile Processing.

Desizing of cotton and micropoly fabrics was done using thermostable xylanase from Bacillus pumilus ASH. Micropoly fabric showed better desizing than cotton under same conditions. Violet scale readings from the TEGEWA test after enzymatic desizing for 90 min at pH 7.0 and at 60°C showed the readings falling in the range of 4-5, indicating good desizing efficiency. During bioscouring the weight loss values and liberation of reducing sugars were highest when EDTA was used along with xylanase. The weight loss value of 1.5% was observed for dry cotton fabric after 1 h in case of agitated system at pH 7.0 and at an optimal enzyme dosage of 5 IU/g. The weight loss values and the liberation of reducing sugars were higher in case of cotton fabrics. Wetting time of fabrics was lowered significantly after 60 min of bioscouring using xylanase. Increase in temperature or concentration of surfactant led to further reduction in the wetting time. The whiteness values of fabrics after bioscouring were 0.9% higher than the chemically scoured fabrics indicating good efficacy of xylanase during the scouring process.

Novel insights into host responses to Japanese Encephalitis Virus infection: Reanalysis of public transcriptome and microRNAome datasets.

PURPOSE: Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV), is the principal cause of viral encephalitis in South-East Asian and Western Pacific countries; accounting for 68,000 cases, and up to 20,400 fatalities, annually across the world. Despite being a high-risk condition, there is no specific treatment for JE. Given rapid additions in genomics databases and the power of data reanalysis in addressing critical medical questions, the present study was designed to identify novel host factors that might have potential roles in JEV infection. METHODS: We extracted microarray and RNA-Seq data sets from NCBI-GEO and compared mock and JEV-infected samples. Raw data from all the studies were re-analyzed to identify host factors associated with JEV replication. RESULTS: We identified several coding and non-coding host factors that had no prior known role in viral infections. Of these, the coding transcripts: Myosin Heavy Chain 10 (MYH10), Progestin and AdipoQ Receptor Family Member 8 (PAQR8), and the microRNAs: hsa-miR-193b-5p, hsa-miR-3714 and hsa-miR-513a-5p were found to be novel host factors deregulated during JEV infection. MYH10 encodes a conventional non-muscle myosin, and mutations in MYH10 have been shown to cause neurological defects. PAQR8 has been associated with epilepsy, which exhibits symptoms similar to JEV infection. JE is a neuro-degenerative disease, and the known involvement of MYH10 and PAQR8 in neurological disorders strongly indicates potential roles of these host factors in JEV infection. Additionally, we observed that MYH10 and PAQR8 had a significant negative correlation with Activating transcription factor 3 (ATF3), which is a previously validated modulator of JEV infection. ATF3 is a transcription factor that binds to the promotors of genes encoding other transcription factors or interferon-stimulated genes and negatively regulates host antiviral responses during JE. CONCLUSION: Our findings demonstrate the significance of data reanalysis in the identification of novel host factors that may become targets for diagnosis/ therapy against viral diseases of major concern, such as, JE. The deregulated coding and non-coding transcripts identified in this study need further experimental analysis for validation.

Xylanase production using agro-residue in solid-state fermentation from Bacillus pumilus ASH for biodelignification of wheat straw pulp.

Two stage statistical design was used to optimize xylanase production from Bacillus pumilus ASH under solid-state fermentation. Initially, Plackett-Burman designing (PB) was used for the selection of crucial production parameters. Peptone, yeast extract, incubation time, moisture level and pH were found to be the crucial factors for the xylanase production. Crucial variables were further processed through central composite designing (CCD) of response surface methodology (RSM) to maximize the xylanase yield. Each significant factor was investigated at five different levels to study their influence on enzyme production. Statistical approach resulted in 2.19-fold increase in xylanase yield over conventional strategy. The determination coefficient (R (2)) as shown by analysis of variance (ANOVA) was 0.9992, which shows the adequate credibility of the model. Potential of cellulase-free xylanase was further investigated for biobleaching of wheat straw pulp. Xylanase aided bleaching through XCDED(1)D(2) sequence resulted in 20 and 17% reduction in chlorine and chlorine dioxide consumption as compared to control. Significant increase in pulp brightness (%ISO), whiteness and improvement in various pulp properties was also observed.

SARS-CoV-2 in Pregnancy: Fitting Into the Existing Viral Repertoire.

The risk of viral infection during pregnancy is well-documented; however, the intervention modalities that in practice enable maternal-fetal protection are restricted by limited understanding. This becomes all the more challenging during pandemics. During many different epidemic and pandemic viral outbreaks, worse outcomes (fetal abnormalities, mortality, preterm labor, etc.) seem to affect pregnant women than what has been evident when compared to non-pregnant women. The condition of pregnancy, which is widely understood as "immunosuppressed," needs to be re-understood in terms of the way the immune system works during such a state. The immune system gets transformed to accommodate and facilitate fetal growth. The interference of such supportive conversion by viral infection and the risk of co-infection lead to adverse fetal outcomes. Hence, it is crucial to understand the risk and impact of potent viral infections likely to be encountered during pregnancy. In the present article, we review the effects imposed by previously established and recently emerging/re-emerging viral infections on maternal and fetal health. Such understanding is important in devising strategies for better preparedness and knowing the treatment options available to mitigate the relevant adverse outcomes.

A study elucidating the relation between cellulose dissolution and crystallinity after cellulase treatment at different doses.

Cellulose is the most abundant renewable resource which has found a diverse range of applications. Cellulose dissolution is a significant property for manufacturing man-made cellulosic fiber through viscose process. Crystalline microfibrillar structure and relatively high ordered packing of polymeric chains contribute to recalcitrance and poor reactivity of cellulose. One of the most common methods to improve cellulose dissolution is cellulase treatment. Herein, cellulase treatment at different doses was studied to explore the correlation of cellulose dissolution with crystallinity. Pulp showed improvement in Fock reactivity and other properties related to viscose application. But contrary to previous studies, cellulose crystallinity as determined by XRD and FTIR did not correlate with Fock reactivity at a higher dose of cellulase. The results indicated some complex mechanism to be involved between the cellulose dissolution and crystallinity than a simple negative correlation. Cellulase treatment at 150 HCU/g resulted in the upgraded pulp suitable for viscose application.

Clinical Predictors of COVID-19 Severity and Mortality: A Perspective.

The COVID-19 pandemic has caused huge socio-economic losses and continues to threat humans worldwide. With more than 4.5 million deaths and more than 221 million confirmed COVID-19 cases, the impact on physical, mental, social and economic resources is immeasurable. During any novel disease outbreak, one of the primary requirements for effective mitigation is the knowledge of clinical manifestations of the disease. However, in absence of any unique identifying characteristics, diagnosis/prognosis becomes difficult. It intensifies misperception and leads to delay in containment of disease spread. Numerous clinical research studies, systematic reviews and meta-analyses have generated considerable data on the same. However, identification of some of the distinct clinical signs and symptoms, disease progression biomarkers and the risk factors leading to adverse COVID-19 outcomes warrant in-depth understanding. In view of this, we assessed 20 systematic reviews and meta-analyses with an intent to understand some of the potential independent predictors/biomarkers/risk factors of COVID-19 severity and mortality.

Partial purification of bacterial cellulo-xylanolytic enzymes and their application in deinking of photocopier waste paper.

The potential of alkaline cellulo-xylanolytic enzymes from non-pathogenic Bacillus subtilis strain was tested for deinking of photocopier waste paper. Cellulase and xylanase play a crucial role in deinking of different types of waste paper. Partial purification of cellulo-xylanolytic enzymes was carried out using ultrafiltration followed by ammonium sulfate precipitation. The ultrafiltered enzyme was used for deinking the photocopier waste paper along with chemical deinking. An enzyme dose of 0.6 IU/g and reaction time of 60 min for ultrafiltered cellulo-xylanolytic enzyme significantly increased deinking efficiency, tear index (9.52%) and folding endurance (5±2%) as compared to chemical deinking. There was improvement in strength properties such as tear index and double-fold along with freeness of pulp (18%). There was slight decrease in tensile index (0.6%) and burst index (16%) while ISO brightness remained unaffected. Enzymatic deinking (74.3%) by ultrafiltered cellulo-xylanolytic from Bacillus subtilis was found significant over conventional chemical deinking.

Effect of electron-phonon interaction and valence band edge shift for carrier-type reversal in layered ZnS/rGO nanocomposites.

The artificial stacking of nanohybrid films helps to enhance their properties and thus intrigues researchers to explore this possibility in emerging technologies. The layer-by-layer approach was used to fabricate samples of zinc sulfide/reduced graphene oxide (ZnS/rGO) by using spin coating technique. The structure and optoelectronic properties has been extensively studied by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), UV-VIS-NIR spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and Hall measurements. Raman spectrum elucidates the phonon contribution of ZnS and breathing mode of κ-point phonons and sp2 bonds of carbon atoms of rGO. The electron-phonon interactions reveal reduction in electron mobility and enhancement in holes contribution with rGO content leading to surface charge transfer doping (SCTD). XPS results explain the valence band edge and conduction band edge to form type-I band alignment to reconfirm carrier-type reversal. A change in the dispersion of refractive indices along with a small rise in the value of absorption coefficient in terahertz (THz) region for ZnS/rGO nanocomposite films has been observed. These results will open up new opportunities to furthering the science of this technologically important class of materials for future electronics.

Identification, characterization, and regulation of a novel antifungal chitosanase gene (cho) in Anabaena spp.

Two contrasting cyanobacterial species (Anabaena fertilissima and Anabaena sphaerica) were selected based on differences in antifungal behavior in order to study the mechanism for production of an antifungal enzyme and the genes responsible for this production. In A. fertilissima, chitosanase and antifungal activities were increased significantly under of growth-limiting conditions (8 of light and 16 h of darkness). The lack of such activities in A. sphaerica was associated with high levels of protein that accumulated during the stationary phase (at 28 days) under the same light conditions. The gene putatively responsible for chitosanase and antifungal activities was amplified using specific primers, and sequence analysis of the amplified products (1.086 and 1.101 kb in A. sphaerica and A. fertilissima, respectively) showed that they belong to the glycoside hydrolase 3 (GH3)-like family of Anabaena variabilis ATCC 29413. Pairwise alignment of the corresponding protein sequences identified a putative signal peptide (amino acids 1 to 23) and some amino acid changes in the sequence of A. fertilissima which may be responsible for functioning of the chitosanase and the observed antifungal activity. Hydrolysis of the chitosan oligosaccharide (GlcN)(5) to (GlcN)(2) and (GlcN)(3) confirmed the presence of chitosanase activity in A. fertilissima. Site-directed mutagenesis of the A. fertilissima chitosanase-encoding gene (cho) led to identification of catalytic residues (Glu-121 and Glu-141) important for the antifungal effect of the cho product. The level of expression of cho was monitored by quantitative real-time reverse transcription-PCR, which indicated that transcription of this gene is significantly enhanced under conditions that retard growth, such as a long dark period.

Antibacterial and antifungal studies of macrocyclic complexes of trivalent transition metal ions with their spectroscopic approach.

A new series of complexes of the type [M(C24H16N4)X]X2, where M = Cr(III), Fe(III), and Mn(III), X = Cl-, NO3-, and CH3COO-, has been synthesized by template condensation of 1,8-diaminonaphthalene and glyoxal in the presence of trivalent metal salts in methanolic medium. The complexes have been characterized with the help of elemental analysis, conductance measurements, magnetic measurements, and electronic, NMR, IR, and mass spectral studies. On the basis of these studies, a five-coordinate square pyramidal geometry for all of these complexes has been proposed. All the synthesized metal complexes were also tested for their in vitro antimicrobial activities against some bacterial strains, viz. Bacillus subtilis, Bacillus stearothermophilus (gram-positive bacteria), Escherichia coli, and Pseudomonas putida (gram-negative bacteria), and some fungal strains, viz. Aspergillus flavus and Aspergillus niger. The results obtained were compared with standard antibiotics: chloramphenicol, streptomycin, and the antifungal drug cyclohexamide. Some of the tested complexes showed remarkable antimicrobial activities.

Water quality assessment of river Hindon at Ghaziabad, India: impact of industrial and urban wastewater.

River Hindon is a major source of water to the highly populated and predominantly rural population of western Uttar Pradesh, India. The main goal of the present study was to assess the impact of urban and industrial activities on the water quality of river Hindon at the Ghaziabad. For this, river water samples were collected from six different sites all along the route of Hindon main streamline and its branch and were analyzed for pH, turbidity, electrical conductivity (EC), total dissolved solids (TDS), total alkalinity (TA), total hardness (TH) and calcium hardness (Ca-H), chemical oxygen (COD) demand, biochemical oxygen demand (BOD), dissolved oxygen (D.O.), sulphate (as SO(4)(2-)), nitrate (as NO(3)(-)) and chloride (Cl-) levels. There were drastic variations for EC (0.83-5.04 ms), turbidity (28.7-109.3 NTU), TDS (222.2-2426.3 mg l(-1)), SO(4) (36.4-162.4 mg l(-1)), NO(3) (106-245 mg l(-1)), TA (347.0-596.3 mg l(-1)), TH (235.1-459.9 mg l(-1)), Ca-H (64.5-402.2 mg l(-1)), BOD (27-51 mg l(-1)) and COD (85.0-337.4 mg l(-1)) levels at different sites. Water pollution indicating parameters were manifold higher than the prescribed limit by the National Pollution Control Agency, i.e. CPCB. This is the first study on itself and the interrelationship of human activities and river water quality makes the study significant and interesting to assess the pollution load discharges in catchments of Hindon at Ghaziabad. Overall, the water quality of Hindon was relatively poor with respect to its use for domestic purposes.

Influence of endosulfan on microbial biomass and soil enzymatic activities of a tropical alfisol.

The effect of endosulfan at normal residue level (1 and 10 ppm) and elevated level (100 ppm) on microbial biomass and enzymatic activities of a tropical alfisol was studied. Dehydrogenase, Fluorescein diacetate hydrolase, acid phosphatase, aryl sulphatase activities and microbial biomass of the soil increased by 2.4, 1.7, 1.4, 1.8 and 3.7 times, respectively by the 14th day of incubation with 1 ppm endosulfan, indicating the possible involvement of soil microorganisms and their enzymes in degradation of endosulfan. Soil nitrogenase activity decreased by 8.0 times by the 14th day of application of 1 ppm endosulfan, suggesting that endosulfan or its metabolites may pose toxicological threat to nitrogen fixers in soil.